Immunomodulatory properties of bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent progenitor cells of mesodermal originpossessing multilineage differentiation potential and ease of expansion in vitro. Over the years, these cells havegained attention owing to their potential in cell-based therapies in treating various diseases. In particular, thewide spectrum of immunoregulatory/immunomodulatory role of MSCs in various clinical conditions hasgained immense attention. The immunomodulatory properties of BM-MSCs are mediated by either cell–cellcontact (interactions with various immune cells in a context-dependent manner), paracrine mode of action orextracellular vesicles, making them a potential option as immunosuppressants/immunomodulators in treatingvarious clinical conditions. A plethora of studies have demonstrated that MSCs do so by exhibiting a profoundeffect on various immune cells for example they can inhibit the proliferation of T cells, B cells, and naturalkiller cells; modulate the activities of dendritic cells and induce regulatory T cells both in vitro and in vivo. Inthis review we aim at briefly elucidating the characteristics of BM-MSCs, specifically addressing the currentunderstanding on the hypoimmunogeneticity and immunomodulatory properties of the same with specificreference to their interactions with B cells, T cells, Dendritic cells and natural killer cells. We also aim atreviewing the secretory profile and their role in some clinical conditions that have shown promising outcomes.

Oral epithelial cells transplanted on to corneal surface tend to adapt to the ocular phenotype.

To understand the response of oral epithelial cells, transplanted on corneal surface to the ocular cues in vivo. The corneal bu􀄴 on obtained after penetrating keratoplasty (PK) of an eye of a patient with total limbal stem cell defi ciency (LSCD), previously treated with cultured oral mucosal epithelial transplantation (COMET) was examined by immunohistochemistry for the expression of keratins, p63, p75, PAX6, Ki-67, CD31, and CD34. COMET followed by optical-PK has improved visual acuity to 20/40 and rendered a stable ocular surface. The excised corneal tissue showed the presence of stratifi ed epithelium with vasculatures. The epithelial cells of the corneal bu􀄴 on expressed K3, K19, Ki- 67, p63, p75 and the cornea-specifi c PAX6 and K12. This study confi rms that the oral cells, transplanted to corneal surface, survive and stably reconstruct the ocular surface. They maintain their stemness at the ectopic site and acquire some of the corneal epithelial-like characters.

Human lacrimal gland regeneration: perspectives and review of literature

The human lacrimal gland is an essential component of the lacrimal functional unit [LFU]. Any perturbation of this unit can lead to the debilitating morbid condition called the dry eye syndrome [DES]. The current line of therapy available for dry eye remains supportive and palliative with the patient being dependent on life long and frequent administration of lubricating eye drops. Even advanced therapies like punctual plugs, cyclosporine B administration, and salivary gland auto-transplantation have led to a limited success. Under these scenarios, the option of cell based therapy needs to be explored to provide better and long term relief to these patients. This review gives an overview of the efforts in lacrimal gland regeneration and examines the past and ongoing research in cell based therapies in animals as well as human lacrimal gland cultures. The authors discuss their first of its kind functionally viable human lacrimal gland in vitro culture system from fresh exenteration specimens. A brief overview of research in near future and the potential implications of lacrimal gland regenerative therapies have been discussed

Outcome of Very Low Birth Weight Infants with Abnormal Antenatal Doppler Flow Patterns: A Prospective Cohort Study.

Background: Fetal growth restriction and abnormal Doppler flow studies are commonly associated. Neonatal outcomes are not well known particularly in developing countries, where the burden of the disease is the highest. Objective: To determine outcomes of preterm infants with history of absent/reversed end-diastolic umbilical artery Doppler flow (AREDF) vs. infants with forward end-diastolic flow (FEDF). Design: Cohort study. Setting: Tertiary care perinatal center in India. Participants: 103 AREDF very low birth weight (<1500 gm) (VLBW) infants and 117 FEDF VLBW infants were prospectively enrolled. Results: At 40 weeks adjusted post-menstrual age, AREDF vs. FEDF group had a higher risk for death in the NICU (12% vs. 1%), respiratory distress syndrome (33% vs. 19%), and cystic periventricular leukomalacia (12% vs. 1%). At 12-18 months corrected age, AREDF vs. FEDF group had a trend towards increased risk for cerebral palsy (7% vs. 1%, P=0.06). After logistic regression analysis, adjusting for confounders, AREDF was independently associated only with mortality in the NICU. Conclusion: AREDF is an independent predictor of adverse outcomes in preterm infants in a developing country setting.

