Edel Figueiredo Barbosa-Stancioli

HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) Inflammatory Network

HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) is a systemic immune-mediated inflammatory disease and tissues other than nervous can be damaged, mainly ocular, rheumatic and dermatologic. Over 90% of HTLV-1-infected individuals remain lifelong asymptomatic and this retrovirus persists indefinitely in their CD4+ T-lymphocytes. The infection is maintained due to the proliferation of lymphocytes that harbor a provirus and express HTLV-1 proteins, particularly Tax, promoting an active and selective expansion of infected T cells. High proviral load is related to disease progression, which is correlated to disequilibrium between host and virus. Cytotoxic T lymphocytes are abundant and chronically activated in asymptomatic carriers and in HAM/TSP patients. The asymptomatic carriers were shown to have a high frequency of pro-inflammatory monocytes and anti-inflammatory IL-10+CD4+ and IL-10+CD8+ T-cells, as an immunoregulatory mechanism to counterbalance the monocyte-derived TNF-alpha. A putative immunomodulatory event would be the key to control their overall immunological status. In HAM/TSP, a pro-inflammatory microenvironment is the hallmark of the immunological profile. Enhanced frequency of activated CD8+ T-cells (HLA-DR+) in combination with high CD18 surface expression has been seen. In blood and cerebrospinal fluid, increased levels of Type-1 cytokines, as interferon-(IFN)-gamma, Tumor Necrosis Factor (TNF)-alpha, Interleukin (IL)-2, and pro-inflammatory IL-6, can be found. Concerning the progression, HLA polymorphisms may influence HAM/TSP and the allele HLA-A*2 has been associated with protection. The authors showed that HAM/TSP is strongly associated with a decreased percentage of B-cells, with enhanced T/B-cell ratio and activated CD8+ T-cells. These immunological parameters have been proposed as a prognostic biomarker for HAM/TSP.

Monitoring the HTLV-1 proviral load in the peripheral blood of asymptomatic carriers and patients with HTLV-associated myelopathy/tropical spastic paraparesis from a Brazilian cohort: ROC curve analysis to establish the threshold for risk disease.

Human T‐lymphotropic virus 1 (HTLV‐1) infection is associated with HTLV‐associated myelopathy/tropical spastic paraparesis (HAM/TSP), which affects approximately 5% of carriers. High proviral load is a risk marker for HAM/TSP, although there is an overlap of proviral load levels in peripheral blood between asymptomatic carriers and HAM/TSP patients. In this study, receiver operating characteristic curve analysis was used to define a set point of HTLV‐1 proviral load that better indicates an increased risk for HAM/TSP. Proviral load was quantified in 75 asymptomatic carriers and 78 HAM/TSP patients in a Brazilian cohort. The cut‐off of proviral load was defined as 114 copies/104 cells, with 78.2% sensitivity to identify true HAM/TSP patients. The mean proviral load levels were not significantly different between males and females with the same clinical status, and there was no significant correlation between proviral load and age at blood sampling, age at the onset of illness, or duration of disease. In HAM/TSP patients, proviral load was significantly higher in wheelchair‐bound patients than in individuals able to walk without support and in those with the worst spinal cord injuries. Follow‐up of HTLV‐1‐infected individuals showed that proviral load was more stable in asymptomatic carriers than in HAM/TSP patients. In a cohort study, periodically quantifying proviral load in asymptomatic carriers is necessary to identify those at risk for developing neurological disease, and it is necessary for HAM/TSP patients to monitor spinal injury and progression to walking disability. The measure of proviral load in clinical practice implicates the definition of the cut‐off of proviral load and its validation during follow‐up. J. Med. Virol. 84:664–671, 2012.

Bovine Herpesvirus 5 (BoHV-5) in Bull Semen: Amplification and Sequence Analysis of the US4 Gene

Bovine herpesvirus type 5 (BoHV-5), which is potentially neuropathogenic, was detected in clinical samples of bovine semen, both directly and after isolation in cell culture, using a nested PCR system for amplifying the US4 gene. Nucleotide sequences generated from the amplicons were analysed and deposited at GenBank (NCBI, Bethesda, MD, USA) under the accession numbers AF298174 and AF330157. Alignment of these sequences and previously deposited sequences of BoHV-1 and BoHV-5 showed 82% and 98% similarity, respectively. The bulls, which were maintained at an artificial insemination centre, had presented no clinical signs, indicating that bovine semen should be screened for BoHV-5 to prevent transmission of the virus.

Detecção de herpesvírus bovino 5 (BoHV-5) em bovinos do Sudeste Brasileiro

This paper reports the detection of bovine herpesvirus type 5 (BoHV-5) by a specific nested PCR assay. Samples were collected from the central nervous system (CNS) of cattle from Minas Gerais and Sao Paulo States, Brazil. All animals died presenting neurological symptoms. Nineteen frozen CNS samples analyzed had been previously tested by fluorescence antibody test for rabies virus and showed negative results. Three paraffin-embedded brain tissue samples were examined by histopatology and the observed alterations suggested nonsuppurative meningoencephalitis. BoHV-5 was detected in five (22.7%) among 22 tested samples. The occurrence of BoHV-5 infection is reported in the Southeast region of Brazil, indicating that epidemiological studies should be carried out.

