Adriana Malheiro

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.

Inhibition of the inflammatory and coagulant action of Bothrops atrox venom by the plant species Marsypianthes chamaedrys.

AIM OF THE STUDY This study investigated the efficacy of Marsypianthes chamaedrys Vahl (Lamiaceae) inflorescence and leaf extracts in inhibiting the inflammatory and coagulant actions of Bothrops atrox venom. MATERIALS AND METHODS Marsypianthes chamaedrys, which is used in Brazil as a folk medicine to treat snakebites and local inflammatory reactions, was tested in vitro to determine its ability to block indirect phospholipase A(2) and direct coagulant activities and in vivo to determine its ability to inhibit leukocyte migration and cytokine release. RESULTS In vitro, Marsypianthes chamaedrys showed antiphospholipase A(2) and anticoagulant activities; the latter activity was also confirmed by prothrombin time (PT) and activated partial thromboplastin time (aPTT) in the absence of venom. Of the extracts used, those obtained from the crushed plant had the greater inhibitory activity in in vitro tests, showing that biological activity is affected by the way extracts are obtained. In vivo, Marsypianthes chamaedrys inhibited leukocyte migration and the release of the proinflammatory cytokines IL-6 and TNF-α without altering the concentration of the anti-inflammatory cytokine IL-10. CONCLUSIONS As specific antivenoms are not effective in neutralizing the local action of Bothrops venoms, characterization of the anti-inflammatory mechanisms induced by Marsypianthes chamaedrys is of vital importance if the extracts of this plant species are to be used in future as adjuvants in the treatment of snakebites.

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.

Methotrexate-loaded lipid-core nanocapsules are highly effective in the control of inflammation in synovial cells and a chronic arthritis model

Background Rheumatoid arthritis (RA) is the most common autoimmune disease in the word, affecting 1% of the population. Long-term prognosis in RA was greatly improved following the introduction of highly effective medications such as methotrexate (MTX). Despite the importance of this drug in RA, 8%–16% of patients must discontinue the treatment because of adverse effects. Last decade, we developed a promising new nanocarrier as a drug-delivery system, lipid-core nanocapsules. Objective The aim of the investigation reported here was to evaluate if methotrexate-loaded lipid-core nanocapsules (MTX-LNC) reduce proinflammatory and T-cell-derived cytokines in activated mononuclear cells derived from RA patients and even in functional MTX-resistant conditions. We also aimed to find out if MTX-LNC would reduce inflammation in experimentally inflammatory arthritis at lower doses than MTX solution. Methods Formulations were prepared by self-assembling methodology. The adjuvant arthritis was induced in Lewis rats (AIA) and the effect on edema formation, TNF-α levels, and interleukin-1 beta levels after treatment was evaluated. Mononuclear cells obtained from the synovial fluid of RA patients during articular infiltration procedures were treated with MTX solution and MTX-LNC. For in vitro experiments, the same dose of MTX was used in comparing MTX and MTX-LNC, while the dose of MTX in the MTX-LNC was 75% lower than the drug in solution in in vivo experiments. Results Formulations presented nanometric and unimodal size distribution profiles, with D[4.3] of 175±17 nm and span of 1.6±0.2. Experimental results showed that MTX-LNC had the same effect as MTX on arthritis inhibition on day 28 of the experiment (P<0.0001); however, this effect was achieved earlier, on day 21 (P<0.0001), by MTX-LNC, and this formulation had reduced both TNF-α (P=0.001) and IL-1α (P=0.0002) serum levels by the last day of the experiment. Further, the MTX-LNC were more effective at reducing the cytokine production from mononuclear synovial cells than MTX. Conclusion The MTX-LNC were better than the MTX solution at reducing proinflammatory cytokines and T-cell-derived cytokines such as interferon-gamma and interleukin-17A. This result, combined with the reduction in the dose required for therapy, shows that MTX-LNC are a very promising system for the treatment of RA.

