Genomic characterization of Haemophilus influenzae: a focus on the capsule locus.

BACKGROUND: Haemophilus influenzae (Hi) can cause invasive diseases such as meningitis, pneumonia, or sepsis. Typeable Hi includes six serotypes (a through f), each expressing a unique capsular polysaccharide. The capsule, encoded by the genes within the capsule locus, is a major virulence factor of typeable Hi. Non-typeable (NTHi) does not express capsule and is associated with invasive and non-invasive diseases. METHODS: A total of 395 typeable and 293 NTHi isolates were characterized by whole genome sequencing (WGS). Phylogenetic analysis and multilocus sequence typing were used to characterize the overall genetic diversity. Pair-wise comparisons were used to evaluate the capsule loci. A WGS serotyping method was developed to predict the Hi serotype. WGS serotyping results were compared to slide agglutination (SAST) or real-time PCR (rt-PCR) serotyping. RESULTS: Isolates of each Hi serotype clustered into one or two subclades, with each subclade being associated with a distinct sequence type (ST). NTHi isolates were genetically diverse, with seven subclades and 125 STs being detected. Regions I and III of the capsule locus were conserved among the six serotypes (≥82% nucleotide identity). In contrast, genes in Region II were less conserved, with only six gene pairs from all serotypes showing ≥56% nucleotide identity. The WGS serotyping method was 99.9% concordant with SAST and 100% concordant with rt-PCR in determining the Hi serotype. CONCLUSIONS: Genomic analysis revealed a higher degree of genetic diversity among NTHi compared to typeable Hi. The WGS serotyping method accurately predicted the Hi capsule type and can serve as an alternative method for Hi serotyping.

Whole-Genome Sequencing for Characterization of Capsule Locus and Prediction of Serogroup of Invasive Meningococcal Isolates.

Invasive meningococcal disease is mainly caused by Neisseria meningitidis serogroups A, B, C, X, W, and Y. The serogroup is typically determined by slide agglutination serogrouping (SASG) and real-time PCR (RT-PCR). We describe a whole-genome sequencing (WGS)-based method to characterize the capsule polysaccharide synthesis (cps) locus, classify N. meningitidis serogroups, and identify mechanisms for nongroupability using 453 isolates from a global strain collection. We identified novel genomic organizations within functional cps loci, consisting of insertion sequence (IS) elements in unique positions that did not disrupt the coding sequence. Genetic mutations (partial gene deletion, missing genes, IS insertion, internal stop, and phase-variable off) that led to nongroupability were identified. The results of WGS and SASG were in 91% to 100% agreement for all serogroups, while the results of WGS and RT-PCR showed 99% to 100% agreement. Among isolates determined to be nongroupable by WGS (31 of 453), the results of all three methods agreed 100% for those without a capsule polymerase gene. However, 61% (WGS versus SASG) and 36% (WGS versus RT-PCR) agreements were observed for the isolates, particularly those with phase variations or internal stops in cps loci, which warrant further characterization by additional tests. Our WGS-based serogrouping method provides comprehensive characterization of the N. meningitidis capsule, which is critical for meningococcal surveillance and outbreak investigations.

Poly (ADP) ribose polymerase enzyme inhibitor, veliparib, potentiates chemotherapy and radiation in vitro and in vivo in small cell lung cancer.

Poly (ADP) ribose polymerase (PARP) plays a key role in DNA repair and is highly expressed in small cell lung cancer (SCLC). We investigated the therapeutic impact of PARP inhibition in SCLC. In vitro cytotoxicity of veliparib, cisplatin, carboplatin, and etoposide singly and combined was determined by MTS in 9 SCLC cell lines (H69, H128, H146, H526, H187, H209, DMS53, DMS153, and DMS114). Subcutaneous xenografts in athymic nu/nu mice of H146 and H128 cells with relatively high and low platinum sensitivity, respectively, were employed for in vivo testing. Mechanisms of differential sensitivity of SCLC cell lines to PARP inhibition were investigated by comparing protein and gene expression profiles of the platinum sensitive and the less sensitive cell lines. Veliparib showed limited single-agent cytotoxicity but selectively potentiated (≥ 50% reduction in IC50 ) cisplatin, carboplatin, and etoposide in vitro in five of nine SCLC cell lines. Veliparib with cisplatin or etoposide or with both cisplatin and etoposide showed greater delay in tumor growth than chemotherapy alone in H146 but not H128 xenografts. The potentiating effect of veliparib was associated with in vitro cell line sensitivity to cisplatin (CC = 0.672; P = 0.048) and DNA-PKcs protein modulation. Gene expression profiling identified differential expression of a 5-gene panel (GLS, UBEC2, HACL1, MSI2, and LOC100129585) in cell lines with relatively greater sensitivity to platinum and veliparib combination. Veliparib potentiates standard cytotoxic agents against SCLC in a cell-specific manner. This potentiation correlates with platinum sensitivity, DNA-PKcs expression and a 5-gene expression profile.

