Recurrent leishmaniasis infection isolated in the pleural fluid.

We present a rare case of recurrent leishmaniasis infection in a female in her 80s who re-presented with a pleural effusion. The patient was initially investigated as an outpatient for cytopenia and underwent a bone marrow biopsy which subsequently diagnosed visceral leishmaniasis. Following full treatment, and apparent recovery, she re-presented with pleural effusion, hypoalbuminaemia and cytopenia. Leishmania was eventually isolated in a pleural fluid sample obtained on therapeutic drainage, and she was treated for a recurrence at a tertiary infectious disease unit. This interesting and challenging case demonstrates the importance of suspecting leishmaniasis recurrence in previously treated cases and the diagnostic benefit of pleural fluid analysis in the context of suspected leishmaniasis.

Principal diagnostic features of paediatric foreign body aspiration.

OBJECTIVES: The aim of this study is to identify the most common and important features within the presenting history, clinical examination and chest radiograph that are associated with foreign body (FB) aspiration in the paediatric population, to support rationalised decision making in regards to proceeding with diagnostic bronchoscopy. METHODS: A retrospective notes review was conducted of 70 patients over a 12-year period at our tertiary referral centre. Their presenting history, clinical and radiographic signs were documented and univariate logistic regression model used to calculate odds ratios. RESULTS: The main features identified within our cohort with a positive FB finding at bronchoscopy were history of a cough (OR 5.1, p = 0.008) and radiographic evidence of hyperinflation or air trapping (OR 7.1, p = 0.016). Zero patients with a FB presented with only a positive history in the absence of other clinical or radiological signs. History of a witnessed choking episode neither increased or decreased the likelihood of as aspirated FB (OR 1, p = 0.967). CONCLUSIONS: We have identified two principal features, as described above, which are associated with paediatric FB aspiration. Reliance on a positive clinical history alone, but specifically the history of a witnessed choking episode, did not support the presence of a FB and other associated signs need to be considered in deciding to proceed to bronchoscopy.

The Economic Burden of Heart Failure with Reduced Ejection Fraction: Living Longer but Poorer?

Treatment of heart failure with reduced ejection fraction (HFrEF) has benefitted from a proliferation of new medications and devices. These treatments carry important clinical benefits, but also come with costs relevant to payers, providers, and patients. Patient out-of-pocket costs have been implicated in the avoidance of medical care, nonadherence to medications, and the exacerbation of health care disparities. In the absence of major health care policy and payment redesign, high-quality HFrEF care delivery requires transparent integration of cost considerations into system design, patient-clinician interactions, and medical decision making.

Are we cross-matching too much blood for elective open abdominal aortic aneurysm repair?

OBJECTIVES: This study aims to identify current blood transfusion requirements in elective open abdominal aortic aneurysm repair and to compare this to an existing maximum surgical blood order schedule. METHODS: We retrospectively identified patients who underwent elective open abdominal aortic aneurysm repair over a 40-month period in our institution. Pre-operative number of units cross-matched and the number of units actually transfused were identified. The cross-match to transfusion ratio was then calculated. RESULTS: Blood transfusion at any time post-operatively was required in 23 (48.9%) cases. Patients needing an intra-operative blood transfusion had a median of 2 units. Of the pre-operative cross-matched units (123), only 43 were used, giving a cross-match to transfusion ratio of 2.86. CONCLUSION: Our current maximum surgical blood order schedule is poorly followed and a cross-match to transfusion ratio of 2.86 indicates we are cross-matching too many units for elective open abdominal aortic aneurysm repair. A carefully considered individualised management of blood products, with the requirement of at least a valid group and save sample, may be more appropriate.

In Vitro Generation of Human NK Cells Expressing Chimeric Antigen Receptor Through Differentiation of Gene-Modified Hematopoietic Stem Cells.

NK cells represent a very promising source for adoptive cellular approaches for cancer immunotherapy, and extensive research has been conducted, including clinical trials. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer techniques is very limited. Here we describe two protocols designed to generate mature human NK cells from gene-modified hematopoietic stem cells. These protocols use chimeric antigen receptor as the transgene, but could potentially be modified for the expression any particular transgene in human NK cells.

Cocaine-mediated impact on HIV infection in humanized BLT mice.

Cocaine abuse has been shown to have broad-ranging effects on human immunity. With regards to HIV infection, in vitro studies have shown that cocaine enhances infection of stimulated lymphocytes. Moreover, cohort studies in the pre- and post-HAART era have linked stimulant abuse with increased HIV pathogenesis. The latter data, however, have been undermined by a series of confounding factors underscoring the importance of controlled in vivo models to fully assess the impact of cocaine use and abuse on HIV infection and pathogenesis. Here, we have infected humanized mice with HIV-1 following acute cocaine exposure to assess the impact on infection. Stimulant exposure resulted in increased inflammatory cytokine expression, accelerated HIV infection, while blunting effector function of cytotoxic T lymphocytes. These data demonstrate cocaine's multifactorial impact on HIV infection that extends beyond high-risk behavior.

Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.

We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.

Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4(+) T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+) T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+) T-cells ex vivo. Furthermore, levels of gene-marked CD4(+) T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease.

