Herpes simplex keratitis (HSK) often presents either as dendritic or geographic ulcers [1]. Atypical presentations of HSK have been reported [1,2]. The diagnosis of this condition can pose difficulties if patients present at later stages of the disease, especially when associated with corneal perforation. We report here an unusual case of perforated corneal ulcer with a large infiltrate, caused by HSV-1 in a contact lens wearer, initially diagnosed as bacterial keratitis. A simple investigation such as microscopic examination of Giemsa stained corneal scraping provided a clue to the diagnosis.

An eighteen-year-old female student presented to us with complaints of pain, redness and gradual decrease in vision in the right eye of 20 days duration. She was treated with 0.3% ciprofloxacin eye drops, by a local ophthalmologist. She had discontinued using the medication prior to presentation to us. She was not treated with steroids at any point of time. She was a myope using daily wear monthly disposable hydrogel contact lenses since eight months which she discontinued wearing with the onset of the present symptoms.

On examination, her visual acuity was restricted to perception of light and accurate projection of rays in the right eye and 20/20 in the left eye. Slit lamp examination of left eye was within normal limits. The right eye showed congestion of bulbar conjunctiva. Cornea showed a paracentral solitary, large, well circumscribed, full thickness, and yellowish white stromal infiltrate measuring 4.5 mm × 5.5 mm with a central 2-mm perforation. Anterior chamber was shallow (Fig. 1A).

Based on the history and clinical findings a clinical diagnosis of perforated corneal ulcer, probably of bacterial origin (Pseudomonas spp.) was made. Corneal scrapings were collected for microscopic examination, bacterial, fungal and acanthamoeba cultures as described elsewhere [3]. Contact lenses and the cleaning solution were also collected for microbiological investigations. Gram, Giemsa stained smears and the potassium hydroxide preparation of the scraping did not reveal any organisms. However, Giemsa stained smear showed the presence of multinucleated giant cells, with characteristic molding of nuclei (Fig. 1B), suggesting infectious keratitis, probably of a viral etiology. Scrapings obtained from the lower palpebral conjunctiva, on the following day (corneal scrapings could not be collected due to the application of tissue adhesive and a bandage contact lens), was positive for HSV-1 antigen (Fig. 1C) by an immunoperoxidase assay. Patient was prescribed 3% acyclovir ointment application 5 times daily. None of the cultures yielded any growth.

She returned to the clinic after a week. On examination, the tissue adhesive and bandage contact lens were dislodged. Repeat corneal scrapings were obtained for microbiological investigations. HSV-1 antigen was detected by an immunoperoxidase assay (Fig. 1D) in the repeat corneal scrapings while bacterial and viral cultures (shell vial and conventional tube cultures for HSV-1) were negative.

Clinical condition did not improve during the subsequent week. Hence, the patient underwent a therapeutic penetrating keratoplasty (PK). Corneal tissue was obtained at PK. She received oral acyclovir 400 mg, 5 times a day and 1% prednisolone acetate eye drops, 6 times a day. Corneal tissue was positive for HSV-1 antigen by immunohistochemistry (Fig. 2A, B) and for HSV DNA by PCR (Fig. 2D). Histopathological examination of the formalin fixed corneal tissue revealed polymorpho nuclear neutrophils admixed with mononuclear cells, multinucleated giant cells in H & E and PAS (Fig. 2C) stained sections. Stromal melting was seen in the central cornea. The visual acuity improved to 20/20. There was no recurrence and the graft was clear (14 months post PK).

Clinical diagnosis of atypical HSV keratitis can often be difficult. This case was diagnosed initially as bacterial keratitis considering the rapid progression of the symptoms and a history of contact lens wear. A simple stain like Giemsa revealed the presence of multinucleated giant cells in the corneal scraping. The presence of multinucleated giant cells with characteristic nuclear morphology (molding), may suggest a viral etiology. It may be seen in infections caused by HSV- 1, HSV-2 and VZV. However, this staining technique is not as sensitive as other methods like viral antigen detection, culture or rapid assays like HerpChek . Subsequent virological investigations confirmed the etiology (HSV-1) in this case. Virus cultures were negative probably because the specimens were collected following antiviral therapy.

We performed the PCR to confirm the presence of HSV DNA, since all viral cultures were negative. PCR for HSV has been shown to be most useful to the clinician in atypical presentations of herpetic ocular disease [1,4,5]. Predisposing factor for an atypical presentation as seen in this case could be due to the contact lens wear, for poor fitting lenses, poor lens hygiene and micro-trauma may all lead to the reactivation of herpetic keratitis.

It is interesting to note that it is very unusual for a herpes ulcer alone to present with a large yellowish-white infiltrate. Concurrent bacterial infection could have contributed to such a presentation. The possibility of a mixed infection (Bacterial: Herpetic infection) cannot be ruled out entirely in this case since the patient had received topical antibiotics elsewhere before presenting to us. The negative bacterial cultures may be attributed to this finding.

This case highlights the following: Atypical HSK can present as a perforated corneal ulcer associated with a large infiltrate. A "Low Tech" method like "Giemsa stain" of corneal scraping may offer a clue to the diagnosis. Nevertheless, other tests like viral antigen detection, culture or HerpChek should be done if HSV is specifically suspected. Conjunctival scrapings may be useful for the detection of viral antigen where corneal scrapings cannot be collected. PCR may be useful in the laboratory diagnosis of such an atypical presentation of HSK.

We believe that diagnosis of such cases would be beneficial in the prompt institution of prophylactic antivirals, especially following penetrating keratoplasty to prevent complications.

The pre-publication history for this paper can be accessed here:

Herpes simplex keratitis (HSK) is a sight threatening ocular infection and is a leading cause of corneal blindness [1]. Clinical presentation of HSK is often protean. While a typical and common presentation of HSK is usually a dendritic or geographic ulcer, atypical presentations are not uncommon [2]. We report here an unusual presentation of HSK. The patient presented to us with bullous keratitis, which was misdiagnosed as a case of pseudophakic bullous keratopathy (PBK) with secondary glaucoma.

A sixty-year-old male presented to our cornea services with a complaint of progressive diminution of vision, in the right eye, of 20 days duration. There were no other ocular or systemic complaints. He gave a history of having undergone extracapsular cataract extraction with posterior chamber intraocular lens implantation 4.5 years earlier in the RE and phacoemulsification with posterior chamber intraocular lens implantation 2 years earlier in the LE. Surgery and postoperative period were uneventful on both occasions with a final visual acuity of 6/6 in both eyes. On examination, visual acuity in RE was restricted to perception of light with accurate projection of light in all the four quadrants. Conjunctiva was congested and cornea showed epithelial bullae all over the surface with mild to moderate epithelial and stromal edema. Anterior chamber was deep and quiet. Intraocular lens was in place and fundus details were within normal limits. Intraocular pressure in the RE was 30 mm of Hg and LE was 14 mm Hg. A diagnosis of PBK with secondary glaucoma, was made. Patient was immediately treated with intravenous mannitol (350 cc) and a single oral dose of 250 mg acetazolamide, followed by 250 mg three times daily and 0.5% timolol eye drops twice daily for the right eye. Intraocular pressure was 16 mm of Hg following mannitol administration and he was discharged. Patient presented to us four days later. Visual acuity in the RE had improved to counting fingers at 1 m and 6/6 in LE. On examination, cornea showed few epithelial bullae with mild to moderate epithelial and stromal edema. The intraocular pressure in the right eye was 16 mm Hg. Two dendritic lesions were seen (Figure 1) in the cornea. A clinical diagnosis of PBK with HSK was made. Corneal scrapings were collected for virological investigations. The patient was treated with 3% acyclovir eye ointment five times daily and cyclopentolate eye drops twice daily for the RE. The patient was lost to follow up.

Papanicolaou stained smear of the corneal scraping showed multinucleated giant cells and intranuclear eosinophilic inclusion (Figure 2). HSV-1 antigen was detected in the epithelial cells of the corneal scraping and the smear revealed multinucleated giant cells (Figure 3). HSV-1 was isolated in culture and PCR was positive for HSV DNA using primers, which amplified a 179 bp region of the DNA polymerase gene of HSV 1/2.

This case was initially diagnosed as PBK with secondary glaucoma based on a history of cataract surgery, presence of diffuse corneal stromal and epithelial edema, epithelial bullae and a raised intraocular pressure in the affected eye. The subsequent appearance of typical dendritic lesions and virological investigations confirmed a diagnosis of herpetic bullous keratitis. Further, the present event occurred after 4 years after the cataract surgery. Since corneal latency of HSV has been described [4], it is perfectly reasonable to assume that herpes rather than the previous endothelial damage caused the whole syndrome.

The presence of glaucoma possibly suggests HSV trabeculitis in the affected eye. It is difficult to ascertain this finding since we did not perform any investigation. A PCR assay for the detection of HSV DNA using aqueous humor would have provided supporting evidence.

This event was possibly a syndrome of HSV stromal and epithelial keratitis with trabeculitis, which explains the signs of a diffuse corneal stromal and epithelial edema and an acute rise in the intraocular pressure in the affected eye. The formation of epithelial bullae due to corneal edema could have predisposed the cornea for the development of dendritic ulcers. It has earlier been shown that the presence of corneal epithelial bullae has a statistically significant effect on the rate of ulcer development [3].

This case highlights the fact that HSK can present initially as a more diffuse corneal stromal and epithelial edema with epithelial bullae mimicking bullous keratopathy.

To the best of our knowledge and based on a MEDLINE search, such a presentation of HSK has not been documented.

Herpetic bullous keratitis, although unusual, should be considered in the differential diagnosis under such circumstances to prevent further complications and for the prompt institution of specific antiviral therapy.

None declared

The pre-publication history for this paper can be accessed here:

Herpes Simplex Keratitis (HSK) is a sight threatening ocular infection often caused by HSV-1. It is a leading cause of corneal blindness and occurs worldwide [1]. HSK usually occurs in its typical form as a dendritic or geographical corneal ulcer. However, there are reports of atypical HSK [2]. There can be a high degree of overlap between the ocular manifestations of HSV-1 and those of other infections [3]. A specific and rapid laboratory diagnosis of HSK is essential for the initiation of specific antiviral therapy. Further, complications arising from misdiagnosis and inappropriate treatment can be avoided [3]. A variety of techniques have been employed for the rapid diagnosis of HSK [4,5]. Many of these techniques are expensive, time consuming and may not be available in all settings. Impression cytology has been employed for the rapid diagnosis of superficial viral infections [6]. It is one of the most preferred techniques in ocular surface sampling in dry eye, keratitis and conjunctivitis [7]. Conventionally, impression cytology is collected using cellulose acetate filter paper [8], Biopore membrane [6] or Nitrocellulose membrane [9]. These membranes are expensive and may not be available in all laboratories. We describe here an economical, simple and rapid technique for the collection of impression cytology, for the detection of viral antigen.