Predictors of depression in breast cancer patients treated with radiation: role of prior chemotherapy and nuclear factor kappa B.

BACKGROUND: Depression is common during and after breast cancer treatment. However, the role of specific therapeutic modalities and related biologic mechanisms remains unclear. Radiation is an essential component of breast-conserving therapy and may contribute to depression in patients with breast cancer through the activation of inflammatory pathways. METHODS: Depressive symptoms and inflammatory mediators, including nuclear factor kappa B (NF-κB), were assessed at baseline (before radiation), during radiation, and 6 weeks after radiation in 64 women who had stage 0 through IIIA breast cancer. RESULTS: No significant increases in depressive symptoms occurred during or after radiation, although a number of patients exhibited moderate-to-severe depression throughout the study. Multivariate analyses of baseline factors predictive of depression revealed that educational status, perceived stress, prior chemotherapy, and peripheral blood NF-κB DNA binding all were independent predictors of persistent depressive symptoms after radiation (all P < .05). Of these factors, only prior chemotherapy was associated with inflammatory mediators, including NF-κB DNA binding, soluble tumor necrosis factor-alpha receptor 2, and interleukin-6, which, in univariate analyses predicted depressive symptoms after radiation (all P < .05). Chemotherapy-treated patients also exhibited an over-representation of gene transcripts regulated by NF-κB. CONCLUSIONS: Radiation was not associated with increased depressive symptoms in the current study, but of disease and treatment-related factors, prior chemotherapy predicted significant depression after radiation. Longitudinal studies are warranted to investigate the relationship among prior chemotherapy, inflammation, and persistent depression after breast cancer treatment.

Phenotype, function, and gene expression profiles of programmed death-1(hi) CD8 T cells in healthy human adults.

T cell dysfunction is an important feature of many chronic viral infections. In particular, it was shown that programmed death-1 (PD-1) regulates T cell dysfunction during chronic lymphocytic choriomeningitis virus infection in mice, and PD-1(hi) cells exhibit an intense exhausted gene signature. These findings were extended to human chronic infections such as HIV, hepatitis C virus, and hepatitis B virus. However, it is not known if PD-1(hi) cells of healthy humans have the traits of exhausted cells. In this study, we provide a comprehensive description of phenotype, function, and gene expression profiles of PD-1(hi) versus PD-1(lo) CD8 T cells in the peripheral blood of healthy human adults as follows: 1) the percentage of naive and memory CD8 T cells varied widely in the peripheral blood cells of healthy humans, and PD-1 was expressed by the memory CD8 T cells; 2) PD-1(hi) CD8 T cells in healthy humans did not significantly correlate with the PD-1(hi) exhausted gene signature of HIV-specific human CD8 T cells or chronic lymphocytic choriomeningitis virus-specific CD8 T cells from mice; 3) PD-1 expression did not directly affect the ability of CD8 T cells to secrete cytokines in healthy adults; 4) PD-1 was expressed by the effector memory compared with terminally differentiated effector CD8 T cells; and 5) finally, an interesting inverse relationship between CD45RA and PD-1 expression was observed. In conclusion, our study shows that most PD-1(hi) CD8 T cells in healthy adult humans are effector memory cells rather than exhausted cells.

Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37.

The sole human cathelicidin peptide, LL-37, has been demonstrated to protect animals against endotoxemia/sepsis. Low, physiological concentrations of LL-37 (< or =1 microg/ml) were able to modulate inflammatory responses by inhibiting the release of the proinflammatory cytokine TNF-alpha in LPS-stimulated human monocytic cells. Microarray studies established a temporal transcriptional profile and identified differentially expressed genes in LPS-stimulated monocytes in the presence or absence of LL-37. LL-37 significantly inhibited the expression of specific proinflammatory genes up-regulated by NF-kappaB in the presence of LPS, including NFkappaB1 (p105/p50) and TNF-alpha-induced protein 2 (TNFAIP2). In contrast, LL-37 did not significantly inhibit LPS-induced genes that antagonize inflammation, such as TNF-alpha-induced protein 3 (TNFAIP3) and the NF-kappaB inhibitor, NFkappaBIA, or certain chemokine genes that are classically considered proinflammatory. Nuclear translocation, in LPS-treated cells, of the NF-kappaB subunits p50 and p65 was reduced > or =50% in the presence of LL-37, demonstrating that the peptide altered gene expression in part by acting directly on the TLR-to-NF-kappaB pathway. LL-37 almost completely prevented the release of TNF-alpha and other cytokines by human PBMC following stimulation with LPS and other TLR2/4 and TLR9 agonists, but not with cytokines TNF-alpha or IL-1beta. Biochemical and inhibitor studies were consistent with a model whereby LL-37 modulated the inflammatory response to LPS/endotoxin and other agonists of TLR by a complex mechanism involving multiple points of intervention. We propose that the natural human host defense peptide LL-37 plays roles in the delicate balancing of inflammatory responses in homeostasis as well as in combating sepsis induced by certain TLR agonists.