Toll-like receptor 2 signaling protects mice from tumor development in a mouse model of colitis-induced cancer.

Inflammatory bowel disease (IBD) is a disorder of chronic inflammation with increased susceptibility to colorectal cancer. The etiology of IBD is unclear but thought to result from a dysregulated adaptive and innate immune response to microbial products in a genetically susceptible host. Toll-like receptor (TLR) signaling induced by intestinal commensal bacteria plays a crucial role in maintaining intestinal homeostasis, innate immunity and the enhancement of intestinal epithelial cell (IEC) integrity. However, the role of TLR2 in the development of colorectal cancer has not been studied. We utilized the AOM-DSS model for colitis-associated colorectal cancer (CAC) in wild type (WT) and TLR2(-/-) mice. Colons harvested from WT and TLR2(-/-) mice were used for histopathology, immunohistochemistry, immunofluorescence and cytokine analysis. Mice deficient in TLR2 developed significantly more and larger colorectal tumors than their WT controls. We provide evidence that colonic epithelium of TLR2(-/-) mice have altered immune responses and dysregulated proliferation under steady-state conditions and during colitis, which lead to inflammatory growth signals and predisposition to accelerated neoplastic growth. At the earliest time-points assessed, TLR2(-/-) colons exhibited a significant increase in aberrant crypt foci (ACF), resulting in tumors that developed earlier and grew larger. In addition, the intestinal microenvironment revealed significantly higher levels of IL-6 and IL-17A concomitant with increased phospho-STAT3 within ACF. These observations indicate that in colitis, TLR2 plays a protective role against the development of CAC.

Identification of a novel human MD-2 splice variant that negatively regulates Lipopolysaccharide-induced TLR4 signaling.

Myeloid differentiation factor 2 (MD-2) is a secreted gp that assembles with TLR4 to form a functional signaling receptor for bacterial LPS. In this study, we have identified a novel alternatively spliced isoform of human MD-2, termed MD-2 short (MD-2s), which lacks the region encoded by exon 2 of the MD-2 gene. Similar to MD-2, MD-2s is glycosylated and secreted. MD-2s also interacted with LPS and TLR4, but failed to mediate LPS-induced NF-kappaB activation and IL-8 production. We show that MD-2s is upregulated upon IFN-gamma, IL-6, and TLR4 stimulation and negatively regulates LPS-mediated TLR4 signaling. Furthermore, MD-2s competitively inhibited binding of MD-2 to TLR4. Our study pinpoints a mechanism that may be used to regulate TLR4 activation at the onset of signaling and identifies MD-2s as a potential therapeutic candidate to treat human diseases characterized by an overly exuberant or chronic immune response to LPS.

Involvement of innate and adaptive immunity in a murine model of coronary arteritis mimicking Kawasaki disease.

Kawasaki disease (KD) is the most common cause of acquired cardiac disease and acute vasculitis in children in the developed world. Injection of a cell wall extract isolated from Lactobacillus casei (LCCWE) into mice causes a focal coronary arteritis that histopathologically mimics the coronary lesions observed in KD patients. In this study we used this model to investigate the participation of T cells, B cells, and dendritic cells (DC) in the development of coronary arteritis. RAG1(-/-), B cell(null), and wild-type (WT) mice were injected with a single dose of LCCWE (500 microg/mouse i.p.). None of the RAG1(-/-) mice developed coronary arteritis, whereas 70% of WT and 100% of B cell(null) mice developed coronary lesions, indicating that T cells were required for lesion formation. When splenocytes isolated from LCCWE-treated mice were restimulated with LCCWE, we observed significant IFN-gamma secretion in WT but not in RAG1(-/-) mice. Immunohistochemical staining showed F4/80(+) macrophages, activated MIDC-8(+) myeloid DCs (mDC), plasmacytoid DCs, and colocalization of CD3(+) T cells with mDCs in coronary artery lesions, suggesting an Ag-driven process. T cells but not B cells are required for LCCWE-induced coronary arteritis. Similar to human lesions, the coronary lesions contain macrophages, activated mDCs, and plaslmacytoid DCs all in close proximity to T cells, further strengthening the relevance of this mouse model to the immunopathology of coronary disease in KD. These studies are consistent with the interpretation that macrophages and DCs may collaborate with T cells in the pathological mechanisms of coronary arteritis.

Ubiquitination and de-ubiquitination: role in regulation of signaling by Toll-like receptors.

Signaling by Toll-like receptors (TLRs) has attracted accelerating attention over the past decade because of the central role of TLR signaling in both innate and adaptive immunity. In addition, TLR signaling is now increasingly implicated in a remarkably wide range of diseases that are either caused, or accompanied, by dysregulated inflammation. Much has been learned about the basic signaling framework and participants, as well as how signaling is turned off and fine-tuned. Here, we summarize key aspects of TLR signaling, focusing on interaction with the anti-inflammatory TGF-beta signaling network. We propose that ubiquitination and de-ubiquitination of TLR pathway components may be a mechanism by which predominantly anti-inflammatory input is integrated into the host response to fine-tune inflammation in accordance with the needs of host defenses.