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.

Experimental protection of diabetic mice against Lethal P. aeruginosa infection by bacteriophage.

The emergence of antibiotic-resistant bacterial strains has become a global crisis and is vulnerable for the exploration of alternative antibacterial therapies. The present study emphasizes the use of bacteriophage for the treatment of multidrug resistant P. aeruginosa. P. aeruginosa was used to induce septicemia in streptozotocin (STZ) induced diabetic and nondiabetic mice by intraperitoneal (i.p.) injection of 3 × 10(8) CFU, resulting in a fatal bacteremia within 48 hrs. A single i.p. injection of 3 × 10(9) PFU phage GNCP showed efficient protection in both diabetic (90%) and nondiabetic (100%) bacteremic mice. It was further noted that the protection rate was reduced in diabetic mice when phage GNCP was administered after 4 h and 6 h of lethal bacterial challenge. In contrast, nondiabetic bacteremic mice were rescued even when treatment was delayed up to 20 h after lethal bacterial challenge. Evaluation of results confirmed that a single intraperitoneal injection of the phage dose (3 × 10(9) PFU/mL) was more effective than the multiple doses of imipenem. These results uphold the efficacy of phage therapy against pernicious P. aeruginosa infections, especially in cases of immunocompromised host.

Possible significance of anti-heat shock protein (HSP-65) antibodies in autoimmune myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disorder caused by antibody-mediated attack against skeletal muscle nicotinic acetylcholine receptor (AchR) at neuromuscular junction. A close relationship exists between heat shock proteins (HSPs) and numerous autoimmune diseases. HSPs are over expressed to protect the host against various insults. Antibodies to HSP-65 (IgG, IgA, IgM) are evaluated in 40 MG sera by ELISA. About 80% of MG cases showed anti-HSP antibodies (62.5% IgG, 55% IgA, 40% IgM). The result suggests that, expression of HSP-65 increases in MG and most individuals produce antibodies to it. These antibodies might play a significant role in the pathogenesis of MG.

Microwave-assisted synthesis of medicinally relevant indoles.

Indoles represent an important structural class in medicinal chemistry with broad spectrum of biological activities. The synthesis of indoles, therefore, has attracted enormous attention from synthetic chemists. Microwave methods for the preparation of indole analogs have been developed to speed up the synthesis, therefore, microwave assisted organic synthesis (MAOS) in controlled conditions is an invaluable technique for medicinal chemistry. In this review, indole forming classical reactions such as Fischer, Madelung, Bischler-Mohlau, Batcho-Leimgruber, Hemetsberger-Knittel, Graebe-Ullmann, Diels-Alder and Wittig type reactions using microwave radiation has been summarized. In addition, metal mediated cyclizations along with solid phase synthesis of indoles have been discussed.

Widespread Emergence of Multidrug Resistant Pseudomonas aeruginosa Isolated from CSF Samples.

Bacterial infections of the central nervous system, especially acute infections such as bacterial meningitis require immediate, invariably empiric antibiotic therapy due to the widespread emergence of resistance among bacterial species. Nosocomial infections by Pseudomonas aeruginosa have been described with an increasing trend towards multidrug resistance. P. aeruginosa isolates n = 53 (66%) isolated from the cerebrospinal fluid (CSF) were used for this study. Antibiotic resistance in 53 P. aeruginosa clinical isolates from 80 CSF samples were evaluated. Of these, n = 42 (80%) of the isolates showed multidrug resistance to more than eight antibiotics and n = 17 (32%) isolates were found to be imipenem resistant P. aeruginosa (IMPR-Pa). Genotypical examination by ERIC based PCR revealed minor genetic variations. Polymicrobial infections are common in the CSF samples. However, high prevalence of P. aeruginosa as an opportunistic pathogen has been developing with increased resistance to antimicrobial agents and thus becoming a significant threat.

Do cytokines have any role in epilepsy?

OBJECTIVE: We analyzed cytokines levels in patients with epilepsy and new onset seizure and correlated it with various parameters. MATERIALS AND METHODS: After obtaining consent, serum samples from 100 patients with epilepsy or new onset seizure were prospectively collected in the immediate post-ictal phase. In 16 patients, a second sample was collected during the seizure-free period. The serum cytokine levels [TNF-alpha, IFN-gamma, IL-1beta, IL-2, IL-4, and IL-6] were assessed (ELISA) in these patients and 100 matched healthy controls. CSF analysis was carried out in 9 patients of this cohort, when clinically indicated. RESULTS: The type of seizures (n=100) was major (45), partial (41) and status epilepticus (SE=14), while the epilepsy syndromes were idiopathic generalized (53) and localization related (47). The detectable serum cytokines in the patient group (n=100) were: IL-6 (42), TNF-alpha (36), IL-2 (22), IL-4 (22), IFN-gamma (20) and IL-1 (11) compared to the controls. CSF IL-6 and IL-1 was detectable in 4/9 and 2/9 patients, respectively while, IL-2, IL-4, IFN-gamma was detectable 1 in each patient. Correlations were noted between male gender and IL-1beta (p=0.04), positive family history and IL-1beta (p=0.001), "no alcohol use" and TNF-alpha (p=0.05), more than one year history of epilepsy and IL-1beta (p=0.02), status epilepticus (SE) and IL-6 (p=0.04). There was no difference between the new onset seizures vs. epilepsy group. Serial analysis during the seizure-free period revealed a decrease in cytokine levels: TNF-alpha (25% to 12.5%), IFN-gamma (12.5% to 0%), IL-1 (25% to 0) and IL-2 (6.2% to 6.2%), IL-4 (18.8% to 0%) and IL-6 (18.8% to 6.2%). CONCLUSIONS: We found increased post-ictal serum cytokine levels in patients with several epilepsy syndromes.

Do cytokines have any role in Wilson's disease?

The aim of this study was to determine the serum cytokine levels in patients with Wilson's disease (WD) and correlate with phenotype, therapeutic status and laboratory data. In this cross-sectional study, the serum levels of cytokines were estimated in 34 patients (M : F, 23 : 11; drug-naive, 11) with WD (mean age: 13.8 +/- 8.6 and 19.6 +/- 9.03 years) and compared with 30 controls. The following serum cytokines were analysed using enzyme-linked immunosorbent assay: (i) tumour necrosis factor (TNF)-alpha, (ii) interferon (IFN)-gamma, (iii) interleukin (IL)-2, (iv) IL-6 and (v) IL-4. Serum TNF-alpha (P < 0.001), IFN-gamma (P = 0.005) and IL-6 (P < 0.001) were detectable in WD compared with controls. However, serum level elevation of IL-4 (P = 0.49) and IL-2 (P = 0.11), although detectable compared with controls, was statistically insignificant. The disease severity and therapeutic status did not affect the cytokines. Presence of anaemia, leucopenia, thrombocytopenia, pancytopenia and hepatic dysfunction did not influence cytokine levels. There was a significant negative correlation between IL-6 and ceruloplasmin (P = 0.04) and anti-inflammatory cytokines (IL-4) and copper level (P = 0.01). Serum cytokines, both proinflammatory and anti-inflammatory subtypes, were elevated significantly in patients with WD. Further studies would establish their role in its pathogenesis.

Sequential interpenetrating polymer network hydrogel microspheres of poly(methacrylic acid) and poly(vinyl alcohol) for oral controlled drug delivery to intestine.

Sequential interpenetrating networks of poly(methacrylic acid) and poly(vinyl alcohol) have been prepared and cross-linked with glutaraldehyde to obtain pH sensitive microspheres by a water-in-oil emulsification method. Microspheres have been used to deliver the chosen model anti-inflammatory drug viz., ibuprofen to the intestine. Ibuprofen was encapsulated up to 70% within polymeric matrices. The interpenetrating polymer network formed was analysed by Fourier transform infrared spectroscopy. Differential scanning calorimetry and X-ray diffraction analyses were done on drug-loaded microspheres to confirm the polymorphism of ibuprofen. Results of this study indicated the molecular level dispersion of ibuprofen in the developed microspheres. Scanning electron microscopy confirmed the spherical nature and smooth surfaces of the microspheres produced. Mean particle size of the microspheres as measured by laser light scattering ranged between 51-176 microm. Swelling was performed in the simulated gastric as well as the intestinal conditions. Microspheres showed a pulsatile swelling behaviour when pH of the swelling media was altered. The swelling data have been fitted to an empirical equation to understand water transport trends as well as to calculate the diffusion coefficients. Values of diffusion coefficients in acidic media were lower than those found in the basic media. Values of diffusion coefficients decrease with increasing cross-linking of the matrix. In vitro release studies have been performed in 1.2 and 7.4 pH media to simulate the gastric and intestinal conditions. The in vitro release results indicated a dependence on the pH of the release media, extent of cross-linking and the amount of drug loading. The release data were fitted to an empirical relation to estimate the transport parameters and thereby to understand the transport mechanism.

Aminoglycoside-resistance mechanisms in multidrug-resistant Staphylococcus aureus clinical isolates.

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6')-aph(2''), and aph(3')-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3')-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.

Metabolomics.

Metabolomics is based on the simultaneous analysis of multiple low-molecular-weight metabolites from a given sample. The goals of metabolomics are to catalog and quantify the myriad small molecules found in biological fluids under different conditions. The metabolomics represents the collection of all metabolites in a biological organism, and metabolic profiling can give an instantaneous 'snapshot' of the physiology of that cell. Together with the other more established omics technologies, metabolomics will strengthen its claim to contribute to the detailed understanding of the in vivo function of gene products, biochemical analysis, regulatory networks and more ambitious, the mathematical description and simulation of the whole cell in the systems biology approach. This phenomenon will allow the construction of designer organisms for process application using biotransformation and fermentative approaches making effective use of single enzymes, whole microbial and even higher cells and allows the connection of data from genomics, proteomics to enables coordinating the timing of the analysis to physiologically important windows.

Impact assessment of coal mines in Erai watershed of Chandrapur district using geoinformatics.

The industrial development and growing population in India is in demand of more energy. Coal based thermal power generation is a major source of energy and is expanding at a very high rate leading to over exploitation of coal reserves, which is causing adverse impacts on the environment. Remote sensing and GIS techniques have been found to be useful in mapping and monitoring of dynamic changes taking place due to mining activity. Satellite based environmental impact assessment involves various aspects, such as land use, water resources, land degradation, etc. These studies help in formulating environmental management plan for the mining sector. Coal mines in Erai watershed of Chandrapur district so far have lost 2139.68 hectares of land constituting a fertile agriculture land, reserve forest, protected forest and natural river course of Erai river, Upsa nala and Motaghat nala severely affecting the watershed eco-system. Therefore, an in-depth impact assessment study of coal mines in Erai watershed of Chandrapur district was carried out using geoinformatics and the results are presented in this paper.

Myasthenia gravis and acetylcholine receptor antibodies: a clinico immunological correlative study on South Indian patients.

UNLABELLED: Acquired myasthenia Gravis (MG), a disorder of impaired neuromuscular transmission is recognized as an autoimmune disorder, with a majority of the patients having antibodies against acetylcholine receptor (AChR antibodies) in the serum. AIM AND OBJECTIVES: To correlate the AChR antibody status with the clinical characteristics of patients with myasthenia gravis. STUDY DESIGN: Retrospective and prospective study. MATERIALS AND METHODS: This study was carried out in patients with definite MG, attending the Neurology services of the National Institute of Mental Health and Neurosciences, Bangalore, India, during the period 1999-2003. The AChR antibody status was determined using the direct and indirect enzyme linked immunosorbent assay (ELISA) technique. RESULTS: There were 165 patients in this study (M : F :: 1.5 : 1). The overall seropositivity rate was 59.4%. Seropositive patients had higher age of onset and presentation, and more frequent occurrence of crises, both at presentation and at any time during the course. Other parameters, viz. gender of the patient, Osserman staging, thymic enlargement on CT and remission during follow-up did not differ between the two groups. CONCLUSION: This communication reports the result of AChR antibody assay in a large cohort of patients, using a simple diagnostic tool, namely direct and indirect ELISA technique. In addition, the characteristics of a large cohort of patients with seronegative myasthenia gravis are described.

High-level oxacillin and gentamycin resistance with reduced susceptibility to vancomycin in Staphylococcus aureus-carrying mecA and femA gene complex.

Staphylococcus aureus oxiva 10 and oxiva 14 strains clinically isolated from diabetic patients were resistant to gentamycin and oxacillin The minimal inhibitory concentrations (MICs) of oxacillin and gentamycin were 720 and >2048 microg/mL, respectively, for oxiva 10 and 680 and 400 microg/mL. respectively, for oxiva 14; both strains carry mecA and femA genetic determinants in their genomes. In addition, both are vancomycin-intermediate Staphylococcus aureus (VISA) isolates. The addition of vancomycin led to significant decreases in oxacillin resistance of both oxiva 10 and oxiva 14 strains, whereas the addition of vancomycin to gentamycin plates showed a decrease in gentamycin resistance of non-high-level gentamycin-resistant (non-HLGR) oxiva 14 and indifference in gentamycin resistance in HLGR oxiva 10. Transmission electron microscopy of representative strains unveils a remarkable increase in the thickness of the cell wall, indicating that thickening of the cell wall is a common phenotype associated with vancomycin resistance in VISA isolates. The present study reports that the rate of synergism and synergistic effect in the combination vancomycin-gentamycin vary according to the MICs of gentamycin.

Disseminated crytococcosis with extensive cutaneous involvement in AIDS.

Cutaneous infections is observed in 15% of patients with disseminated cryptococcosis with AIDS. We present here a case of a 34 years old female with AIDS. She presented with multiple skin coloured umbilicated over face, neck, trunk and limbs, which mimicked molluscum contagiosum and kaposi sarcoma. The tissue from cutaneous lesions was collected by excision biopsy and processed by standard mycological methods. Cryptococcus neoformans was isolated and identified. Cerebrospinal fluid (CSF) also yielded the growth of C. neoformans . Cryptococcal antigen was detected with a titre of 1024 by Latex agglutination, is serum and CSF. Her serum was reactive for HIVI and 2 antibodies. The CD4 lymphocytes count was 80/cmm. The HIV viral load was 2,48,084 copies/mL. She was treated with amphotericin B injectable and oral fluconazole. She responded well and lesions regressed.