Augmenting Vaccine Efficacy against Delta Variant with 'Mycobacterium-w'-Mediated Modulation of NK-ADCC and TLR-MYD88 Pathways.

Mycobacterium-w (Mw) was shown to boost adaptive natural killer (ANK) cells and protect against COVID-19 during the first wave of the pandemic. As a follow-up of the trial, 50 healthcare workers (HCW) who had received Mw in September 2020 and subsequently received at least one dose of ChAdOx1 nCoV-19 vaccine (Mw + ChAdOx1 group) were monitored for symptomatic COVID-19 during a major outbreak with the delta variant of SARS-CoV-2 (April-June 2021), along with 201 HCW receiving both doses of the vaccine without Mw (ChAdOx1 group). Despite 48% having received just a single dose of the vaccine in the Mw + ChAdOx1 group, only two had mild COVID-19, compared to 36 infections in the ChAdOx1 group (HR-0.46, p = 0.009). Transcriptomic studies revealed an enhanced adaptive NK cell-dependent ADCC in the Mw + ChAdOx1 group, along with downregulation of the TLR2-MYD88 pathway and concomitant attenuation of downstream inflammatory pathways. This might have resulted in robust protection during the pandemic with the delta variant.

Indistinct retinal outer layers in the walls of the idiopathic full-thickness macular hole - A potential predictive biomarker for surgical outcomes.

Purpose: To evaluate the absence of external limiting membrane (ELM) and ellipsoid zone (indistinct retinal outer layers, I-ROL) in the walls of idiopathic full-thickness macular holes (FTMHs) circumferentially on optical coherence tomography (OCT) and its correlation with surgical outcome. Methods: In this retrospective observational study, OCT images of patients undergoing vitrectomy for FTMHs with at least 3-months of postoperative follow-up were analyzed for preoperative circumferential extent of I-ROL. Derived macular hole indices such as hole form factor (HFF), macular hole index (MHI), tractional hole index (THI), and hole diameter ratio (HDR) were also calculated. The circumferential extent of I-ROL was correlated with derived hole indices as well as anatomical closure, foveal architecture, and restoration of ELM following surgery. Results: All nine eyes (eight patients) with FTMH (mean size: 610.11 ± 122.95 microns) in the study showed I-ROL in ≥1 quadrant. The mean HFF, MHI, THI, and HDR values were 0.72 ± 0.09, 0.35 ± 0.05, 0.71 ± 0.24, and 0.53 ± 0.14, respectively. All eyes achieved type-1 hole closure with improvement in best-corrected visual acuity to 0.58 ± 0.32 LogMAR from 0.81 ± 0.26 LogMAR. Regular foveal architecture was achieved in six eyes. Out of these, five eyes had I-ROL in ≥2 quadrants, and one eye had I-ROL in <2 quadrants (P = 0.0476). Restoration of ELM was seen in aforementioned six eyes (complete = 5, partial = 1). Out of the five eyes with complete ELM restoration, four had a circumferential extent of I-ROL in ≥2 quadrants (P = 0.0476). Complete restoration of ELM was associated with the complete restoration of the ellipsoid zone in three eyes. Conclusion: Preoperative circumferential extent of I-ROL in FTMH walls can be a potential predictive OCT marker for the type of closure, postoperative foveal architecture, and ELM restoration.

Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19.

The kinetics of NKG2C+ adaptive natural killer (ANK) cells and NKG2A+inhibitory NK (iNK) cells with respect to the incidence of SARS-CoV-2 infection were studied for 6 months in a cohort of healthcare workers following the administration of the heat-killed Mycobacterium w (Mw group) in comparison to a control group. In both groups, corona virus disease 2019 (COVID-19) correlated with lower NKG2C+ANK cells at baseline. There was a significant upregulation of NKG2C expression and IFN-γ release in the Mw group (p=0.0009), particularly in those with a lower baseline NKG2C expression, along with the downregulation of iNK cells (p<0.0001). This translated to a significant reduction in the incidence and severity of COVID-19 in the Mw group (incidence risk ratio-0.15, p=0.0004). RNA-seq analysis at 6 months showed an upregulation of the ANK pathway genes and an enhanced ANK-mediated antibody-dependent cellular cytotoxicity (ADCC) signature. Thus, Mw was observed to have a salutary impact on the ANK cell profile and a long-term upregulation of ANK-ADCC pathways, which could have provided protection against COVID-19 in a non-immune high-risk population.

