Immunopathology in ocular toxoplasmosis: facts and clues

Although parasite-mediated host cell lysis is deemed to be an important cause of tissue destruction in ocular toxoplasmosis (OT), the severity of the disease is probably correlated with hypersensitivity and inflammation. Notwithstanding, the mechanisms that regulate the inflammatory process in recurrent OT are poorly understood. Recent evidence has identified interleukin (IL) 17 as a marker for disease severity. The ocular and cerebral presence of this cytokine is generally associated with the induction of autoimmune responses in the brain and the eye. Indeed, there are indications that autoimmunity may contribute to clinical variability in the activity of OT. IL-23, which induces the proliferation of IL-17-producing cells and IL-27, which is a counterplayer to IL-17, may regulate T(H)-1-cell-mediated responses in OT. The importance of these cytokines in experimental models of uveitis and encephalitis has been recently reported. CD25(+) regulatory T-cells may control the local inflammatory response and protect the host against collateral inflammatory tissue damage. The responses of these cells to OT may be suitably tailored to cope with either an acquired or a congenital aetiology. Knowledge relating to immunoreactivity in OT has grown impressively during the past few years. Its characteristic and variable features have been identified and the potential relevance of autoimmunity has been assessed. In light of this knowledge, potential future treatment options have been considered.

Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1·8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Nosocomial bacteremia due to an as yet unclassified acinetobacter genomic species 17-like strain.

We describe a case of bacteremia due to an as yet unclassified Acinetobacter genomic species 17-like strain. The recognition of this microorganism as non-Acinetobacter baumannii may have important epidemiological implications, as it relieves the hospital of the implementation of barrier precautions for patients infected or colonized as may be necessary with a multiresistant A. baumannii epidemic.

Serosurvey Study of Toscana Virus in Domestic Animals, Granada, Spain

Toscana virus (TOSV) is transmitted by infected sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis in humans. It remains unclear if there are animal reservoirs able to maintain the virus through the cold months of the year, when the vector is not circulating. From May to October of 2006 and 2007, we conducted a serosurvey study on domestic animals from Granada province (southern Spain). TOSV was investigated in 1186 serum samples from horses, goats, pigs, cats, dogs, sheep, and cows by serology (indirect fluorescence assay), viral culture, and RT-polymerase chain reaction. Specific anti-TOSV antibodies were detected in 429 (36.2%) serum samples. The highest seropositivity rates were observed in cats (59.6%) and dogs (48.3%). These results suggest that an important percentage of the domestic animals have been infected by TOSV. Significantly different seroprevalence rates were detected in goats among distinct geographical areas. All viral cultures were negative. TOSV was detected by RT-polymerase chain reaction in only one serum sample from a goat. Thus, the studied animals do not seem to act as reservoirs for TOSV; otherwise, they could be amplifying hosts for the virus.

Canakinumab for acute gouty arthritis in patients with limited treatment options: results from two randomised, multicentre, active-controlled, double-blind trials and their initial extensions.

OBJECTIVES: Gouty arthritis patients for whom non-steroidal anti-inflammatory drugs and colchicine are inappropriate have limited treatment options. Canakinumab, an anti-interleukin-1β monoclonal antibody, may be an option for such patients. The authors assessed the efficacy/safety of one dose of canakinumab 150 mg (n=230) or triamcinolone acetonide (TA) 40 mg (n=226) at baseline and upon a new flare in frequently flaring patients contraindicated for, intolerant of, or unresponsive to non-steroidal anti-inflammatory drugs and/or colchicine. Core study co-primary endpoints were pain intensity 72 h postdose (0-100 mm visual analogue scale and time to first new flare. METHODS: Two 12-week randomised, multicentre, active-controlled, double-blind, parallel-group core studies with double-blind 12-week extensions (response in acute flare and in prevention of episodes of re-flare in gout (β-RELIEVED and β-RELIEVED-II)). RESULTS: 82.6% patients had comorbidities. Mean 72-h visual analogue scale pain score was lower with canakinumab (25.0 mm vs 35.7 mm; difference, -10.7 mm; 95% CI -15.4 to -6.0; p<0.0001), with significantly less physician-assessed tenderness and swelling (ORs=2.16 and 2.74; both p≤0.01) versus TA. Canakinumab significantly delayed time to first new flare, reduced the risk of new flares by 62% versus TA (HR: 0.38; 95% CI 0.26 to 0.57) in the core studies and by 56% (HR: 0.44; 95% CI 0.32 to 0.60; both p≤0.0001) over the entire 24-week period, and decreased median C-reactive protein levels (p≤0.0001 at 72 h and 7 days). Over the 24-week period, adverse events were reported in 66.2% (canakinumab) and 52.8% (TA) and serious adverse events were reported in 8.0% (canakinumab) and 3.5% (TA) of patients. Adverse events reported more frequently with canakinumab included infections, low neutrophil count and low platelet count. CONCLUSION: Canakinumab provided significant pain and inflammation relief and reduced the risk of new flares in these patients with acute gouty arthritis.