Granulomatous inflammation in Acanthamoeba sclerokeratitis.

This report describes the histopathological findings in a patient with Acanthamoeba sclerokeratitis (ASK). A 58-year-old patient with ASK underwent enucleation and sections of the cornea and sclera were subjected to histopathology and immunohistochemistry with monoclonal mouse antihuman antibodies against T cell CD3 and B cell CD20 antigens. Hematoxylin and Eosin stained sections of the cornea revealed epithelial ulceration, Bowman's membrane destruction, stromal vascularization, infiltration with lymphocytes, plasma cells, and granulomatous inflammation with multinucleated giant cells (MNGC). The areas of scleritis showed complete disruption of sclera collagen, necrosis and infiltration with neutrophils, macrophages, lymphocytes, and granulomatous inflammation with MNGC. No cyst or trophozoites of Acanthamoeba were seen in the cornea or sclera. Immunophenotyping revealed that the population of lymphocytes was predominantly of T cells. Granulomatous inflammation in ASK is probably responsible for the continuance and progression of the scleritis and management protocols should include immunosuppressive agents alongside amoebicidal drugs.

Coculture of autologous limbal and conjunctival epithelial cells to treat severe ocular surface disorders: Long-term survival analysis.

Background: Cultivated limbal epithelium for reconstruction of corneal surface is a well-established procedure; however, it is not adequate for damage which also extensively involves the conjunctiva. In severe cases of ocular surface damage that warrant additional conjunctival transplantation apart from cultivated limbal stem cell transplantation, we describe the long-term survival of a novel method of cocultivating autologous limbal and conjunctival epithelium on a single substrate. Materials and Methods: Forty eyes of 39 patients with severe limbal stem cell deficiency and conjunctival scarring or symblepharon underwent transplantation of autologous cocultivated epithelium on human amniotic membrane. A ring barrier was used to segregate the central limbal and peripheral conjunctival epithelia in vitro. Patients were followed up at regular intervals to assess stability of the ocular surface, defined by absence of conjunctivalization into the central 4 mm of the cornea and absence of diffuse fluorescein staining. Penetrating keratoplasty (PKP) was subsequently performed, where indicated, in patients with surface stability. Results: The cumulative survival probability was 60% at 1 year and 45% at 4 years by Kaplan–Meier analysis (mean follow-up duration: 33 ± 29 months, range: 1–87 months). Best-corrected visual acuity improved to greater than 20/200 in 38% eyes at the last follow-up, compared with 5% eyes before surgery. Immunohistochemistry in five of the corneal buttons excised for PKP showed an epithelial phenotype similar to cornea in all five. Conclusions: Synchronous use of cultured limbal and conjunctival epithelium offers a feasible alternative and a simpler one-step surgical approach to treat severe ocular surface disorders involving limbus and conjunctiva.

Microsporidial spores can cross the intact descemet membrane in deep stromal infection

We report a rare case of a deep stromal keratitis with a chronic indolent course, diagnosed as microsporidial keratitis from corneal scrapings. The patient's condition worsened despite medical therapy and penetrating keratoplasty was performed. The histopathology of the corneal tissue revealed multiple microsporidial spores in the posterior stroma and the endothelial exudates, whereas there was no clinical or histopathological breach in Descemet's membrane. This is the second report in the literature to report that micropsoridial spores can cross the intact Descemet's membrane

