Increased but error-prone nonhomologous end joining in immortalized lymphoblastoid cell extracts from adult cancer patients with late radionecrosis.

PURPOSE: To study nonhomologous end joining in extracts of two lymphoblastoid cell lines derived from patients with late radionecrosis after radiotherapy. Both cell lines were previously shown to exhibit impaired rejoining of DNA double-strand breaks in a pulse-field gel electrophoresis assay. METHODS AND MATERIALS: We used a cell-free system and quantitative real-time polymerase chain reaction, as well as sequencing analysis of end joining products. RESULTS: Paradoxically, extracts of the two cell lines display increased rates of in vitro end joining of noncohesive termini compared with normal cell extracts. This increase was seen in the absence of added deoxyribonucleoside triphosphates and was sensitive to inhibition by wortmannin. Sequencing of the joined products revealed that, despite increased rates of end joining, the process was error prone with a greater frequency of deletions compared with that observed in normal controls. CONCLUSION: These findings are consistent with the suggestion that a promiscuous, deletion-prone abnormality of nonhomologous end joining might underpin the predisposition of certain radiotherapy patients to late radionecrosis. We hypothesize that some individuals might harbor subclinical defects in nonhomologous end joining that clinically manifest on challenge with high-dose radiation. Because both quantitative and qualitative aspects of end joining have demonstrably been influenced, we recommend that the study of patient samples should involve a combination of quantitative methods (e.g., quantitative real-time polymerase chain reaction), sequencing analysis, and a comparison of multiple join types.

The innate interferon gamma response of BALB/c and C57BL/6 mice to in vitro Burkholderia pseudomallei infection.

BACKGROUND: Burkholderia pseudomallei is the causative agent for melioidosis. For many bacterial infections, cytokine dysregulation is one of the contributing factors to the severe clinical outcomes in the susceptible hosts. The C57BL/6 and BALB/c mice have been established as a differential model of susceptibility in murine melioidosis. In this study, we compared the innate IFN-gamma response to B. pseudomallei between the C57BL/6 and BALB/c splenocytes and characterized the hyperproduction of IFN-gamma in the relatively susceptible BALB/c mice in vitro. RESULTS: Naïve BALB/c splenocytes were found to produce more IFN-gamma in response to live bacterial infection compared to C57BL/6 splenocytes. Natural killer cells were found to be the major producers of IFN-gamma, while T cells and Gr-1intermediate cells also contributed to the IFN-gamma response. Although anti-Gr-1 depletion substantially reduced the IFN-gamma response, this was not due to the contribution of Gr-1high, Ly-6G expressing neutrophils. We found no differences in the cell types making IFN-gamma between BALB/c and C57BL/6 splenocytes. Although IL-12 is essential for the IFN-gamma response, BALB/c and C57BL/6 splenocytes made similar amounts of IL-12 after infection. However, BALB/c splenocytes produced higher proinflammatory cytokines such as IL-1beta, TNF-alpha, IL-6, IL-18 than C57BL/6 splenocytes after infection with B. pseudomallei. CONCLUSION: Higher percentages of Gr-1 expressing NK and T cells, poorer ability in controlling bacteria growth, and higher IL-18 could be the factors contributing to IFN-gamma hyperproduction in BALB/c mice.

Reduced IL-17A Secretion Is Associated with High Levels of Pneumococcal Nasopharyngeal Carriage in Fijian Children.

Streptococcus pneumonia (the pneumococcus) is the leading vaccine preventable cause of serious infections in infants under 5 years of age. The major correlate of protection for pneumococcal infections is serotype-specific IgG antibody. More recently, antibody-independent mechanisms of protection have also been identified. Preclinical studies have found that IL-17 secreting CD4+ Th17 cells in reducing pneumococcal colonisation. This study assessed IL-17A levels in children from Fiji with high and low pneumococcal carriage density, as measured by quantitative real-time PCR (qPCR). We studied Th17 responses in 54 children who were designated as high density carriers (N=27, >8.21x10(5) CFU/ml) or low density carriers (N=27, <1.67x10(5) CFU/ml). Blood samples were collected, and isolated peripheral blood mononuclear cells (PBMCs) were stimulated for 6 days. Supernatants were harvested for cytokine analysis by multiplex bead array and/or ELISA. Th17 cytokines assayed included IL-17A, IL-21, IL-22 as well as TNF-α, IL-10, TGF-ß, IL-6, IL-23 and IFNγ. Cytokine levels were significantly lower in children with high density pneumococcal carriage compared with children with low density carriage for IL-17A (p=0.002) and IL-23 (p=0.04). There was a trend towards significance for IL-22 (p=0.057) while no difference was observed for the other cytokines. These data provide further support for the role of Th17-mediated protection in humans and suggest that these cytokines may be important in the defence against pneumococcal carriage.

Janus kinase 3-activating mutations identified in natural killer/T-cell lymphoma.

UNLABELLED: The molecular pathogenesis of natural killer/T-cell lymphoma (NKTCL) is not well understood. We conducted whole-exome sequencing and identified Janus kinase 3 (JAK3) somatic-activating mutations (A572V and A573V) in 2 of 4 patients with NKTCLs. Further validation of the prevalence of JAK3 mutations was determined by Sanger sequencing and high-resolution melt (HRM) analysis in an additional 61 cases. In total, 23 of 65 (35.4%) cases harbored JAK3 mutations. Functional characterization of the JAK3 mutations support its involvement in cytokine-independent JAK/STAT constitutive activation leading to increased cell growth. Moreover, treatment of both JAK3-mutant and wild-type NKTCL cell lines with a novel pan-JAK inhibitor, CP-690550, resulted in dose-dependent reduction of phosphorylated STAT5, reduced cell viability, and increased apoptosis. Hence, targeting the deregulated JAK/STAT pathway could be a promising therapy for patients with NKTCLs. SIGNIFICANCE: Gene mutations causing NKTCL have not been fully identified. Through exome sequencing, we identified activating mutations of JAK3 that may play a significant role in the pathogenesis of NKTCLs. Our findings have important implications for the management of patients with NKTCLs.

Model of differential susceptibility to mucosal Burkholderia pseudomallei infection.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with protean clinical manifestations. The major route of infection is thought to be through subcutaneous inoculation of contaminated soil and water, although ingestion and inhalation of contaminated aerosols are also possible. This study examines infection through the intranasal route in a murine model to mimic infection through inhalation. Two strains of mice, C57BL/6 and BALB/c, exhibit differential susceptibilities to the infection, with the C57BL/6 mice being considerably more resistant. To examine host factors that could contribute to this difference, bacterial loads and cytokine profiles in the two strains of mice were compared. We found that infected BALB/c mice exhibited higher bacterial loads in the lung and spleen and that they produced significantly higher levels of gamma interferon (IFN-gamma) in the serum than C57BL/6 mice. Although tumor necrosis factor alpha and interleukin-1 could be detected in the nasal washes and sera of both strains of mice, the production in serum was transient and much lower than that of IFN-gamma. C57BL/6 mice also exhibited memory responses to bacteria upon reinfection, with the production of serum immunoglobulin G (IgG) and mucosal IgA antibodies. Thus, it is possible that the production of systemic and mucosal antibodies is important for protection against disease in C57BL/6 mice.