Clear cell renal cell carcinoma cells show diffuse expression of phagocytotic markers through the identification of lysosome-rich eosinophilic granules.

BACKGROUND: Paneth cell-like granules (PCLG) in clear cell renal cell carcinomas (RCC) have previously been reported but were not found to express neuroendocrine markers. This study was to investigate if the eosinophilic granules (so called PCLG) were enlarged lysosomes. METHODS: A retrospective review of 72 different renal tumors was conducted which included 42 clear cell RCC, 16 papillary RCC, 6 chromophobe RCC, 5 clear cell papillary RCC, 2 urothelial carcinomas and 1 unclassified RCC. All tumors were evaluated for the eosinophilic granules on hematoxylin and eosin-stained sections. In addition, PAS-D staining, immunohistochemical stains, and electron microscopy were performed. RESULTS: The eosinophilic granules were found in 19% (8 out of 42) clear cell RCC, but not in the other renal tumor types. The granules stained positively for PAS-D and were also positive for lysosomal protein markers CD68 and lysozyme. Electron microscopy revealed that the eosinophilic granules were smooth ball-shaped structures in the cytoplasm, ranging in size from 0.8 to 1.4 µm. The overall findings indicate that the eosinophilic granules were best correlated with lysosomes. CONCLUSIONS: The eosinophilic granules in clear cell RCC are expanded lysosomes, and this may be used as a unique feature for confirming the pathologic diagnosis of clear cell RCC. The findings further support the view that clear cell RCC have phagocytic capacity due to their containing abundant lysosomes in the cytoplasm.

Punctate IgG staining particles localize in the budding ballooning clusters of reactive foot processes in minimal change disease.

The etiology of minimal change disease (MCD) remains a mystery as the only characteristic findings are the diffuse effacement of foot processes seen on electron microscopy (EM). Punctate IgG staining found floating outside glomerular capillary loops in MCD cases was recently identified as autoimmune antibodies against nephrin of podocytes. We hypothesized that the punctate IgG staining is located on budding ballooning clusters (BBC) of reactive foot processes in Bowman's space found on EM. We identified seven patients with MCD cases showing IgG staining that were subsequently evaluated for BBC on EM. We concurrently examined 12 negative controls, either unremarkable cases or tubulointerstitial diseases, by EM. Immunogold labeling was performed to confirm the presence of IgG and determine localization. In seven MCD cases, there were positive punctate IgG staining particles outside of the glomerular basement membranes (GBM) along with concurrent punctate staining for C3, kappa, and lambda. By EM, all seven (100%) MCD cases revealed BBC that was characterized by ballooning foot processes ranging from 1 to 6 µm and was either budding or detached from the GBM in 3-7 clusters; no electron-dense materials were seen in BBC. BBC was also seen in only 1 of 12 (8%) negative controls. Immunogold labeling identified IgG particles within BBC of MCD by EM, but not in the negative control. Our data suggest that BBC are EM structures of reactive foot processes that are most likely correlated with punctate IgG staining seen in cases of MCD, supported by immunogold labeling for IgG.

Impact of Covishield Vaccination in Terms of SARS CoV-2 Neutralizing Antibody Expression.

The vaccination efficacy can indirectly be assessed through the quantification of neutralizing antibodies. Very few data are available on Covishield efficacy in terms of neutralizing antibody expression upon vaccination. This study is focused on profiling of neutralizing antibody expression during and after the Covishield two shot vaccination and observing COVID-19 infection in vaccinated participants during the period. SARS CoV-2 neutralizing antibody concentrations in samples were estimated using electrochemiluminescence immunoassay kit for Lifotronics eCL8000. The sampling had been done sequentially at 45th, 85th day after 1st dose and 15th day after 2nd dose Covishield vaccination. Parallelly, in order to confirm the total SARS CoV-2 IgG response in COVID-19 infection, measured the IgG using SARS CoV-2 IgG lateral flow immunoassay test kit. The subjects previously infected with COVID-19 before 1st dose vaccination demonstrated high neutralizing antibody (> 10AU/ml). In COVID-19 uninfected subjects, there was a sudden incline in neutralizing antibody after the 2nd dose. Infection with SARS CoV-2 between 1st and 2nd dose of Covishield vaccination implicate that the level of neutralizing antibody in serum after 1st dose was not adequate to combat the virus and prevent infection. We observed COVID-19 infection in participants even after 2nd dose of vaccination. Interestingly, there was no protection against SARS CoV-2 even with a high neutralizing antibody expression of 188.5 AU/mL after the 2nd dose. Findings of Covishield efficacy in different cohort samples before and after 2 doses of Covishield vaccination provide impetus for improvement or development of next generation vaccines.