Fifteen patients with a clinical diagnosis of HSK (either dendritic or geographic ulcers) and five patients with other corneal infections (Mycotic keratitis, n = 3, Bacterial keratitis, n = 2) were included in the study. Informed consent was obtained from all the patients included in this study and the study conformed to the guidelines stipulated by our institutional research forum. Samples were collected following instillation of a topical anaesthetic (4% Lignocaine hydrochloride or 0.5% Proparacaine hydrochloride). Corneal impression cytology specimens were collected using a sterile glass slide (Blue Star Micro Slides, Polar Industrial Corporation, Bombay, India). These slides are made from selected optically flat micro-glass, the edges are polished (therefore, the edges are not sharp), available in lint free packing and are packed under controlled conditions. The slides were initially washed with distilled water and immersed for 24 h in potassium dichromate-sulphuric acid cleaning solution (This solution was prepared by adding 63 gms of potassium dichromate to 35 ml of distilled water. Concentrated sulphuric acid (1 L) was then added slowly down the sides of the bottle held in an ice bath with intermittent shaking to dissipate the heat generated). Slides were removed and thoroughly washed in tap water, rinsed in distilled water, and air-dried. They were packed in aluminium foil and sterilized by hot air oven for 1 h at 160°C. This method of cleaning slides ensures that the charge of the glass surface is not alkaline and therefore, renders the surface suitable for cell adhesion (an alkaline surface is unsuitable for cell adhesion and is caused by alkaline detergents). Impression cytology smears were obtained by pressing the surface of one end of the slide firmly, but gently on the corneal lesion (Fig. 1). A single impression cytology smear was collected from all the patients except in two cases with classical dendritic ulcers and a case of bacterial keratitis, wherein additional smears were collected for Papanicolaou staining. Corneal scrapings were collected from these patients following the impression cytology procedure for the detection of viral antigen [6] and viral cultures [5]. Impression cytology specimens and smears made from corneal scrapings were air dried for 30 minutes at room temperature and fixed in acetone for 30 minutes at -20°C. Smears were stained by an immunoperoxidase or immunofluorescence assay for the detection of HSV-1 antigen using a polyclonal antibody to HSV-1, as described elsewhere [6]. The entire smear was screened for positively stained cells (infected cells) and smears were graded as "Positive" for viral antigen only when >40-50% of the cells/HPF were stained. Viral cultures were performed employing the shell vial assay [5] using vero/ A 549 or BHK-21 cell line. Corneal scrapings collected from patients with infectious keratitis of non-viral origin were processed for etiological agents as described earlier [10].

Statistical analysis was performed on results using a computer assisted statistical program (Epi Info, revision 6.04 b, CDC, USA). Chi square test for proportions (with Yates correction when necessary) was used and P value was considered significant if less than 0.05.

This simple technique allowed the collection of adequate corneal epithelial cells (10-20 epithelial cells/HPF) for the detection of HSV-1 antigen in a majority of the patients. Most of the smears showed superficial corneal epithelial cells. Some smears showed rounded up corneal epithelial cells. Adequate cells could not be collected in one patient. HSV-1 antigen was detected in 12/15 (80%) cases while virus was isolated from 5/15 (33.3%) patients with HSK. Virus isolation alone was positive in 3/15 patients. All the patients with a clinical diagnosis of HSK (n = 15) were confirmed by virological investigations (viral antigen detection and/or viral cultures).

HSV-1 antigen was detected in the impression cytology smears (Figs. 2, 3) and corneal scrapings (Fig. 4) in 11/15 (73.3%) and 12/15 (80%) patients, respectively (P = 1.00). All the corneal scraping specimens which were found positive for the presence of viral antigen (n = 12) were also positive by impression cytology (n = 11) except in one case. Adequate cells could not be collected in this case. Minimal background staining was seen in impression cytology smears while there was a disturbing background in corneal scrapings stained by the immunological methods.

Papanicolaou stained impression cytology smears showed presence of multinucleated giant cell (Fig. 5), koilocytic changes (Fig. 6) and inflammatory cells (Fig. 7).

None of the patients in the control group (non-HSV keratitis) were positive for viral antigen (Fig. 8) or virus isolation.

Impression cytology is a non-invasive technique for the diagnosis of external eye diseases. It has not been extensively used despite its diagnostic potential because of technical inconvenience in the use of conventional membranes and its non-availability in many settings.

We have described here a highly economical, simple and rapid technique for the collection of impression cytology for the diagnosis of HSK, by using an ordinary glass slide. Our preliminary data shows that this technique is comparable to collection of corneal scrapings, especially for the detection of viral antigen (11/15 [73.3%] versus 12/15 [80%], P = 1.00). The result was not statistically significant.

Qualitatively, interpretation of antigen detection in impression cytology smears was very easy, since the background staining was virtually negligible. Interpretation of smears stained by the immunofluorescence assay was easier than smears stained by the immunoperoxidase aassay, since the background staining was somewhat lesser in the former. Additional advantage of this technique is that the glass slides are totally devoid of background fluorescence in comparison to cellulose acetate and biopore membranes. The specificity and sensitivity of this technique was 100% and 73.3% respectively and was comparable to that of corneal scrapings (specificity: 100% and sensitivity: 80%), for the detection of viral antigen. Earlier reports using membranes have shown better sensitivity [6,11]. This could be attributed to the inherent property of the membrane devices to which the cells adhere effectively resulting in the collection of a large number of cells[6]. In contrast, most of the smears made from corneal scrapings which were positive for viral antigen (9/12) showed a troublesome background staining, though the antigen positive cells (virus infected cells) could be identified from uninfected cells.

Quantitatively, adequate number of cells (10-20 epithelial cells/HPF) could be collected in most of the cases. However, the disadvantage of impression cytology was that in a small proportion of cases (2/15 [13.3%]), the material obtained was inadequate. This may contribute to antigen negativity. Similar findings have been reported by Yagmur et al. These authors have shown that the cell number was inadequate for diagnosis in 21% of the samples observed by impression cytology [7]. The numbers of cells, which can be collected using a glass slide, are probably small when compared to a membrane. Nevertheless, the results of our preliminary study are encouraging. This may be attributed to the method we have adopted for cleaning the slides, which improves cell adhesion.

Considering the ease of performance and cost per patient, this technique was very easy to perform and proved to be very economical. The advantage of this technique is that the cells are collected directly onto the slide. Handling the slide for various staining procedures is easy and permanent mounts can be stored conveniently when immunoperoxidase assays are used. It is worthwhile to note that the cost (figures are based on the actual cost of procuring these utilities in a developing country like India) of collecting an impression cytology on a glass slide is approximately $0.01 whereas the cost of a millipore membrane is $2.00.

The impression cytology smears can be used for other staining procedures like PAP and Giemsa stain. Characteristic features of a viral infection, for example, the presence of multinucleated giant cell (Fig. 6), koilocytic changes in epithelial cells (Fig. 7) and inflammatory cells (Fig. 8), all can be well-delineated in corneal scrapings and impression cytology smears. We have collected impression cytology for such staining procedures in three patients. Further studies are warranted to assess these procedures. Further, impression cytology from other sites like conjunctiva and skin vesicles can be collected by this method, especially for direct staining techniques. This needs further evaluation.

Based on our preliminary results, the technique we have described may be as useful as collection of impression cytology using Biopore membranes and cellulose acetate papers, as described by other authors[6,8,9]. We believe that this procedure is as safe as collection of corneal scraping which is routinely obtained using blade no. 15 on a Bard Parker handle. We did not encounter any difficulty nor untoward incidents during the collection process using the method we have described. It is worthwhile to note that we have used special glass slides with polished edges. Nevertheless, the glass slides can further be modified wherein the square edges can be rounded off. Alternatively, Perspex cover slips can be used for the collection of cells [12]. However, handling a Perspex cover slip may not be convenient when compared to a glass slide, especially for the collection of corneal impression cytology. This technique can be considered as a suitable alternative procedure where facilities for such devices (Biopore membranes, cellulose acetate paper) are not available. However, our results should be considered with caution, since the sample size is small.

In conclusion, collection of impression cytology on a sterile glass slide followed by viral antigen detection is an inexpensive, simple and rapid technique for the diagnosis of HSK. Immunological techniques can be applied on such smears, which can provide virological results within 2-5 hours. This technique could be modified for use in the diagnosis of other external eye diseases and various staining procedures, which needs further evaluation.

None declared

The pre-publication history for this paper can be accessed here:

The primary cause of senile cataract development is still unclear. To date the involvement of α-crystallin, a molecular chaperone for β- and γ-crystallin, has principally been considered in senile cataract development [1,2], the decrease in the chaperone ability of α-crystallin with age being implicated. Xanthurenic acid is produced from a metabolite of tryptophan (3-hydroxy-kynurenine) [3] in the presence of 2,3-dioxygenase [4-7]. While 3-hydroxykynurenine [3] is photochemically inert [8] and serves as a protective UV filter of retina, xanthurenic acid is a photosensitizer [9,10]. Xanthurenic acid accumulates with aging in mammalian lenses [11,12] and is involved in the increased fluorescence of the lens with aging [13]. The glucoside of xanthurenic acid is also present in aged lenses [14]. Xanthurenic acid is an example of an endogenous ER stressor provoking an accumulation of unfolded proteins which in turn leads to an overexpression of Grp 94 and calreticulin in the lens epithelial cells of young mammals [15]. We have previously reported that porcine lens epithelial cells in culture respond to xanthurenic acid exposure by an overexpression of stress chaperone proteins, Grp 94 and calreticulin, in [15]. Here, we report that xanthurenic acid leads to human lens epithelial cells (HuLEC) death associated with caspase-3 activation, intracellular Ca²⁺ increase and calpain Lp82 induction. Previously, lens epithelial cell apoptosis was observed in an in vitro model of the cataract [16-18].

