Malaria over-diagnosis in Cameroon: diagnostic accuracy of Fluorescence and Staining Technologies (FAST) Malaria Stain and LED microscopy versus Giemsa and bright field microscopy validated by polymerase chain reaction.

BACKGROUND: Malaria is a major world health issue and its continued burden is due, in part, to difficulties in the diagnosis of the illness. The World Health Organization recommends confirmatory testing using microscopy-based techniques or rapid diagnostic tests (RDT) for all cases of suspected malaria. In regions where Plasmodium species are indigenous, there are multiple etiologies of fever leading to misdiagnoses, especially in populations where HIV is prevalent and children. To determine the frequency of malaria infection in febrile patients over an 8-month period at the Regional Hospital in Bamenda, Cameroon, we evaluated the clinical efficacy of the Flourescence and Staining Technology (FAST) Malaria stain and ParaLens AdvanceTM microscopy system (FM) and compared it with conventional bright field microscopy and Giemsa stain (GS). METHODS: Peripheral blood samples from 522 patients with a clinical diagnosis of "suspected malaria" were evaluated using GS and FM methods. A nested PCR assay was the gold standard to compare the two methods. PCR positivity, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined. RESULTS: Four hundred ninety nine samples were included in the final analysis. Of these, 30 were positive via PCR (6.01%) with a mean PPV of 19.62% and 27.99% for GS and FM, respectively. The mean NPV was 95.01% and 95.28% for GS and FM, respectively. Sensitivity was 26.67% in both groups and specificity was 92.78% and 96.21% for GS and FM, respectively. An increased level of diagnostic discrepancy was observed between technicians based upon skill level using GS, which was not seen with FM. CONCLUSIONS: The frequency of malarial infections confirmed via PCR among patients presenting with fever and other symptoms of malaria was dramatically lower than that anticipated based upon physicians' clinical suspicions. A correlation between technician skill and accuracy of malaria diagnosis using GS was observed that was less pronounced using FM. Additionally, FM increased the specificity and improved the PPV, suggesting this relatively low cost approach could be useful in resource-limited environments. Anecdotally, physicians were reluctant to not treat all patients symptomatically before results were known and in spite of a negative microscopic diagnosis, highlighting the need for further physician education to avoid this practice of overtreatment. A larger study in an area with a known high prevalence is being planned to compare the two microscopy methods against available RDTs.

Activation of the extrinsic apoptotic pathway by TNF-alpha in human salivary gland (HSG) cells in vitro, suggests a role for the TNF receptor (TNF-R) and intercellular adhesion molecule-1 (ICAM-1) in Sjögren's syndrome-associated autoimmune sialadenitis.

UNLABELLED: Studies suggest that apoptosis plays a major role in destruction of salivary glands in Sjögren's syndrome. We hypothesise that apoptosis results in the exposure of cryptic T cell epitopes and an autoimmune response which is pathway-specific. OBJECTIVE: To activate the extrinsic or intrinsic apoptotic pathway to examine morphology, adhesion molecules, markers of antigen processing, and autoantigens on apoptotic bodies in vitro. METHODS: Tumor necrosis factor-alpha (TNF-alpha) or staurosporine, a protein kinase inhibitor, was used to trigger the extrinsic or intrinsic apoptotic pathway in a Human Salivary Gland cell line (HSG), in vitro. Activated genes were profiled by cDNA array. Apoptotic bodies were visualised using light and SEM. Proteins were evaluated by immunofluorescence and confirmed by functional binding assays with Jurkat lymphocytes. RESULTS: TNF-alpha-triggered extrinsic apoptosis resulted in "sticky" aggregated, apoptotic bodies which displayed cleaved alpha-fodrin autoantigen. In contrast, intrinsic apoptosis induced by staurosporine, resulted in dispersed cell blebs which were alpha-fodrin-negative. cDNA arrays revealed that TNF-alpha, but not staurosporine, upregulated transcriptional expression of Intercellular Adhesion Molecule-1 (ICAM-1) and Macrophage Inflammatory Protein-3 (CCL20). CONCLUSION: The apoptotic pathway controls morphological, structural and functional properties of apoptotic bodies. Collectively, TNF-alpha-dependent activation of the extrinsic apoptotic pathway leads to upregulation of ICAM-1 and CCL20 in HSG in vitro. This suggests that pathogenesis in Sjögren's syndrome may involve a TNF-controlled cross-talk between apoptotic ductal and CCL20-attracted dendritic cells via the CD137/CD137L signalling pathway.