Animal models in retinoblastoma research

Advances in animal models of retinoblastoma have accelerated research in this field, aiding in understanding tumor progression and assessing therapeutic modalities. The distinct pattern of mutations and specific location of this unique intraocular tumor have paved the way for two types of models- those based on genetic mutations, and xenograft models. Retinoblastoma gene knockouts with an additional loss of p107, p130, p53 and using promoters of Nestin, Chx10, and Pax6 genes show histological phenotypic changes close to the human form of retinoblastoma. Conditional knockout in specific layers of the developing retina has thrown light on the origin of this tumor. The use of xenograft models has overcome the obstacle of time delay in the presentation of symptoms, which remains a crucial drawback of genetic models. With the advances in molecular and imaging technologies, the current research aims to develop models that mimic all the features of retinoblastoma inclusive of its initiation, progression and metastasis. The combination of genetic and xenograft models in retinoblastoma research has and will help to pave way for better understanding of retinoblastoma tumor biology and also in designing and testing effective diagnostic and treatment modalities

Late occurrence of granular dystrophy in bilateral keratoconus: Penetrating keratoplasty and long-term follow-up.

We report a rare case of keratoconus with granular dystrophy with a follow-up of two decades, documenting the sequential presentation of two diseases confirmed by histology and genetic studies. A 13-year-old boy was diagnosed in 1988 with keratoconus in both eyes (BE) based on slit-lamp biomicroscopy findings of corneal ectasia in BE accompanied by Fleischer's ring, Vogt's striae, a small, old, healed hydrops. The left eye (LE) had central corneal thinning and scar in the central area involving the mid and posterior stroma secondary to healed hydrops. Penetrating keratoplasty (PKP) was advised. The boy was lost to follow-up till 1991 and presented with white, dot-like opacities in the central cornea in the RE only, suggestive of granular corneal dystrophy. Similar findings of white, dot-like opacities were noted in the LE in 1995 and the patient subsequently underwent PKP in BE. Histopathology of corneal buttons confirmed the presence of patchy, crystal-like orange deposits, which stained bright red with Masson's trichrome. Mutational analysis of the TGFBI gene in patient's DNA revealed a heterozygous mutation corresponding to a change in Arg555Trp in the keratoepithelin protein. Granular dystrophy recurred after 8 years in the RE.

Splicing aberrations caused by constitutional RB1 gene mutations in retinoblastoma.

Analysis of RB1 mRNA from blood leukocytes of patients with retinoblastoma identified the effects of mutations involving consensus splice site, exonic substitution and whole-exon deletions identified in genomic DNA of these patients. In addition, this study identified mutations in cases in which no mutations were detectable in the genomic DNA. One proband had mutation at the canonical splice site at +5 position of IVS22, and analysis of the transcripts in this family revealed skipping of exon 22 in three members of this family. In one proband, a missense substitution of c.652T>G (g.56897T>G; Leu218Val) in exon 7 led to splicing aberrations involving deletions of exons 7 and 8, suggesting the formation of a cryptic splice site. In two probands with no detectable changes in the genomic DNA upon screening of RB1 exons and flanking intronic sequences, transcripts were found to have deletions of exon 6 in one, and exons 21 and 22 in another family. In two probands, RNA analysis confirmed genomic deletions involving one or more exons. This study reveals novel effects of RB1 mutations on splicing and suggests the utility of RNA analysis as an adjunct to mutational screening of genomic DNA in retinoblastoma.

Update on pathologic diagnosis of corneal infections and inflammations

One of the most frequent types of corneal specimen that we received in our pathology laboratory is an excised corneal tissue following keratoplasty. Several of these cases are due to corneal infections or the sequelae, like corneal scar. Advances in the histological and molecular diagnosis of corneal infections and inflammations have resulted in rapid and accurate diagnosis of the infectious agent and in the overall understanding of the mechanisms in inflammatory diseases of the cornea. This review provides an update of histopathological findings in various corneal infections and inflammations

Orbital solitary fibrous tumor: a clinicopathologic correlation and review of literature

Orbital solitary fibrous tumor [SFT] is a rare tumor originating from the mesenchyme. Initially described in the pleura and subsequently in other mesenchymal structures, orbit continues to be one of the uncommon extrapleural sites. The diagnosis of orbital SFT cannot be made with certainty on clinical or radiological evaluation and requires histologic studies with immunohistochemical confirmation for which CD 34 is the most specific diagnostic test. We describe clinical presentations, radiological and operative findings, and pathological features of a patient with orbital SFT along with a review of literature

Isolated giant xanthogranuloma of the orbit.

