Ocular surface sphingolipids associate with the refractory nature of vernal keratoconjunctivitis: newer insights in VKC pathogenesis.

BACKGROUND: The etiopathogenesis of vernal keratoconjunctivitis (VKC) is incompletely understood. Bioactive lipids play a key role in allergic disorders. This study focused on the sphingolipid metabolism on the ocular surface of VKC and to explore if it has a contributory role in the refractoriness of the disease. METHODS: Active VKC cases, (n=87) (classified as mild/moderate and severe/very severe based on the disease symptoms) and age-matched healthy controls (n=60) were recruited as part of a 2-year prospective study at a tertiary eye care centre in South India. Conjunctival imprint cytology was used to assess gene expression of enzymes of sphingolipids metabolism. Sphingolipids were estimated in the tears by LC-MS/MS analysis. In vitro study was done to assess IgE-induced alterations in sphingosine-1-phosphate (S1P) receptor expression and histone modification in cultured mast cells. RESULTS: Significantly altered gene expression of the sphingolipids enzymes and S1P receptor (SIP3R) were observed in conjunctival imprint cells of VKC cases. Pooled tears analysis revealed significantly lowered levels of S1P(d17:0), S1P(d20:1) (p<0.001) and S1P(d17:1) (p<0.01) specifically in severe/very severe VKC compared with both mild/moderate VKC and control. Cer(d18:/17:0) (p<0.001), ceramide-1-phosphate (C1P)(d18:1/8:0) (p<0.01) and C1P(d18:1/2.0 (p<0.05) were lowered in severe/very severe VKC compared with mild/moderate VKC. Cultured mast cells treated with IgE revealed significantly increased gene expression of S1P1 and 3 receptors and the protein expression of histone deacetylases (1, 6). CONCLUSION: Altered sphingolipid metabolism in the ocular surface results in low tear ceramide and sphingosine levels in severe/very severe VKC compared with the mild/moderate cases. The novel finding opens up fresh targets for intervention in these refractory cases.

Altered retinoid metabolism gene expression in chronic Stevens-Johnson syndrome.

BACKGROUND: Stevens-Johnson syndrome (SJS), a blistering disorder of the skin and mucous membrane, leads to ocular morbidity in >60% of cases. Retinoids are vital micronutrients for vision, regulating corneal and conjunctival cell proliferation, differentiation and immune function. This prospective case-control study probed for alterations in retinoid metabolism by evaluating retinoic acid receptor signalling in the conjunctival cells of patients with SJS. METHODS: Imprints were collected from the bulbar conjunctiva of patients with chronic SJS. The gene expression of retinoic acid receptors, namely, RXRA, RARA, RARG, RORA; the fibrosis marker TGFß and its receptor TGFßRII; the transcription factors PPAR-γ, STRA6 and Stat3; the enzymes aldehyde dehydrogenase (ALDH1a1), alpha-1 antitrypsin (A1AT); and the Cyp genes Cyp26a1 and Cyp26b1 were assessed by quantitative PCR in patients with SJS pre-mucous (n = 34) and post-mucous membrane graft (MMG) intervention (n=19) in comparison with age-matched/sex-matched healthy controls (n=20). Western blot analysis of ALDH1a1, RARA and RARG were done in the conjunctival imprint cells. RESULTS: The transcript levels of ALDH1a1, RXRA, RORA, STRA6, Cyp26a1 and Cyp26b1 were decreased around 4, 26, 17, 129, 9 and 8 folds, respectively, and RARA, RARG, PPAR-γ, TGFß, TGFßRII were increased by 12, 15, 51, 16 and 87 folds, respectively, in SJS conjunctiva at the pre-MMG stage. The changes in RORA, Cyp26a1, Cyp26b1, RARA and Stat3 were statistically significant (p<0.05). Changes in protein expression of ALDH1a1, RARA and RARG supported the gene expression changes. CONCLUSIONS: The study provides the first experimental insight into the role of retinoid metabolism in the ocular sequelae of chronic SJS.