Diagnostic Accuracy of SARS-CoV-2 Nucleocapsid Antigen Self-Test in Comparison to Reverse Transcriptase-Polymerase Chain Reaction.

BACKGROUND: Currently, the rapid antigen test (RAT) and reverse transcriptase-polymerase chain reaction (RT-PCR) are considered the main stakeholders in COVID-19 diagnosis. In RT-PCR, any of at least 2 evolutionary conserved genes (RdRP, E-, N-, ORF1ab gene) and S-gene of SARS-CoV-2 are endorsed, and in RAT, the nucleocapsid antigen (N-Ag) of SARS-CoV-2 is considered due to its stability and fewer chances of mutation effects. In the present work, we evaluated the performance of the AG-Q COVID-19 N-Ag self-test kit and conducted a validation study in comparison with the RT-PCR. METHODS: AG-Q COVID-19 N-Ag rapid test kit is an Indian Council of Medical Research (ICMR) approved product developed and marketed by Agappe Diagnostics Limited. The RT-PCR assay was performed with a COVIPATH COVID-19 RT-PCR kit from Thermo Fisher Scientific. RESULTS: We observed 19 false-negative results in antigen self-tests, including samples of threshold cycle (Ct) values 22/22 (N-gene/ORF1ab-gene) in RT-PCR, indicating inadequate sampling by the patients in self-tests, leading to false-negative results and increased chances of the disease spreading. Based on the RT-PCR Ct value vs antigen self-test comparison, it is evident that proper sampling is crucial in performing antigen self-tests. Also, there were weak positive results in antigen self-tests with a Ct value of 18/19 in RT-PCR. CONCLUSIONS: Although the sensitivity and diagnostic accuracy offered by the AG-Q COVID-19 N-Antigen self-test in comparison with RT-PCR fulfills the ICMR tenets for RAT, this study recommends the laboratory/hospital-based RAT execution would be appropriate, rather than the self-test.

Sequential Profiling of Anti-SARS CoV-2 IgG Antibody in Post COVID-19 Patients.

Upon SARS CoV-2 infection, humoral immune system triggers production of anti-SARS CoV-2 IgM and IgG antibodies. Currently, antibodies against SARS CoV-2 spike protein receptor binding domain play a central role in disease protection, making them potential target for in vitro diagnostics applications. This study determines the expression level and sustainability of anti-receptor binding domain (RBD) SARS CoV-2 IgG in post COVID-19 patients. Anti-RBD SARS CoV-2 IgG antibodies in patient serum were analysed by standardised indirect ELISA using SARS CoV-2 spike receptor binding domain protein and HRP conjugated anti-human IgG antibody (anti-h IgG). The study was conducted using 35 adult patient samples with confirmed SARS CoV-2 infection. Additionally, correlation between antibody response after each stage and disease symptoms in post COVID-19 patients were studied. Maximum antibody titre was seen at Day 40 and decreased relatively to Day 180 in antibody positive samples when compared with controls. Overall, more IgG antibody expression is observed in patients who suffered from loss of smell and taste at Day 40. 71% of the positive subjects in this study showed high SARS CoV-2 IgG antibody concentration of above 10 ng/mL and 37% showed strong antibody concentration above 20 ng/mL at the peak of seroconversion.

Development and Troubleshooting in Lateral Flow Immunochromatography Assays.

The development of Lateral Flow Immunochromatography Assay can be divided into two levels; standardizing membrane characteristics and optimizing molecular level immunoassay reaction between analyte and detector molecules. In the preliminary phase the reaction specificity of capture and detector antibodies with the analyte has to be checked with other techniques like ELISA. Molarity and pH of conjugation buffer have prime importance in the immunoreaction among analyte and antibodies. Epitope mapping of the capture and detector antibodies is also recommended to confirm the specificity of the assay. Standardization of membrane characteristics directly relates to the sensitivity of the assay through its porosity, hydrophobicity, protein holding/releasing capacity and wicking rate. Under optimised condition a perfect Lateral Flow Immunochromatography Assay should have high on-rate (target binding efficiency), low off-rate (target releasing efficiency) and low Cross-reactivity. In this manuscript, we share our experience, especially on developmental strategies and troubleshooting, that we have experienced during Lateral Flow Immunochromatography Assay kit development.