Our previous studies in cell culture showed that xanthurenic acid is a potent endogenous pathological substance in retinal pigment epithelium and smooth muscle cells [24]. In this study, we observed that xanthurenic acid leads to lens epithelial cells death associated with an induction of caspase-3 and calpain Lp82. Apoptosis was observed in models of cataract upon lens treatment with staurosporine, diamide, and ionophore [16-18]. In the selenite cataract model caspase-3 and calpain were induced [25]. In the normal apoptosis, such as observed with development, cells disappear because of caspase-3-dependent cleavage of DNA and the cytoskeleton [26,27]. The caspase remodeling of the cytoskeleton was indicated as a possible mechanism leading to the aging of lenses [28,29]. Apoptosis is considered as a common cellular basis for non-congenital cataract in mammals [30]. Recent data indicate that senile cataract is a result of proteolysis of crystalins by calpains [31]. Calpains are activated by Ca²⁺[32].

Previously, it was reported that ER Ca²⁺ homeostasis affects the cells' sensitivity to apoptosis [33]. Lens epithelial cells overloaded by Ca²⁺ showed vimentin cleavage and opacification [34] Thapsigargin, a plant alkaloid, which depletes Ca²⁺ from the ER, was used to stop lens epithelial cell growth [35]. Here, we show that xanthurenic acid, an endogenous molecule, lead to induction of calpain Lp82 and caspase-3 activation. The simultaneus activation of caspase and calpain may lead to an abnormality of apoptosis because calpain cleaves caspases [36]. The calpain Lp82 is involved in cataract formation in connexin α-3 knockout mice [37]. The induction of calpain Lp82 leads to the cleavage of crystalins in the lenses and is involved in the senile cataract development [38]. Xanthurenic acid leads to formation of unfolded proteins [15].

An accumulation of unfolded protein can lead to Ca²⁺ release from intracellular stores as well to caspase induction [39,40]. The observed death of the lens epithelial cells has apoptotic characteristics because release of cytochrome c and caspase-3 activation were observed. However, the apoptosis-like process does not lead to a collapse of the cytoskeleton driven by caspase-3 cleaved gelsolin in the presence of Fas-induced apoptosis [20]. In our study, in the presence of xanthurenic acid cells look normal when observed by the light microscopy. However, when visualized by fluorescence microscopy with Hoechst and propidium iodide a nuclear dysfunction is evident. We have previously reported that xanthurenic acid leads to an abnormal cleavage of gelsolin. The gelsolin stains cytoskeleton of the dead cells. Ca²⁺ and bis-phosphatidylinositol (PPI2) regulate, respectively, association and dissociation of gelsolin to actin [41]. Thus, also the increase in Ca²⁺ may play a role in the association of gelsolin to cytoskeleton.

The mitochondrial damage observed in the presence of xanthurenic acid associated with cytochrome c release could lower energy necessary for the lens enzymes activation observed in senile cataract development.

In this study we observed that xanthurenic acid accumulation can be an upstream event leading to an induction of the lens proteases: caspase and calpain. An accumulation of xanthurenic acid in the human lenses with aging can change the intracellular Ca²⁺ homeostasis. In summary, xanthurenic acid can induce the pathology of the lens epithelial cells without participation of light.

All authors contributed equally to realize this work

None declared

The pre-publication history for this paper can be accessed here:

Until a few years ago, metabolic diseases, were thought to be controversial entities with exotic names, only a few people were involved in their research. The fact that the majority of these diseases were considered incurable reduced the interest of their investigation. However, recently the situation has changed dramatically and metabolic diseases have received serious attention and considerable progress has been made in prevention and therapeutic management. It is now clear that metabolic diseases are far from rare. The incidence of metabolic diseases in North America and in Central and Northern Europe is 1:5000.

Metabolic diseases mainly affect the neural tissue, but may also affect others such as the heart, liver, kidneys, blood and eyes. The ocular symptoms and signs are found in a large number of these diseases [4]. They frequently represent an already allocated symptom of a systemic disorder. The ocular finding can rarely be so characteristic as to render itself the key to the diagnosis, as for example in Wilson's disease [5,6].

A pilot study has been implemented in rural Crete and its first results and experiences have been reported in a recent publication [7]. There are very few written reports on ocular findings in subjects with inborn errors of metabolism (IEM), thus it was interesting to study the frequency of ocular findings in the studied population and explore their contribution to the early diagnosis of IEM.

Our pilot study was carried out during the period from January of 1997 to January 2000. The predominant source of the study's data was derived from the eight Primary Health Care Centres in rural Crete, which accounts for 225,000 inhabitants, and are taking part in the activities of the Cretan Primary Healthcare Network [7]. An invitation was extended to all of the primary care physicians of these Health Centres, of which eight medical doctors participated in an intensive educational programme for IEM and their early diagnosis. A checklist with specific illness conditions, symptoms and clinical signs was drawn up (see additional file 1). Primary care physicians checked the presence of at least one condition, sign or symptom in all children or adults who visited the participating Health Centres and who had been symptomatic since childhood and were not related to other conditions. They referred all eligible subjects to the Department of Child Care at the University Hospital of Heraklion, Crete. Three of children who had examined at the Department of Ophthalmology at the University Hospital of Heraklion had fulfilled the inclusion criteria were added in our study sample. A full and analytical physical examination as well as a detailed laboratory investigation was made in this department. Patients with history of perinatal asphyxia, infection, injury or tumor of the central nervous system were excluded of the study. An ophthalmologic evaluation was part of this assessment. The ophthalmologic evaluation included the following:

(1) Case History (Personal and Family). (2) Visual acuity measurements for near and/or distant vision. In selecting the method of estimation of visual acuity we took into consideration factors such as age, existence of mental retardation, and collaboration of the patient. (3) Color Perception (using the Ishihara Isochromatic Cards). (4) Pupil reflexes (direct and indirect). (5) Near reflex. (6) Orthoptic evaluation including conjugate eye movements; cover-uncover test; cover-test and prism cover test where it was necessary. (7) Examination for presence of nystagmus. (8) Slit lamp examination of the anterior segment of the eye. (9) Retinoscopy. (10) Indirect ophthalmoscopy. (11) Photography when applicable.

The ocular investigation was performed every year on each patient included in the study.

During the 3-year period the study involved the examination of 125 subjects which fulfilled the inclusion criteria of this study. There were 75 males (60%) and 50 females (40%). The ages were between 0 to 30 years. The mean age of the examined patients was 6.34 (S.D: ± 5.75) years. Of the 125 subjects examined, a diagnosis of IEM was established in 23 (18.4%). Subsequently 63 (50.4%) had ocular findings.

From the 23 diagnosed patients, 10 (43.5%) had ocular findings, and 8 of these (34.8%) had findings specific for the diagnosed diseases (see additional file 2). In one patient diagnosed with glycogenosis type 1b a rare finding of large preretinal spherical deposits near the ora serrata was documented. These deposits probably consisted of glycogen and/or triglycerides. To the best of our knowledge, this finding has not previously been reported in any publication.

Thirteen (56.5%) of the diagnosed patients had no ocular findings (5 patients with organic acidurias, 2 with glycogenosis type la, 2 b-oxidation defects, one with lysosomal disorder, one mitochondriopathy and two with aminoacid disorders).

From the 102 non-diagnosed patients 40 (39.21%) had no ocular findings and 9 (8.82%) had only a very low refractive error (-2.0D to + 2.0D). Fifty – three (51.96 %) undiagnosed patients presented various ophthalmic findings that are shown in (see additional file 3)

This study attempted to show the significance of the ocular investigation in the early diagnosis and management of IEM. It is known that IEM affect vision because of the damage to the brain, visual pathways and other eye structures such as the lens, the cornea and the retina. The existence of the ocular finding was critical for the suspicion of the disease and the definitive diagnosis in several of the diagnosed patients (18.4%). Of the 23 diagnosed patients, 10 (43, 47%) presented ocular symptoms (Table 2). In 4 patients (with Canavan's disease, metachromatic leucodysrtophy and adrenoleucodystrophy) the suspicion of the disease was set because of the other clinical symptoms in spite of the fact that atrophy of the optic nerve was a specific finding [8]. In 8 (34,78%) of the 10 diagnosed patients the ocular symptoms were specific for the underlying disease. This means that 80% (8/10) of the patients diagnosed with ocular symptoms had specific ocular findings for the diagnosed disease.

The diagnosis of IEM represents one of the most difficult aspects of medicine and their differential diagnosis from perinatal lesions is not always easy. In this case, the ocular findings can be of great value in directing the diagnosis to a specific disease. This was the case with one of our patients with Niemman-Pick type C who had a very slow progression. It was the ocular findings (vertical gaze paresis, doll's eye movements and retinitis pigmentosa) which led to the suspicion of the diagnosis [9,11].

In the cases of the patients with Kearns-Sayre syndrome, mannosidosis and Lowe's syndrome the ocular findings were characteristic for the disease and contributed to the fast diagnosis of the underlying disease. The patient with the Kearns-Sayre syndrome exhibited the typical triad of progressive external ophthalmoplegia, retinal pigmentary degeneration, and heart block [12,14]. The patient with Mannosidosis presented a progressively developing wheel-like cataract and optic disk pallor [15,16] which was probably related to demyelination and associated gliosis involving the parieto-occipital white matter [17]. The patients mother was at the first trimester of her 2^nd pregnancy. Prenatal screening was ordered which revealed the some diagnosis in the fetus [18]. The patient in Lowe's syndrome (oculocerebrorenal syndrome) presented congenital discoid cataracts [19,20], miosis, congenital glaucoma [21]. The mother had flake like opacities of the lenses, a typical finding in a female carrier [22]. The child was treated for cataracts, and glaucoma. Currently, he is under topical therapy with a b-blocker.

In biotinidase deficiency, strabismus is a non-specific ocular finding. However, it is described in 30% of the patients in various research studies [23,24].

The case of a patient with glycogenosis type 1b is of interest. This patient presented large preretinal spherical deposits near the ora serrata, probably consisting of glycogen and/or triglycerides. This patient was under no therapeutic scheme until the age of 25. Three years later in a new fundus examination the large preretinal deposits were absorbed and replaced with small drussen-like preretinal deposits. These new deposits were mainly concentrated in the peripheral retina of the inferior nasal quadrant of each eye. Our ocular finding in glycogenosis type 1b may be correlated with ocular findings reported by other authors for this disease. [25].

It is an interesting fact that although 18.4% were finally diagnosed, but ocular findings existed in 50.4% of them.

The findings in undiagnosed patients were notable. A high percentage of ocular findings were documented for these patients (51.96%) A tight surveillance of the undiagnosed patients is important as some of these findings may be related to a metabolic disease, the existence of which can not be established at the present time although it is certain that some of these findings should be very helpful in the future.

