Understanding the killer-cell immunoglobulin like receptor polymorphism in retinoblastoma.

BACKGROUND: The KIR receptors present on the natural killer (NK) cells play a crucial role by exercising cytotoxicity to eliminate tumor cells. Both KIR and class-I HLA molecules exhibit extensive polymorphism. Although RB1 inactivation triggers the initiation of retinoblastoma; however additional immune alterations trigger tumor development. The aim was to explore the KIR/HLA polymorphism and its role in the pathogenesis of retinoblastoma. METHODS: Patients with unilateral, non-familial retinoblastoma were enrolled as cases. Healthy individuals matched for ethnicity were enrolled as controls. KIR genotyping was performed by sequence-specific primer assay. The investigated KIR genes included: inhibitory (2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DL5B), activating (2DS1, 2DS2, 2DS3, 2DS4*FUL, 2DS4*DEL, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1) and pseudogenes (2DP1, 3DP1*FUL, 3DP1*DEL). In addition, HLA ligands were investigated by sequence-specific oligonucleotide assay for HLA-A, B, and C locus. RESULTS: KIR genotyping was performed in 48 cases and 107 controls. The mean age of cases was 2.9 ± 2.2 years (range: 0.25-10). Among the 19 KIR genes, the frequency of KIR2DS4*FUL (p = 0.0019) and 2DS5 (p = 0.0095) was increased among cases. HLA ligands were investigated in 25 cases and 50 controls. The frequency of HLA ligands (C1/C2, Bw4, A3/A11) was similar among cases and controls. However, the KIR/HLA combination frequency for KIR3DS1/HLA-Bw4 was decreased in cases (p = 0.006). CONCLUSION: It is the pioneer study to report the association of killer cell immunoglobulin-like receptors in retinoblastoma. KIR2DS4*FUL and KIR2DS5 had a susceptible, and KIR3DS1/HLA-BW4 had a protective role in retinoblastoma. The results will aid in exploring the therapeutic potential of NK cell-based therapy for retinoblastoma.

Deciphering the TLR transcriptome and downstream signaling pathway in cerebrospinal fluid in pediatric meningitis.

OBJECTIVE/DESIGN: Pediatric meningitis is characterized by a colossal inflammatory response to the pathogen in the central nervous system (CNS). This unabated inflammatory response persists even after the removal of the pathogen by antibiotics/steroids causing collateral damage to CNS tissue. Toll-like receptors (TLRs) are the key players in the recognition and elicitation of innate-immune response against bacterial/viral components in cerebrospinal fluid (CSF). Till date, the precise understanding of TLR-triggered inflammatory response in pediatric meningitis is lacking. The present study was designed to delineate the role of TLR transcriptome and downstream signaling pathways in CSF of pediatric meningitis. METHODS: Children in the age group of > 3 months to 12 years with pediatric meningitis were included. A total of 249 cases of pediatric meningitis (bacterial = 89, viral = 160) were included. In addition, 71 children who tested negative to the pathogen in CSF tap and did not have signs of infection clinically constituted the controls. RNA was extracted from the CSF samples of both cases and controls. The relative gene expression profile of 42 TLR signaling pathway genes was performed. For the analysis of secretory cytokines and chemokines in CSF, Luminex assay was performed. RESULTS: We report global upregulation of TLR genes in patients with acute bacterial meningitis (ABM). The downstream signaling molecules were upregulated as well. The CSF of pediatric ABM patients revealed a predominant pro-inflammatory milieu marked by increased levels of pro-inflammatory cytokines. A significant correlation between poor clinical outcomes of patients and an increased expression of TLR/pro-inflammatory cytokine genes was observed. CONCLUSION: Our findings provide support for future studies exploring TLR-based adjunct therapy to limit the neurological sequelae, owing to persistent inflammation in pediatric ABM patients.

Understanding HLA-G driven journey from HPV infection to cancer cervix: Adding missing pieces to the jigsaw puzzle.

