Profile of total IgG, IgG1, IgG2, IgG3 and IgG4 levels in sera of patients with paracoccidioidomycosis: treatment follow-up using Mexo and rPb27 as antigens in an ELISA

The levels of total of IgG, IgG1, IgG2, IgG3 and IgG4 were evaluated in 54 patients with chronic paracoccidioidomycosis (PCM) before, during and after treatment using an enzyme-linked immunosorbent assay with Mexo and recombinant Pb27 (rPb27) as the antigens. Mexo was effective in distinguishing PCM patients from individuals in the negative control group (NC) based on total IgG and rPb27 performed worse than Mexo when these two groups were compared. IgG1, IgG2, IgG3 and IgG4 could not be used to clearly distinguish PCM patients from those in the NC group using either antigen. There was no clear relationship between antibody levels and the period of treatment. The majority of patients presented with decreased antibody levels during treatment, with no statistically significant differences among the different periods of treatment. Only IgG4 presented a negative correlation between its levels and clinical improvement during treatment. In total, 65% of untreated PCM patients showed reactivity against IgG4 when the Mexo antigen was used and this reactivity decreased over the course of treatment. There was a tendency towards decreasing antibody levels during treatment, but these antibody levels did not necessarily clear after the treatment was stopped. Mexo was useful for PCM diagnosis using total IgG; however, more studies are necessary before this antigen can be used in measuring the levels of total IgG and its subclasses for monitoring patients during treatment.

Genetic polymorphism in Taenia solium metacestodes from different Brazilian geographic areas

The aim of the present study is to investigate genetic polymorphisms in Taenia solium metacestodes from different Brazilian geographical areas and to relate them to antibody recognition in serum samples of neurocysticercosis (NC) patients. Metacestodes were obtained from the Distrito Federal (DF), Bahia, Minas Gerais (MG) and São Paulo (SP) regions of Brazil. Samples of human sera from 49 individuals with NC, 68 individuals with other helminthiasis and 40 healthy volunteers were analysed (157 individuals in total). Antigens were prepared and used in enzyme-linked immunosorbent assay and western blotting assays to detect specific immunoglobulin G antibodies. Genetic distances between metacestode populations were analysed using random amplified polymorphic DNA (RAPD) analysis. Our results show that there was a higher frequency of reactivity in the DF region in the sera from NC patients (p < 0.05), while discrimination between active and inactive NC was seen only in extracts from the MG and SP regions (p < 0.05). Using RAPD, the sample from the DF region presented a greater increase compared to the other regions. A relationship between genetic polymorphisms among T. solium metacestodes from different areas in Brazil and the differences in antibody detection in patients with NC were established.

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 µg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85% and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.

Parvovirus B19 seroconversion in a cohort of human immunodeficiency virus-infected patients

Erythrovirus B19 (B19V) infection may cause red cell aplasia in patients infected with human immunodeficiency virus (HIV). The introduction of highly active antiretroviral therapy (HAART) has improved the immune function of these patients by modifying the course of B19V infection. The purpose of this study was to estimate the frequency of B19 seroconversion in a cohort of HIV-infected patients and evaluate the occurrence of B19V-related anaemia during the seroconversion period. Adult HIV-infected patients were studied at a public hospital in Niterói, state of Rio de Janeiro, Brazil. IgG and IgM antibodies against B19V were detected by an enzyme-linked immunosorbent assay and B19 viraemia was assayed by polymerase chain reaction. Medical records were reviewed for any clinical evaluation of anaemia. Seroconversion was detected in 31.8% of the 88 individuals who began the study as anti-B19V IgG-negative. No clinical manifestations of B19V infection were detected during the period of seroconversion. Patients who seroconverted were 5.40 times more likely to have anaemia than those who did not [odds ratio 5.40 (95% confidence interval: 1.33-22.93)]. Anaemia was detected in eight patients. All patients recovered from anaemia by either beginning or continuing HAART, without requiring blood transfusions. In the HAART era, B19V infection may only be associated with a course of disease characterised by less severe chronic anaemia. This milder course of B19V-associated disease is likely due to the increased immune function of HAART-treated patients.

The combination of three faecal parasitological methods to improve the diagnosis of schistosomiasis mansoni in a low endemic setting in the state of Ceará, Brazil

Laboratory diagnosis of intestinal schistosomiasis mansoni can be accomplished through various methods of stool examination to detect parasites, ranging from the most classic tests (Kato-Katz) to several methods that are still undergoing validation. This study was conducted to assess two new parasite identification methods for diagnosing schistosomiasis mansoni in residents of a low endemic area in the municipality of Maranguape, in the state of Ceará, Brazil using the Kato-Katz method as a reference and serology (enzyme-linked immunosorbent assay) for the screening of patients. The Kato-Katz, the saline gradient method and the Helmintex® method parasite identification methods were employed only in subjects who exhibited positive serologic tests. The test results were then analysed and treatment of positive individuals was subsequently performed. After comparing the test results, we observed that the saline gradient method and the Helmintex® method were more effective in diagnosing schistosomiasis mansoni in the study area compared with the Kato-Katz method.