IL-10 produced by CD4+ and CD8+T cells emerge as a putative immunoregulatory mechanism to counterbalance the monocyte-derived TNF-α and guarantee asymptomatic clinical status during chronic HTLV-I infection

Although it is believed widely that distinct patterns of the host immune response are associated with the outcome of chronic human T cell lymphotropic virus type 1 (HTLV‐I) infection toward asymptomatic or symptomatic neurodegenerative myelopathy (HAM/TSP), the exact mechanism underlying these immunological events still remains unknown. In this study, we have evaluated the cytokine pattern [interleukin (IL)‐12, interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α, IL‐4 and IL‐10] of innate and adaptive immunity cells present at the peripheral blood from non‐infected (NI) and HTLV‐I infected individuals [asymptomatic (AS), oligosymptomatic (OL) and HAM/TSP‐HT], following in vitro short‐term incubation in the absence/presence of phorbol myristate acetate (PMA) pan‐leucocyte stimulation. In the absence of PMA stimulation, our data demonstrate that despite the overall immunological profile of AS mimicry that observed for NI, the high frequency of IL‐12+ neutrophils and TNF‐α+ monocytes are also a hallmark of this group of individuals. However, the outstanding positive correlation between the high frequency of TNF‐α+ monocytes and high levels CD4+ IL‐10+ and CD8+ IL‐10+ T cells suggests the establishment of immunoregulatory mechanisms that guarantee their asymptomatic clinical status. On the other hand, OL and HT did not present any association between the high frequency and TNF‐α+ neutrophils and monocytes and this immunoregulatory profile at their adaptive immunity cells. Upon PMA‐index analysis, high levels of type 1 CD4+ T cells, as well as higher IFN‐γ/IL‐10 and TNF‐α/IL‐10 ratios, were observed in HT, and re‐emphasize the role of Th1‐cytokines from CD4+ cells to HTLV‐I immunity and disease. Moreover, increasing frequency of CD8+ IFN‐γ+ and CD8+ TNF‐α+ cells were observed in the HT, which corroborates the marked inflammatory profile underlying this pathological condition and the role of CD8+ T cells in the pathogenesis of HAM/TSP.

Genotyping of Chlamydia trachomatis from endocervical specimens in Brazil.

Background: There is no data concerning genotyping of Chlamydia trachomatis from Brazilian samples. Goal: To characterize the genotype of C. trachomatis detected in women assisted at a STD public clinic and establish the prevalence of this infection in that population. Study Design: Endocervical samples of a group of 100 women were tested for chlamydial infection with PCR directed to C. trachomatis cryptic plasmid. Genotyping of positive samples were done after omp1 amplification and sequencing. Results: The overall prevalence of C. trachomatis infection was 19%, with the highest prevalence in women between 15 and 25 years old (68.4%). Four genotypes were found associated with endocervical infections: D, E, F, and K. Sequence analysis revealed a coinfection of genotypes D and E in 1 woman. Conclusions: To our knowledge this is the first study to characterize Brazilian C. trachomatis endocervical samples and Brazilian C. trachomatis genotype coinfection. Our results also emphasize the importance of routine diagnosis of C. trachomatis for the control of this STD.

Dermatological Findings in 3 Generations of a Family with a High Prevalence of Human T Cell Lymphotropic Virus Type 1 Infection in Brazil

BACKGROUND Dermatologic manifestations are quite common in patients with adult T cell leukemia and lymphoma and patients with myelopathy and/or tropical spastic paraparesis associated with human T cell lymphotropic virus type 1 (HTLV-1). The aim of this study was to investigate dermatological findings presented by 30 members of a Brazilian family, half of whom are infected with HTLV-1 (as confirmed by enzyme-linked immunosorbent assay and Western blot). METHODS The subjects underwent dermatologic examination and laboratory assessment, which included the search for the HTLV-1 genome in peripheral blood mononuclear cells (PBMCs) by qualitative and semiquantitative polymerase chain reaction (PCR) and in skin samples by nested qualitative PCR and immunofluorescence assay. RESULTS We found that cases of xerotic dermatological alterations, including 3 cases of acquired ichthyosis, were more frequent among the infected patients (7 cases vs. none among the uninfected individuals; P=.0063). Other lesions observed in this group included impetigo, scabies, epidermal nevus, herpes zoster scar, rosacea, and juvenile acne. One HTLV-1-infected individual presented with concurrently acquired ichthyosis, impetigo, scabies, dermatophytosis, and seborrheic dermatitis. The PCR performed on PBMCs and skin samples from 24 patients confirmed the serological results in all cases. Additionally, the HTLV-1 proviral load was higher in patients with >1 skin lesion. Finally, HTLV-1 could be identified in the skin by immunofluorescence assay, which, by use of PCR as the gold standard, showed a sensitivity and specificity of 61.5% and 100%, respectively. CONCLUSIONS Altogether, these clinical and laboratory findings point to an HTLV-1 tropism toward the skin, even in HTLV-1 carriers without adult T cell leukemia/lymphoma or HTLV-1-associated myelopathy and/or tropical spastic paraparesis.

