João Paulo Diniz Pimentel

Thrombocytopenia in Plasmodium vivax Malaria Is Related to Platelets Phagocytosis

Background Although thrombocytopenia is a hematological disorder commonly reported in malarial patients, its mechanisms are still poorly understood, with only a few studies focusing on the role of platelets phagocytosis. Methods and Findings Thirty-five malaria vivax patients and eight healthy volunteers (HV) were enrolled in the study. Among vivax malaria patients, thrombocytopenia (<150,000 platelets/µL) was found in 62.9% (22/35). Mean platelet volume (MPV) was higher in thrombocytopenic patients as compared to non- thrombocytopenic patients (p = 0.017) and a negative correlation was found between platelet count and MPV (r = −0.483; p = 0.003). Platelets from HV or patients were labeled with 5-chloromethyl fluorescein diacetate (CMFDA), incubated with human monocytic cell line (THP-1) and platelet phagocytosis index was analyzed by flow cytometry. The phagocytosis index was higher in thrombocytopenic patients compared to non-thrombocytopenic patients (p = 0.042) and HV (p = 0.048). A negative correlation was observed between platelet count and phagocytosis index (r = −0.402; p = 0.016). Platelet activation was assessed measuring the expression of P-selectin (CD62-P) in platelets’ surface by flow cytometry. No significant difference was found in the expression of P-selectin between thrombocytopenic patients and HV (p = 0.092). After evaluating the cytokine profile (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17) in the patients’ sera, levels of IL-6, IL-10 and IFN-γ were elevated in malaria patients compared to HV. Moreover, IL-6 and IL-10 values were higher in thrombocytopenic patients than non-thrombocytopenic ones (p = 0.044 and p = 0.017, respectively. In contrast, TNF-α levels were not different between the three groups, but a positive correlation was found between TNF-α and phagocytosis index (r = −0.305; p = 0.037). Conclusion/Significance Collectively, our findings indicate that platelet phagocytosis may contribute to thrombocytopenia found in vivax malaria. Finally, we believe that this study opens new avenues to explore the mechanisms involved in platelet dysfunction, commonly found in vivax malaria patients.

Hepatitis C virus in blood donors, Brazil.

To the Editor: The Fundacao de Hematologia e Hemoterapia do Amazonas is a public health service in Manaus, Brazil, that is responsible for serologic screening of all blood donations in the region. In the state of Amazon, 9.0% of donated blood is discarded on the basis of serologic findings; discarding because of hepatitis C virus (HCV) antibodies declined from 1.25% in 1995 to 0.32% in 2007. The aim of this study was to characterize the serologic and molecular profile of HCV-antibody–positive blood donors from the Fundacao de Hematologia e Hemoterapia do Amazonas. For the study, 154 donors were selected from a routine database of voluntary blood donors who had donated from September 2005 through April 2007 (82,851 donations). Fresh plasma samples were sent to the laboratory in Manaus through the usual transportation systems for regular donations; i.e., samples from 27 cities are transported by air for ≈2 hours, and samples from 21 localities are transported by boat or road, all under refrigerated conditions. An in-house standardized nested-PCR was used to detect HCV RNA (1). Genotype assignment was based on type-specific motifs on the sequenced amplicons delimited by primers HC11/HC18 from the 5′ untranslated region (2). Viral load was determined by commercial assay (HCV Monitor, Roche Molecular Systems, Inc., Branchburg, NJ, USA). An association was observed between HCV RNA and donor age; the same trend was seen in the first-time blood donor group. Associations between HCV-RNA detection and gender (p = 0.875) and place of donation (p = 0.989) were not significant. Using 18–25 years of age as the reference group, we found that odds ratios (ORs) for having HCV viremia were higher for those 45–55 years of age (OR 8.19, p<0.001) and 35–45 years of age (OR 3.49, p = 0.003). We observed increasing rates of RNA detection according to the signal-to-cutoff (S/CO) ratio. However, some donors had a weak S/CO ratio (between 1 and 2) with positive nested-PCR tests (Figure). Although adopting an S/CO ratio as a criterion for referring for further testing by recombinant immunoblot assay (RIBA) has been advocated by some groups (3), our data show that this criterion may be misleading and would deny a confirmatory diagnosis by giving false-negative results for many persons. Figure Distribution of hepatitis C virus (HCV) enzyme immunoassay signal-to-cutoff (S/CO) values by recombinant immunoblot assay (RIBA) interpretations among HCV-RNA–positive [PCR (+)] and HCV-RNA–negative [PCR (−)] donated blood samples. ... A total of 113 samples were analyzed by RIBA; among 48 RIBA-reactive samples, 9 (18.8%) were negative for HCV RNA in plasma. However, because PCR results may sometimes be negative for persons who are actually infected, a single negative PCR result should not be relied on as evidence that virus has cleared from plasma. Such patients must be observed for years before they may be declared cured (4). Among 97 RIBA-positive or -indeterminate samples, viral load was detectable in only 33 samples: 27 (81.8%) RIBA-positive samples and 6 (18.2%) RIBA-indeterminate samples. Only HCV genotypes 1 (87.1%) and 3 (12.9%) were found. Geographic distribution shows genotypes 1 and 3 in Manaus and only genotype 1 in other Amazon cities. This genotype geographic distribution is similar to that found for many Brazilian cities and Eastern countries and may reflect the route of HCV introduction into the Amazon; the virus was probably brought to the Amazon region by European immigrants and blood-derived medicines imported to Brazil. This hypothesis is corroborated by the finding of genotype 3 exclusively in Manaus, suggesting that this city is the point of arrival of HCV and that new strains were disseminated from Manaus to inner localities. Historical reconstruction of HCV in Amazon could be attempted by using these isolates as well as others from hepatitis patients in the region, including genotype 2 (5). We found a higher-than-expected rate of 50% for indeterminate immunoblot results among samples that were HCV-RNA positive by nested PCR. The presence of HCV RNA in plasma samples from 70%–75% of blood donors with indeterminate immunoblot results has also been reported by other groups in Brazil (6,7); however, in contrast, other investigators have reported RNA prevalence in such samples to be ≈2.5% (1,8). Indeterminate RIBA test results can indicate seroconversion or seroreversion or, occasionally, a chronic infection when RNA HCV is detected in plasma (9,10). To provide better understanding of the meaning of these indeterminate results, ongoing follow-up studies are examining the immune status of these persons. Our data offer insights for counseling of donors who have repeatedly HCV-reactive results. We suggest that Amazon region blood banks screen by enzyme immunoassay and use molecular testing as the first supplemental test and that immunoblot be applied to the remaining HCV-RNA nonreactive samples to distinguish between true and false anti-HCV carriers. This new algorithm would save considerable resources currently spent on immunoblot-indeterminate persons in addition to HCV-RNA reactive persons who do not require further testing for confirmation. Moreover, according to current policy, those with false-positive results may be reinstated as donors if they have negative retesting results after 6 months.