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.

Orodigital pemphigus vulgaris : a pathogenic role of anti-desmoglein-3 autoantibodies in pemphigus paronychia?

Paronychia is a rare complication of pemphigus vulgaris (PV), and the immunological profile of patients with digital disease has not been assessed so far. We report 2 cases of PV with oral mucosa and periungual involvement, who had high titers of anti- desmoglein (Dsg)-3 circulating antibodies. These observations raise the possibility that expression of Dsg-1 and Dsg-3 in distinct areas of periungual skin is disease specific and that anti-Dsg-3 antibodies alone may have an as yet unrecognized importance for the development of paronychia in PV.

Antigenic proteins involved in occupational rhinitis and asthma caused by obeche wood (Triplochiton scleroxylon).

BACKGROUND Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. OBJECTIVE To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma MATERIALS AND METHODS An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. RESULTS Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA-), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases. CONCLUSIONS Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens.

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

In vitro methods for diagnosing nonimmediate hypersensitivity reactions to drugs.

Nonimmediate drug hypersensitivity reactions (DHRs) are difficult to manage in daily clinical practice, mainly owing to their heterogeneous clinical manifestations and the lack of selective biological markers. In vitro methods are necessaryto establish a diagnosis, especially given the low sensitivity of skin tests and the inherent risks of drug provocation testing. In vitro evaluation of nonimmediate DHRs must include approaches that can be applied during the different phases of the reaction. During the acute phase, monitoring markers in both skin and peripheral blood helps to discriminate between immediate and nonimmediate DHRs with cutaneous responses and to distinguish between reactions that, although they present similar clinical symptoms, are produced by different immunological mechanisms and therefore have a different treatment and prognosis. During the resolution phase, in vitro testing is used to detect the response of T cells to drug stimulation; however, this approach has certain limitations, such as the lack of validated studies assessing sensitivity. Moreover, in vitro tests indicate an immune response that is not always related to a DHR. In this review, members of the Immunology and Drug Allergy Committee of the Spanish Society of Allergy and Clinical Immunology (SEAIC) provide an overview of the most widely used in vitro tests for evaluating nonimmediate DHRs.

Cerebrospinal fluid biomarkers of central nervous system dysfunction in HIV-infected patients

Background :¦In addition to opportunistic infections of the central nervous system (CNS), which are due to immunosuppression related to HIV, the latter virus, itself, can cause neuropathological abnormalities which are located mainly in the basal ganglia and are characterized by microglial giant cells, reactive astrocytosis and perivascular monocytes. This HIV encephalopathy is characterized, clinically, by psycho-motor slowing, memory loss, difficulties in complex tasks requiring executive functions, as well as motor disorders .These cognitive deficits are grouped under the acronym of HIV-associated neurocognitive disorders (HAND). In fact, HANDs are subdivided in three groups in accordance with the severity of the cognitive impairment: Asymptomatic Neurocognitive Impairment (ANI), Mild/moderate Neurocognitive Disorders (MND) and HIV Associated Dementia (HAD).¦While the incidence of HAD has significantly decreased in the era of combined antiretrobiral therapy (cART), the prevalence of milder forms of HIV-associated neurocognitive disorders HAND seem to have increased. There are many potential reasons to explain this state of facts.¦An important question is to understand how soon the brain may be affected by HIV. Since performing a biopsy in these patients is not an issue, the study of the CSF represents the best available way to look at putative biomarkers of inflammation/neurodegeneration in the CNS. Here, we wanted to examined the putative usefulness of different biomarkers as early indicators of anti-retroviral failure at the level of the CNS. We chose to study the CSF levels of:¦Amyloid-β 1-42 (Aβ42), Tau total (tTau), phosphorylated Tau (pTau), Neopterin and S100-β.¦Indeed, these molecules are representative biomarkers of the major cells of the CNS, i.e. neurons,¦macrophages/microglia and astrocytes.¦To examine how sensitive were these CSF biomarkers to indicate CNS insults caused by HIV, we proposed to take advantage of the MOST (Monotherapy Switzerland/Thailand study) study, recently published in AIDS. Thus, we collaborated with Prof. Pietro Vernazza in St-Gall. In MOST study, monotherapy (MT) consisting in ritonavir-boosted lopinavir (LPV/r) was compared to continuous conventional antiretroviral therapy including several molecules, hereafter referred as CT¦Methods :We tested 61 cerebrospinal fluid (CSF) samples from 52 patients enrolled in MOST, including 34 CSF samples of CT and 27 of MT (mean duration on MT: 47+20 weeks) in patients who maintained full VL suppression in blood (<50cps/ml). Using enzyme-linked immunosorbent assay (ELISA), we determined the CSF concentration of S100-beta (astrocytosis), neopterin (microglia, inflammation), total Tau (tTau), phosphorylated Tau (pTau), and amyloid-beta 1-42 (Abeta), the latter three markers indicating neuronal damages. The CSF samples of 37 HIV-negative patients with Alzheimer dementia (AD) served as controls. Results are expressed in pg/ml and reported as median ± interquartile range. Mann Whitney-U test was used to compare the results of a given biomarker between two groups and the Fisher test to compare frequencies.¦Results: We found a higher concentration of S100-beta (570±1132) and neopterin (2.5±2.9) in the CSF of MT versus CT (0±532, p=0.002 and 1.2±2.5, p=0.058, respectively). A cutoff of 940 pg/ml for S100-beta allowed to discriminate MT (11 above versus 16 below) from CT (1 vs 33, p=0.0003). At a lesser extent, a cutoff of 11 pg/ml for neopterin separated MT (4 above versus 23) from CT (0 vs 34, p=0.034) (Figure).¦In AD, tTau was higher (270±414) and Abeta lower (234±328) than in CT (150±153, p=0.0078, and 466±489, p=0.007, respectively). Such as for CT, Abeta was lower in AD than in MT (390±412, p=0.01). However, contrasting with CT, the levels of tTau were not different between AD and MT (199±177, p=0.11). S100b (173±214; p=0.0006) and neopterin (1.1±0.9; p=0.0014) were lower in AD than MT.¦Conclusions: Despite full VL-suppression in blood, HIV monotherapy is sufficient to trigger inflammation and, especially, astrocytosis. CSF markers of patients on CT have the same profile as reported for healthy subjects, suggesting that CT permits a good control of HIV in the brain. Finally, the levels of tTau, which are relatively similar between AD and MT patients, suggest that neurons are damaged during monotherapy.

Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll-like receptor 4 agonist LPS.

Broad-spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti- and proinflammatory effects on macrophages in vitro. We report here that several broad-spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS-induced mRNA expression of the proinflammatory mediators Edn-1, Ccl-7/MCP-3, and Il-12p40 but amplified the expression of the proatherogenic factors Cox-2 and Pai-1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS-stimulated TEPM and HMDM. The pro- and anti-inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1-selective inhibitor, MS-275, retained proinflammatory effects (amplification of LPS-induced expression of Cox-2 and Pai-1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6-selective inhibitor) retained anti-inflammatory but not proinflammatory properties. Despite this, HDAC6(-/-) macrophages showed normal LPS-induced expression of HDAC-dependent inflammatory genes, arguing that the anti-inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Rituximab in treatment-resistant CIDP with antibodies against paranodal proteins.

OBJECTIVE To describe the response to rituximab in patients with treatment-resistant chronic inflammatory demyelinating polyneuropathy (CIDP) with antibodies against paranodal proteins and correlate the response with autoantibody titers. METHODS Patients with CIDP and IgG4 anti-contactin-1 (CNTN1) or anti-neurofascin-155 (NF155) antibodies who were resistant to IV immunoglobulin and corticosteroids were treated with rituximab and followed prospectively. Immunocytochemistry was used to detect anti-CNTN1 and anti-NF155 antibodies and ELISA with human recombinant CNTN1 and NF155 proteins was used to determine antibody titers. RESULTS Two patients had a marked improvement; another patient improved slightly after 10 years of stable, severe disease; and the fourth patient had an ischemic stroke unrelated to treatment and was lost to follow-up. Autoantibodies decreased in all patients after rituximab treatment. CONCLUSIONS Rituximab treatment is an option for patients with CIDP with IgG4 anti-CNTN1/NF155 antibodies who are resistant to conventional therapies. CLASSIFICATION OF EVIDENCE This study provides Class IV evidence that rituximab is effective for patients with treatment-resistant CIDP with IgG4 anti-CNTN1 or anti-NF155 antibodies.

Neutrophil defensins: their possible role in allergic asthma.

BACKGROUND Neutrophil defensins, originally identified as broad-spectrum antimicrobial peptides, have been implicated in the regulation of inflammatory and immunological processes. OBJECTIVES To investigate whether the in vitro challenge of neutrophils from patients with bronchial asthma with allergens stimulated the release of alpha-defensins and whether levels released were dependent on lung infections. METHOD The neutrophils were cultivated with different agonists and the concentration of alpha-defensin in cell-free supernatant was measured with enzyme-linked immunosorbent assay (ELISA). RESULTS Neutrophils from allergic patients released alpha-defensins via an allergen-dependent mechanism. Our results indicate that the in vitro activation of neutrophils is highly allergen-specific. In this context, allergens other than those which produced clinical symptoms did not elicit alpha-defensin release, and allergens had no effect on neutrophils from healthy donors. However, neutrophils from both allergic patients and healthy controls were able to release alpha-defensins upon treatment with PMA. In the allergen-stimulated neutrophils, cells from asthmatic patients stimulated with a sensitizing allergen showed a significantly higher production of alpha-defensin under respiratory tract infection than cells from the same patients without such an infection. CONCLUSION Neutrophils from allergic patients release alpha-defensins via an allergen-dependent mechanism.