Increased parasite surface antigen-2 expression in clinical isolates of Leishmania donovani augments antimony resistance.

Resistance to sodium antimony gluconate (SAG) is a major cause of therapeutic failure in a large proportion of visceral leishmaniasis (VL) cases. Determinants of SAG resistance have been widely studied; however, the mechanism operating in clinical isolates is poorly understood. In the present study, expression of parasite surface antigen-2 (PSA-2) gene was studied in clinical isolates of Leishmania donovani comprising of antimony resistant (n=10) and sensitive (n=4) parasites. The expression of PSA-2 gene was found to be consistently high in SAG resistant clinical isolates (≥1.5-fold) at both transcript and protein level. Further, over-expression of PSA-2 in L. donovani isolates (LdPSA-2(++)) resulted in conversion of SAG sensitive phenotype to resistant. The LdPSA-2(++) parasites showed significantly decreased susceptibility towards SAG (>12-fold), amphotericin B (>4-fold) and miltefosine (>2.5-fold). Marked decrease in antimony accumulation and enhanced tolerance towards complement mediated lysis was evident in LdPSA-2(++) parasites. The study established the role of PSA-2 gene in SAG resistance and its potential as a biomarker to distinguish resistant and sensitive clinical isolates of L. donovani.

Diagnosis of visceral leishmaniasis: developments over the last decade.

Diagnostic parameters for visceral leishmaniasis (VL), a potentially fatal parasitic disease caused by Leishmania donovani, have been redefined in the last decade with the development of serological and molecular tests, though a definitive diagnosis still banks on the century-old parasitological methods in many areas. Recombinant antigens have improved performance of serodiagnostic methods. Serology-based tests, rk39 antigen dipstick, and direct agglutination test commonly employed in the field are highly sensitive methods, however, fail to distinguish past infections. Molecular approaches have become increasingly relevant due to remarkable sensitivity, specificity, and flexibility in choice of samples. Quantitative polymerase chain reaction is a highly sensitive and specific tool used in referral labs for detection/assessment of parasite load in VL patients and subsequently in monitoring treatment response to antileishmanial agents. The method displays potential to provide threshold for distinguishing asymptomatics in endemic areas. Currently, improvement in VL diagnostics is required for successful decentralized (point-of-care) testing in field conditions and to detect VL-HIV co-infection. Techniques such as loop-mediated isothermal amplification offer a reliable molecular diagnostic method for field application. The diagnosis based on bioanalytics/biosensors promise frontiers for point-of-care VL detection after adequate standardization. This review summarizes the recent developments in VL diagnostics, drawing attention towards the need for standardization of the diagnostics across the affected regions.

Presence of anti-Lepp12 antibody: a marker for diagnostic and prognostic evaluation of visceral leishmaniasis.

The diagnostic potential of recombinant Lepp12 (rLepp12) antigen cloned from Leishmania infantum was assessed in L. donovani infections by Western blotting. Ninety-two serum samples, including 30 patients with active kala-azar (KA), 17 post-treated KA patients (KA-PT), 20 post-kala-azar dermal leishmaniasis (PKDL) patients and 25 controls, were analysed for rLepp12, rK39 and DAT positivity. All KA samples taken at pre-treatment stage were positive for Lepp12 antibodies. Seventeen of these were evaluated post treatment (KA-PT), 10 of which were found to be negative. Nine of these 10 negative cases corresponded to clinically cured patients with regressed spleen. Seven post-treatment cases were rLepp12-positive; all of them corresponded to patients who were considered clinically cured but continued to have an enlarged spleen (> or =5 cm). The majority of PKDL patients (18/20) were found to be seronegative by immunoblot test using rLepp12 antigen. The rLepp12-based Western blot diagnosed 100% of patients with visceral disease, whilst none of the control cases were found to be reactive to rLepp12. rLepp12 protein provides a useful reagent for highly sensitive and specific diagnosis of KA. Additionally, rLepp12 appears to have potential as a prognostic marker for the infection.