COVID-19: Current Trends in Invitro Diagnostics.

The novel coronavirus SARS-CoV-2 is the seventh known species of coronavirus, infectious to human beings. The pandemic COVID-19 spread all over the world with an unprecedented spreading rate after its first appearance in Wuhan, China. As a novel viral disease there in no antiviral treatment or vaccine for the COVID-19. At present, the early detection and the quarantine of infected patients are the ways to stop the spreading of the disease. This review will discuss about the current invitro diagnostic methods used worldwide for the early and accurate diagnosis of COVID-19. Currently the nucleic acid based polymerase chain reaction is used as the reliable diagnostic platform and antigen/antibody detection immunoassays are playing the role of screening tests for early detection and prognosis in COVID-19 treatment.

Removal of malachite green from aqueous solutions using a modified chitosan composite.

The synthesis of a novel composite adsorbent prepared from coir pith activated carbon (CPAC), chitosan and sodium dodecyl sulphate (SDS, an anionic surfactant) is reported. The characterisation of the composite was done using SEM, XRD, UV-visible and IR spectroscopy studies. The effectiveness of the composite was made for the removal of a toxic cationic dye, malachite green (MG) from waste water based on adsorption studies. The reaction conditions for the adsorption studies were optimized based on initial dye concentration, dose rate, reaction time, pH and temperature. Langmuir and Freundlich isotherm models were adopted to study the mechanism of adsorption. The adsorption process was found to follow pseudo second order kinetics. The results of the present study indicate that the CPAC based composite could be an effective low cost adsorbent for the removal of MG from waste water.

Platelet parameters in children with chromosome 22q11 deletion and conotruncal heart defects.

BACKGROUND AND OBJECTIVES: The 22q11 deletion syndrome is associated with a wide spectrum of phenotypic features, hence clinical diagnosis is difficult. Individuals with this syndrome are found to have a risk of developing transfusion associated graft-versus-host reaction, if they are given nonirradiated blood. Our aim was to elucidate whether chromosome 22q11 deletion in children with syndromic conotruncal heart defects is associated with platelet abnormality. MATERIALS AND METHODS: The genetic analysis was performed by standard cytogenetic and Fluorescence in situ hybridization technique. The platelet parameters in 39 patients with chromosome 22q11 deletion were compared with 154 cases without deletion. RESULTS: In deletion versus no deletion group, the mean of mean platelet volume (MPV) was 10.5 ± 2.5 vs 7.6 ± 1.5 fL, platelet count was 225 ± 80.7 and 339 ± 127.3 × 10 9 /L and frequency of high MPV was 49% vs 7% (P < .0001). The MPV was associated with a sensitivity of 90.9% and a specificity of 79.6% at a cutoff value of 8.32 fL, (area under the ROC curve 91%). A nonsignificant negative correlation was found between MPV and platelet count (r = -0.152; P = .361) in children with deletion. CONCLUSION: A cutoff value of 8.32 fL for MPV can be an indicator of high risk of chromosome 22q11 deletion in individuals with syndromic conotruncal defects. Individuals with chromosome 22q11 deletion should be given irradiated blood especially during cardiac surgery. Further investigation should clarify the etiology behind variation in frequency of high MPV in different conotruncal lesions.

Dietary Fats and Oxidative Stress: A Cross-Sectional Study Among Coronary Artery Disease Subjects Consuming Coconut Oil/Sunflower Oil.

Coconut oil has been used by the people of Kerala as a cooking medium for several decades. Due to its alleged hypercholesterolemic activity, general population in recent times is shifting to cooking oils rich in polyunsaturated fats, the most popular being sunflower oil. The effect of long-term consumption of sunflower oil on oxidative stress in humans is not well investigated. We studied oxidative stress among coronary artery disease (CAD) patients who were consuming coconut oil or sunflower oil as a part of their routine diet. Men, aged 35-70 years, with established CAD, who presented to the hospital for routine cardiac evaluations, were enrolled in this observational study. Group 1 and 2 consisted of 73 and 80 subjects consuming coconut oil and sunflower oil respectively for over a period of 2 years. Lipid profile and parameters for oxidative stress were evaluated among them. Conventional lipid parameters did not differ significantly between the two groups. Mean vitamin C concentration was significantly reduced for subjects on sunflower oil compared to those consuming coconut oil (P = 0.044). Malondialdehyde was higher for sunflower oil consumers compared to coconut oil consumers (P < 0.0001). Other parameters such as oxidized LDL, GSH, GPx and SOD were not found to be significantly different between the two groups. The results of the present study show that coconut oil did not induce hypercholesterolemia compared to sunflower oil. On the other hand, sunflower oil group had elevated oxidative stress compared to coconut oil group.

Tissue-engineered tendon constructs for rotator cuff repair in sheep.

Current rotator cuff repair commonly involves the use of single or double row suture techniques, and despite successful outcomes, failure rates continue to range from 20 to 95%. Failure to regenerate native biomechanical properties at the enthesis is thought to contribute to failure rates. Thus, the need for technologies that improve structural healing of the enthesis after rotator cuff repair is imperative. To address this issue, our lab has previously demonstrated enthesis regeneration using a tissue-engineered graft approach in a sheep anterior cruciate ligament (ACL) repair model. We hypothesized that our tissue-engineered graft designed for ACL repair also will be effective in rotator cuff repair. The goal of this study was to test the efficacy of our Engineered Tissue Graft for Rotator Cuff (ETG-RC) in a rotator cuff tear model in sheep and compare this novel graft technology to the commonly used double row suture repair technique. Following a 6-month recovery, the grafted and contralateral shoulders were removed, imaged using X-ray, and tested biomechanically. Additionally, the infraspinatus muscle, myotendinous junction, enthesis, and humeral head were preserved for histological analysis of muscle, tendon, and enthesis structure. Our results showed that our ETC-RCs reached 31% of the native tendon tangent modulus, which was a modest, non-significant, 11% increase over that of the suture-only repairs. However, the histological analysis showed the regeneration of a native-like enthesis in the ETG-RC-repaired animals. This advanced structural healing may improve over longer times and may diminish recurrence rates of rotator cuff tears and lead to better clinical outcomes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:289-299, 2018.

Facile synthesis of carbon dot and residual carbon nanobeads: Implications for ion sensing, medicinal and biological applications.

Synthesis of carbon dots (Cdots) via chemical route involves disintegration of carbon materials into nano-domains, wherein, after extraction of Cdots, the remaining carbon material is discarded. The present work focuses on studying even the leftover carbon residue namely, carbon nanobeads (CNBs) as an equally important material for applications on par with that of carbon dot. It employs oxidative treatment of carbonised gum olibanum resin (GOR) to produce the carbons namely Cdots and CNBs (as the residue). The Cdots (~5-10nm) exhibit blue-green fluorescence with an optical absorption at ~300nm unlike the CNBs (40-50nm) which fail to exhibit fluorescence. The fluorescence behaviour exhibited by Cdots were utilized for heavy metal ion sensing of Pb2+, Hg2+ and Cd2+ ions in aqueous media. Interestingly, both Cdots and CNBs are biocompatible to normal cell lines but cytotoxic to cancer cell lines, observed during several in vitro experiments (cell viability assay, cell cycle assay, apoptosis assay, ROS determination assay, caspase-9 activity assay). Additionally, Cdots exhibit bright green fluorescence in B16F10 cells. The Cdots and CNB's demonstrate multifunctional activities (sensor, cellular imaging and cancer therapy) in biomedical applications.

An efficient removal of crystal violet dye from waste water by adsorption onto TLAC/Chitosan composite: A novel low cost adsorbent.

A composite of Typha latifolia activated carbon (TLAC) (a novel, low cost absorbent) and chitosan (TLAC/Chitosan composite) was prepared. The composite was characterised using IR spectra, XRD, FESEM and Pore size studies. Its effectivity was tested for the removal of crystal violet dye from aqueous solutions. The effect of pH, dose rate and initial dye concentration was evaluated. The adsorption isotherm, kinetics and thermodynamic parameters were studied. Langmuir and Freundlich isotherm models were found fit effectively for the dye adsorption data in the present study. The adsorption followed pseudo-second order kinetics. The evaluated thermodynamic parameters show a spontaneous and exothermic reaction. Overall, this study indicates TLAC/Chitosan composite as an effective adsorbent for the removal of crystal violet dye from aqueous solutions.

A randomized study of coconut oil versus sunflower oil on cardiovascular risk factors in patients with stable coronary heart disease.

BACKGROUND AND RATIONALE: Coronary artery disease (CAD) and its pathological atherosclerotic process are closely related to lipids. Lipids levels are in turn influenced by dietary oils and fats. Saturated fatty acids increase the risk for atherosclerosis by increasing the cholesterol level. This study was conducted to investigate the impact of cooking oil media (coconut oil and sunflower oil) on lipid profile, antioxidant mechanism, and endothelial function in patients with established CAD. DESIGN AND METHODS: In a single center randomized study in India, patients with stable CAD on standard medical care were assigned to receive coconut oil (Group I) or sunflower oil (Group II) as cooking media for 2 years. Anthropometric measurements, serum, lipids, Lipoprotein a, apo B/A-1 ratio, antioxidants, flow-mediated vasodilation, and cardiovascular events were assessed at 3 months, 6 months, 1 year, and 2 years. RESULTS: Hundred patients in each arm completed 2 years with 98% follow-up. There was no statistically significant difference in the anthropometric, biochemical, vascular function, and in cardiovascular events after 2 years. CONCLUSION: Coconut oil even though rich in saturated fatty acids in comparison to sunflower oil when used as cooking oil media over a period of 2 years did not change the lipid-related cardiovascular risk factors and events in those receiving standard medical care.

In vivo structural and cellular remodeling of engineered bone-ligament-bone constructs used for anterior cruciate ligament reconstruction in sheep.

Anterior cruciate ligament (ACL) ruptures rank among the most prevalent and costly sports-related injuries. Current tendon grafts used for ACL reconstruction are limited by suboptimal biomechanical properties. We have addressed these issues by engineering multiphasic bone-ligament-bone (BLB) constructs that develop structural and mechanical properties similar to native ACL. The purpose of this study was to examine the acute remodeling process that occurs as the BLB grafts advance toward the adult ligament phenotype in vivo. Thus, we implanted BLB constructs fabricated from male cells into female host sheep and allowed 3, 7, 14, or 28 days (n = 4 at each time point) for recovery. To address whether or not graft-derived cells were even necessary, a subset of BLB constructs (n = 3) were acellularized, implanted, and allowed 28 days for recovery. At each recovery time point, the following histological analyses were performed: picrosirius red staining to assess collagen alignment and immunohistochemistry to assess both graft development and host immune response. Polymerase chain reaction (PCR) analysis, performed on every explanted BLB, was used to detect the presence of graft-derived male cells remaining in the constructs and/or migration into surrounding host tissue. The analysis of the PCR and histology samples revealed a rapid migration of host-derived macrophages and neutrophils into the graft at 3 days, followed by increased collagen density and alignment, vascularization, innervation, and near complete repopulation of the graft with host cells within 28 days. This study provides a greater understanding of the processes of ligament regeneration in our BLB constructs as they remodel toward the adult ligament phenotype.

Protective role of extracts of grape skin and grape flesh on ethanol-induced oxidative stress, inflammation and histological alterations in rat brain.

Ethanol consumption has deleterious effects on all organs and especially on brain. In our study, the radical scavenging properties of grape skin and grape flesh (contains natural antioxidants like polyphenols) was determined in vitro and for in vivo studies, male Wistar rats (16-18 week-old) and 100-120 gm weight were divided in five groups of six animals each. One group were fed with ethanol (1.6 gm/kg body weight), while second group were fed with ethanol (1.6 g/kg body weight) and grape skin extract (2.5 g/kg body weight), the third group were fed with ethanol (1.6 g/kg body weight) and grape flesh extract, daily once for 12 weeks, while the fourth group were fed with ethanol (1.6 g/kg body weight) and tocopheryl acetate (80 mg/kg/day) daily once for 12 weeks. Results of in vitro studies indicate that grape skin extracts showed significant radical scavenging properties (ROS). There was also significantly alteration of serum cytokines in our study.

Allogeneic versus autologous derived cell sources for use in engineered bone-ligament-bone grafts in sheep anterior cruciate ligament repair.

The use of autografts versus allografts for anterior cruciate ligament (ACL) reconstruction is controversial. The current popular options for ACL reconstruction are patellar tendon or hamstring autografts, yet advances in allograft technologies have made allogeneic grafts a favorable option for repair tissue. Despite this, the mismatched biomechanical properties and risk of osteoarthritis resulting from the current graft technologies have prompted the investigation of new tissue sources for ACL reconstruction. Previous work by our lab has demonstrated that tissue-engineered bone-ligament-bone (BLB) constructs generated from an allogeneic cell source develop structural and functional properties similar to those of native ACL and vascular and neural structures that exceed those of autologous patellar tendon grafts. In this study, we investigated the effectiveness of our tissue-engineered ligament constructs fabricated from autologous versus allogeneic cell sources. Our preliminary results demonstrate that 6 months postimplantation, our tissue-engineered auto- and allogeneic BLB grafts show similar histological and mechanical outcomes indicating that the autologous grafts are a viable option for ACL reconstruction. These data indicate that our tissue-engineered autologous ligament graft could be used in clinical situations where immune rejection and disease transmission may preclude allograft use.

Fresh versus frozen engineered bone-ligament-bone grafts for sheep anterior cruciate ligament repair.

Surgical intervention is often required to restore knee instability in patients with anterior cruciate ligament (ACL) injury. The most commonly used grafts for ACL reconstruction are tendon autografts or allografts. These current options, however, have shown failure rates requiring revision and continued instability in the long term. The mismatched biomechanical properties of the current tendon grafts compared with native ACL tissue are thought to contribute to these poor outcomes and potential risk of early onset osteoarthritis. As a possible solution to these issues, our laboratory has fabricated tissue-engineered ligament constructs that exhibit structural and functional properties similar to those of native ACL tissue after 6 months implantation. In addition, these tissue-engineered grafts achieve vascular and neural development that exceeds those of patellar tendon grafts. However, the utility of our tissue-engineered grafts is limited by the labor-intensive method required to produce the constructs and the need to use the constructs fresh, directly from the cell culturing system. Ideally, these constructs would be fabricated and stored until needed. Thus, in this study, we investigated the efficacy of freezing our tissue-engineered constructs as a method of preservation before use for ACL reconstruction. We hypothesized that frozen constructs would have similar histological and biomechanical outcomes compared with our fresh model. Our results showed that 6 months postimplantation as an ACL replacement graft, both our tissue-engineered fresh and frozen grafts demonstrated similar mechanical and histological outcomes, indicating that freezing is a suitable method for preserving and storing our graft before ACL reconstruction. The ability to use frozen constructs significantly increases the versatility of our graft technology expanding the clinical utility of our graft.

Efficacy and safety of methylcobalamin, alpha lipoic acid and pregabalin combination versus pregabalin monotherapy in improving pain and nerve conduction velocity in type 2 diabetes associated impaired peripheral neuropathic condition. [MAINTAIN]: Results of a pilot study.

BACKGROUND AND OBJECTIVE: To assess whether methylcobalamin and alpha lipoic acid (ALA) added to pregabalin provide additional benefit compared to pregabalin alone in type 2 diabetes mellitus associated peripheral neuropathy. SETTING AND DESIGN: An open label, randomized, controlled parallel-group pilot study. MATERIALS AND METHODS: Thirty adult patients with type 2 diabetes mellitus with symptoms of peripheral neuropathy for ≥6 months were randomized to receive pregabalin 75 mg, methylcobalamin 750 µg, and ALA 100 mg (PMA, n = 15); or pregabalin 75 mg (PG, n = 15) for 12 weeks. Assessment variables were numeric rating scale (NRS), sleep interference scores (SIS), response rate to pain, and global assessment for the usefulness of therapy. The nerve conduction velocity was assessed for sensory and motor nerves. Safety assessment included adverse events reported by the patients, clinical laboratory, and general medical, neurological examinations. STATISTICAL ANALYSIS: Efficacy analyses were done on per-protocol (PP) population, whereas safety analyses were done on intent-to-treat (ITT) population. RESULTS: Significant improvement was seen in pain and sleep interference in both groups. Mean nerve conduction velocity of left common peroneal nerve (CPN) showed significant improvement in PMA group at week 12 compared to baseline (P = 0.018). For right CPN both groups showed significant improvement. (PMA, P = 0.002, PG, P = 0.007). For sensory testing, at week 12, right superficial peroneal nerve showed reduction in nerve conduction velocity in PG group compared to baseline (P = 0.043). CONCLUSION: Methylcobalamine, ALA and pregabalin combination provides pain relief and improves sleep interference. Addition of methylcobalamin and ALA to pregabalin improves the nerve function. Due to small sample size, most of the efficacy parameters could not reach significant difference between groups; hence benefit of the 3-drug-combimation should be interpreted with reservation.