The high percentage of ocular findings (43.47% in the diagnosed and 51.96% in the undiagnosed patients) in both diagnosed and undiagnosed patients is indicative of the significant involvement of the eye in problems raising a suspicion of an IEM disease. The ocular evaluation of these patients seems to be obligatory.

The results of this study demonstrate that a thorough ophthalmologic examination can be extremely useful for the pediatricians and the neurologist concerning the diagnosis of metabolic neurogenetic diseases. For the establishment of the diagnosis, the collaboration of various institutes is usually necessary in some cases the diagnosis can be facilitated by the ophthalmologic findings. Moreover, there are cases where an ophthalmologic examination is the main examination that leads to the early recognition of these diseases. Finally, the amelioration of the ocular symptoms can offer a better quality of life to those patients.

None declared.

Dr Tsagaraki Daria and Dr Evangeliou Athanasios had primary responsibility for protocol development, patient screening, enrolment, outcome assessment, preliminary data analysis and writing of the manuscript. Dr Tsilibaris Miltiadis, Dr Spilioti Martha and Dr Lionis Christos participated in the development of protocol and analytic framework of the study and contributed to the writing of the manuscript. Dr Michailidou Eleni was responsible for patient screening and Dr Pallicaris Ioannis supervised the design and execution of the study and contributed to the writing of the manuscript.

The pre-publication history for this paper can be accessed here:

Herpes simplex keratitis (HSK), a leading cause of corneal blindness, is a sight threatening ocular infection often requiring a specific and prompt laboratory diagnosis [1]. A variety of techniques have been employed for the rapid diagnosis of HSK [2-5]. Isolation of Herpes simplex virus-1(HSV-1) in culture provides the most reliable and specific method and is considered as the "Gold Standard" in the laboratory diagnosis of HSK. However, the sensitivity of this technique has been found to be low [6]. The reasons for this low sensitivity, especially for the isolation of HSV-1 from corneal scrapings, may be attributed to the sensitivity of the cell line used and the small volume of the specimen available. A number of cell lines are currently being used for the rapid isolation of HSV-1 in cell culture including primary rabbit kidney cells, human fetal foreskin fibroblasts, human embryonic lung fibroblasts and Vero [7].

A recent study has reported that isolates derived from herpetic keratitis grow better in corneal epithelial cells and rabbit corneal epithelial cells may be more suitable for isolating HSV from the cornea [8]. Therefore, we performed a prospective study comparing the efficacy of a recently described novel immortalized human corneal epithelial cell line [9] and Vero cells in the isolation of HSV-1 from corneal scrapings obtained from patients with HSK.

This study was performed to assess the relative sensitivity of a recently described HCE and the Vero cells based on an earlier observation that corneal epithelial cells may be more suitable for the isolation of HSV from the cornea [8].

Vero cells were chosen for comparison since our earlier observations over a two-year period (unpublished data) suggested that this cell line performs better than HEp2/ BHK-21/ HeLa or A549 in the isolation of HSV-1 from patients with HSK. We employed the shell vial assay since this technique has been shown to be a rapid and sensitive method for the isolation of HSV [11] and adenovirus [12] from ocular specimens when compared to the conventional tube cultures.

Assays done to determine the performance characteristics of HCE show that this cell line is susceptible to HSV-1 and suitable for the isolation of the virus from corneal scrapings in a shell vial assay.

Our results show that the HCE is superior to Vero in the isolation of HSV-1, both in the qualitative and quantitative analysis. Considering the qualitative analysis, it is interesting to note that HCE yielded the maximum number of isolations. There were no cases where HSV-1 was isolated only in Vero cells (Table 2), which suggests the increased sensitivity of the HCE. While there was a statistically significant difference in the sensitivity of these two cell lines for virus isolation, the specificity and PPV were very good (100 %, Table 1). However, the low NPV of both the cell lines (Table 1) suggests that there is a need for better cell lines, especially for the isolation of virus from corneal scrapings. Further, it is worthwhile to note that CPE can be observed in HCE earlier than in conventional tube cultures, in some specimens, as seen in this study. Such specimens can be processed for imunofluorescence/ immunoperoxidase assay immediately leading to a "reduced turn around time" of reporting the results.

Analysis of the quantitative results showed that HCE had the greatest sensitivity irrespective of the viral load, while vero had similar sensitivity to that of HCE only when the load was high (Table 3). HCE proved to be very beneficial, especially when the viral load was low (< 10 IF) (Table 3).

It is obvious from the results that HSV-1 can be more often isolated in HCE than Vero. This has been shown both by qualitative and quantitative analysis.

In general, the isolation rates of HSV-1 in cultures from corneal specimens have been low [6], irrespective of the cell line used. Our earlier report showed a similar trend wherein we could isolate HSV-1 in Vero cells only in 5/15 (33.3%) specimens obtained from patients with HSK [13]. This study demonstrates that using HCE can further increase the virus isolation rate.

The rate of isolation of HSV-1 from corneal scrapings has increased from 33.3% to 58.82% in our laboratory employing the HCE, as seen in this study. We have used an inoculum size of 0.5 ml of the specimen in our study. It has earlier been shown that increasing the inoculum size of the specimen can further increase the sensitivity of the shell vial assay [14]. Such studies are being done at our laboratory.

We have shown the superiority of HCE over Vero cells in the isolation of HSV-1 from corneal scrapings for the laboratory diagnosis of HSK, in this preliminary study. However, the results should be interpreted with caution, since the sample size is small. Further studies are warranted using an adequate sample size. Such a study is being pursued in our laboratory.

To the best of our knowledge based on a "MEDLINE" search, this is the first report of the HCE being used for the isolation of HSV-1 in a shell vial assay for the laboratory diagnosis of HSK. We believe that the inclusion of this cell line, when available, in the routine culture protocols of ocular virology laboratories would result in a significant increase in the diagnostic yield.

None declared

The pre-publication history for this paper can be accessed here:

In previous studies changes in tear protein patterns of diabetic patients suffering from dry-eye disease could be found [1-3]. The occurrence of the dry eye disease and other ocular surface diseases is increased in diabetic patients [4].

The dry eye syndrome has a very high frequency of occurrence in the industrial word. In the United States, 1 of 5 people, i.e. 59 millions of patients, suffer from symptoms of this disease (Eagle Vision, Yankelovich und Partners, 1997), and the number of dry eye patients has been doubled since the last ten years [5-8]. Dry eye patients typically suffer from discomfort, burning, irritation, photophobia, blurred vision, and have an increased risk of corneal infection and resulting irreversible tissue damages [9,10]. This is mostly caused by aqueous, mucin or lipid deficiencies in tears.

Until today, no causative treatment of the disease is available. Patients are symptomatically treated with lubricant eye drops.

Worldwide about 100 millions of people suffer from Diabetes mellitus [11]. Diabetes is a systemic disease with multiple severe very known related disorders such as the diabetic angiopathy, polyneuropathy, and nephropathy. Many ocular complications such as the inflammation of lids, acute orbital infections, cataract, and the diabetic retinopathy are known to be associated with diabetes mellitus and many of them may lead to blindness. Moreover, the risk of cataract is 2–4 times greater than in healthy people [12-15]. In diabetic patients a significantly increased corneal thickness [16] and a decreased corneal sensitivity [17-20] was demonstrated. Interestingly, the severity of the dry eye disease correlates with the severity of the diabetic retinopathy [21], which represents a main reason for blindness in diabetic patients.

The tear film quantity (Schirmer test=basal secretory test, BST) is decreased in diabetic patients [22]. In this study, we attempt to investigate the changes in the composition of tears in diabetic patients compared to healthy subjects.

There were no age- or gender-related statistical differences between patients with diabetes and control subjects.

In the electrophoretic separations, the known main protein peaks could be detected (Fig. 1). The quantitative analysis of the volume of the 5 main protein peaks (lactoferrin, sIgA, albumin, lipocalin, and lysozyme) revealed no statistical significant difference between the clinical groups (healthy persons and patients suffering from diabetes mellitus; p > 0.05).

However, a significant increase in the number of peaks could be detected in diabetic patients compared to controls (P < 0.0003; Fig. 2).

A complete electrophoretic pattern analysis was performed – represented by the data vector derived from the densitographic data. The multivariate analysis of discriminance revealed a highly significant discrimination between diabetic patients and controls (Wilks lambda: 0.27; P < 0.000001).

For each diabetic patient, a BST was performed. According to the results of this test, 140 of the diabetic patients suffered from dry-eye (DIDRY) and 120 of them did not (DICTRL). The protein patterns of DIDRY and DICTRL were compared and a significant difference in the protein pattern and number of peaks of diabetic patients could be detected between those suffering from dry eye disease or not (P < 0.002).

To investigate the correlation between the changes in protein patterns and the duration of the diabetic disease, the diabetic patients were subgrouped between those, who had a known diabetic history longer than 10 years (DIA2) or shorter (DIA1).

The changes in the protein patterns of diabetic patients increased with the duration of the diabetic disease. In diabetic patients with a disease duration longer than 10 years the alterations in protein patterns were significantly more expressed than in patients with a shorter diabetic history (P < 0.003). Furthermore, the changes were more pronounced in DIA2 compared to healthy subjects (P < 0.0001) (Fig. 3). A correlation analysis (spearman rank correlation test) revealed a r²-value of 0.9 (p < 0.03).

In previous studies a significant difference in the tear protein patterns of patients suffering from dry eye, diabetic patients suffering from dry eye, and healthy volunteers could be demonstrated [3]. This was done by means of one-dimensional electrophoretical separations of the tear proteins.

In the present study, the electrophoretic patterns from tear proteins of non-diabetic and diabetic patients were analyzed and compared to controls. The main protein peaks, lactoferrin, lysozyme, lipocalin, and albumin were detected and quantified. No statistical significant difference between the main protein peaks of the groups could be found. However, the number of peaks was significantly increased in the tear protein pattern of diabetics. By including all peaks of the electrophoretic separations in the comparison, the multivariate approach could demonstrate a significant difference between both groups (diabetics and healthy volunteers). Thus, this must be due to changes in tear protein patterns, e.g. the development of new protein peaks, not only by slight differences between the concentrations of the main protein peaks. The most important differences in protein patterns were found in the molecular weight range of 30–50 kDa. We could not identify one single peak that was consistent in all patients. However, an increase of peaks in that molecular weight range was highly specific.