Leptospirosis serosurvey in bovines from Brazilian Pantanal using IGG ELISA with recombinant protein LipL32 and microscopic agglutination test

This investigation was carried out in Brazilian Pantanal: region with important biodiversity. This regions climatic conditions, hydrology and geomorphology as well as the existence of great variety of wild species favor the maintenance of the Leptospira in the environment. The aim of this study was to evaluate IgG ELISA with recombinant protein LipL32 in comparison with microscopic agglutination test (MAT) and additionally contribute to the knowledge of the distribution of the one of most important worldwide zoonotic infection, assessing the seropositivity of bovine leptospirosis in beef cattle herds of Brazilian Pantanal, an important ecological preserved area, where cattle constitute not only the most important economic resource but also the major activity compatible of the conservation of natural resource of the region. Out of 282 samples of cattle serum analyzed, 143 (50.71%) were positive in MAT. The serovar Hardjo (genotypic Hardjoprajitno and Hardjobovis), Wolffi and Ballum showed the largest frequency of reactive samples. In the IgG ELISA rLipL32, 161 samples (57.09%) were positive. This result was higher than obtained by MAT (p<0.001). The sensitivity of the ELISA test was 99.30% and the specificity was 86.33%, based on the MAT. This test was shown to be a more sensitive, specific and accurate test for the diagnosis of bovine leptospirosis compared to the MAT.

Strong correlation between tax and HBZ mRNA expression in HAM/TSP patients: Distinct markers for the neurologic disease

BACKGROUND HTLV-1 proviral load is a risk marker for HAM/TSP, but it is insufficient to determine the disease outcome. HTLV-1 Tax and HBZ proteins have been implicated in HAM/TSP pathogenesis in inducing cell proliferation and cytotoxic T lymphocytes response. OBJECTIVES To quantify the expression of tax and HBZ mRNA in asymptomatic carriers (AC) and HAM patients, and to investigate their association with HAM/TSP. STUDY DESIGN We quantified the expression of HTLV-1 tax and HBZ mRNA in 37 AC and 26 HAM patients classified according to proviral load as low (AC(L) and HAM(L): <1% infected cells) or high (AC(H) and HAM(H): >1%). RESULTS The AC(L) subgroup showed the lowest frequency of individuals expressing tax mRNA in comparison with AC(H), HAM(L) and HAM(H), and tax mRNA load normalized by proviral load was significantly lower in the AC(L). In turn, normalized HBZ mRNA expression was similar in all subgroups. Both tax and HBZ mRNA expression were moderately correlated with proviral load in AC (r=0.6, p<0.001) and were weaker in HAM (r=0.4, p<0.05). In contrast, the correlation between tax and HBZ mRNA load was moderate in AC (r=0.5, p=0.001) and was much stronger in HAM (r=0.8, p<0.001). In addition, HBZ mRNA load, but not tax, was significantly associated with motor disability in HAM patients (p=0.036). CONCLUSIONS The expression of tax mRNA seems to be best to estimate the risk of HAM/TSP, whereas HBZ mRNA appears to be a surrogate marker to disease progression, indicating that they have important but distinct roles in HAM/TSP pathogenesis.

Vaginal Microbiome Characterization of Nellore Cattle Using Metagenomic Analysis.

Understanding of microbial communities inhabiting cattle vaginal tract may lead to a better comprehension of bovine physiology and reproductive health being of great economic interest. Up to date, studies involving cattle microbiota are focused on the gastrointestinal tract, and little is known about the vaginal microbiota. This study aimed to investigate the vaginal microbiome in Nellore cattle, heifers and cows, pregnant and non-pregnant, using a culture independent approach. The main bacterial phyla found were Firmicutes (~40–50%), Bacteroidetes (~15–25%) and Proteobacteria (~5–25%), in addition to ~10–20% of non-classified bacteria. 45–55% of the samples were represented by only ten OTUs: Aeribacillus, Bacteroides, Clostridium, Ruminococcus, Rikenella, Alistipes, Bacillus, Eubacterium, Prevotella and non-classified bacteria. Interestingly, microbiota from all 20 animals could be grouped according to the respiratory metabolism of the main OTUs found, creating three groups of vaginal microbiota in cattle. Archaeal samples were dominated by the Methanobrevibacter genus (Euryarchaeota, ~55–70%). Ascomycota was the main fungal phylum (~80–95%) and Mycosphaerella the most abundant genus (~70–85%). Hormonal influence was not clear, but a tendency for the reduction of bacterial and increase of archaeal populations in pregnant animals was observed. Eukaryotes did not vary significantly between pregnant and non-pregnant animals, but tended to be more abundant on cows than on heifers. The present work describes a great microbial variability in the vaginal community among the evaluated animals and groups (heifers and cows, pregnant and non-pregnant), which is significantly different from the findings previously reported using culture dependent methods, pointing out the need for further studies on this issue. The microbiome found also indicates that the vaginal colonization appears to be influenced by the gastrointestinal community.