Xanthogranuloma is an uncommon tumor in the orbit and is usually associated with systemic diseases or blood abnormalities. We report an extremely rare presentation of isolated orbital xanthogranuloma unassociated with any systemic disease, hematological or biochemical abnormalities. A 47-year-old physician presented with proptosis of the left eye of three years duration with yellowish skin plaques. The CT scan revealed a well-defined heterogeneous mass in the medial orbit. There was no evidence of systemic, serum or biochemical abnormalities. The mass was removed by a medial orbitotomy. Histopathology confirmed the diagnosis based on the presence of inflammatory infiltrates, histiocytes and Touton giant cells.

Clinical outcome of autologous cultivated limbal epithelium transplantation.

PURPOSE: To report the clinical outcome of autologous cultivated limbal epithelial transplantation. METHODS: Eighty-six patients' records and their clinical photographs were reviewed for demographics, primary etiology, type of limbal transplantation, ocular surface stability, visual acuity, final outcome and possible factors affecting outcome and complications. RESULTS: Eighty-eight eyes of 86 patients with limbal stem cell deficiency (LSCD) underwent autologous cultivated limbal epithelium transplantation between March 2001 and May 2003, with a mean follow-up of 18.3 months. The etiology of LSCD was alkali burns in 64% patients. Sixty-one eyes had total LSCD. Thirty-two of the 88 eyes had undergone amniotic membrane transplantation and 10 eyes had previously undergone limbal transplantation with unfavorable outcome. Nineteen eyes underwent penetrating keratoplasty, of which 11 grafts survived at the final follow-up. Finally, 57 eyes (73.1%, 95% CI: 63.3-82.9) had a successful outcome with a stable ocular surface without conjunctivalization, 21 eyes (26.9%, 95%CI: 17.1-36.7) were considered failures and 10 patients were lost to follow-up. CONCLUSION: LSCD can be successfully treated by autologous cultivated limbal epithelium transplantation in majority of the cases.

Primary adult human retinal pigment epithelial cell cultures on human amniotic membranes.

PURPOSE: Retinal pigment epithelial (RPE) cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM) as substrate. MATERIALS AND METHODS: Human cadaver eyeballs (16) were obtained from the eye bank after corneal trephination. RPE cells were harvested by a) mechanical dissection of the inner choroid surface (10, group 1) or by b) enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2). The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students' 't' test. RESULTS: Eleven samples (68.75%) showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days) in the mechanical group, significantly earlier (P < 0.025) than in the enzymatic group (day 29-35, average 31.6 days). CONCLUSIONS: Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes. Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion.

Early results of penetrating keratoplasty following limbal stem cell transplantation.

PURPOSE: To describe the early results of penetrating keratoplasty (PKP) in patients who had earlier received limbal transplantation (LT). METHODS: Prospective, non-comparative interventional case series comprising of four patients with limbal stem cell deficiency (LSCD) due to chemical injury (Cases 1, 2, 4) and xeroderma pigmentosum (Case 3). Cadaveric kerato-limbal allografts or living-related conjunctival-limbal allografts were done in four eyes followed by PKP for visual rehabilitation 3-4.5 months later. The following details were noted: demographics, primary aetiology, type of limbal transplant (cadaveric or living-related), immunosuppression, vision and ocular surface stability before and after LT and PKP, surgical complications and outcome of PKP. RESULTS: Three eyes received living-related conjunctival-limbal allotransplantation and one received cadaveric kerato-limbal allograft. Duration of follow up after PKP ranged from 4 to 11 months. Visual acuity improved in the early postoperative period in all patients but reduced in 2 due to endothelial rejection and after trans-scleral cyclophotocoagulation for medically uncontrolled glaucoma. The ocular surface remained stable in all patients. All patients were started on immunosuppression on the first postoperative day. This was continued till the last follow-up visit. Post-PKP complications were punctate epithelial keratopathy, corneal allograft rejection and secondary glaucoma (one patient each). CONCLUSION: Satisfactory visual rehabilitation is possible after PKP following LT without compromising ocular surface stability. However, a prolonged and close follow-up is warranted to avert complications.