Those "new" proteins, which do not exist in healthy subjects, could play a role in the pathogenesis of the diabetic disease and/or the development of eye-related complications of the diabetic disease. The present study demonstrates that changes in protein patterns are strongly correlated with the duration of the diabetic disease. The longer the disease duration, the more changes were expressed. Thus, it is promising to evaluate the use of these proteins as early-onset or follow-up marker of the diabetic disease. Further study has to prove if the differences in tear proteins found in this study have a prognostic value for the development of diabetic retinopathy. The differences found might not be caused by the diabetes itself, but by the trophic or autonomic disturbances common to that condition. However, an increased knowledge about the protein changes found in this study could be helpful, e.g., for identifying diabetics at risk of becoming blind.

It is still not clear why diabetic patients develop dry eyes more often than healthy subjects [27]. One possible explanation could be an exocrine dysfunction of the main lacrimal gland in patients with diabetes mellitus or perhaps the development of additional unknown proteins in the tear fluids. Another cause could be the reduction of stimulatory signals from the ocular surface to the lacrimal gland as consequence of the reduced corneal sensation and the influence on regulatory systems.

However, this study could prove that the tear protein patterns of diabetics are different from those diabetic patients who suffer additionally from the dry-eye disease. These changes were clearly distinguishable from each other. Thus, it can be excluded that the method used to determine the changes in protein pattern just recognizes in general any changes as significant. In this study, the tear composition of diabetics was found to be changed in comparison to other diabetic patients suffering additionally from an ocular surface disease that is already known to have an influence on the protein patterns in tears [2,3].

This preliminary study could clearly and surprisingly demonstrate that there are marked alterations in the diabetic tear protein patterns. Further studies will evaluate whether the changes in the tear protein patterns of the diabetic patients can be used as a new non-invasive diagnostic tool. Furthermore, identifying these proteins could contribute to learn more about the pathogenesis of diabetic related ocular surface disease.

None declared.

PS carried out the biochemical procedures. FHG conceived of the study and was responsible for the study design and computer analysis. BHD, AJA, and NP was involved in study design, discussion of the results and work with patients included in that study.

The pre-publication history for this paper can be accessed here:

The normal human ocular lens exhibits a steady increase in size due to growth throughout life, harboring some the oldest cells in the body [1-3]. Overall, from birth to 65 years of age, the human lens will increase 63% in equatorial diameter, while increasing only 22% in A-P axial thickness [4]. Cell growth dramatically influences the contours of the anterior and posterior lens surfaces, but is not the sole factor affecting lens form.

Previous studies investigating the hydration of transparent human lenses revealed an age-related dependence between water content and lens region [5]. With advancing age, the human lens cortex shows a slight increase in water composition (+0.0087%/year), whereas the nucleus decreases its total water content (-0.077%/year), setting up a gradient of hydration [5]. Studies of human diabetic lenses have revealed greater total water content in comparison to transparent, non-diabetic lenses, with swelling reported to extend into the adult and perhaps the fetal nuclei [6,7]. The result is a characteristically larger lens in comparison to transparent, age-matched, non-diabetic lenses. Previous documentation by Scheimpflug analysis demonstrated that the annual expansion rate of the lenticular A-P length is accelerated 70% in early-onset diabetics [8]. Steepening of the anterior and posterior surface curvatures was detected but little difference in lens nuclear size was found. The osmotic imbalance observed in the diabetic lens may be responsible for the pronounced influx of water into the epithelial and cortical areas, swelling and damaging the cells [9].

In our previous morphological study of non-diabetic aging human lenses by SEM it was concluded that the innermost nuclear fibers of the human lens significantly decrease in size and change shape with age and cataractogenesis [4], a process termed compaction. In concordance with previous investigations [10], it was found that the rate of lens compaction was not constant over time, and that the majority of compaction was observed before middle age. Changes in the A-P length of the embryonic nucleus impact the curvature angles of the inner fetal nuclear fibers at the equator, possibly affecting the overall lenticular form. Employing transmission electron microscopy (TEM) in an additional study [9], extensive fiber cell damage was noted in the outer adult nucleus of late-onset diabetic cataractous lenses, with minimal morphological disturbances in the inner nuclei.

In this study, SEM was used to search for evidence of osmotic swelling and/or fiber cell compaction in the nucleus of non-cataractous and clinically diagnosed nuclear cataractous human lenses from persons with diabetes. Due to the manner in which we receive our specimens, the nature and medical history of the donors' diabetes are unknown. This study aims to compare the transparent lenses of diabetic patients to those of with nuclear cataract, without conclusions based on disease type or treatment. For further comparisons, previously documented non-diabetic compaction data was utilized [4].

Comparisons of transparent diabetic and diabetic lenses with nuclear cataract yielded statistically significant differences in each measured parameter, indicating a detectable and real difference in inner nuclear size between the groups. Based on the strengths of the statistical tests used, these results should be interpreted as trends between the tissues. As observed in non-diabetic aged transparent and age-related human cataractous lenses, it appears that nuclear fiber cell compaction is responsible for these differences.

It should be noted that any study that utilizes tissue processing for electron microscopy is subject to potential morphological artifacts due to fixation. Every effort has been made in this study to minimize the potential occurrences of such artifacts by utilizing proven preservation methods for lens tissue [12,13]. It is possible that the processing may dehydrate the presbyopic lens nuclei; however, the potential structural changes may occur on too small a scale to significantly affect the gross measurements made here.

To relate these variations in compaction to diabetes and/or cataractogenesis, statistical testing was performed using previously collected non-diabetic data. In Brown and Hungerford's review of the size of the lens in ocular disease he comments that the rise in overall lens growth rate observed in the diabetic lens can be almost entirely attributed to increased cortical thickness, with relatively insignificant width changes in the nucleus [19]. Later postulations concerning the mechanism of this growth included increased fiber formation, enlarged fiber cell volume, and decreased nuclear compaction [8,20]. Parameter comparisons of transparent aged human lenses to those of diabetics did not yield statistically significant results, indicating little difference in average nuclear compaction between the tissues. Our previous study demonstrated a 12% average decrease in elliptical angle and A-P axis measurements between aged transparent lenses and those of age-related nuclear cataracts [4]; this is roughly the same amount of change reported here between transparent and nuclear cataractous diabetic lenses (11% decrease). This suggests that the compaction changes observed in this study are not necessarily a direct effect of diabetes itself, but are perhaps due to morphological changes in the nuclei undergoing cataractogenesis. The role that diabetes does appear to play, however, involves the onset age of cataract. Though we cannot deduce the age of cataract onset in our samples, we do know the age at which the cataract was considered advanced and removed by an ophthalmologist. In our comparisons, the average age for the age-related nuclear cataract group was 70 years [4] whereas that for the cataractous diabetic group in this study was only 55 years old. These results suggest an age-related acceleration of cataract formation in persons with diabetes. Several population studies have documented a link between diabetes and senile cataract formation in persons aged 65 years and younger [21-26]. Conversely, a number of studies have shown little association of nuclear cataracts with diabetes [27-29]. Though this relationship continues to be debated, as are the underlying physiological mechanisms, premature opacification may occur due to an increased susceptibility to oxidative damage.

Although the exact details concerning the cause of compaction remain unknown, some features of the multifactorial process are understood. The morphological changes described in the inner nuclear areas are a direct result of the change in A-P axis length. This change in length has an observable effect on the reduction of both anterior and posterior FN elliptical angles, and manifests itself in the EN as accordion-like membrane folds along the straight fibers. This phenomenon suggests a loss of cytoplasmic water and a decrease in nuclear cytoplasmic osmolarity [30,31]. The tendency of lens proteins to self-associate and the release of bound water to the bulk water pool can lead to greater cellular dehydration [5,32]. The end result is a decrease in cell volume without the loss of cell surface area, producing an EN with measurably shorter fiber cells with increased membrane complexity.

In conclusion, this study is in agreement with our earlier finding that nuclear fiber cell compaction in the lens is a normal and measurable process with advancing age; the process of cataractogenesis appears to have a profound effect on the extent and severity of compaction in the nucleus. The osmotic swelling of the cortex in typical diabetic lenses is not matched by comparable water uptake in the nucleus; in fact, compaction occurs in similar fashion to that observed in non-diabetic lenses. Diabetes may elicit an increased compaction rate as well as an earlier onset of cataract formation, though there may be multiple mechanisms of action dependent on disease type, duration, or treatment strategy. Further investigations employing a broad population of samples with extensive medical histories will yield additional, more conclusive results about the relationship of diabetes and nuclear cataract.

None declared.

CDF and KJA were responsible for tissue preparation, SEM examination, morphometric measurements, and data analysis. CDF drafted the manuscript. JRK and MJC conceived of the study, and participated in its design and coordination.

The pre-publication history for this paper can be accessed here:

Allergic conjunctivitis (AC) is an ocular surface inflammatory disease that affects approximately 25% of the general population [1-3] and has a significant impact on the social and economic aspects of life. It can appear alone or associated with other allergic diseases, especially allergic rhinitis. AC is an immunopathological disease which the number of mast cells in the substantia propria increase, and also the epithelium becomes densely infiltrated [4,5]. Activation of mast cells by IgE bound-receptor crosslinking by allergens promotes the release of several mediators such as histamine, prostaglandins, leukotrienes, tryptase and cytokines, all of them responsible for the symptoms of AC and the inflammatory changes in conjunctival cells [6-8].

Conjunctival epithelial cells (EC) play an important proinflammatory role in chronic ocular allergic diseases, the expression profile levels of different cellular adhesion molecules and surface inflammatory antigens have been reported in normal and altered human conjunctival epithelium [9-11]. Recent results have demonstrated that expression of ICAM-1 may allow EC to recruit, retain and locally concentrate leukocytes and the presence of HLA-DR raises the question of conjunctival EC antigen presentation [12].

Although the level of expression of CD29 (the common chain of the beta 1 integrins) on normal conjunctival ECs is already known [10], there is not information about changes in its expression during AC. In addition, different epithelial cytokines/chemokines, which are upregulated actively, participate in allergic inflammation [13]. Among those chemokines eotaxin not only play an important role in eosinophilic recruiting and in damaging the tissue, but also maintain this type of immune response [14].

AC can be treated with local anti-allergic agents such as antihistamines, either alone or in combination with alpha-adrenergic agents and mast cell stabilizers [15-18]. Ketotifen fumarate is derived from cyproheptadine, a serotonin and histamine antagonist [19]. The efficacy and safety of this drug treatment in AC management is well known [20,21] and its clinical use has also been widely studied in relation to bronchial asthma where have been demonstrated that ketotifen treatment significantly decreased EG2+ activated eosinophils, CD3+ and CD4+ T cells in the bronchial mucosa and inhibit the expression of E-selectin and ICAM-1 on vascular endothelial cells [22-24].