Human Papillomavirus (HPV) is a vital risk-factor for cancer cervix. However, persistent HPV infection results in cervical cancer in only a minority. Probably, HPV subdues the host immune response for persistence, which includes augmentation of HLA-G and plausibly aids in progression to cervical cancer. HLA-G, which comprises of membrane and soluble form, downregulates the host's immune response and generate tolerance. The current study aimed to analyze both forms of HLA-G in fresh tissue and plasma of women with HPV-infected and uninfected cervix and cancer cervix using Western blot and ELISA. The study cohort included 30 women with cervical carcinoma and equal number with normal cervix and 6 with HPV infected cervix. We observed a significant upregulation of membranous HLA-G expression in HPV infected cervix and cervical carcinoma (P < 0.001). Interestingly, the pairwise comparison of HLA-G tissue protein expression of the normal cervix and cervical carcinoma, as well as the normal cervix with HPV infected cervix, was significant (P < 0.001). Levels of soluble HLA-G were significantly raised in carcinoma cervix. We observed a progressive increase in HLA-G protein expression in HPV infected cervix and cervical carcinoma. These findings compel us to hypothesize that the upregulation of HLA-G expression favors the persistence of HPV in a microenvironment of a submissive host response. This progressive upregulation further leads to cervical cancer. Thus elimination of HPV infection seems to be a desirable proposition to prevent cervical cancer. In the absence of antiviral therapy for HPV, exploration of HLA-G antibody-based therapeutic strategies appear promising.

Evaluation of Effect of Probiotics on Cytokine Levels in Critically Ill Children With Severe Sepsis: A Double-Blind, Placebo-Controlled Trial.

OBJECTIVES: To evaluate the effect of probiotics on cytokines in children with severe sepsis. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: ICU of a tertiary care teaching hospital in North India. PATIENTS: Children 3 months to 12 years old with severe sepsis. INTERVENTIONS: Enrolled children were randomized to probiotic (n = 50) and placebo (n = 50) groups. Probiotic group received VSL#3 (Danisco-Dupont USA, Madison, WI) (Lactobacillus paracasei, L. plantarum, L. acidophilus, L. delbrueckii, Bifidobacterium longum, B. infantis, B. breve, Streptococcus salivarius; maltose and silicon dioxide), and placebo group received maltose and silicon dioxide. Dose was 1 sachet twice daily for 7 days. Blood was collected on days 1 and 7 for estimation of interleukin-6, interleukin-12p70, interleukin-17, tumor necrosis factor-α, interleukin-10, and transforming growth factor -ß1. "Primary outcome": Change in cytokine levels in probiotic and placebo groups from day 1 to 7. "Secondary outcomes": Sequential Organ Failure Assessment score, healthcare-associated infections, ICU stay, and mortality. MEASUREMENTS AND MAIN RESULTS: On day 7, probiotic group had significantly lower levels of proinflammatory cytokines (interleukin-6 [80 vs 186 pg/mL, p = 0.001]; interleukin-12p70 [44 vs 79 pg/mL, p = 0.001]; interleukin-17 [217 vs 293 pg/mL, p = 0.01]; and tumor necrosis factor-α [192 vs 348 pg/mL, p = 0.01]) and higher levels of antiinflammatory cytokines (interleukin-10 [320 vs 240 pg/mL, p = 0.02] and transforming growth factor-ß1 [311 vs 221 ng/mL, p = 0.01]) than placebo group. From day 1 to 7, probiotic group showed significant decrease in proinflammatory cytokines (interleukin-6 [196-80 pg/mL, p = 0.001]; interleukin-12p70 [71-44 pg/mL, p = 0.01]; interleukin-17 [258-217 pg/mL, p = 0.01]; and tumor necrosis factor-α [347-192 pg/mL, p = 0.001]) and increase in antiinflammatory cytokines (interleukin-10 [198-320 pg/mL, p = 0.001] and transforming growth factor-ß1 [216-311 ng/mL, p = 0.001]) as compared to placebo group. Sequential Organ Failure Assessment score on day 7 was significantly less in probiotic group (1 vs 3). There was a nonsignificant trend toward lower incidence of healthcare-associated infections (14% vs 20%) and duration of ICU stay (6.5 vs 9 d) in probiotic group. Mortality was similar in two groups. CONCLUSIONS: Probiotics supplementation for 7 days resulted in significant decrease in proinflammatory and increase in antiinflammatory cytokines in children with severe sepsis.