The Robust and Modulated Biomarker Network Elicited by the Plasmodium vivax Infection Is Mainly Mediated by the IL-6/IL-10 Axis and Is Associated with the Parasite Load

Background. Recent studies have shown that the inflammatory process, including the biomarker production, and the intense activation of innate immune responses are greater in the malaria caused by Plasmodium vivax than other species. Here, we examined the levels of serum biomarkers and their interaction during acute malaria. Material and Methods. Blood samples were collected from P. vivax-infected patients at admission and from healthy donors. Levels of serum biomarkers were measured by Cytometric Bead Assay or ELISA. Results. P. vivax infection triggered the production of both inflammatory and regulatory biomarkers. Levels of IL-6, CXCL-8, IFN-γ, IL-5, and IL-10 were higher in P. vivax-infected patients than in healthy donors. On the other hand, malaria patients produced lower levels of TNF-α, IL-12p70, and IL-2 than healthy individuals. While the levels of IL-10 and IL-6 were found independent on the number of malaria episodes, higher levels of these cytokines were seen in patients with higher parasite load. Conclusion. A mixed pattern of proinflammatory and regulatory biomarkers is produced in P. vivax malaria. Analysis of biomarker network suggests that IL-10 and IL-6 are a robust axis in malaria patients and that this interaction seems to be associated with the parasite load.

CD11c+CD123Low dendritic cell subset and the triad TNF-α/IL-17A/IFN-γ integrate mucosal and peripheral cellular responses in HIV patients with high-grade anal intraepithelial neoplasia: a systems biology approach.

Background:The incidence of anal cancer has increased over the past 25 years, and HIV/HPV coinfection is the most important risk factor for anal squamous cell carcinoma. In this study, we demonstrated that the evaluation of systemic and compartmentalized anal mucosa immune response is relevant to differentiating HIV(+) patients at risk of anal intraepithelial neoplasia (AIN). Methods:A systems biology approach was used to integrate different immunological parameters from anal mucosal tissue and peripheral blood assessed by phenotypic and intracytoplasmic analysis of lymphocytes and dendritic cell subsets. Results:Our data demonstrated that anal mucosal mononuclear cells from AIN(+)HIV(+) patients showed a robust capacity in producing proinflammatory/regulatory cytokines, mainly mTNF-&agr; > IL-4 > IL-10 > IL-6 = IL-17A. Mucosal TNF-&agr;/IFN-&ggr;/IL-17A are selective high-grade squamous intraepithelial lesion (HSIL)-related biomarkers. Higher levels of circulating CD11c+CD123Lowcells and CD1a+ cells along with elevated levels of IFN-&ggr;+CD4+ T cells are major features associated with HSIL in AIN(+)HIV(+) patients. Regardless of the presence of AIN, HIV(+) patients presented a complex biomarker network, rich in negative connections. Among those patients, however, HSIL+ patients displayed stronger positive links between peripheral blood and anal mucosa environments, exemplified by the subnet of IL-17A/TNF-&agr;/CD4+IFN-&ggr;+/CD11c+CD123Low cells. Conclusions:The significant association between HSIL and the levels of TNF-&agr;/IL-17A/IFN-&ggr; along with the different subsets of DCs present in the anal mucosa milieu should be studied in more detail as a way to identify and categorize HIV(+) patients vis à vis the high risk of anal cancer outcome.