The effects of different H1 antihistamines on ICAM-1 expression have been extensively studied [24-29], but since there is not too much information about the effects of ketotifen on expression of CD29, HLA-DR and eotaxin on conjunctival EC during AC, we performed an open, uncontrolled study on patients with AC to investigat the effect of this drug on clinical features and those markers of inflammation.

After 7 days of treatment, 53% of patients showed improvements of their symptoms and signs (p < 0.0001). With continued treatment through day 14, symptoms control was achieved in 76% of patients (p < 0.0001). Moreover, administration of 0,05% ketotifen eye drops for thirty days significantly (p < 0.0001) reduced the TSSS for each patient (Figure 1) between days 0 and 30.

Treatment with this drug has clinical effect on burning, watery discharge, and swelling, particularly, at the end of the treatment. Every other symptom were significantly reduced (p < 0.016) at any time of clinical evaluation (Figure 2).

The effect of the treatment was also studied on the expression of different molecules on CD45 negative cells (EC) by FACS. It is worth to note that cells studied which were obtained by conjunctival scraping never contain more than 3 % of CD45 + cells (data not shown).

When the expression of HLA-DR, CD29 and eotaxin on EC was evaluated after the treatment we found a drop in the percentage of those positive EC in 58%, 68% and 73% of patients, respectively. Although the variation in percentage of HLA-DR+ EC was not significant, the percentage of CD29+ and eotaxin + EC significantly decreased (p < 0.0062 and <0.0082, respectively) at the end of the treatment (Table 1). In 9/19 patients a simultaneous drop in EC positive for CD29 and eotaxin was observed. Figure 3 shows a representative case from this group of patients.

We did not find any correlation between variations in mean fluorescent intensity and percentage of positive EC for these markers before and after the treatment.

AC is a common, prevalent, and clinically significant IgE mediate hypersensitivity response in which signs and symptoms associated with the progression from early to late phase reaction start four to eight hours after challenge and persist up to 24 hours. The clinical reaction is accompanied by a significant recruitment of inflammatory cells (mainly neutrophils) in tears 20 minutes after challenge, and eosinophils and lymphocytes 6 to 24 hours after challenge [32]. In the chronic and more severe forms of AC, other mechanisms and cells contribute to a more complex clinical profile [33]. In addition, the mast cell is considered to play a pivotal role in causing signs and symptoms since Bacon AS et al found increased levels of histamine, tryptase, PGD2, and leukotriene C4 in the tears of patients with Seasonal Allergic Conjunctivitis after conjunctival allergen challenge [34].

It is now becoming clear that EC may be viewed as active participants in the regulation of inflammation and leukocyte behavior. EC from a number of mucosal sites have been shown to be capable of: 1) expressing adhesion molecules like ICAM-1 (CD54) or beta 1 integrin (CD29), which are important for leukocyte emigration into tissue; 2) expressing MHC class II molecules and 3) synthesizing and releasing prostaglandins and leukotrienes which attract and activate leukocytes and 4) producing a variety of pro-inflammatory cytokines and chemokines. The adhesion molecules expressed on EC not only allow interactions between cells and cells with matrix but also they might be involved in intracellular signals [12,13,35-37].

Although we did not found significant variations in the percentage of HLA-DR+ EC before and after treatment, the majority of patients exhibited a significant decrease in the percentage of CD29+ cells suggesting that the expression of these molecules are controlled by different mechanisms.

Eotaxin, a CC chemokine, is a potent and selective eosinophil chemoattractant to the site of inflammation but also plays an important role in damaging tissue by its capacity to induce the release of reactive oxygen species [38,39]. It has been previously demonstrated that eotaxin mRNA and protein are constitutively expressed by bronchial and nasal epithelium from normal individuals and increased within the airways of asthmatic individuals, and in nasal biopsy specimens from individuals with allergic rhinitis. In addition eotaxin immunoreactivity was found in macrophages, eosinophils, T cells, mast cells, and neutrophils, suggesting that this chemokine is an important mediator in the physiological and pathological trafficking of eosinophils [40,41].

It has been reported that in EC from normal conjunctival biopsies there is a weak cytoplasmic expression of eotaxin [13]. In our study we found a strong expression of eotaxin on EC from patients with AC which decrease significantly after treatment with ketotifen.

Current therapy of ocular allergic disease focuses on allergen elimination, modulation of the immune system and pharmacological inhibition of the chemical mediators involved in the immune response. The most commonly used therapeutic option is the pharmacological inhibition of chemical mediators. Mast cells stabilizers and antihistamines are two of the most commonly used group of therapeutic agents; they stabilize the mast cell membranes by preventing calcium influx across the mast cell membranes, thereby preventing mast cell degranulation and mediator release and the new antihistamines have been demonstrated to be capable of affecting several phenomena of the allergic inflammation, including mediator release, cellular activation and adhesion molecule expression [42,43]. Among these drugs, ketotifen fumarate, attenuated the local allergic reaction in patients with AC [20,21] and the efficacy and safety of this drug in AC management has also been shown in human conjunctival allergen challenge model [44,45].

Here, we corroborated the effectiveness of ketotifen fumarate in decreasing the symptoms and signs of AC in the majority of patients, but more importantly we showed that this drug even though was not effected in reducing the expression of HLA-DR on EC, it significantly decreased the percentage of CD29+ and eotaxin+ EC.

The mechanism of action by which this drug produce this effects are probably related to the decrease amounts of inflammatory cytokines/chemokines released by effector cells at the conjunctival level. Clearly, additional studies are needed to fully understand the complex mechanisms of the anti-inflammatory effect of Ketotifen fumarate in patients with AC.

AC = Allergic Conjunctivitis.

EC = Epithelial cells.

TSSS = Total symptoms and signs score.

mAb = Monoclonal antibody.

None declared.

Martín AP: collected conjunctival scrapings and carried out immunofluorescence studies, performed the statistical analysis and drafted the manuscript.

Urrets-Zavalia J: performed the ophthalmological evaluation of the patients with AC.

Berra A: performed conjunctival cytology studies and quantification of tear IgE.

Mariani AL: carried out the quantification of serum IgE, secretory IgA and lisozime.

Gallino N: carried out the prick tets and clinical evaluation of the patients with AC.

Gomez Demel E: performed the ophthalmological evaluation of the patients with AC.

Gagliardi J: carried out the prick tets and clinical evaluation of the patientswith AC.

Baena-Cagnani CE: carried out the prick tets and clinical evaluation of the patientswith AC.

Urrets-Zavalia E: conceived of the study, and participated in its design and coordination.

Serra, HM: conceived of the study, and participated in its design and coordination.

The pre-publication history for this paper can be accessed here:

Alpha-Crystallin is comprised of two polypeptides, alphaA-crystallin (alphaA) and alphaB-crystallin (alphaB), which share 55% amino acid sequence homology [1]. They are the most abundant proteins in lens fiber cells [2,3] and exist as heteroaggregates of approximately 800 kDa that can undergo inter-aggregate subunit exchange [4]. The expression of these two proteins in the lens epithelium, however, is not uniform throughout different regions of the anterior epithelium. AlphaB expression is detected in central epithelium and increases from the central to elongation zones, where epithelial cells differentiate into fiber cells. AlphaA, however, is not detected in the central epithelium. The relative proportion of alphaA and alphaB changes from a molar ratio of 1:3 in the pericentral and germative zones to a molar ratio of 3:1 in the elongation zone and fiber cells [2,3]. These differences in the relative proportions of alphaA and alphaB within the lens suggest different functions for the two subunits in the developing lens.

Prior to the 1990's, alpha-crystallin was thought to be a structural protein whose major cellular function was to produce a dense solution necessary for the refraction of light in the lens. During the early 1990's, alpha-crystallin was shown to act as a molecular chaperone, binding to partially denatured proteins, both in vitro [5] and probably in vivo [6], to inhibit further denaturation and aggregation of lens proteins. AlphaA and alphaB were also shown to have sequence homology with several other proteins that are members of the small heat shock protein (hsp) family [7]. Moreover, expression of alpha-crystallin, particularly alphaB, was shown to not be restricted to the lens. AlphaB was found to be expressed at significant levels in a variety of nonlenticular tissues, while alphaA has only been detected in small amounts in a few other tissues such as retina, spleen and thymus [8-12]. Collectively, these findings have challenged the dogma that alpha-crystallin is purely a structural protein necessary for light refraction, and have led to the realization that alpha-crystallin may have a variety of biological functions in the lens.

A much broader scope of cellular functions of alpha-crystallin in lens is inferred from in vitro observations. Both alphaA and alphaB can bind specifically to actin, in vitro [13] and in vivo [14]. Although actin filament formation has been shown to be necessary for differentiation of lens epithelial cells [15], the significance of alpha-crystallin's interaction with actin in differentiation is not known. In the lens, alpha-crystallin also associates with type III intermediate filament proteins and the beaded filament proteins CP49 and CP115, and correct beaded filament assembly has been shown depend on the presence of alpha-crystallin [16]. Beaded filament mRNA levels are greatly increased in differentiating lens epithelium and have been suggested as a pan-specific marker for lens fiber development [17]. Alpha-crystallin has also been shown to interact directly with DNA [18]. In transfected CHO cells, alphaB has also been shown to ectopically localize to interphase nuclei, suggesting a role for this protein in the nucleus [19]. A nuclear role for alphaB in the lens was supported by the findings that a subset of lens epithelial cells derived from alphaB knockout mice demonstrated hyperproliferation and genomic instability [20]. In addition, the administration of exogenous alpha-crystallin to primary bovine lens epithelial cell cultures resulted in the formation of lentoid bodies, consistent with a role for these proteins in lens differentiation [21]. These findings indicate that alpha-crystallin may have a multitude of in vivo functions.

One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. Brady et al. [22] demonstrated, by targeted disruption of the mouse alphaA gene, that this protein was essential for the maintenance of lens transparency, possibly by maintaining the solubility of alphaB, or associated proteins, in the lens. These lenses were also reported to be smaller in equatorial and light axial dimensions than age matched wild type lens. It was not possible using the techniques employed in the study to determine if the smaller lenses were due to reduced volume or number of fiber cells. Targeted disruption of the mouse alphaB gene resulted in lenses similar in size to aged-matched wild type lens. Moreover, no cataract formation was observed in the alphaB knockout lenses. These animals have muscle cell abnormalities, severe postural anomalies, selective muscle degeneration, and shorter life spans compared to normal controls [23].