Proteomic analysis of differentially expressed proteins in vitreous humor of patients with retinoblastoma using iTRAQ-coupled ESI-MS/MS approach.

There is close proximity of vitreous humor with the tumor bulk in eyes with retinoblastoma. This renders vitreous humor a promising source to evaluate disease-specific protein targets in retinoblastoma. We studied the differential proteome of vitreous fluid in retinoblastoma tumors (n = 4) as compared to controls (n = 4). The vitreous humor was depleted off the high abundant fraction using MARS-6 affinity column. Subsequently, the tryptic peptides were derivatised with iTRAQ labels. The labelled peptides were pooled and subjected to fractionation using bRPLC. This was followed by protein identification and quantification using electrospray ionisation mass spectrometry (ESI-MS/MS) approach. The identified proteins were subjected to bioinformatics analysis utilizing PANTHER 7.0 and IPA software. Four hundred and thirty-one non-redundant (362 upregulated and 69 downregulated) proteins (≥2 unique peptides, ± 1.5 folds, p < 0.05) were identified. The majority of the proteins were cytoplasmic (40 %), majorly involved in catalytic (32.7 %) and binding activities (26.3 %). Highly deregulated proteins included MMP2, TNC, CD44, SUZ12 and CRABP1. The protein expression of GFAP, CRABP1, MMP2 and TNC was validated by western blotting. Pathway and network analyses revealed p38MAPK and Akt signalling to be the most significantly regulated pathways in retinoblastoma. This is the first report of differential vitreous proteome of retinoblastoma and highlights novel protein targets, such as MMP2, TNC and CRABP1. Further investigations into unravelling the biological role of the proteins and their prospects of being utilised as potential candidates in therapeutics are warranted.

Characterization of Toll-like receptor transcriptome in squamous cell carcinoma of cervix: A case-control study.

OBJECTIVE: Human papillomavirus (HPV) is a proven etiological agent for cervical cancer However, not all HPV infections result in cervical cancer. The mechanisms of host immune system to prevent/control HPV infection remain poorly understood. Toll-like receptors (TLRs) are a system of innate immune defense. HPV has been demonstrated to modulate TLR expression and interfere in TLR signaling pathways, leading to persistent viral infection and carcinogenesis. The aim was to study the relative gene expression of TLRs in cervical squamous cell carcinoma (SCC). METHODS: Gene expression profile of TLRs 1 to 9 was examined in 30 cervical SCCs and an equal number of normal cervical tissue samples using a PCR array platform. Gene expression studies for TLRs 3 and 7 were validated by western blotting. RESULTS: HPV was detected in all cases and in none of the controls (p<0.0001). HPV16 was the preponderant (83.3%) subtype. A significant downregulation in the relative gene expression of TLR3 (p<0.0001), TLR4 (p<0.0005) and TLR5 (p<0.0001) was observed in cases. A significant upregulation for TLR1 was observed (p=0.006). Although TLRs 2, 7, 8 and 9 were upregulated and TLR6 was downregulated, it was not significant. The western blot performed with antibodies against TLRs 3 and 7 confirmed the findings of the gene expression studies. CONCLUSIONS: A significant downregulation in the gene expression of TLRs 3, 4 and 5 and upregulation of TLR1 was observed in cervical SCC as compared to controls. Study results evoke the proposition for investigating TLRs 3, 4 and 5 agonists for therapeutic exploration.