Cerebrospinal fluid inflammatory cytokine profiles of patients with neurotropic parasitic infections

@#The pathogenesis of chronic parasitic central nervous system (CNS) infections, including granulomatous amoebic meningoencephalitis (GAE), cerebral toxoplasmosis (CT), and neurocysticercosis (NCC), is primarily due to an inflammatory host reaction to the parasite. Inflammatory cytokines produced by invading T cells, monocytes, and CNS resident cells lead to neuroinflammation which underlie the immunopathology of these infections. Immune molecules, especially cytokines, can therefore emerge as potential biomarker(s) of CNS parasitic infections. In this study, cerebral spinal fluid (CSF) samples from suspected patients with parasitic infections were screened for pathogenic free-living amoebae by culture (n=2506) and PCR (n=275). Six proinflammatory cytokines in smear and culture-negative CSF samples from patients with GAE (n = 2), NCC (n = 7), and CT (n = 23) as well as control (n = 7) patients were measured using the Multiplex Suspension assay. None of the CSF samples tested was positive for neurotropic free-living amoebae by culture and only two samples showed Acanthamoeba 18S rRNA by PCR. Of the six cytokines measured, only IL-6 and IL-8 were significantly increased in all three infection groups compared to the control group. In addition, TNFa levels were higher in the GAE and NCC groups and IL-17 in the GAE group compared to controls. The levels of IL-1b and IFNg were very low in all the infection groups and the control group. There was a correlation between CSF cellularity and increased levels of IL-6, IL-8, and TNFa in 11 patients. Thus, quantifying inflammatory cytokine levels in CSF might help with understanding the level of neuroinflammation in patients with neurotropic parasitic diseases. Further studies with clinico-microbiological correlation in the form of reduction of cytokine levels with treatment and the correlation with neurological deficits are needed.

Cerebrospinal fluid inflammatory cytokine profiles of patients with neurotropic parasitic infections.

The pathogenesis of chronic parasitic central nervous system (CNS) infections, including granulomatous amoebic meningoencephalitis (GAE), cerebral toxoplasmosis (CT), and neurocysticercosis (NCC), is primarily due to an inflammatory host reaction to the parasite. Inflammatory cytokines produced by invading T cells, monocytes, and CNS resident cells lead to neuroinflammation which underlie the immunopathology of these infections. Immune molecules, especially cytokines, can therefore emerge as potential biomarker(s) of CNS parasitic infections. In this study, cerebral spinal fluid (CSF) samples from suspected patients with parasitic infections were screened for pathogenic free-living amoebae by culture (n=2506) and PCR (n=275). Six proinflammatory cytokines in smear and culture-negative CSF samples from patients with GAE (n = 2), NCC (n = 7), and CT (n = 23) as well as control (n = 7) patients were measured using the Multiplex Suspension assay. None of the CSF samples tested was positive for neurotropic free-living amoebae by culture and only two samples showed Acanthamoeba 18S rRNA by PCR. Of the six cytokines measured, only IL-6 and IL-8 were significantly increased in all three infection groups compared to the control group. In addition, TNFa levels were higher in the GAE and NCC groups and IL-17 in the GAE group compared to controls. The levels of IL-1b and IFNg were very low in all the infection groups and the control group. There was a correlation between CSF cellularity and increased levels of IL-6, IL-8, and TNFa in 11 patients. Thus, quantifying inflammatory cytokine levels in CSF might help with understanding the level of neuroinflammation in patients with neurotropic parasitic diseases. Further studies with clinico-microbiological correlation in the form of reduction of cytokine levels with treatment and the correlation with neurological deficits are needed.

Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing.

