Kishan K. Nyati

Evaluation of the MTT lymphocyte proliferation assay for the diagnosis of neurocysticercosis

Neurocysticercosis (NCC) is caused by the larval form of the pork tapeworm Taenia solium when lodged in the central nervous system (CNS). Clinical diagnosis of NCC is complicated due to its polymorphic manifestations with no specific signs or symptoms. A wide range of serological assays and neuroimaging modalities are used for its diagnosis. The aim of the present study was to evaluate the MTT assay for the diagnosis of NCC and to determine its sensitivity, specificity and accuracy. MTT assay was based upon the cellular reduction of the tetrazolium salt by the proliferating cells and quantification of the colored product. Total 59 patients with NCC-related active epilepsy (AE), 30 with AE other than NCC (disease controls) and 64 healthy volunteers were enrolled for the study. Lymphocytes were freshly isolated from the enrolled subjects and cultured on cyst fluid antigen coated tissue culture plates. MTT assay was performed according to the standard protocol. The mean values of proliferation index (PI) with cyst fluid antigens were 2.13+/-0.72, 0.622+/-0.31 and 0.71+/-0.36 for NCC patients, disease controls and healthy volunteers respectively. PI values for NCC patients were higher than the cut-off value (mean of controls+2 standard deviations; 1.31). The sensitivity, specificity and accuracy of the MTT assay for the diagnosis of NCC were 87.93%, 94.68% and 91.5% respectively. For single cyst infection the sensitivity of the assay was found to be 86.4%. The present study shows that MTT is an adaptable technique which can be used for diagnosis of NCC.

Toll-Like Receptor 4 Polymorphism and Its Association with Symptomatic Neurocysticercosis

BACKGROUND Symptoms and signs of neurocysticercosis (NCC) are nonspecific and depend upon several factors, including the host immune response to the parasite. Toll-like receptors (TLRs) play an important role in innate immunity. Susceptibility of humans to NCC in relation to TLR polymorphism is unknown. The present study examines TLR4 polymorphism in human NCC and its role in symptomatic disease. METHODS A total of 140 patients with NCC (82 symptomatic [ie, with active epilepsy] and 58 asymptomatic) and 150 healthy control subjects were examined for TLR4 Asp299Gly and Thr399Ile polymorphisms by means of polymerase chain reaction and restriction fragment-length polymorphism. RESULTS TLR4 Asp299Gly and Thr399Ile were significantly associated with the occurrence of NCC (P < .001 for Asp299Gly; P = .003 for Thr399Ile) and progression to symptomatic NCC, compared with control subjects (P < .001 for Asp299Gly; P < .001 for Thr399Ile) or asymptomatic NCC (P < .001 for Asp299Gly; P = .002 for Thr399Ile). Frequency of haplotype Gly/Thr (P <.001) was observed to be a risk factor for susceptibility to NCC. Gly and Ile carriers had a statistically significant association with NCC (P < .001 for Gly; P = .003 for Ile) and symptomatic NCC (P < .001 for Gly; P <or= .001 for Ile), compared with control subjects. Both carriers were also associated with symptomatic NCC (P < .001 for Gly; P < .004 for Ile), when compared with asymptomatic NCC.

TH1 and TH2 Response to Campylobacter jejuni Antigen in Guillain-Barré Syndrome

OBJECTIVES To determine the expression of proinflammatory and anti-inflammatory cytokines in lymphocytes from the progressive and recovery phases of Guillain-Barré syndrome (GBS) after stimulation with Campylobacter jejuni outer membrane proteins. DESIGN Case-control study. SETTING Sanjay Gandhi Postgraduate Institute of Medical Sciences. PARTICIPANTS Sixty-five patients with GBS, 60 age- and sex-matched disease control individuals, and 68 healthy control individuals were included in the study. MAIN OUTCOME MEASURES Lymphocytes from patients with GBS were stimulated with C jejuni outer membrane proteins, and the levels of different proinflammatory (T(H)1 [helper T cell, subtype 1]) and anti-inflammatory (T(H)2) cytokines were determined by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS In the progressive phase of the disease, the expressions of interferon γ (IFN-γ), interleukin 1β (IL-1β), tumor necrosis factor (TNF), IL-6, IL-10, and the IFN-γ:IL-4 ratio were significantly upregulated, but expressions of transforming growth factor (TGF)-β1 and IL-4 were lower in patients compared with disease and healthy controls. In contrast, the levels of IFN-γ, IL-1β, TNF, IL-6, IL-10, and the IFN-γ:IL-4 ratio were significantly lower, but TGF-β1 and IL-4 were upregulated in the recovery phase of GBS patients compared with controls. CONCLUSIONS Upregulation of T(H)1 cytokines in the early disease course may be associated with immune-mediated disease progression due to neuronal inflammation, but upregulation of T(H)2 immune response during the later phase aids recovery from the disease.

Immune response in symptomatic and asymptomatic neurocysticercosis

Innate immune system is crucial in the pathogenesis of neurocysticercosis (NCC) and helminth glycans can induce anti-inflammatory milieu via toll-like receptor 4 (TLR4) dependent mechanisms. The association of TLR4 and cytokines is yet to be explored in NCC. Therefore, the present study detected the serum levels of cytokines and soluble intercellular adhesion molecule (sICAM)-1 in asymptomatic and symptomatic NCC and their association with TLR4 expression. Sixty eight patients with NCC (asymptomatic, 36 and symptomatic, 32), and age and gender matched 37 healthy controls were enrolled to determine the levels of different pro- and anti-inflammatory cytokines, sICAM-1 in the serum by ELISA and expression of TLR4 in peripheral blood mononuclear cells (PBMCs) by flow cytometry. In asymptomatic NCC cases, the levels of IL-10 and IL-4 were significantly elevated compared to healthy controls and symptomatic NCC patients whereas the levels of IFN-γ, TNF-α, IL-17, IL-23 and sICAM-1 were higher in symptomatic NCC patients compared to healthy controls and asymptomatic NCC individuals. Frequency of TLR4 expressing PBMCs and CD14 positive cells were significantly higher in both groups of NCC. Although the number of TLR4 expressing cells was almost similar in both asymptomatic and symptomatic groups, the median fluorescence intensity was significantly higher in symptomatic group indicating that higher levels of TLR4 expression in symptomatic patients correlated with enhanced pro-inflammatory cytokine production.

Association of TLR4 Asp299Gly and Thr399Ile polymorphisms with Guillain-Barré syndrome in Northern Indian population

Molecular mimicry between Campylobacter jejuni lipopolysaccharide and host gangliosides induces an immune response leading to axonal damage and Guillain-Barré syndrome (GBS). TLR polymorphisms are associated with many autoimmune diseases. The role of the TLR4 gene in GBS susceptibility largely remains unknown. We investigated TLR4 polymorphism in GBS. One hundred and twenty GBS patients and 150 healthy controls were included. TLR4 (Asp299Gly and Thr399Ile) genes were studied by PCR-RFLP. TLR4 (Asp299Gly) polymorphism was significantly associated with GBS (p, 0.045; OR, 8.75; 95% CI, 1.05-72.88); only acute motor axonal neuropathy (AMAN) was associated with Gly299Gly homozygote (p, 0.027; OR, 12.40; 95% CI, 1.33-115.77) and Thr399Ile (p, 0.019; OR, 3.42; 95% CI, 1.22-9.54) heterozygote, and TLR4-399Ile allele (p, 0.045; OR, 2.63; 95% CI, 1.02-6.75) compared to controls. In conclusion, TLR4 (Asp299Gly) polymorphism is associated with an increased susceptibility to GBS. Besides Asp299Gly, AMAN subtype is also associated with Thr399Ile polymorphism.

Association of MMP-2 and MMP-9 with clinical outcome of neurocysticercosis

Matrix metalloproteinases (MMPs) are the major endopeptidases involved in proteolysis of blood brain barrier (BBB) during central nervous system (CNS) infections. The present study detected serum levels and activities of MMP-2 and MMP-9 in patients with neurocysticercosis (NCC) and their association with symptomatic disease. In total, 68 individuals with NCC (36 symptomatic patients with active seizures and 32 asymptomatic individuals) and 37 healthy controls were enrolled for the study. Serum MMP-2 and MMP-9 levels and their activities were measured by ELISA and gel zymography respectively. Mean serum MMP-2 levels (ng/ml) were higher both in asymptomatic and symptomatic NCC cases compared to healthy controls. However, significantly higher levels of serum MMP-9 (ng/ml) were detected only in symptomatic NCC patients compared to asymptomatic NCC cases and healthy controls. Levels of both MMPs positively correlated with symptomatic NCC. Serum MMP-2 activities were significantly higher in symptomatic and asymptomatic NCC compared to healthy controls whereas serum MMP-9 activity was significantly associated with symptomatic NCC compared to healthy controls and asymptomatic NCC. In conclusion, the elevated level of MMP-9 in serum appears to play an important role in the development of symptoms i.e. active seizures in patients with NCC. However, further studies are needed to elucidate its precise role in disease pathogenesis.

Tumor necrosis factor–α polymorphisms and expression in Guillain–Barré syndrome

Tumor necrosis factor-alpha (TNF-alpha) polymorphisms with increased expression is associated with many autoimmune and inflammatory diseases. Possible role of TNF-alpha polymorphism in the pathogenesis of Guillain-Barré syndrome (GBS) largely remains unknown. We investigated polymorphisms in the promoter region of TNF-alpha gene and its expression in GBS patients and healthy controls. TNF-alpha (-308 G>A, -857 C>T, and -863 C>A) polymorphisms in 140 GBS patients and 206 healthy controls were studied using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele specific-PCR. TNF-alpha level in serum by ELISA was determined in 60 patients and an equal number of controls. Prevalence of TNF-alpha -308 G > A polymorphic A allele was associated with increased risk of GBS (p < 0.001; OR = 2.58, 95% CI = 1.61-4.14). Heterozygous genotype (G/A) had an association with acute motor axonal neuropathy (p < 0.001; OR = 4.23, 95% CI = 2.00-8.95) and variant genotype A/A with both axonal subtypes, acute motor axonal neuropathy (p = 0.015, OR = 7.00, 95% CI = 1.46-33.57) and acute motor sensory axonal neuropathy (p = 0.017; OR = 7.73, 95% CI = 1.44-41.37). Variant genotype T/T of TNF-alpha -857 C>T polymorphism was also significantly associated with acute motor axonal neuropathy (p = 0.034; OR = 3.93, 95% CI = 1.11-13.91). Patients with A and T alleles had higher TNF-alpha level in serum. TNF-alpha -308 G > A and -857 C>T (only T/T) polymorphisms with increased TNF-alpha level may predict susceptibility to axonal subtypes of GBS.

Correlation of matrix metalloproteinases-2 and -9 with proinflammatory cytokines in Guillain-Barré syndrome.

The role of matrix metalloproteinases (MMPs) and cytokines in the pathogenesis of Guillain‐Barré syndrome (GBS) largely remains unknown. We studied the role of MMP‐2, MMP‐9, tumor necrosis factor‐α (TNF‐α), and interleukin‐1β (IL‐1β) in disease progression and recovery of patients with GBS. Sixty‐five patients with GBS and 68 healthy controls were enrolled in the study. Serum levels of MMP‐2, MMP‐9, TNF‐α, and IL‐1β were analyzed by ELISA, and activities of MMPs were measured by zymography. Expression of MMP‐9, TNF‐α, and IL‐1β was higher in the progressive phase and lower in the recovery phase of GBS than in controls. A positive correlation of MMP‐2 with IL‐1β and MMP‐9 with TNF‐α and IL‐1β was observed with progressive‐phase GBS. The study shows that up‐regulation of MMP‐9 along with proinflammatory cytokines (TNF‐α and IL‐1β) in the early course appears to be associated with immune‐mediated disease progression resulting from inflammation in the peripheral nervous system, whereas, during the later phase, down‐regulation of MMP‐9 and proinflammatory cytokines is implicated in recovery from the disease.

Role of Cytokines and Toll-Like Receptors in the Immunopathogenesis of Guillain-Barré Syndrome

Guillain-Barré syndrome (GBS) is an autoimmune disease of the peripheral nervous system, mostly triggered by an aberrant immune response to an infectious pathogen. Although several infections have been implicated in the pathogenesis of GBS, not all such infected individuals develop this disease. Moreover, infection with a single agent might also lead to different subtypes of GBS emphasizing the role of host factors in the development of GBS. The host factors regulate a broad range of inflammatory processes that are involved in the pathogenesis of autoimmune diseases including GBS. Evidences suggest that systemically and locally released cytokines and their involvement in immune-mediated demyelination and axonal damage of peripheral nerves are important in the pathogenesis of GBS. Toll-like receptors (TLRs) link innate and adaptive immunity through transcription of several proinflammatory cytokines. TLR genes may increase susceptibility to microbial infections; an attenuated immune response towards antigen and downregulation of cytokines occurs due to mutation in the gene. Herein, we discuss the crucial role of host factors such as cytokines and TLRs that activate the immune response and are involved in the pathogenesis of the disease.

Campylobacteriosis and Water: An Overview

Campylobacteriosis is an acute infectious diarrheal disease caused by the genus Campylobacter. The infection is mainly due to consumption of raw or undercooked poultry meat, unpasteurized milk, and contaminated water. In recent decades, Campylobacter (especially Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and more recently Campylobacter upsaliensis) have acquired great public health importance worldwide. The incidence of Campylobacter infection varies in both industrialized and developing countries. Campylobacter species are isolated more frequently than combined isolation rates by Salmonella and Shigella species from diarrheic stool samples. Serological and nucleic acid-based assays are used to study the epidemiology of Campylobacter infection. Antibiotic resistance in C. jejuni is considered as an emerging public health problem. Various virulence factors such as flagellin, lipopolysaccharides (LPSs), adhesins, and invasins have been implicated in the pathogenesis of Campylobacter infection. In this review, we present information available through literature search on epidemiology, clinical presentations, and bacteriology of campylobacteriosis.