Immunospecific Responses to Bacterial Elongation Factor Tu during Burkholderia Infection and Immunization

Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection

EBV-positive, HHV8-negative large B-cell lymphoma with an unusual germinotropic growth pattern in an immunocompetent patient.

Clinicopathologic and cytogenetic findings of an unusual EBV+ve, HHV8-ve germinotropic lymphoma, with a nongerminal center immunophenotype occurring in an immunocompetent individual, are presented. A comprehensive literature search revealed a single report of three similar cases. These may represent a unique subset of EBV-positive large B-cell lymphomas in immunocompetent individuals

EBV-Positive Nodal T- and NK-Cell Lymphoma Mimicking Anaplastic Large Cell Lymphoma: A Case Report

EBV-positive nodal T- and NK-cell lymphoma (EBV+ NT/NKCL) is a recently recognized entity in the 5th edition of the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues. Notably, CD30 positivity is frequently observed in (EBV+ NT/NKCL), creating diagnostic challenges to distinguish it from ALK-negative anaplastic large cell lymphoma (ALCL). Furthermore, cases of EBV+ ALCL have been documented in the literature, predating the inclusion of EBV+ nodal cytotoxic T-cell lymphoma as a variant of peripheral T-cell lymphoma. We present a case of a 47-year-old male presenting with multiple lymphadenopathies. The histomorphologic and immunophenotypic features of the lymph node closely resemble ALK-negative ALCL, characterized by uniform CD30 expression and a subcapsular distribution of lymphoma cells. However, the lymphoma cells exhibit diffuse positivity for EBV, consistent with EBV+ NT/NKCL. A case of ALK-negative ALCL with an immunophenotype identical to the EBV-positive case is included for comparison. Given that EBV+ NT/NKCL represents an aggressive neoplasm requiring unique clinical management compared to ALK-negative ALCL, it is critical to accurately differentiate EBV+ NT/NKCL from ALK-negative ALCL with a cytotoxic T-cell immunophenotype

Post-Exposure Therapeutic Efficacy of COX-2 Inhibition against <i>Burkholderia pseudomallei</i>

<div><p><i>Burkholderia pseudomallei</i> is a Gram-negative, facultative intracellular bacillus and the etiologic agent of melioidosis, a severe disease in Southeast Asia and Northern Australia. Like other multidrug-resistant pathogens, the inherent antibiotic resistance of <i>B. pseudomallei</i> impedes treatment and highlights the need for alternative therapeutic strategies that can circumvent antimicrobial resistance mechanisms. In this work, we demonstrate that host prostaglandin E2 (PGE₂) production plays a regulatory role in the pathogenesis of <i>B. pseudomallei</i>. PGE₂ promotes <i>B. pseudomallei</i> intracellular survival within macrophages and bacterial virulence in a mouse model of pneumonic melioidosis. PGE₂-mediated immunosuppression of macrophage bactericidal effector functions is associated with increased arginase 2 (Arg2) expression and decreased nitric oxide (NO) production. Treatment with a commercially-available COX-2 inhibitor suppresses the growth of <i>B. pseudomallei</i> in macrophages and affords significant protection against rapidly lethal pneumonic melioidosis when administered post-exposure to <i>B. pseudomallei</i>-infected mice. COX-2 inhibition may represent a novel immunotherapeutic strategy to control infection with <i>B. pseudomallei</i> and other intracellular pathogens.</p></div

PGE₂ promotes <i>B.</i> pseudomallei intracellular survival.

<p>BMDM were incubated in the presence or absence of NS398 (100 µM) +/− PGE₂ (1 µM) for 30 minutes then infected with <i>Bps</i> at MOI 1 <b>A</b>) Percent intracellular survival of <i>Bps</i> and <b>B</b>) corresponding nitrite levels in BMDM supernatants. The data represent biological triplicates per time point. Error bars represent the SEM. Statistical significance was determined using a two way ANOVA with Bonferroni post-test. *p<0.05, *** p<0.001. Data is representative of two independent experiments.</p

<i>B.</i> pseudomallei rapidly induces COX-2 and PGE₂ production by macrophages.

<p><b>A</b>) Bone-marrow derived macrophages (BMDM) were treated with viable <i>B. thailandensis</i> (Bt), <i>B. pseudomallei</i> (Bp) or heat-inactivated Bp (iBp) at MOI 1 and COX-2 mRNA expression was measured by RT-PCR. <b>B</b>) COX-2 enzyme was detected by Western blot in BMDM infected with Bp at MOI 1. <b>C</b>) BMDM were treated with Bp at MOI 0.1 or 1 and iBp at MOI 1 and PGE₂ was measured in culture supernatants by ELISA. The data represent biological triplicates per time point. Error bars represent the standard deviation (SD). Statistical significance was determined using a two way ANOVA with Bonferroni post-test. *p<0.05, *** p<0.001. Data is representative of two independent experiments.</p

Arginase 2 enhances <i>B.</i> pseudomallei survival in macrophages.

<p>BMDM were incubated in the presence or absence of NS398 (100 µM) +/− PGE₂ (1 µM) for 30 minutes, then infected with <i>Bps</i> at MOI 1 for 4 h <b>A</b>) Fold-change in mRNA expression for iNOS, arginase 1 (Arg1) and Arg2 in response to <i>Bps</i> was measured by RT-PCR. Error bars represent the SEM. <b>B</b>) Intracellular survival of <i>Bps</i> in BMDM pre-treated with 100 µM nor-NOHA for 30 minutes and <b>C</b>) corresponding nitrite production by <i>Bps</i>-infected cells in the presence or absence of nor-NOHA. The data represent biological triplicates per time point. Error bars represent the SEM. Statistical significance was determined using a two way ANOVA with Bonferroni post-test. *** p<0.001. Data is representative of two independent experiments.</p

Arg2 is expressed in the lungs of <i>B.</i> pseudomallei-infected mice and decreases upon COX-2 inhibition.

<p>Arg1 and Arg2 expression was examined by Western blot in lung homogenates of uninfected and <i>Bps</i>-infected mice (n = 3 per group) treated with NS398 or mock control. Mouse liver extract and mouse Arg2-transfected 293T cell lysate were used as positive controls for Arg1 and Arg2 respectively. β-actin was used as a loading control and for normalization in densitometry analysis using the ImageJ program: <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>. Statistical significance was determined using one-way ANOVA with Bonferroni post test. *** p<0.001.</p

COX-2 inhibition provides significant protection against lethal pulmonary melioidosis.

<p>BALB/c mice (n = 8 per group) were infected with 3×10³ cfu (4 LD₅₀) of <i>Bps</i> 1026b intranasally. Three h post-exposure, mice were administered 15 mg/kg of COX-2 inhibitor (NS398) or DMSO (Mock treatment) intraperitoneally, then again daily for two consecutive days. Survival was monitored for 10 days. Statistical significance was determined using Kaplan Meier analysis. p<0.0001. Data is representative of two independent bacterial challenge experiments.</p