Divergent cerebrospinal fluid cytokine network induced by non-viral and different viral infections on the central nervous system

BackgroundMeningoencephalitis is one of the most common disorders of the central nervous system (CNS) worldwide. Viral meningoencephalitis differs from bacterial meningitis in several aspects. In some developing countries, bacterial meningitis has appropriate clinical management and chemotherapy is available. Virus-associated and virus not detected meningoencephalitis are treatable, however, they may cause death in a few cases. The knowledge of how mediators of inflammation can induce disease would contribute for the design of affordable therapeutic strategies, as well as to the diagnosis of virus not detected and viral meningoencephalitis. Cytokine-induced inflammation to CNS requires several factors that are not fully understood yet.MethodsConsidering this, several cytokines were measured in the cerebrospinal fluid (CSF) of patients with undiagnosed and viral meningoencephalitis, and these were correlated with cellularity in the CSF.ResultsThe results demonstrate that an altered biochemical profile alongside increased cellularity in the cerebrospinal fluid is a feature of patients with meningoencephalitis that are not associated with the detection of virus in the CNS (P < 0.05). Moreover, HIV-positive patients (n = 10) that evolve with meningoencephalitis display a distinct biochemical/cytological profile (P < 0.05) in the cerebrospinal fluid. Meningoencephalitis brings about a prominent intrathecal cytokine storm regardless of the detection of virus as presumable etiological agent. In the case of Enterovirus infection (n = 13), meningoencephalitis elicits robust intrathecal pro-inflammatory cytokine pattern and elevated cellularity when compared to herpesvirus (n = 15) and Arbovirus (n = 5) viral infections (P < 0.05).ConclusionDifferences in the cytokine profile of the CSF may be unique if distinct, viral or presumably non-viral pathways initially trigger the inflammatory response in the CNS.

Combined impact of hepatitis C virus genotype 1 and interleukin-6 and tumor necrosis factor-α polymorphisms on serum levels of pro-inflammatory cytokines in Brazilian HCV-infected patients

We investigated the association between hepatitis C virus (HCV) genotypes and host cytokine gene polymorphisms and serum cytokine levels in patients with chronic hepatitis C. Serum IL-6, TNF-α, IL-2, IFN-γ, IL-4, IL-10, and IL-17A levels were measured in 67 HCV patients (68.2% genotype 1 [G1]) and 47 healthy controls. The HCV patients had higher IL-6, IL-2, IFN-γ, IL-10, and IL-17A levels than the controls. HCV G1 patients had higher IL-2 and IFN-γ levels than G2 patients. The -174IL6G>C, -308TNFαG>A, and -1082IL10A>G variants were similarly distributed in both groups. However, HCV patients with the -174IL6GC variant had higher IL-2 and IFN-γ levels than patients with the GG and CC variants. Additionally, HCV patients with the -308TNFαGG genotype had higher IL-17A levels than patients with the AG genotype, whereas patients with the -1082IL10GG variant had higher IL-6 levels than patients with the AA and AG variants. A significant proportion of HCV patients had high levels of both IL-2 and IFN-γ. The subgroup of HCV patients with the G1/IL6CG/TNFαGG association displayed the highest proportions of high producers of IL-2 and IFN-γ whereas the subgroup with the G1/TNFαGG profile showed high proportions of high producers of IL-6 and IL-17A. HCV patients with other HCV/cytokine genotype associations showed no particular cytokine profile. Our results suggest that HCV genotype G1 and IL-6 and TNF-α polymorphisms have a clinically relevant influence on serum pro-inflammatory cytokine profile (IL-2 and IFN-γ) in HCV patients.

Liver and blood cytokine microenvironment in HCV patients is associated to liver fibrosis score: a proinflammatory cytokine ensemble orchestrated by TNF and tuned by IL-10

BackgroundIn this study, we have evaluated the immunological status of hepatitis C virus (HCV)-infected patients aiming at identifying putative biomarkers associated with distinct degrees of liver fibrosis. Peripheral blood and tissue T-cells as well as cytokine levels were quantified by flow cytometry.ResultsData analysis demonstrated higher frequency of circulating CD8+ T-cells and Tregs along with a mixed proinflammatory/IL-10-modulated cytokine pattern in HCV patients. Patients with severe liver fibrosis presented lower frequency of circulating CD8+ T-cells, higher levels of proinflammatory cytokines, but lower levels of IL-10, in addition to the higher viral load. Despite the lower frequency of intrahepatic T-cells and scarce frequency of Tregs, patients with severe liver fibrosis showed higher levels of proinflammatory cytokines (TNF and IFN-γ). The tissue proinflammatory cytokine pattern supported further studies of serum cytokines as relevant biomarkers associated with different liver fibrosis scores. Serum cytokine signature showed that mild liver fibrosis is associated with higher IL-10 serum levels as compared to severe liver disease. There was a clear positive connection of IL-10 with the TNF node in patients with mild liver fibrosis, whereas there is an evident inverse correlation between IL-10 with all other cytokine nodes.ConclusionsThese results suggest the absence of modulatory events in patients with severe liver damage as opposed to mild fibrosis. Machine-learning data mining pointed out TNF and IL-10 as major attributes to differentiate HCV patients from non-infected individuals with highest performance. In conclusion, our findings demonstrated that HCV infection triggers a local and systemic cytokine ensemble orchestrated by TNF and tuned by IL-10 in such a manner that mirrors the liver fibrosis score, which highly suggests the relevance of these set of biomarkers for clinical investigations.

Anti-inflammatory/regulatory cytokine microenvironment mediated by IL-4 and IL-10 coordinates the immune response in hemophilia A patients infected chronically with Hepatitis C virus†

In the past decades patients with hemophilia were infected commonly by hepatitis C virus (HCV) and a significant number of patients are infected chronically. Focusing on the role of the immune system for controlling and or maintaining HCV infection, the leukocyte and cytokine profiles of peripheral blood from hemophilia A patients and other patients with and without HCV infection were studied. The results demonstrated that hemophilia A is characterized by a general state of circulating leukocytes activation along with an overall increase in the frequency of IL‐6 and IL‐10 with decrease of IL‐8 and IL‐12. HCV infection of patients with hemophilia A does not influence further the activation state of circulating leukocytes but is accompanied by lower levels of alanine transaminase (ALT) and a prominent anti‐inflammatory/regulatory serum cytokine pattern, mediated by IL‐4 and IL‐10. Additionally, the results demonstrated that hemophilia A patients infected with HCV displaying No/Low antibody response to C33c and C22 have significant lower viral load and higher serum levels of IL‐12 and IL‐4. This finding suggests that the differential RIBA reactivity to C33c/C22 HCV core proteins may have a putative value as a prognostic biomarker for the infection in hemophilia A patients. J. Med. Virol. 85: 1009–1018, 2013.

A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivax developed from two lactate dehydrogenase protein segments

BackgroundImmunoassays for Plasmodium detection are, presently, most frequently based on monoclonal antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum.MethodsTwo recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies (“Test 1”) and another which uses anti-LDH 35-305aa PAbs (“Test 2”) as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients (confirmed by light microscopy) were analysed using both tests.Results“Test 2” performed better at detecting microscopy-positive blood samples when compared to “Test 1”, identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using “test 1”. “Test 1” produced one false positive sample (from the 20 malaria-free control) blood samples; “test 2” produced none. Kappa coefficient analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with “test 1”, but 0.734 when microscope-positive blood smears were compared with the results from “test 2”. Positive predictive value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for “Test 1”, and 99% and 45%, for “test 2”. No cross reactivity was detected with P. falciparum positive blood samples (n = 15) with either test assay.ConclusionBoth tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum. Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general.

Kinetics of mesenchymal and hematopoietic stem cells mobilization by G-CSF and its impact on the cytokine microenvironment in primary cultures.

In this study, we demonstrate that G-CSF administration triggers distinct kinetics of stem cell-SC mobilization with early raise of hematopoietic-HSC and late increase of mesenchymal-MSC in bone marrow-BM and peripheral blood-PB. The cytokine microenvironment observed following primary cultures showed an overall G-CSF dose-dependent profile with a clear mixed pro-inflammatory/regulatory pattern. Moreover, primary cultures performed at the peak of MSC/HSC ratio, showed distinct cytokine patterns, with higher IL-10, TNF-α and IL-17A observed for BM and enhanced IL-10, IL-2 and IFN-γ for PB harvested cells. Positive correlation was observed between BM-MSC and the levels of TNF-α, IL-10 and IL-17A whereas negative correlation was found between IL-10 and BM-HSC. An opposite association was observed between IL-10 and PB-HSC. Our results support the hypothesis that MSC and HSC harvested from BM and PB display differential functional properties that should be considered when electing the SC sources available for cell therapy applied in clinical protocols.