BLIMP-1 Mediated Downregulation of TAK1 and p53 Molecules Is Crucial in the Pathogenesis of Kala-Azar.

Precise regulation of inflammasome is critical during any pathogenic encounter. The whole innate immune system comprising of pattern recognition receptors (PRRs) relies on its ability to sense microbes. The fate of cellular death in infected cells depends mostly on the activation of these inflammasome, the dysregulation of which, due to functional manipulation by various pathogens, leads to be the cause of many human diseases. Here, an interesting finding has been observed which is related to how Leishmania donovani parasites exploit various host mediator molecules to cause immunosuppression. Here we report for the first time that the parasites check pyroptosis in the infected cells in-vitro by BLIMP-1 mediated suppression of TAK1 and p53 proteins. This might be one of the reasons how parasites evade the pro-inflammatory response of the host cells. Further understandings and validations are required to come up with better therapeutic approaches against kala-azar.

BLIMP-1 Plays Important Role in the Regulation of Macrophage Pyroptosis for the Growth and Multiplication of Leishmania donovani.

Visceral leishmaniasis, one of the fatal forms of the disease, is caused by Leishmania donovani and presents morbid clinical manifestations. The parasite evades pro-inflammatory immune responses by several reported mechanisms and modulates the host immune system to cause fatal symptoms. A plethora of reports related to the role of BLIMP-1 and its involvement in suppressing the immune response in various infectious diseases have been documented. Higher parasitic burden due to increased BLIMP-1 production has been reported earlier for malaria and leishmaniasis with no detailed information. We report for the first time the role of BLIMP-1 in suppressing macrophage pyroptosis during L. donovani infection and thereby tweaking the tight regulation of the NFκß-NLRP3 signaling pathway. Expression analyses of BLIMP-1 and NFκß have been measured using real-time PCR and Western blotting. The importance of BLIMP-1 has been validated using a siRNA-mediated experiment along with caspase 1 activity, LDH release assay, and infectivity index analyses. An inverse relationship between BLIMP-1 and NFκß expression has been highlighted during L. donovani infection, which is reversed in blimp-1 deficient cells infected with promastigotes. The above fact has been further validated with caspase 1 activity assay, and LDH release along with IFNγ and TNF-α release assay. Finally, resumption of pyroptosis has been concluded in infected blimp-1 deficient cells in contrast to wild type infected cells. We conjecture that parasites modulate the NFκß-NLRP3 signaling pathway by taking advantage of BLIMP-1 dependent IL-10 production and finally disrupting an inflammation-mediated pyroptosis cell death pathway in infected cells.

Leishmania donovani evades Caspase 1 dependent host defense mechanism during infection.

Significant advances have been made in understanding the regulation of inflammasomes and its involvement in innate immunity during pathogenic infections. Inflammasome activation is a tightly regulated process that provides defense against pathogenic infection and important for inflammatory response. Very few studies on the involvement of NLRP3 inflammasome protein complex have been reported in leishmanial infections with contradictory results and without much mechanistic insights. However, the role of NLRP3 inflammasome and its components has not been well deciphered in Leishmania donovani infection. Here we report for the first time a detailed mechanism and plausible impairment of caspase 1 activation during L. donovani infection leading to the survival of these parasites inside the host cells. Low mRNA expression of pro-caspase 1 and lack of caspase 1 maturation were observed after infection, hindering the processing of pro-IL-1ß and pro-IL-18 into their mature counter parts. Further, siRNA mediated knock-down of caspase 1 in macrophage cells (THP-1) resulted in significantly higher parasitic burden validating the importance of caspase 1 in the host defense mechanism. Taken together, our data suggests that the parasite inhibits caspase 1 activation to evade the inflammatory nature of pyroptosis.

Efficacy and Safety of Mycobacterium indicus pranii as an adjunct therapy in Category II pulmonary tuberculosis in a randomized trial.

