TLR2 and TLR4 co-activation utilizes distinct signaling pathways for the production of Th1/Th2/Th17 cytokines in neonatal immune cells.

Co-activation of TLR2 and TLR4 by gram negative and gram positive bacterial ligands induces a robust pro-inflammatory response in inflammatory cells. In order to understand the signaling mechanism, we aimed to delineate the signaling molecules involved in TLR2 and TLR4 co-activation in neonatal immune cells for the production of Th1/Th2/Th17 inflammatory cytokines. For this, we pretreated cord blood and peripheral blood mononuclear and human mast cells with specific signaling molecule inhibitors such as BAY117082, PD98059 and LY294002 and then stimulated with LPS and PGN and assayed for cytokines IL-6, IL-12/IL-23p40 (Th1), IL-13 (Th2), IL-23 (Th17) and RANTES secretion. We found that upon co-stimulation the phosphorylation of NFκBp65, ERK1/2 and Akt was found to be higher than when stimulated with individual ligands in CBMCs. Also, when compared to adult cells, neonatal cells were more potent in the activation of ERK and Akt through TLR2 and TLR4 co-activation. In addition, neonatal cells possess similar capacity to activate NFκB as that of adult cells for IL-6 secretion. Furthermore, all three signaling molecules were found to be involved in the production of Th17 cytokines which is detrimental during inflammation induced by infection in neonates whereas NFκB is mainly involved in the induction of pro-inflammatory response and Th2 cytokines production. In conclusion, different signaling molecules were utilized for the production of different cytokines in immune cells.

TLR-mediated inflammatory response to neonatal pathogens and co-infection in neonatal immune cells.

Neonates heavily depend on the innate immune system for defence against invading pathogens. Toll-like receptors (TLRs) represent a primary line of host defence and play an important role in orchestrating the inflammatory response to invading pathogens. The most commonly infecting pathogens in neonates are E. coli, Klebsiella pneumoniae and Staphylococcus aureus. Also, co-infection with more than one organism is common in neonatal sepsis. Therefore, we aimed to study the TLR2 and TLR4 mediated neonatal inflammatory response to these pathogens. For this, we stimulated mononuclear cells from cord blood with LPS, PGN, E. coli, K.pneumoniae andS.aureus and analyzed the surface expression of TLR2 and TLR4 on CD14(+)CD16(+) and CD14(dim)CD16(+) and its inflammatory response in comparison with peripheral blood. We found that the TLR2 and TLR4 were differentially expressed on both monocyte subpopulations. Cytokines such as IL-6, IL-1ß, IL-23, IL-10, IL-13, MCP-1 and IL-8 were measured using ELISA and we observed that although, neonatal cells were able to produce similar levels of the classical pro-inflammatory (IL-6, IL-1ß) and anti-inflammatory (IL-10, IL-13) cytokines as that of adult cells, the amounts of IL-23 and MCP-1 were lower in CBMCs while the chemokine IL-8 was higher in CBMCs when compared with PBMCs. In addition, using Human Inflammation Antibody array technique we found that multiple cytokine production was impaired in cord blood when cells were co-infected with LPS and PGN. In conclusion, the TLR-mediated inflammatory response to neonatal pathogens is differentially regulated by different pathogens.

Functional characterization of histamine H4 receptor on human mast cells.

Among the four different types of histamine receptors (H1-H4), H4R is predominantly expressed in immune cells and involved in immunomodulatory response. Here, in this study we determined the expression of H4R in human mast cells (HMC-1, LAD-2 and primary cord blood derived CD34+ human mast cells) and characterized its functional properties. Interestingly, we found that human mast cells responded to both histamine (natural ligand) and 4-methylhistamine (selective H4R agonist) for sustained intracellular calcium mobilization, degranulation and cytokine production. However, only histamine induced the release of cAMP, but 4-methylhistamine down regulates cAMP indicating that H4R mediates its effect through Gαi/o protein and H1R via Gαq protein. Furthermore, both histamine and 4-methylhistamine induced the production of cysteinyl leukotrienes and LTB4. Using human inflammation antibody array membrane, we found that H4R induced the expression of various inflammatory proteins, involving pro-inflammatory cytokines and chemokines and these are TGF-ß1, TNF-α, TNF-ß, PDGF-BB, TIMP-2, M-CSF, IP-10, IL-16, IL-6, IL-3, IL-10, MIP-1α, IL-1α, ICAM-1, Eotaxin-2, RANTES, IL-8, MCP-1, and IL-6sR. We also quantified the level of various inflammatory cytokines produced by human mast cells through H4R. It was observed that, the production level of Th2 cytokines IL-4(401.34 pg/ml), IL-5 (64.21 pg/ml) and IL-13 (1044 pg/ml) and classical proinflammatory cytokines IL-6 (221.27 pg/ml) and IL-1ß (34.24 pg/ml) and chemokines MCP-1(106 pg/ml) and IL-8 (818.32 pg/ml). Furthermore, activation of H4R caused the phosphorylation of ERK and PI3K in a time dependent manner. Taken together these data demonstrate that, the activation of H4R in human mast cells produced not only inflammatory mediators that are associated with allergic reactions but also other inflammatory conditions.

Inflammatory mediators of systemic inflammation in neonatal sepsis.

OBJECTIVE AND DESIGN: Sepsis refers to severe systemic inflammation in response to invading pathogens. To understand the molecular events that initiate the systemic inflammatory response, various inflammatory mediators were analyzed in neonatal sepsis samples and compared with normal samples. MATERIALS AND METHODS: We initially measured the levels of the various classical inflammatory mediators such as acute phase proteins [C-reactive protein (CRP) and procalcitonin (PCT)], granule-associated mediators (NE, MPO and NO), proinflammatory cytokines [tumour necrosis factor-α (TNFα), IL-1ß and IL-6), antiinflammatory cytokines (IL-10 and IL-13) and chemokines [IL-8 and monocyte chemotactic protein (MCP-1)] and novel cytokines (IL-12/IL-23p40, IL-21 and IL-23) using ELISA. We also used the human inflammation antibody array membrane to profile the inflammatory proteins that are involved in neonatal sepsis. RESULTS: There were significantly higher levels of CRP (5.4 ± 0.70 mg/L), PCT (1.500 ± 0.2400 µg/L); NE (499.2 ± 22.01 µg/L), NO (54.22 ± 3.131 µM/L); TNFα (396.6 ± 37.40 pg/mL), IL-1ß (445.3 ± 34.25 pg/mL), IL-6 (320.9 ± 43.38 pg/mL); IL-8 (429.5 ± 64.08 pg/mL) MCP-1 (626.25 ± 88.91 pg/mL), IL-10 (81.80 ± 9.45 pg/mL), IL-12/IL-23p40 (30.25 ± 0.6 pg/mL), IL-21 (8,263.3 ± 526.8 pg/mL) and IL-23 (6,083 ± 781.3 pg/mL) in neonates with sepsis compared to normal. The levels of MPO (21.20 ± 3.099 ng/mL) were downregulated, whereas there was no change in IL-13 (188.7 ± 10.63 pg/mL) levels in septic neonates when compared with normal. Using the human inflammation antibody array membrane, we detected the presence of 17 inflammatory proteins such as IL-3, IL6R, IL12p40, IL-16, TNFα, TNFß, TNF R1, chemokines I-309, IP-10 (IFN-γ inducible protein 10), MCP-1, MCP-2, MIP 1ß (macrophage inflammatory protein), MIP-1δ, eotaxin-2, growth factors TGFß1 (transforming growth factor beta), PDGF (platelet derived growth factor), and cell adhesion molecule ICAM-1 (intracellular adhesion molecule) that were upregulated whereas RANTES which was downregulated in neonatal sepsis. CONCLUSION: The simultaneous secretion and release of multiple mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors were found to be involved in the initiation of systemic inflammation in neonatal sepsis. Therefore, measuring the concentration of multiple mediators may help in the early detection of neonatal sepsis and help to avoid unnecessary antibiotic treatment.

Reliability of "Modified timed up and go" test in children with cerebral palsy.

BACKGROUND: Timed up and go (TUG) is a quick test used in clinical practice as an outcome measure to assess functional ambulatory mobility or dynamic balance in adults. However, little information is available on TUG test used in cerebral palsy. Hence, the purpose of our study was to assess the intra-rater reliability of TUG test in cerebral palsy children. AIM AND OBJECTIVE: To assess within-session and test-retest reliability after 1 week of TUG test in cerebral palsy children. SETTING AND DESIGN: It was an a cross-sectional observational study conducted in a neurorehabilitation unit, with 30 cerebral palsy children of 4-12 years, within Gross Motor Function Classification System (GMFCS) level I, II, III, and with an IQ ≥50.The sampling technique used was purposive sampling excluding children with cognitive deficit. MATERIALS AND METHODS: Subjects performed TUG on three occasions - Initial assessment (time 1), 30 min after initial assessment (time 2), and 1 week after initial assessment (time 3). Three trails were conducted for each of the three occasions. The mean score of three trials was documented as the final score. Within-session and test-retest reliability were analyzed using scores of time 1 and 2, and time 1 and 3, respectively. STATISTICAL ANALYSIS: The documented data were analyzed for within-session and test-retest reliability after 1 week of TUG test by using intraclass correlation coefficient (ICC). RESULTS: Reliability of TUG test was high, with ICC of 0.99 for within-session reliability and 0.99 for test-retest reliability. CONCLUSION: Intra-rater reliability of TUG test in cerebral palsy children was found to be high.

A unique human rotavirus (non vaccine) G9P4 genotype infection in a child with gastroenteritis.

Group A Rotaviruses with serotypes G1-G4 and G9 are the common Rotavirus types of clinical importance. This study aimed at determining the different Rotavirus genotypes in stool sample of children below 5 years. A total of 300 children with acute gastroenteritis were tested for group specific VP6 antigen of group A Rotaviruses by Enzyme Linked Immunosorbent Assay. 47 of these samples were positive for Rotavirus antigen. Out of these, 20 positive samples were subjected to Reverse Transcriptase Polymerase Chain Reaction for genotyping. The identified genotypes were G9P8, G1P8, G2P4, G9P4 (non-vaccine genotype), G1P6, and G1 (P types not identified in 5 samples).

Sonographic depiction of the fetal anus and its utility in the diagnosis of anorectal malformations.

OBJECTIVES: The purpose of this study was to describe the sonographic appearance of the fetal anus and its usefulness in diagnosis of anal atresia. METHODS: An anomaly scan was performed in 13,150 patients over 8 years. Gestational ages ranged from 16 to 38 weeks. A tangential scan of the fetal perineum was performed. The anus was seen as a hypoechoic ring representing the wall of the anal canal with a central echogenic dot representing the lumen. This appearance was seen posterior to the external genitalia. If there was failure to see this appearance, a coronal scan of the fetal pelvis was done to look for the anal canal in contiguity with the rectum and to confirm its extension up to perineum. Failure to see these two features was considered diagnostic of anal atresia. RESULTS: On the basis of these criteria, anal atresia was diagnosed in 17 fetuses. Sixteen of these fetuses also had other associated anomalies. The appearance of the anus was useful for ruling out anal atresia in 2 fetuses with a dilated colon containing echogenic meconium balls. CONCLUSIONS: The anus is visible on a tangential scan of the fetal perineum. It can be seen routinely during an anomaly scan to diagnose or rule out anal atresia.

Health effects of consanguinity in Pondicherry.

Of 1000 pregnant women studied, consanguinity was observed in 30.8%, with a higher frequency among women from rural areas, and among Hindus. In the consanguineous group, first cousin marriages were present in 47.4%, and uncle niece marriages in 23.4% of women. Coefficient of inbreeding was highest in Harijans (0.0258), followed by non-Brahmins (0.0220) and Brahmins (0.0204). Fertility was not influenced by consanguinity. There was a significantly higher rate of still births and infant mortality in consanguineous matings as compared with non-consanguineous. Total morbidity was higher in the consanguineous group as compared with the non-consanguineous (p less than 0.01), especially that due to neonatal infections and jaundice. There was no significant difference in the prevalence of congenital malformations, chromosomal and genetic disorders between the two groups, although the number of abnormal births in this group was small.

PIP: In 1978, pediatricians analyzed data on 1000 mothers at the Jawaharlal Institute of Medical Education and Research Hospital in Pondicherry, India, and followed their newborns for 3 months to determine prevalence and type of consanguinity and its effects on morbidity and mortality. Consanguinity prevalence was 30.8%. It was more common among the rural population than the urban population (35.6% vs. 25.9%; p .001) and among Hindus than Christians or Muslims (32.2% vs. 14.1% and 13.3%, respectively; p = .004). Consanguinity occurred more often among the Harijan caste (37.5%) than the non-Brahmins (30.8%) and Brahmins (27.7%). The inbreeding coefficient was highest among the Harijans (0.0258), followed by non-Brahmins (0.0220) and Brahmins (0.0204). First cousin marriages predominated (47.4%), especially boy with mother's brother's daughter, followed by marriages between more distant relatives (29.2%) and uncle-niece marriages (23.4%). Neonatal mortality was greater, but not significantly so, in consanguineous marriages than nonconsanguineous marriages (63.8 vs. 48.5). Mortality of infants less than 1 year old was significantly greater in consanguineous marriages than nonconsanguineous marriages (97.8 vs. 59.7; p = .007). Further, fetal deaths occurred more often in consanguineous marriages than nonconsanguineous marriages (4.2 vs. 2.8; p .01). Total morbidity, especially infections/septicemia and jaundice, was significantly higher in consanguineous marriages than nonconsanguineous marriages (176.1 vs. 67.2; p .001). No significant difference occurred in the prevalence of congenital malformations and chromosomal and genetic disorders. the number of abnormal births was small, though. Consanguinity did not affect fertility (total fertility for both groups, 2.8). These results suggested that consanguinity contributes considerably to infant mortality and morbidity.

Effect of aspirin on serum lipoprotein profile in rats subjected to myocardial stress by isoproterenol.

The effect of isoproterenol on the levels of serum lipoprotein profile were studied in rats. Rats were treated with isoproterenol (200 mg/100 g body weight, sc twice at an interval of 24 hr) for 2 days. Aspirin was administered orally 1.2 mg/100 g body weight, daily for 60 days. Isoproterenol treated rats showed decrease in the activities of hepatic and extrahepatic lipoprotein lipase. HDL cholesterol level was found to be decreased, significantly with increase in LDL cholesterol in isoproterenol treated rats. Aspirin treated rats showed marked reversal of these metabolic changes. The lipoprotein changes were minimum in rats treated with both aspirin and isoproterenol.

Invertase activity in the gut of 6th instar larvae of Spodoptera mauritia Boisd. (Noctuidae, Lepidoptera).

Invertase activity has been studied in the fore-, mid- and hindgut of the 6th instar larva of Spodoptera mauritia. The highest activity was in the midgut except during the early hours of the larva period when the foregut showed comparatively increased activity. The hindgut invertase activity may be from the voiding of enzyme along with the undigested food.