In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The objectives of the current report were to characterize gross morphology of young (5 wk) and old (54 wk) mouse lenses with targeted disruption of both the alphaA and alphaB genes, in comparison to age matched wild type lenses, using scanning electron microscopy (SEM) and confocal microscopy, to elucidate the possible functions of alpha-crystallin in the lens. The results indicate that alpha-crystallin is necessary for proper fiber cell formation and resulting lens transparency.

Equatorial and axial (sagittal) dimensions of lenses, and gross lenticular appearance, were recorded (Table 1). AlphaA/BKO lenses at 5 wks of age were significantly smaller (p < 0.05), both equatorially and axially, than 5 wk old wild type lenses and remain smaller throughout life. Fifty-four wk old alphaA/BKO lenses were much smaller than similarly aged normal lenses, and were similar in size to 5 wk old wild type lenses. Both the 5 wk old wild type lenses and the 54 wk old alphaA/BKO lenses were significantly smaller (p < 0.01) than the 46 or 72 wk old wild type lenses. AlphaA/BKO lenses at 5 wks of age exhibited vacuoles at the equatorial region (Figure 1A see arrows) and an area of slight light scatter at the posterior subcapsular regions (Figure 1B see arrowheads). By 54 wks of age, alphaA/BKO lenses exhibited dense cortical and nuclear cataracts (Figure 1C). No posterior sutures were observed in any of the alphaA/BKO lenses examined. All wild type lenses were transparent upon removal from the eye (Figure 1D,1E and 1F) and had well defined posterior sutures.

Examination of mid-axial sections stained with DiI and SYTOX green revealed unique differences in gross morphology and nucleic acid staining between alphaA/BKO lenses and wild type lenses (Figure 2). In five week old alphaA/BKO lenses, Sytox green stained nuclei (green) in the anterior epithelium, equatorial/bow, and posterior subcapsular regions (Figure 2A). Cell nuclei localized to the equatorial/bow region were disorganized as compared to wild type. There were enlarged extracellular spaces between cells in the equatorial/bow region in 5 wk old alphaA/BKO lenses. Fifty-four wk old alphaA/BKO lenses (Figure 2B) did not stain for nucleic acid in the posterior subcapsular region, however, the entire anterior subcapsular lens region stained for nucleic acid (refer to Figure 3I for high magnification/resolution image of this area). A defined equatorial/bow region was not observed in these older lenses, and fiber cells extending to the posterior capsule of the lens were not found. Disorganized cellular material capped the anterior region of the embryonic/fetal nucleus, and a distinctive indentation of the lens mass at the equatorial region was apparent in mid-axial sections as well as whole lenses. In alphaA/BKO lenses the embryonic/fetal nucleus (primary lens fibers) had migrated posteriorly and appeared to be in contact with the posterior capsule. In 5 wk, 46 wk or 72 wk old wild type lenses (Figure 2C,2D and 2E), however, there was no posterior or anterior subcapsular nucleic acid staining and the embryonic-fetal nucleus was centrally located, not in contact with the posterior capsule.

At higher magnification, examination of sections stained with DiI and SYTOX green revealed ultrastructural differences between alphaA/BKO lenses and wild type lenses (Figure 3). Anterior epithelial staining in 5 wk old alphaA/BKO (Figure 3A) and 5 wk old wild type lenses were similar (Figure 3K) with respect to nuclear staining with SYTOX green. However, some differences in 54 wk old alphaA/BKO lenses (Figure 3F) were observed in the central anterior epithelium compared to 5 wk (Figure 3K), 46 wk (Figure 3P), and 72 wk (data not shown) wild type lenses. These differences included changes in central epithelial nuclear staining, nuclear shape and epithelial thickness and continuity. Superficial and deep anterior cortical staining was grossly different between alphaA/BKO (Figure 3A,3D,3F and 3I) and wild type lenses (Figure 3K,3N,3P and 3S). In 5 and 54 wk old alphaA/BKO lenses, superficial and deep anterior cortical regions, stained for lipid membranes, did not reveal any patterns typical of fiber cells cut in cross-section or longitudinal section. In 5 wk alphaA/BKO lenses there was a high density of stained membranes per unit area throughout the cortex, with no nucleic acid staining in these regions. In 54 wk alphaA/BKO lenses, large, irregularly shaped cells were observed, interspersed among regions of high membrane staining density per unit area. These large objects were not vacuoles, because examination of the interior of these structures by transmitted and reflected light microscopy showed that the membranes encompassed cellular material (data not shown). In addition, nucleic acid staining was observed (Figure 3F and 3I) within many of these cells exhibiting large cross-sectional profiles. In the wild type lenses, typical cross-sectional patterns of fiber cells organized in radial columns were observed (Figure 3K,3N,3P and 3S).

Ultrastructural differences at the equatorial/bow region were also observed between alphaA/BKO lenses (Figure 3B and 3G) and wild type lenses (Figure 3L and 3Q). In contrast to wild type lenses (Figure 3L and 3Q), the alphaA/BKO lenses (Figure 3B and 3G) contained large areas devoid of cellular material, their nuclei were not limited to a well defined equatorial/bow region, and ordered radial columns of elongating fiber cells extending from the posterior capsule to the anterior epithelium were not observed. In addition to the equatorial region, ultrastructural differences between alphaA/BKO lenses (Figure 3C and 3H) and wild type lenses (Figure 3M and 3R) were observed in fiber cells of the embryonic and fetal nucleus. In 5 wk alphaA/BKO lenses (Figure 3C), a greater variation in cross sectional diameter was observed, with cell diameters ranging from less than 2 microns up to approximately 25 microns, compared to the 5 wk and 46 wk wild type lenses, whose diameters ranged from less than 2 microns up to approximately 10 microns (Figure 3M and 3R). At 54 wk, these cells in the alphaA/BKO lenses were larger in cross sectional diameter than wild type, and exhibited a much greater variation in the cross sectional size and cell shape.

Cells adjacent to the posterior capsule of 5 wk alphaA/BKO lenses (Figure 3E) exhibited nucleic acid staining, were small and non-elongated, with numerous membrane projections., These were, however, not observed in 54 wk alphaA/BKO lenses. In 5 wk alphaA/BKO lenses, deep to the posterior capsule, larger diameter cells, similar to those observed in the embryonic/fetal nucleus were observed (data not shown). Fiber cells of similar dimension and appearance to embryonic/fetal nuclear fibers were seen at the posterior capsule in 54 wk alphaA/BKO lenses (Figure 3J). In both 5 and 54 wk alphaA/BKO lenses, no posterior sutures could be found in any axial or equatorial vibratome sections examined. Posterior sutures were readily found in vibratome sections of wild type lenses (Figure 3O, arrow). Fiber cells attached to the posterior capsule and extending anteriorly were observed in 5 wk (Figure 3O), 46 wk (Figure 3T) and 72 wk (data not shown) wild type lenses.

SEM confirmed many of the observations made by confocal microscopy. In the equatorial/bow region, a disorganized array of relatively small irregular shaped cells were observed in 5 wk (Figure 4A) and 54 wk (Figure 4D) alphaA/BKO lenses, while elongated fiber cells organized into radial columns, with ball and socket interdigitations, were observed in 5 wk (Figure 4G), 46 wk (Figure 4J) and 72 wk (data not shown) wild type lenses. In the anterior cortical region of 5 wk alphaA/BKO lenses, radial columns of cells with numerous cell surface projections were observed (Figure 4B), but these radial columns were no longer present at 54 weeks of age (Figure 4E). In these older lenses, irregularly shaped, convoluted cells, with no recognizable pattern of organization, were present. In contrast, radial columns of elongated fiber cells of uniform size and shape, with ball and socket interdigitations, were present in the anterior cortical region of 5 wk (Figure 4H), 46 wk (Figure 4K) and 72 week (data not shown) wild type lenses. The posterior subcapsular regions of 5 wk (Figure 4C) and 54 wk (Figure 4F) alphaA/BKO lenses were filled with irregularly shaped cells with numerous cell surface projections. In the fetal/embryonic nucleus, elongated fiber cells with numerous long, finger-like projections and furrowed membranes were evident (not shown). No elongated cells extending from the bow region to the posterior capsule of alphaA/BKO lenses were observed. In contrast, radial columns of elongated fiber cells of uniform size and shape containing ball and socket interdigitations were observed in the posterior subcapsular region in 5 wk (Figure 4I), 46 wk (Figure 4L) and 72 wk (data not shown) wild type lenses, and fiber cells in contact with the posterior capsule could be traced back to the equatorial bow region and anterior epithelium.

One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. Brady et al. [22] demonstrated, by targeted disruption of the mouse alphaA gene, that this protein was essential for the maintenance of lens transparency, possibly by maintaining the solubility of alphaB, or associated proteins, in the lens. These lenses were also reported to be smaller in equatorial and axial dimensions than age matched wild type lens, which was very similar to that which was observed with the double knockout lens. Targeted disruption of the mouse alphaB gene, however, resulted in lenses similar in size to aged-matched wild type lens with no cataracts reported [23]. This indicates that alphaA may play a greater role in maintaining the transparency of the lens then alphaB. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. This was supported by the morphological observation made in this study of no posterior sutures or fiber cells extending to the posterior capsule of the lens, ectopically staining nucleic acids in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk, gross morphological differences in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. None of these morphological differences have been reported in the single alphaA or alphaB knockout mice. It must be noted, however, that the alphaA/BKO mice also lack the HSPB2 gene product [23] and the contribution of this protein to normal lens morphology and functions should not be overlooked. Future studies should address the possible functions of HSPB2 in normal lens.

The results of the current study support the hypothesis that alpha-crystallin plays an active role in the differentiation and growth of lens fiber cells. Normal differentiation of lens fiber cells consists of a progression from a simple cuboidal epithelial cell, containing a nucleus and a minimal numbers of organelles, to a stratified layer of elongated fiber-like cells, devoid of nuclei and organelles. Differentiation of epithelial cells occurs in the equatorial/bow region of the lens, where epithelial cells begin to elongate and differentiate into fiber cells of uniform cellular shape, arranged in radial columns of cells extending from the anterior epithelium to the posterior capsule. This process did not appear to have proceeded normally in lenses lacking alphaA and alphaB. The morphological observations presented in this study demonstrate that fiber cells in lenses lacking alphaA and alphaB fail to elongate symmetrically from the bow region and therefore do not establish the typical "onion skin" conformation in which cells extend from the anterior epithelium to the posterior capsule. Additionally, in lenses from 54 wk alphaA/BKO lenses, there was a persistence of cell nuclei in deeper cortical regions, and ectopic cell nuclei were present in large numbers in the anterior central cortex. At 5 wks of age cell nuclei were present, adjacent to the posterior capsule. These morphological observations are consistent with a defect in the normal differentiation pathway of lens epithelial cells into fiber cells.