HBV markers in haemodialysis Brazilian patients: a prospective 12-month follow-up

The aim of this study was to determine the prevalence and the incidence of hepatitis B virus (HBV) among haemodialysis (HD) subjects and to evaluate whether testing for serological markers at the time of admission is suitable for HBV screening in this population. One hundred twenty-three patients belonging to two HD centres from São Paulo, Brazil, were tested prospectively. HBV DNA was detected by polymerase chain reaction (PCR) in each of the prospective subjects (n = 123) during one year. Additionally, all samples (n = 1,476) were analysed for HBV serological markers. The prevalence of hepatitis B core antibody (anti-HBc), hepatitis B surface antigen (HBsAg) and HBV DNA were 34.1%, 15.4% and 8.1%, respectively, while the incidence was null. Fluctuation in HBV serology was observed in one patient. Only 37.8% (17/45) of cases responded to the HBV vaccine. Our results suggest that employing more than one HBV marker and repeated follow-up evaluations may improve HBV screening in HD units.

Antibody reactivity against potato apyrase, a protein that shares epitopes with Schistosoma mansoni ATP diphosphohydrolase isoforms, in acute and chronically infected mice, after chemotherapy and reinfection

Schistosoma mansoni ATP diphosphohydrolase isoforms and potato apyrase share conserved epitopes. By enzyme-linked immunosorbent assays, elevated levels of IgM, IgG2a and IgG1 antibody reactivity against potato apyrase were observed in S. mansoni-infected BALB/c mice during the acute phase of infection, while only IgM and IgG1 antibody reactivity levels maintained elevated during the chronic phase of infection. Antibody reactivity against potato apyrase was monitored over an 11-month period in chronically-infected mice treated with oxamniquine. Eleven months later, the level of seropositive IgM decreased significantly (~30%) compared to the level found in untreated, infected mice. The level of seropositive IgG1 decreased significantly four months after treatment (MAT) (61%) and remained at this level even after 11 months. The IgG2a reactivity against potato apyrase, although unchanged during chronic phase to 11 MAT, appeared elevated again in re-infected mice suggesting a response similar to that found during the acute phase. BALB/c mouse polyclonal anti-potato apyrase IgG reacted with soluble egg antigens probably due to the recognition of parasite ATP diphosphohydrolase. This study, for the first time, showed that the IgG2a antibody from S. mansoni-infected BALB mice cross-reacts with potato apyrase and the level of IgG2a in infected mice differentiates disease phases. The results also suggest that different conserved-epitopes contribute to the immune response in schistosomiasis.

Inflammatory Markers and Incident Heart Failure Risk in Older Adults: The Health, Aging, and Body Composition Study

Background: Inflammation is associated with heart failure (HF) risk factors and also directly affects myocardial function. However, the association between inflammation and HF risk in older adults has not been adequately evaluated. Methods: The association of baseline serum concentrations of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF- ), and C-reactive protein (CRP) with incident HF was assessed with Cox proportional hazards models among 2610 older persons without prevalent HF enrolled in the Health, Aging, and Body Composition (Health ABC) Study (age, 73.6±2.9 years; 48.3% men; 59.6% white). Results: Median (interquartile range) baseline concentrations of IL-6, TNF- , and CRP were 1.80 (1.23, 2.76) pg/mL, 3.14 (2.41, 4.06) pg/mL, and 1.64 (0.99, 3.04) µg/mL, respectively. On follow-up (median, 9.4 years), 311 participants (11.9%) developed HF. In models controlling for clinical predictors of HF and incident coronary heart disease, doubling of IL-6, TNF- , and CRP concentrations was associated with 34% (95% CI, 18 -52%; P<.001), 33% (95% CI, 9 - 63%; P=.006), and 13% (95% CI, 3-24%; P=.01) increase in HF risk, respectively. In models including all 3 markers, IL-6 and TNF- , but not CRP, remained significant. Findings were similar across sex and race. Post-HF ejection fraction (EF) was available in 239 (76.8%) cases. When only cases with preserved EF were considered (n=105), IL-6 (HR per doubling, 1.57; 95% CI, 1.28 -1.94; P<.001), TNF- (HR per doubling, 1.59; 95% CI, 1.12-2.26; P=.01), and CRP (HR per doubling, 1.23; 95% CI, 1.05-1.44; P=.01) were all associated with HF risk in adjusted models. In contrast, when only cases with reduced EF (n=134) were considered, only IL-6 attained marginal significance in adjusted models (HR per doubling, 1.20; 95% CI, 0.99 -1.46; P=.06). Participants with 2 or 3 markers above median had pronounced HF risk in adjusted models (HR, 1.66; 95% CI, 1.12-2.46; P=.01; and HR, 1.76; 95% CI, 1.16 -2.65; P=.007, respectively). Addition of IL-6 to the clinical Health ABC HF model improved discrimination (C index from 0.717 to 0.734; P=.001) and fit (decreased Bayes information criterion by 17.8; P<.001). Conclusions: Inflammatory markers are associated with HF risk among older adults and may improve HF risk stratification.