Prolonged treatment of tuberculosis (TB) often leads to poor compliance, default and relapse, converting primary TB patients into category II TB (Cat IITB) cases, many of whom may convert to multi-drug resistant TB (MDR-TB). We have evaluated the immunotherapeutic potential of Mycobacterium indicus pranii (MIP) as an adjunct to Anti-Tubercular Treatment (ATT) in Cat II pulmonary TB (PTB) patients in a prospective, randomized, double blind, placebo controlled, multicentric clinical trial. 890 sputum smear positive Cat II PTB patients were randomized to receive either six intra-dermal injections (2 + 4) of heat-killed MIP at a dose of 5 × 108 bacilli or placebo once in 2 weeks for 2 months. Sputum smear and culture examinations were performed at different time points. MIP was safe with no adverse effects. While sputum smear conversion did not show any statistically significant difference, significantly higher number of patients (67.1%) in the MIP group achieved sputum culture conversion at fourth week compared to the placebo (57%) group (p = 0.0002), suggesting a role of MIP in clearance of the bacilli. Since live bacteria are the major contributors for sustained incidence of TB, the potential of MIP in clearance of the bacilli has far reaching implications in controlling the spread of the disease.

Mycobacterium W administration for steroid resistant optic neuritis with long-term follow-up.

BACKGROUND: We report the long-term safety and outcomes of off-label Mycobacterium W (Mw) administration in steroid resistant optic neuritis. METHODS: In a case series, six patients with documented idiopathic corticosteroid refractory unilateral optic neuritis were treated with immuvac (Mycobacterium W extract). The dose was repeated at three months. Outcomes measures included the best corrected visual acuity (BCVA), pupillary reaction, colour vision, visual field(VF) examination (when possible), fundus examination and photography, and visually evoked potential (VEP) testing. BCVA, pupillary reaction, and color vision were monitored immediately prior to steroid therapy on days 1 and 7 post-steroid therapy, pre-Mw administration (i.e., 30 days after the last dose of steroids had been completed) and post-Mw administration on days 1, 7, 30, 90, 120, and 180. VF, VEP, and fundus photography were performed immediately prior to steroid administration, 30 days after the last dose of steroids (i.e., immediately prior to Mw), and at days 30, 90,120 and, 180. The patients were assessed six-month intervals thereafter for visual acuity, colour vision, and visual fields. RESULTS: There were five females and three males in the age range of 30-54 years. Minimum follow-up was five years. All patients showed improvement in visual acuity, colour vision, and pupillary reaction. The patients showed stable improvement. There was no recurrence of the disease and no adverse events till the end of the follow-up period. CONCLUSIONS: Mw appears to be safe in the long term and to improve steroid resistant optic neuritis; future randomized clinical trials would help affirm this observation.

Methanol poisoning: predictors of visual outcomes.

OBJECTIVE: To determine whether laboratory markers of methanol ingestion and subsequent toxicity can serve as predictors of visual outcomes in patients. METHODS: Retrospective medical record review of 122 patients in a cluster outbreak of methanol poisoning. Data collected included history, complete ocular and systemic examination details, time to presentation, amount of alcohol ingested, and results of laboratory investigations, such as hemogram, glucose levels, hematocrit level, arterial pH, methanol levels, potassium and bicarbonate levels, and anion and osmolar gap determination, as well as hepatic and renal function tests. Therapy administered consisted of ethyl alcohol, sodium bicarbonate, and nutritional supplements, with hemodialysis in severe cases. Visual acuity (VA), pupillary reaction, and optic disc findings were assessed at presentation and 3 months after discharge. Patients were classified according to their visual disturbance: transient (group 1) or permanent (group 2). Appropriate statistical analysis was performed. Outcome measures included determining the association between biochemical markers of methanol poisoning and final VA. RESULTS: A total of 122 patients (1 female and 121 male) were admitted for treatment; of these, 10 died. Only 1 patient showed a 2-line drop in VA. pH was the strongest predictor of final VA and improvement in VA among all markers. The odds that a patient with an initial pH greater than 7.2 would have only transient visual disturbances were high (odds ratio, 31; 95% CI, 6-149). CONCLUSIONS: The degree of acidosis at presentation appears to determine final VA; early presentation and treatment did not seem to significantly alter the visual outcome, especially in severe poisoning.

Outcomes of toll-like receptors' antagonism in steroid-resistant optic neuritis; a pilot study.

BACKGROUND: To investigate the safety and outcomes of off-label immunomodulator Mycobacterium w. (Mw), a TLR 9 antagonist in steroid-resistant idiopathic unilateral optic neuritis. METHODS: Case series. Eight patients with documented idiopathic unilateral optic neuritis who did not improve with methyl prednisolone followed by oral steroids as per the Optic Neuritis Treatment Trial (ONTT) were administered Mw 5 ml in 500 ml normal saline, 30 days after the last of dose of steroids had been administered. The dose was repeated at 3 months. Outcome measures monitoring the change in the best-corrected visual acuity (BCVA), pupillary reaction, colour vision, visual field (VF) examination (when possible), fundus examination and photography, visually evoked potential (VEP) testing. BCVA, pupillary reaction, and color vision were monitored immediately prior to steroid therapy, on days 1 and 7 post steroid therapy, pre Mw administration (i.e., 30 days after the last dose of steroids had been completed) and post Mw administration on days 1, 7, 30, 90, 120 and 180. VF, VEP and fundus photography was performed immediately prior to steroid administration, 30 days after the last dose of steroids (i.e., immediately prior to Mw), and days 30, 90,120 and 180 post Mw administration. RESULTS: There were five females and three males in an age range of 30-54 years. Six patients were available for follow-up at 6 months. All patients showed improvement in visual acuity, colour vision & pupillary reaction. Visual field monitoring was possible in four patients; all four had a centrocecal scotoma that persisted post steroid therapy but resolved 1 month post Mw therapy. Three out of five patients who had disc edema were available for all follow-ups, and showed resolution of disc edema post Mw therapy. The disc edema had persisted post steroid therapy. No adverse events were seen. The 2nd dose did not improve any of the said parameters. There was no recurrence of the disease up to the end of the follow-up period. CONCLUSIONS: Mw appears to improve steroid resistant optic neuritis; future randomized clinical trials would help affirm this observation.

Preservation of bioavailability of ingredients and lack of drug-drug interactions in a novel five-ingredient polypill (polycap): a five-arm phase I crossover trial in healthy volunteers.

BACKGROUND: The Polycap (polypill; aspirin [acetylsalicylic acid], ramipril, simvastatin, atenolol, and hydrochlorothiazide) was found to be safe and effective for reducing multiple cardiovascular risk factors in The Indian Polycap Study (TIPS). OBJECTIVE: We evaluated the bioavailability of each ingredient of the Polycap and determined any drug-drug interactions relative to single component reference preparations. METHODS: The bioavailability of the ingredients of the Polycap (T; test) when formulated as a single capsule was compared with that of identical capsules with each of its ingredients administered separately (R; reference) in a five-arm, randomized, single-dose, two-period, two-treatment, two-sequence, crossover trial with at least a 2-week washout period in a total of 195 healthy volunteers. Plasma concentrations of each drug and, where applicable, its active metabolite were measured using validated liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography. Mean pharmacokinetic parameters and their standard deviations were computed for each analyte. RESULTS: Comparative bioavailability was computed and no drug-drug interactions and no difference in comparative bioavailability were concluded for each ingredient based on point estimates of the T/R ratio of the geometric means falling within 80-125% for peak plasma concentration (C(max)), area under the plasma concentration-time curve from time zero to the last measurable concentration (AUC(t)), and AUC from time zero to infinity (AUC(infinity)). The T/R ratio for C(max), AUC(t) and AUC(infinity) was within 80-125% for atenolol, hydrochlorothiazide, ramipril, ramiprilat and dose-normalized salicylic acid. However, for simvastatin, the T/R point estimates for C(max), AUC(t) and AUC(infinity) for Ln-transformed data were significantly lower ( approximately 3-4%) than the lower bound of 80%. For its active metabolite, simvastatin acid, these estimates were significantly higher ( approximately 25-35%) than the higher bound of 125%. Thus, the increased bioavailability of active simvastatin acid appeared to compensate for the loss of bioavailability of simvastatin. CONCLUSION: The Polycap was found to be effective and safe in the previously published TIPS trial. The present study in healthy volunteers establishes that Polycap is safe (no serious adverse events) and well tolerated, and that there is no indication of pharmacokinetic drug-drug interactions for any of the ingredients, with their bioavailabilities being well preserved.