It is unlikely that these alterations in alphaA/BKO mouse lenses result from increased susceptibility of these lenses to light-induced damage in the absence of the molecular chaperone protection afforded by alphaA and alphaB in normal lenses. With the normal time of eye opening at approximately 14 days after birth, the 5 wk old mice had their eyes open and lenses exposed to light for only about 3 weeks prior to morphological analysis. Moreover, these animals had been exposed to only animal facility fluorescent lighting and were protected from UV light by plastic cages. If lack of protection from light-induced damage was the major factor affecting the changes in these lenses, then the bulk of the damage should have resided along the visual axis, particularly in the central anterior epithelium and subcapsular cortex in the 5 wk lenses, but this was not the case. In these lenses, gross morphological changes were apparent in the equatorial and posterior subcapsular regions. These changes included posterior subcapsular nucleic acid staining, absence of posterior sutures, and small irregularly shaped cells, not arranged in any discernable pattern, in the equatorial/bow region. Systemic stress factors crossing the blood/aqueous barrier might explain some morphological changes at the equatorial region, but this would not explain nucleic acid staining in the posterior subcapsular region. In the 5 wk alphaA/BKO lenses, nucleic acid staining in the posterior subcapsular region is consistent with either anterior epithelial cells migrating aberrantly to the posterior pole, or primary fiber cells failing to fully differentiate by 5 wks of age. These two possibilities could not be differentiated in the methods employed, and were beyond the objectives of the current study. Future studies are being designed to address which of these two processes might explain nucleic acid staining in the posterior subcapsular region. Staining in this region was not observed in older alphaA/BKO lenses, suggesting that this pattern was transient. The fate of the nucleic acid-containing cells in the posterior capsular region of younger lenses is not known at this time, nor is the morphology of earlier stage lenses. Future studies with defined objectives to address the development and progression of morphological changes seen in this study are being designed. The morphological differences in alphaA/BKO lenses, compared to age matched wild type lenses, were consistent with the hypothesis that alpha-crystallin plays an active role in the differentiation and growth of lens fiber cells. In addition, it was clearly evident that alpha-crystallin is necessary for lens transparency.

The final biological event in a lens epithelial cell's life is the transformation from an epithelial cell into a fiber cell, which occurs at the equatorial/bow region of the lens. The newly forming fiber cell continues to differentiate in the cortex until a mature fiber cell devoid of organelles with suture formation at the ends of the cell is formed. This entire process from epithelial cell to mature fiber cell is defined as lens differentiation. The precise spatial and temporal expression of the crystallin proteins in the developing lens may not be simply a consequence of the differentiation process, but instead may play an important, if not essential, role in the differentiation process itself. The results of the current study support this hypothesis.

The exact in vivo molecular mechanisms, by which alpha-crystallin might influence lens epithelial cell differentiation, and maintenance of lens transparency, remain to be determined. AlphaA and alphaB are members of the shsp family [7]. Previous studies have shown that the alpha-crystallin possesses molecular chaperone activity, binding to partially denatured proteins, both in vitro [5] and probably in vivo [6], to inhibit further denaturation. Although this property may be a major contributor to the maintenance of lens clarity, the early changes in the alphaA/BKO lenses indicate a much broader cellular function for alpha-crystallin. Stress proteins have been shown to be expressed in non-stressed cells during development and differentiation [25]. Hsps were shown to be expressed during the differentiation of mammalian osteoblasts and promelocytic leukemia cells [26]. In addition, hsp expression has been shown to accompany growth arrest in human B lymphocytes [27] and macrophage differentiation of HL 60 cells [28]. During myogenic differentiation, mRNA for alphaB increases in conjunction with the induction of mRNA for myogenin, the earliest known event in myogenesis [29]. The addition of exogenous alpha-crystallin to primary bovine lens epithelium was shown to induce rapid changes in cell shape, leading to the formation of lentoid bodies [21]. These studies strongly suggest that the hsp family of proteins has other functions in addition to protecting proteins and cells during stress.

Alpha-crystallin may play a functional role in the cell nucleus and may have a role in regulating the cell cycle. Several heat shock proteins have been found in cell nuclei in the absence of stress [30], and alpha-crystallin has been shown to interact directly with DNA [18]. AlphaB, expressed in transfected CHO cells, has been shown to ectopically localize to interphase nuclei, suggesting a regulatory role for this protein in the nucleus [19]. A subset of immortalized lens epithelial cells from alphaBKO mice have been shown to hyperproliferate [20] suggesting that alphaB may be important in maintaining genomic stability. In lens epithelium derived from alphaAKO lenses, cell growth rates were reported to be 50% lower compared to wild type [31], suggesting a role for alphaA in regulating the cell cycle. All of these findings raise many questions as to the possible role(s) of alpha-crystallin in the nucleus and in cell cycle regulation during differentiation. In the current study, the observation of a disorganized pattern of nuclei localized to the equatorial bow region of alphaA/BKO lenses, and nucleic acid staining of structures throughout the anterior cortex of 54 wk alphaA/BKO lenses, is consistent with a role for alpha-crystallin in the nucleus.

There is extensive evidence from previous studies demonstrating that alpha-crystallin plays a role in the cytoskeletal organization. Both alphaA and alphaB can bind specifically to actin, both in vitro [13] and in vivo [14]. Actin filament formation has been shown to be necessary for the differentiation of lens epithelial cells [15], however, the significance of alpha-crystallin interaction with actin in differentiation is not known. In the lens, alpha-crystallin also forms a complex with type III intermediate filament proteins and the lens-specific beaded filament proteins CP49 and CP115, which may be critical for proper filament assembly [16]. Beaded filament mRNA levels increase greatly in differentiating lens epithelial cells, and have been suggested as a pan-specific marker for lens fiber cells [17]. It is therefore possible that increased synthesis of alpha-crystallin in epithelial cells early in the differentiation process may have profound effects upon the cytoskeleton, which in turn may profoundly affect cell shape and migration. The lack of cellar organization and uniform cell shape at the equatorial region observed in alphaA/BKO lenses supports this hypothesis. Studies are currently underway to characterize cytoskeletal organization in the alphaA/BKO lens.

None declared

DLB conceived and designed the study, carried out sample preparation, confocal microscopy, scanning electron microscopy, statistical analysis, and drafted the manuscript. JPB assisted in the production of the Alpha/BKO mice. EFW assisted in the production of the AlphaA/BKO mice, experimental design, and initial fixation of samples. All authors read and approved the final manuscript.

The pre-publication history for this paper can be accessed here:

The excimer laser ablation is currently widely used primarily to correct refractive errors by altering the anterior curvature of the cornea. Due to its potential for accurate ablation of anterior stromal corneal lesions its initial clinical utilization was removal of diseased corneal tissue in a procedure called photo therapeutic keratectomy (PTK). In 1985 Serdareviz et al proposed the first therapeutic use of the excimer laser to treat an experimental Candida infectious keratitis [1]. Despite the lack of controlled clinical trials comparing PTK with other treatment modalities such as superficial keratectomy or lamellar keratoplasty, the US Food and Drug Administration (FDA) identified PTK as an alternative therapeutic approach for the treatment of superficial corneal and epithelial membrane dystrophies, irregular corneal surfaces and corneal scars and opacities.

The major inherent obstacle of PTK for the management of corneal surface irregularities is that through photoablation these irregularities are reproduced deeper within the stroma.

Masking techniques refer to photorefractive procedures utilizing various masking means or so called modulators to protect flatter corneal areas while steeper areas are excised with the excimer laser.

Kornmehl et al [2] have shown that an ideal masking agent should have moderate viscosity (between that of saline and 1% carboxymethylcellulose) and concluded that very viscous fluids would not cover irregular surfaces uniformly whereas fluids of inadequate viscosity would run off quickly exposing both peaks and valleys thus resulting in irregular surfaces after ablation.

Numerous previous investigators have used different masking agents as methylcellulose [3-6] and sodium hyaluronate [5,7,8] at various concentrations or performed transepithelial (so that the epithelium acts as a natural masking agent) treatments [5,6,9,10] and reported the beneficial effect of masked PTK. Fasano et al [3] reported that the ideal masking agent should have the same ablation rate to that of the cornea, be biocompatible and adhere well to the cornea.

These properties have been met with the use of collagen solutions as modulators. Their applicability as masking agents for corneal photoablation was examined in experimental models of animal and cadaver eyes (Scott et al: Collagen modulator fluids for use during photoablation keratectomy: ARVO 1992, Margaritis et al. Properties of a new two component gel material as an aid in PRK corneal remodeling: ARVO 1994) [11-13] as well as in limited clinical trials of partially sighted eyes (Pallikaris et al: Photoablated lenticular modulator PALM technique: A report of ten cases: ARVO 1998) [14,15].

Photo-Ablatable Lenticular Modulator (PALM) technique refers to the use of a modified collagen gel solution for the photorefractive correction of corneal surface irregularities [14]. The PALM gel similarly to other collagen modulators [12,13,15] is thermo reversible. The gel is in liquid state when heated to solidify to a firm gel as its temperature lowers. Its use for masking purposes requires its application onto the corneal stoma at a temperature of 49°C where it can be molded to form a stable lenticule that serves as the final masking agent.

At temperatures of 40° to 53°C, the collagen individual fibril bonds are broken and the collagen denatures to its parent gelatinous form. When the tissue temperature drops to below 40°C, these bonds can reunite with possible rearrangement of the matrix but without any cellular damage [16]. Under this consideration the thermal contact of the 49°C preheated PALM gel onto the bare corneal stroma for the in situ molding of the lenticule, would not be expected to induce any irreversible changes of the corneal collagen chains. However its sequential use as masking agent for PTK could affect the laser-tissue interaction and probably the corneal healing response to irradiation.

In the current study we examined the possible implication of the PALM gel when used as described above, to the corneal wound healing on rabbit corneas.

We examined the reepithelization process of 12 rabbit eyes after PTK through PALM gel lenticules formed in situ. We used a moving slit delivery system in a 5 mm ablation zone confined by means of a metal iris diaphragm. An equal group of rabbit eyes received conventional PTK of the same depth and treatment zone and served as control.