Th1 and Th17 hypercytokinemia as early host response signature in severe pandemic influenza.

INTRODUCTION Human host immune response following infection with the new variant of A/H1N1 pandemic influenza virus (nvH1N1) is poorly understood. We utilize here systemic cytokine and antibody levels in evaluating differences in early immune response in both mild and severe patients infected with nvH1N1. METHODS We profiled 29 cytokines and chemokines and evaluated the haemagglutination inhibition activity as quantitative and qualitative measurements of host immune responses in serum obtained during the first five days after symptoms onset, in two cohorts of nvH1N1 infected patients. Severe patients required hospitalization (n = 20), due to respiratory insufficiency (10 of them were admitted to the intensive care unit), while mild patients had exclusively flu-like symptoms (n = 15). A group of healthy donors was included as control (n = 15). Differences in levels of mediators between groups were assessed by using the non parametric U-Mann Whitney test. Association between variables was determined by calculating the Spearman correlation coefficient. Viral load was performed in serum by using real-time PCR targeting the neuraminidase gene. RESULTS Increased levels of innate-immunity mediators (IP-10, MCP-1, MIP-1beta), and the absence of anti-nvH1N1 antibodies, characterized the early response to nvH1N1 infection in both hospitalized and mild patients. High systemic levels of type-II interferon (IFN-gamma) and also of a group of mediators involved in the development of T-helper 17 (IL-8, IL-9, IL-17, IL-6) and T-helper 1 (TNF-alpha, IL-15, IL-12p70) responses were exclusively found in hospitalized patients. IL-15, IL-12p70, IL-6 constituted a hallmark of critical illness in our study. A significant inverse association was found between IL-6, IL-8 and PaO2 in critical patients. CONCLUSIONS While infection with the nvH1N1 induces a typical innate response in both mild and severe patients, severe disease with respiratory involvement is characterized by early secretion of Th17 and Th1 cytokines usually associated with cell mediated immunity but also commonly linked to the pathogenesis of autoimmune/inflammatory diseases. The exact role of Th1 and Th17 mediators in the evolution of nvH1N1 mild and severe disease merits further investigation as to the detrimental or beneficial role these cytokines play in severe illness.

Activation of peroxisome proliferator-activated receptor-β/-δ (PPAR-β/-δ) ameliorates insulin signaling and reduces SOCS3 levels by inhibiting STAT3 in interleukin-6-stimulated adipocytes.

OBJECTIVE It has been suggested that interleukin (IL)-6 is one of the mediators linking obesity-derived chronic inflammation with insulin resistance through activation of STAT3, with subsequent upregulation of suppressor of cytokine signaling 3 (SOCS3). We evaluated whether peroxisome proliferator-activated receptor (PPAR)-β/-δ prevented activation of the IL-6-STAT3-SOCS3 pathway and insulin resistance in adipocytes. RESEARCH DESIGN AND METHODS First, we observed that the PPAR-β/-δ agonist GW501516 prevented both IL-6-dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes. In addition, this drug treatment abolished IL-6-induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6-induced STAT3 phosphorylation on Tyr(705) and Ser(727) residues in vitro and in vivo. Moreover, GW501516 prevented IL-6-dependent induction of extracellular signal-related kinase (ERK)1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPAR-β/-δ-null mice, STAT3 phosphorylation (Tyr(705) and Ser(727)), STAT3 DNA-binding activity, and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPAR-β/-δ-null mice compared with wild-type mice. CONCLUSIONS Collectively, our findings indicate that PPAR-β/-δ activation prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of cytokine-induced insulin resistance in adipocytes.

Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus.

An indirect immunofluorescence antibody test employing whole eggs as the antigen for the diagnosis of abdominal angiostrongyliasis

Abdominal angiostrongyliasis is a potentially fatal zoonotic disease with a broad geographical distribution throughout Central and South America. This study assessed the performance of Angiostrongylus costaricensis eggs as the antigen in an indirect immunofluorescence assay for the determination of parasite-specific IgG and IgG1 antibodies. For prevalence studies, an IgG antibody titre > 16 was identified as the diagnostic threshold with the best performance, providing 93.7% sensitivity and 84.6% specificity. Cross reactivity was evaluated with 65 additional samples from patients with other known parasitic infections. Cross reactivity was observed only in samples from individuals infected with Strongyloides stercoralis. For clinical diagnosis, we recommend the determination of IgG only as a screening test. IgG1 determination may be used to increase the specificity of the results for patients with a positive screening test.

The tumor necrosis factor-related apoptosis-inducing ligand receptors TRAIL-R1 and TRAIL-R2 have distinct cross-linking requirements for initiation of apoptosis and are non-redundant in JNK activation.

Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-kappaB in cultured cells. In this study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-kappaB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-kappaB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-kappaB activation and apoptosis, whereas TRAIL-R2 signals NF-kappaB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL.