Antibody and memory B cell responses to the dengue virus NS1 antigen in individuals with varying severity of past infection

To further understand the role of NS1-specific antibodies (Abs) in disease pathogenesis, we compared neutralizing antibody levels (Nabs), NS1-Ab levels, IgG antibody subclass profiles and NS1-specific memory B-cell responses (Bmems) in individuals, with varying severity of past dengue. Nabs (Neut50 titres) were assessed using the Foci Reduction Neutralization Test (FRNT) and in-house ELISAs were used to assess NS1-Abs and NS1-Ab subclasses for all four DENV serotypes in individuals with past DF (n = 22), those with past DHF (n = 14) and seronegative (SN) individuals (n = 7). B-cell ELISpot assays were used to assess NS1-specific Bmem responses. 15/22 (68.18%) individuals with past DF and 9/14 (64.29%) individuals with past DHF had heterotypic infections. Neut50 titres were found to be significantly higher for DENV1 than DENV2 (p = 0.0006) and DENV4 (p = 0.0127), in those with past DHF, whereas there was no significant difference seen in titres for different DENV serotypes in those with past DF. Overall NS1-Ab to all serotypes and NS1-specific IgG1 responses for DENV1, 2 and 4 serotypes were significantly higher in those with past DHF than individuals with past DF. Those with past DHF also had higher IgG1 than IgG3 for DENV1 and DENV3, whereas no differences were seen in those with past DF. Over 50% of those with past DF or DHF had NS1-specific Bmem responses to >2 DENV serotypes. There was no difference in the frequency of Bmem responses to any of the DENV serotypes between individuals with past DF and DHF. Although the frequency of Bmem responses to DENV1 correlated with DENV1-specific NS1-Abs levels (Spearman r = 0.35, p = 0.02), there was no correlation with other DENV serotypes. We found that those with past DF had broadly cross-reactive Nabs, while those with past DHF had higher NS1-Ab responses possibly with a different functionality profile than those with past DF. Therefore, it would be important to further evaluate the functionality of NS1-specific antibody and Bmem responses to find out the type of antibody repertoire that is associated with protection against severe disease.     

Reasons for delay in cataract surgery in patients with advanced cataracts during the COVID-19 pandemic

Purpose:To analyze the reasons for delay in cataract surgery in patients with advanced cataracts during the COVID-19 pandemic.Methods:This was a prospective, cross-sectional, multicenter questionnaire study which included patients with mature cataract, nuclear sclerotic cataract grade IV, and cataracts with best corrected visual acuity (BCVA) <5/60, during the COVID-19 pandemic from December 2020 to April 2021. Reasons for delay in presentation to the hospital were analyzed.Results:One thousand four hundred seventy two patients were recruited with advanced cataracts. Absence of ophthalmic care nearby (44.2%), lack of awareness regarding elective surgeries (42.6%), lack of public transportation (37%), fear of contracting COVID-19 (23.4%), and waiting for outreach camps (20.4%) were found to be the reasons behind the delay in cataract surgery. 53.7% of the patients had worsening of defective vision and 55.3% of them had difficulty in carrying out activities of family living. 30.8% of the patients faced difficulty in commuting and 8.4% of the patients suffered a fall during this pandemic due to worsening of the visual acuity.Conclusion:The lockdown imposed during the pandemic has created a significant backlog of patients who are progressing to advanced cataracts due to lack of ophthalmic care nearby, lack of awareness regarding elective surgeries, lack of public transportation, and no outreach camps. Proactive measures to deal with this backlog are of utmost need to prevent blindness due to cataract.     

Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCdelta in platelets

Thrombin has been known to cause tyrosine phosphorylation of protein kinase C delta (PKCdelta) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCdelta and the role of this event in platelet function. PKCdelta was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCdelta did not occur in Galpha(q)-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5'-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ', N '-tetraacetic acid), suggesting a role for Galpha(q) pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCdelta. Inhibition of tyrosine phosphorylation or the kinase activity of PKCdelta dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCdelta in a calcium- and Src-family kinase-dependent manner in platelets, with functional implications in thromboxane A2 generation.     

Organ perfusion and mass spectrometry: a timely merger for drug development

Organ perfusion techniques bridge the methodological gap between in vivo studies on the one hand and in vitro studies on the other. In drug candidate selection and subsequent development the differences between these systems should be considered carefully in study design, as one approach may be more suitable than the other depending on the question(s) being asked and, in particular, how the data will be used. This article is not concerned with the mechanics, the surgery or composition of perfusates as there are numerous reviews/books covering these aspects. Instead, using perfused gut, liver, lung, kidney and brain as examples, the emphasis is on the usefulness (or otherwise) of the data generated with respect to drug absorption, metabolism, pharmacokinetics (PK) and the factors which affect these parameters. Perfusion systems are not difficult to set up but do require 'high maintenance' for routine use. For this reason they have been used sparingly by the pharmaceutical industry mainly for problem solving or mechanistic studies. The latter part of this article shows how simultaneous dosing of numerous compounds followed by multiple--component analysis using LC/MS/MS has proved to be an effective way to improve the throughput of absorption, pharmacokinetics and metabolism screening ex vivo.     

A study on the comparison between clinical and microbiological diagnoses of sexually transmitted diseases

The efficacy of microbiological diagnosis in sexually transmitted diseases (STDs) has been evaluated in comparison with the clinical diagnosis. Amongst the clinical diagnoses of single STDs, syphilis, genital warts, gonorrhoea and herpes genitalis were the predominant ones. Syphilis was the most predominant infection in both the single and mixed STD infections in Chennai. Clinical diagnoses of trichomoniasis, genital chlamydiasis and genital herpes were more accurate and correlated well with laboratory investigations. On the other hand, clinical diagnoses of gonorrhoea, candidiasis and syphilis were less accurate. More over many of these cases, clinically diagnosed as single, infection, were also positive for other STDs in the laboratory investigations. Double infections were clinically diagnosed only in 7 cases as against 11 cases in microbiological tests and one triple infection diagnosed in microbiological tests was diagnosed only as single disease clinically. Therefore, the laboratory/microbiological investigations have been emphasised to have better accuracy of diagnosis of STDs.     

Detection of disease related immune complexes in the serum of leprosy patients. A novel single step method

Mycobacterium leprae antigen and antibody complexes could be detected in the serum of leprosy patients using monoclonal antibody ML34 and anti-BCG antibodies by enzyme-linked immunosorbent assay. This simplified system detects disease related complexes without the need for isolating and purifying them from the serum. Immune complexes captured using monoclonal antibody ML34 revealed positivity in seven out of eight neuritic, two out of nine tuberculoid (TT), five out of ten borderline tuberculoid (BT), four out of ten borderline lepromatous (BL) and four out of ten lepromatous (LL), leprosy cases. One of the controls also showed immune complex of an IgM type. Anti-BCG based IgG immune complexes assay revealed positivity in six out of eight neuritic, one out of nine TT, four out of ten BT, two out of ten BL, four out of ten LL leprosy cases, and two out of 24 healthy controls. IgM type of mycobacterial immune complexes were almost negligible. Capture of complexes using monoclonal antibody ML34 which is against lipoarabinomannan of M. leprae seems to work better than polyclonal anti-BCG antibody. The probable role of immune complexes in nerve damage needs to be evaluated, as very high levels of immune complexes are found in neuritic leprosy by both the assays. The above test would be useful in immunodiagnosis of neuritic leprosy and also in cases where antibody response is not detectable because of the formation of immune complexes.     

Preventive action of food seasoning spices mixture on fructose-induced lipid abnormalities

High fructose feeding in rats induces insulin resistance, hyperinsulinemia, hyperglycemia and dyslipidemia. The present study was undertaken to determine the hypolipidemic effect of food seasoning spices mixture on fructose-fed insulin resistant rats. Male Wistar rats received a daily diet containing either 60% fructose or 60% starch. They were administered with the spices mixture at three different doses (10 mg, 30 mg or 50 mg/day/rat) orally 15 days later. At the end of 45 days of the experimental period fructose-fed rats displayed elevated plasma glucose and insulin levels and dyslipidemia which included elevated levels of cholesterol, triglycerides, free fatty acids, reduced high density lipoprotein cholesterol and increased very low density lipoprotein cholesterol. Alterations in tissue lipid levels were also observed. Simultaneous treatment with spices mixture along with fructose diet resulted in the normalization of plasma glucose and insulin levels and restoration of lipid levels in plasma and tissues. The insulin potentiating action of the active principles in these spices may contribute to the hypolipidemic effect of spices mixture in high fructose-fed rats.     

The enhancement of riboflavin-mediated photo-oxidation of doxorubicin by histidine and urocanic acid

PURPOSE: Previously we have shown that doxorubicin (Adriamycin, ADR) can be inactivated by light-excited riboflavin. The inactivation of the drug results from its direct oxidation by the excited triplet riboflavin in a type I photosensitization reaction, and 3-methoxysalicyclic acid is an ADR breakdown product. In the present study, we investigated the enhancement of this process by histidine and some other imidazole analogs. METHODS: ADR solutions containing various concentrations of riboflavin and other agents were exposed to 365 nm light for various time periods and then the absorbance spectrum of ADR was measured by a double beam spectrophotometer. These measurement were used to calculate the half-time of the ADR degradation process. The degraded ADR solutions were analyzed by HPLC. RESULTS: The rate of bleaching of ADR by light-excited riboflavin was enhanced in the presence of histidine in a concentration-dependent manner. This enhancement was more pronounced at higher riboflavin concentrations. Histidine also enhanced the riboflavin-mediated photobleaching of N,N-dimethyl-4-nitrosoaniline (RNO), a compound known to be resistant to oxidation by singlet oxygen but sensitive to oxidation by the trans-annular peroxide of histidine. RNO was found to block the histidine enhancement of the riboflavin-mediated photobleaching of ADR in a competitive manner. Among the imidazole analogs of histidine tested, urocanic acid was found to be the most efficient enhancer of the riboflavin-mediated photobleaching of ADR. Superoxide anion radicals which retard the oxidation of ADR were quenched by urocanic acid but not by histidine. It was shown that the oxidation of ADR by the trans-annular peroxide of histidine resulted in the formation of 3-methoxysalicylic acid. CONCLUSIONS: In contrast to singlet oxygen, the trans-annular peroxide, formed by the interaction of histidine and the singlet oxygen produced by photoexcited riboflavin, is an efficient oxidizer of ADR. The enhancement of the riboflavin-mediated photobleaching of ADR by histidine analogs depends on the rate of their conversion to a trans-annular peroxide and on the efficiency of these products in oxidizing ADR. However, for some analogs of histidine, as shown for urocanic acid, other mechanisms could also be involved. The presence of urocanic acid in the skin suggests that significant degradation of ADR could occur in the presence of biologically relevant concentrations of riboflavin if patients treated with ADR are exposed to sunlight. The finding that histidine also enhanced the degradation of ADR to 3-methoxysalicylic acid, suggests that the process of ADR oxidation by the trans-annular peroxides is similar to the direct oxidation of ADR by excited triplet riboflavin.     

Development of rapid assessment procedures for the delimitation of lymphatic filariasis-endemic areas

Lymphatic filariasis caused by Wuchereria bancrofti is a major public health problem in 73 tropical and subtropical countries including India. Delimitation of endemic areas is essential to plan control operations. The current method of night blood survey (NBS) for delimitation is cumbersome, time-consuming and expensive. Therefore, there is a need to develop assessment procedures which can rapidly delimit endemic areas. For this purpose we evaluated three procedures: direct interviewing of key informants using structured questionnaires, an indirect method of a self-administered questionnaires to key informants and physical examination by health workers for the presence of chronic filarial disease. Thirty rural communities in a filariasis-endemic region in Cuddalore district in Tamil Nadu State in southern India constituted the study population. The determination of filariasis endemicity in the village communities assessed by the above procedures was compared in terms of rapidity, specificity, sensitivity and cost with the microfilaria rate and disease rate obtained by night blood sample survey and clinical examination by physicians. Prevalence score, control preference score and weighted mean number of cases with filarial disease per village were calculated using the key informant questionnaire techniques. While the prevalence and control preference score showed low sensitivity and moderate specificity, weighted mean number of cases showed high sensitivity and moderate specificity in identifying endemic villages. The prevalence of disease as determined by the physical examination of a sample population by health workers was highly sensitive in identifying communities endemic for filariasis. The degree of association between the disease rates estimated by physician and trained health workers was significant (r = 0.56; P < 0.05). These observations suggest that the weighted mean number of cases per village obtained through key informant techniques may be considered at a primary level to crudely identify endemic areas, followed by physical examination by health workers for filariasis, since it is relatively cheap and rapid.