IFN-γ +875 microsatellite polymorphism as a potential protection marker for leprosy patients from Amazonas state, Brazil

Polymorphisms present in the first intron of IFN-γ may have an important role in the regulation of the immune response, which could have functional consequences for gene transcription. Leprosy patients are characterized by different immune responses in different clinical forms. We investigated a possible association of the +874 polymorphism and CA repeats present in the first intron of IFN-γ with susceptibility to leprosy and with the manifestation of the different clinical forms. Nucleotide sequencing was performed with samples from 108 leprosy patients and 113 controls subjects, as well as immunophenotyping of CD(4)(+), CD(8)(+) and CD(69)(+) T cells by flow cytometry. The data showed that there were no significant differences between patients and control subjects, as well as according classification of Ridley-Jopling. However, the A/A genotype was significantly increased in paucibacillary patients (p=0.028) and the microsatellite encoding 16 CA repeats were significantly associated with paucibacillary compared to multibacillary patients (p=0.019). Individuals homozygous for the +874 A allele, the mean level of CD(4)(+) and CD(69)(+) T cells was higher. Our data suggest that polymorphisms present in the first intron of IFN-γ are not associated with susceptibility to leprosy, nevertheless, the +874 polymorphism and the CA repeats number encoded in IFN-γ gene may be related to a higher cellular immune response in patients and are consistently more frequently detected in PB patients.

Distribution of human immunodeficiency virus type 1 subtypes in the State of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible “homogenous” subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country. Key words: HIV-1; Subtypes; Phylogenetic analysis; Blood donors; Molecular and epidemiological characterization

Immunological/virological peripheral blood biomarkers and distinct patterns of sleeping quality in chronic Hepatitis C patients

The rational of this study we intended to investigate whether the peripheral blood immunological/virological biomarkers were associated with distinct patterns of sleeping quality in patients with chronic hepatitis C‐(HCV). Distinct well‐established indexes/scores were used to categorize the sleeping quality of HCV patients, including the Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale and Fatigue Severity Scores. Our findings demonstrated that HCV patients classified as ‘good sleeper’ displayed an enhanced frequency of circulating CD8+ T cells, lower frequency of activated (CD69+) neutrophils and eosinophils but enhanced frequency of activated lymphocytes besides lower seric levels of IL‐4/IL‐8/IL‐10 but higher levels of IL‐12, besides lower HCV virus load and lower anti‐HCV IgG levels. In contrast, HCV patients classified as ‘poor sleeper’ displayed enhanced levels of activated neutrophils and eosinophils but lower frequency of activated lymphocytes, higher seric levels of IL‐6/TNF‐α/IL‐10 but lower levels of IL‐12 besides higher HCV virus load and increased anti‐HCV IgG levels. Positive correlation was further confirmed by the relationship between the leucocyte activation status, the cytokine levels, the HCV viral load and the anti‐HCV IgG reactivity with the PSQI indexes. Analysis of cytokine signature curves demonstrated that lower frequency of IL‐10 was observed in HCV patients classified as ‘good sleepers’, whereas enhanced frequency of IL‐6 was found HCV patients classified as ‘poor sleepers’. In conclusion, our data suggest that immunological biomarkers (leucocytes activation status and seric cytokines levels) are likely to be associated with sleeping quality patterns in HCV patients, suggesting their putative use for clinical monitoring purposes.

The role of inflammatory, anti-inflammatory, and regulatory cytokines in patients infected with cutaneous leishmaniasis in Amazonas State, Brazil.

The authors discuss in this paper the role of inflammatory, anti-inflammatory, and regulatory cytokines in patients infected with different species of Leishmania in Amazonas State, Brazil. A comparative analysis was made of serum concentrations of these cytokines in the peripheral blood of 33 patients infected with cutaneous leishmaniasis. The isolates were identified as Leishmania guyanensis, L. naiffi, and L. amazonensis. Most (64%) of the patients were male ranging in age from 18 to 58 years. Protein expression profiles of IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α, and IL-17 cytokines were shown to vary significantly between infected and noninfected (control group) individuals and according to the Leishmania species. Infection caused by L. guyanensis accounted for 73% of the cases and patients with this parasite also showed higher concentrations of IL-2, IFN-γ, IL-4, and IL-17 when compared to infection by L. amazonensis. Patients with infection caused by L. naiffi showed higher concentration of the cytokines analyzed when compared to uninfected patients; however, there was no statistically significant difference with the other species analyzed.