Despite clinical suspicion of an infection, brain abscess samples are often culture-negative in routine microbiological testing. Direct PCR of such samples enables the identification of microbes that may be fastidious, non-viable, or unculturable. Brain abscess samples (n = 217) from neurosurgical patients were subjected to broad range 16S rRNA gene PCR and sequencing for bacteria. All these samples and seven formalin-fixed paraffin-embedded tissue (FFPE) samples were subjected to species-specific 18S rRNA PCR for neurotropic free-living amoeba that harbour pathogenic bacteria. The concordance between smear and/or culture and PCR was 69%. One-third of the samples were smear- and culture-negative for bacterial agents. However, 88% of these culture-negative samples showed the presence of bacterial 16S rRNA by PCR. Sanger sequencing of 27 selected samples showed anaerobic/fastidious gram negative bacteria (GNB, 38%), facultative Streptococci (35%), and aerobic GNB (27%). Targeted metagenomics sequencing of three samples showed multiple bacterial species, including anaerobic and non-culturable bacteria. One FFPE tissue revealed the presence of Acanthamoeba 18S rRNA. None of the frozen brain abscess samples tested was positive for 18S rRNA of Acanthamoeba or Balamuthia mandrillaris. The microbial 16/18S rRNA PCR and sequencing outperformed culture in detecting anaerobes, facultative Streptococci and FLA in brain abscess samples. Genetic analyses of 16S/18S sequences, either through Sanger or metagenomic sequencing, will be an essential diagnostic technology to be included for diagnosing culture-negative brain abscess samples. Characterizing the microbiome of culture-negative brain abscess samples by molecular methods could enable detection and/or treatment of the source of infection.

Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing

@#Despite clinical suspicion of an infection, brain abscess samples are often culture-negative in routine microbiological testing. Direct PCR of such samples enables the identification of microbes that may be fastidious, non-viable, or unculturable. Brain abscess samples (n = 217) from neurosurgical patients were subjected to broad range 16S rRNA gene PCR and sequencing for bacteria. All these samples and seven formalin-fixed paraffin-embedded tissue (FFPE) samples were subjected to species-specific 18S rRNA PCR for neurotropic free-living amoeba that harbour pathogenic bacteria. The concordance between smear and/or culture and PCR was 69%. One-third of the samples were smear- and culture-negative for bacterial agents. However, 88% of these culture-negative samples showed the presence of bacterial 16S rRNA by PCR. Sanger sequencing of 27 selected samples showed anaerobic/fastidious gram negative bacteria (GNB, 38%), facultative Streptococci (35%), and aerobic GNB (27%). Targeted metagenomics sequencing of three samples showed multiple bacterial species, including anaerobic and non-culturable bacteria. One FFPE tissue revealed the presence of Acanthamoeba 18S rRNA. None of the frozen brain abscess samples tested was positive for 18S rRNA of Acanthamoeba or Balamuthia mandrillaris. The microbial 16/18S rRNA PCR and sequencing outperformed culture in detecting anaerobes, facultative Streptococci and FLA in brain abscess samples. Genetic analyses of 16S/18S sequences, either through Sanger or metagenomic sequencing, will be an essential diagnostic technology to be included for diagnosing culture-negative brain abscess samples. Characterizing the microbiome of culture-negative brain abscess samples by molecular methods could enable detection and/or treatment of the source of infection.

Adverse childhood experiences and substance misuse in young people in India: results from the multisite cVEDA cohort.

BACKGROUND: Adverse childhood experiences (ACEs) increases vulnerability to externalising disorders such as substance misuse. The study aims to determine the prevalence of ACEs and its association with substance misuse. METHODS: Data from the Consortium on Vulnerability to Externalising Disorders and Addictions (cVEDA) in India was used (n = 9010). ACEs were evaluated using the World Health Organisation (WHO) Adverse Childhood Experiences International Questionnaire whilst substance misuse was assessed using the WHO Alcohol, Smoking and Substance Involvement Screening Test. A random-effects, two-stage individual patient data meta-analysis explained the associations between ACEs and substance misuse with adjustments for confounders such as sex and family structure. RESULTS: 1 in 2 participants reported child maltreatment ACEs and family level ACEs. Except for sexual abuse, males report more of every individual childhood adversity and are more likely to report misusing substances compared with females (87.3% vs. 12.7%). In adolescents, family level ACEs (adj OR 4.2, 95% CI 1.5-11.7) and collective level ACEs (adj OR 6.6, 95% CI 1.4-31.1) show associations with substance misuse whilst in young adults, child level ACEs such as maltreatment show similar strong associations (adj OR 2.0, 95% CI 1.1-3.5). CONCLUSION: ACEs such as abuse and domestic violence are strongly associated with substance misuse, most commonly tobacco, in adolescent and young adult males in India. The results suggest enhancing current ACE resilience programmes and 'trauma-informed' approaches to tackling longer-term impact of ACEs in India. FUNDING: Newton Bhabha Grant jointly funded by the Medical Research Council, UK (MR/N000390/1) and the Indian Council of Medical Research (ICMR/MRC-UK/3/M/2015-NCD-I).

On the estimation of female births missing due to prenatal sex selection.

This research note is prompted by a paper by Kashyap (Is prenatal sex selection associated with lower female child mortality? Population Studies 73(1): 57-78). Kashyap's paper, which provides 40 original estimates of missing female births, relies on an alternative definition of missing female births, leading to estimates of about half the magnitude of other estimates. There appears, therefore, a real need to take stock of the concept of missing female births widely used by statisticians around the world for assessing the demographic consequences of prenatal sex selection. This research note starts with a brief review of the history of the concept and the difference between Amartya Sen's original method and the alternative method found elsewhere to compute missing female births. We then put forward three different arguments (deterministic and probabilistic approaches, and consistency analysis) in support of the original computation procedure based on the number of observed male births and the expected sex ratio at birth.

Synthesis, biological evaluation and molecular modelling of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-imidazoles as ALK5 inhibitors.

A series of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-imidazoles, 7a-c, 11a-h, and 16a-h has been synthesised and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. Incorporation of a quinoxalin-6-yl moiety and a methylene linker at the 4- and 2-position of the imidazole ring, respectively, and a m-CONH2 substituent in the phenyl ring generated a highly potent and selective ALK5 inhibitor 11e. Docking model of ALK5 in complex with 11e showed that it fitted well in the ATP-binding pocket with favourable interactions.

ApoE4 and late onset depression in Indian population.

OBJECTIVE: ApoE4 is a 'risk factor' for cognitive disorders like Alzheimer's dementia, and Late Onset Depression (LOD) is a forerunner of dementia. There is thus a need to study the association between ApoE4 allele and LOD. METHOD: The study assessed the frequency of ApoE4 allele in 31 cases of LOD above the age of 50 years and 31 matched controls. The subjects were assessed on various clinical parameters towards diagnosis. RESULTS: There was a significant association between the ApoE4 allele and LOD in comparison to controls (Odd's ratio=4.7, Confidence Interval=1.12 to 19.79, P=0.035). ApoE4 allele had no association with the age of onset of depression, cognitive functions and severity of LOD. CONCLUSION: Individuals with LOD have a significantly higher frequency of the ApoE4 allele. In other words, elderly in India with an ApoE4 allele have 4.7 times more risk of developing depression in old age. Within LOD group there is no difference between those with and without ApoE4 accordingly in age of onset of depression, cognitive functions and severity of LOD.

Synthesis and biological evaluation of 2-pyridyl-substituted pyrazoles and imidazoles as transforming growth factor-beta type 1 receptor kinase inhibitors.

A series of 2-pyridyl-substituted pyrazoles (16a-d, 17, 18, and 28a-e) and imidazoles (22 and 23) has been synthesized and evaluated for their ALK5 inhibitory activity in cell-based luciferase reporter assays. Among them, 3-(3-(6-methylpyridin-2-yl)-4-(quinolin-6-yl)-1H-pyrazole-1-carbothioamido)benzamide (28c) showed 96% and 93% inhibition at 0.1 microM in luciferase reporter assays using HaCaT cells transiently transfected with p3TP-luc reporter construct and ARE-luc reporter construct, respectively.

Synthesis and biological evaluation of 4(5)-(6-methylpyridin-2-yl)imidazoles and -pyrazoles as transforming growth factor-beta type 1 receptor kinase inhibitors.

A series of 4(5)-(6-methylpyridin-2-yl)imidazoles 16-19 and -pyrazoles 22-29, 33, and 34 have been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in cell-based luciferase reporter assays. The 6-quinolinyl imidazole analogs 16 and 18 inhibited ALK5 phosphorylation with IC(50) values of 0.026 and 0.034 microM, respectively. In a luciferase reporter assay using HaCaT cells transiently transfected with p3TP-luc reporter construct, 18 displayed 66% inhibition at 0.05 microM, while competitor compounds 2 and 3 showed 44% inhibition. The binding mode of 18 generated by flexible docking studies with ALK5:18 complex shows that it fits well into the active site cavity of ALK5 by forming broad and tight interactions.

Synthesis and biological evaluation of benzenesulfonamide-substituted 4-(6-alkylpyridin-2-yl)-5-(quinoxalin-6-yl)imidazoles as transforming growth factor-beta type 1 receptor kinase inhibitors.

A series of benzenesulfonamide-substituted 4-(6-alkylpyridin-2-yl)-5-(quinoxalin-6-yl)imidazoles have been synthesized and evaluated for their ALK5 inhibitory activity in cell-based luciferase reporter assays. Among them, 4-[5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-ylmethyl]benzenesulfonamide and 4-[5-(6-ethylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-ylmethyl]benzenesulfonamide showed more than 90% inhibition at 0.5 microM in a luciferase reporter assay using HaCaT cells transiently transfected with p3TP-luc reporter construct, but inhibited p38alpha MAP kinase activity only 11 and 8% at a concentration of 10 microM, respectively.

Synthesis and biological evaluation of trisubstituted imidazole derivatives as inhibitors of p38alpha mitogen-activated protein kinase.

A series of trisubstituted imidazole derivatives containing a 4-fluorophenyl group, a pyrimidine ring, and a CN- or CONH(2)-substituted benzyl moiety have been synthesized and evaluated for p38alpha MAP kinase inhibitory activity. Among them, compounds 22c, 27b, and 28b inhibited p38alpha MAP kinase with IC(50) values 27.6, 28, and 31 nM, respectively.

Protein tyrosine phosphatases and their inhibitors.

Protein tyrosine phosphatase (PTP) is the family of enzymes that are key players in cellular signal transduction system and perturbation in their functioning is implicated in many disease-states. Diverse chemical compounds are being synthesized and evaluated as PTP inhibitors. This review presents a brief account of various enzymes of the PTP family and their inhibitors. Peculiar features of these enzymes and their roles in various diseases are summarized along with important inhibitors developed in recent years.

Common psychiatric diseases and human genetic variation.

OBJECTIVE: A better understanding of human genetic variation is important in assessing disease epidemiology and phenotypic variation, and may be critical in evaluating genetic aspects of common genetic diseases, such as schizophrenia, bipolar disease and Parkinson's. These diseases are particularly difficult to investigate as there are few peripheral markers, and although a genetic aetiology has long been suspected, robust findings have been hard to establish. METHODS: Variations in alleles at 13 tri-nucleotide gene loci expressed in the brain and implicated in several neurodegenerative diseases, as well as certain other loci, were examined in the Indian population for comparison with other major ethnic groups. RESULTS AND CONCLUSION: In the Indian population, the distribution of alleles at the Machado-Joseph disease locus was similar to the Western European pattern of distribution. Analysis of haplotypes at the locus for Huntington's disease suggested multiple origins, and possible effects of population admixture because of the recent history of the country. At other alleles of neuropsychiatric interest (dopamine receptor, serotonin receptor, serotonin transporter, alcohol dehydrogenase), allele frequencies in the Indian population differed from other populations. Interspecies comparison suggests a gradual expansion in repeat size, with the exception of the CLOCK gene, which displays a contraction of CAG repeat numbers. World-wide differences in disease phenotypes need to be explored, and an appreciation of their genetic basis may provide a window of opportunity for improving our knowledge of the underlying genetic mechanisms.