Amniotic membrane transplantation for reconstruction of corneal epithelial surface in cases of partial limbal stem cell deficiency.

PURPOSE: To assess the efficacy of amniotic membrane for treatment of partial limbal stem cell deficiency (LSCD). METHODS: Medical records of four patients with partial LSCD who underwent pannus resection and amniotic membrane transplantation (AMT) were reviewed for ocular surface stability and improvement in visual acuity. Clinico-histopathological correlation was done with the resected pannus tissue. RESULTS: All the eyes exhibited stable corneal epithelial surface by an average of 7 weeks postoperatively with improvement in subjective symptoms. Best corrected visual acuity improved from preoperative (range: 6/9p-6/120) to postoperative (range: 6/6p-6/15) by an average of 4.5 lines on Snellen visual acuity charts. Histopathological examination of excised tissue showed features of conjunctivalisation. CONCLUSION: Amniotic membrane transplantation appears to be an effective means of reconstructing the corneal epithelial surface and for visual rehabilitation of patients with partial limbal stem cell deficiency. It may be considered as an alternative primary procedure to limbal transplantation in these cases.

Lattice corneal dystrophy type III with corneal fistula. A case report.

Lattice corneal dystrophy is a distinct clinical entity characterised by amyloid deposits in the corneal stroma. We report a patient who presented with a corneal fistula in the right eye and thick lattice lines involving the peripheral cornea in both eyes suggestive of type III lattice dystrophy. The association of corneal fistula with lattice corneal dystrophy type III makes this a unique case.

Ex-vivo potential of cadaveric and fresh limbal tissues to regenerate cultured epithelium.

PURPOSE: To evaluate and compare the ex-vivo growth potential and formation of cultured corneal epithelium from residual corneo-limbal rings obtained from the operating room after penetrating keratoplasty, and fresh limbal tissues from patients undergoing routine cataract surgery. METHODS: With the approval of the Institutional Review Board and informed consent from patients, 1-2 mm of limbal tissues from 15 patients and 31 tissues from the cadaveric limbal ring preserved in MK medium (16 tissues) and Optisol (15 tissues) were used for the study. Donor data included age, time lapse between death and collection, collection and preservation and preservation and culture. Tiny bits of the limbal tissue were explanted on the de-epithelialised human amniotic membrane prepared following standard guidelines, and cultured using Human Corneal Epithelial cell medium. Radial growth from the explant was observed and measured by phase contrast microscopy over 2-4 weeks. After adequate confluent growth, whole mount preparation of the membrane was made and stained with haematoxylin and eosin. Part of the membrane was fixed in formalin and processed for routine histologic examination. The sections were stained with haematoxylin and eosin. RESULTS: Forty-six tissues were evaluated from 42 eyes (15 from patients, 31 from cadaveric eyes) with a mean age of 55.3 years +/- 21.23 years (range 18 years - 110 years). The growth pattern observed was similar in all the positive cases with clusters of cells budding from the explant over 24-72 hours, and subsequent formation of a monolayer over the next 2-3 weeks. The stained whole mount preparation showed a radial growth of cells around explants with diameter ranging from 5 to 16mm. Histologic evaluation of the membrane confirmed the growth of 2-3 cell-layered epithelium over the amniotic membrane. Cultivated epithelium around explant cell cultures was observed in 100% (15/15) of limbal tissue obtained from patients, as against 56% (9/16) of MK medium preserved tissues and 46.7% (7/15) of Optisol preserved tissues. This was statistically significant (P=0.0131) There was no significant statistical difference in the growth properties, i.e, the mean percentage of fragments showing growth (P=0.229) or the mean diameter of growth (P=0.479) in the cultures obtained from fresh and preserved tissues. The time lapse at various stages between death and utilisation and donor age had no significant influence on the growth potential of the limbal tissues. CONCLUSION: The potential for generating cultured corneal epithelium from fresh limbal tissues obtained from living subjects is higher than that observed with preserved tissues. It would also be worthwhile to address the factors that could further enhance the proliferative potential of the cadaveric tissues obtained from eye banks.