Immunotoxicity of perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator-activated receptor alpha.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are environmentally widespread and persistent chemicals with multiple toxicities reported in experimental animals and humans. These compounds can trigger biological activity by activating the alpha isotype of peroxisome proliferator-activated receptors (PPARs), ligand-activated transcription factors that regulate gene expression; however, some biological effects may occur independently of the receptor. Activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) modulates lipid and glucose homeostasis, cell proliferation and differentiation, and inflammation. Reported immunomodulation in experimental animals exposed to PFOA and PFOS has included altered inflammatory responses, production of cytokines and other proteins, reduced lymphoid organ weights, and altered antibody synthesis. Mounting experimental animal evidence suggests PPARalpha independence of some immune effects. This evidence originates primarily from studies with PPARalpha knockout models exposed to PFOA that demonstrate hepatic peroxisome proliferation, reduced lymphoid organ weights, and altered antibody synthesis. As human PPARalpha expression is significantly less than that of rodents, potential PPARalpha independence indicates that future research must explore mechanisms of action of these compounds, including PPARalpha-dependent and -independent pathways. This multiauthored review contains brief descriptions of current and recently published work exploring immunomodulation by PFOA and PFOS, as well as a short overview of other PPARalpha ligands of therapeutic and environmental interest.

Gamma-synuclein as a marker of retinal ganglion cells.

PURPOSE: Previous studies have described gamma-synuclein as a protein highly expressed in retinal ganglion cells (RGCs), and a loss of RGCs correlates with a downregulation of gamma-synuclein gene expression in glaucoma. Here we asked whether gamma-synuclein expression in the retina can be considered a specific marker of RGCs. METHODS: gamma-Synuclein expression was examined with immunohistochemistry in retinal sections from normal and glaucomatous human eyes. Primary cultures of RGCs from Sprague-Dawley rats purified by sequential immunopanning using a monoclonal antibody to Thy1-1, cultures of A7 immortalized optic nerve astrocytes from newborn rats, and the immortalized RGC-5 cell line were studied using immunofluorescence and quantitative RT-PCR. RESULTS: gamma-Synuclein was highly expressed in RGCs in the human retina and was localized in cytoplasm adjacent to the RGC nuclear marker, Brn-3a. Axons of RGCs were immunopositive for gamma-synuclein in the nerve fiber layer (NFL), the lamina cribrosa and the retrobulbar optic nerve. In the optic nerve of glaucoma patients, axon swellings were likewise immunopositive, whereas in the retina of patients with retinoblastoma, NFL staining appeared reduced. In primary rat RGCs and in immortalized RGC-5 cultures, gamma-synuclein was localized predominantly in the perinuclear area and in cell processes. Among rat retinal cells in culture, all Brn-3a positive cells were stained with a gamma-synuclein antibody; rare gamma-synuclein-positive cells were not stained by the Brn-3a antibody. CONCLUSIONS: gamma-Synuclein is selectively and abundantly expressed in human RGCs in vivo, primary rat RGCs in vitro, and immortalized RGC-5 cells. In pathology, gamma-synuclein abundance may vary between RGC somas and axons. Coincident Brn-3a and gamma-synuclein expression suggests that strong gamma-synuclein expression can be considered a marker of RGCs. Future translational approaches might include using a gamma-synuclein promoter for the specific delivery of siRNA or therapeutic proteins to RGCs.

Phthalate-Induced Liver Protection against Deleterious Effects of the Th1 Response: A Potentially Serious Health Hazard.

Infection with Mycobacterium tuberculosis (TB) induces pulmonary immunopathology mediated by classical Th1 type of acquired immunity with hepatic involvement in up to 80% of disseminated cases. Since PPAR agonists cause immune responses characterized by a decrease in the secretion of Th1 cytokines, we investigated the impact of activating these receptors on hepatic pathology associated with a well-characterized model of Th1-type pulmonary response. Male Fischer 344 rats were either maintained on a drug-free diet (groups I and II), or a diet containing diethylhexylphthalate (DEHP), a compound transformed in vivo to metabolites known to activate PPARs, for 21 days (groups III and IV). Subsequently, animals were primed with Mycobacterium bovis purified protein derivative (PPD) in a Complete Freund's Adjuvant. Fifteen days later, animals in groups II and IV were challenged with Sepharose 4B beads covalently coupled with PPD, while animals in groups I and III received blank Sepharose beads. Animals with Th1 response (group II) showed a marked structural disruption in the hepatic lobule. Remarkably, these alterations were conspicuously absent in animals which received DEHP (group IV), despite noticeable accumulation of T cells in the periportal triads. Immunostaining and confocal microscopy revealed hepatic accumulation of IFNgamma+ Th1 and IL-4+ Th2 cells in animals from groups II and IV, respectively. Our data suggest a PPARalpha-mediated suppression of the development of a Th1 immune response in the liver, resulting in hepatoprotective effect. However, potentially negative consequences of PPAR activation, such as decreased ability of the immune system to fight infection and interference with the efficacy of vaccines designed to evoke Th1 immune responses, remain to be investigated.