Ivan Best

SYBR Green–based quantitation of human T-lymphotropic virus type 1 proviral load in Peruvian patients with neurological disease and asymptomatic carriers: Influence of clinical status, sex, and familial relatedness

To evaluate the human T-lymphotropic virus type 1 (HTLV-1) proviral DNA load in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and asymptomatic HTLV-1 carriers, a SYBR Green-based real-time quantitative polymerase chain reaction (qPCR) assay was developed. HTLV-1 proviral DNA in peripheral blood mononuclear cells (PBMCs) was quantified using primers targeting the pX region and the HTLV-1 copy number normalized to the amount of ERV-3 (Endogenous Retrovirus 3) cellular DNA. Thirty-three asymptomatic HTLV-1 carriers (ACs) and 39 patients with HAM/TSP were enrolled. Some participants were relatives of HAM/TSP cases (16 ACs and 7 patients with HAM/TSP). On multiple linear regression analysis, the authors found a significant association between clinical status and HTLV-1 proviral load (P < .01), but only among women. ACs showed a median proviral load of 561 copies per 104 PBMCs (interquartile range: 251–1623). In HAM/TSP patients, the median proviral load was 1783 (1385–2914). ACs related to HAM/TSP patients presented a relatively high proviral load (median 1152); however, the association between relatedness to a HAM/TSP patient and proviral load was not significant (P = .1). In HAM/TSP patients, no association was found between proviral load and disease duration, progression or severity. The fact that the effect of HAM/TSP upon the HTLV-1 proviral load differed between sexes and the finding of a high proviral load among asymptomatic relatives of HAM/TSP patients suggest that not yet identified genetic or environmental factors influence the pathogenesis of HTLV-1 infection.

Proviral load and immune markers associated with human T‐lymphotropic virus type 1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) in Peru

Human T‐lymphotropic virus type 1 (HTLV‐1) is the aetiological agent of HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). The objective of this study is to identify which ex vivo and in vivo markers are associated independently with HAM/TSP in a Peruvian population. Eighty‐one subjects (33 men/48 women) were enrolled: 35 presented with HAM/TSP, 33 were asymptomatic HTLV‐1 carriers (ACs) and 13 were HTLV‐1‐seronegative controls (SCs). Ex vivo markers included T cell proliferation and Th1 [interferon (IFN)‐γ], Th2 [interleukin (IL)‐4, IL‐5], proinflammatory [tumour necrosis factor (TNF)‐α] and anti‐inflammatory (IL‐10) cytokine production in non‐stimulated peripheral blood mononuclear cell (PBMC) cultures. In vivo CD4+ T cell count, markers of Th1 [interferon‐inducible protein (IP)‐10] and Th2 (sCD30) activity in plasma and HTLV‐1 proviral load in PBMCs were also evaluated. In univariate analysis, several markers, including T cell proliferation, IFN‐γ, IP‐10, sCD30 and proviral load were associated with HAM/TSP, but in a multiple logistic regression analysis only the proviral load remained associated significantly with disease manifestation [adjusted OR 9·10 (1·24–66·91)]. Our findings suggest that HAM/TSP is associated primarily with proviral load, whereas the observed association with some immune markers seems secondary.

IFN‐γ production in response to Tax 161–233, and frequency of CD4+ Foxp3+ and Lin− HLA‐DRhigh CD123+ cells, discriminate HAM/TSP patients from asymptomatic HTLV‐1‐carriers in a Peruvian population

Human T‐lymphotropic virus 1 (HTLV‐1) can cause HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). The objective of this study was to gain insight into the pathogenesis of HAM/TSP by focusing on the CD8+ T‐cell response. Twenty‐three HTLV‐1‐seronegative controls (SC), 29 asymptomatic HTLV‐1 carriers (AC) and 48 patients with HAM/TSP were enrolled in the study. We evaluated the production of interferon‐γ (IFN‐γ) in peripheral blood mononuclear cells stimulated with Tax overlapping peptides, the expression of genes related to the CD8+ cytotoxic T‐cell response, the frequency of CD4+ Foxp3+ cells and of dendritic cells, and the HTLV‐1 provirus load (PVL). The frequency of cells producing IFN‐γ in response to Tax 161–233, but not to Tax 11–19, discriminated patients with HAM/TSP from AC. The increased pro‐inflammatory response observed in patients with HAM/TSP was shared by AC with a high PVL, who also exhibited lower levels of granzyme H mRNA in unstimulated CD8+ T cells than AC with a low PVL. Patients with HAM/TSP showed higher frequencies of CD4+ Foxp3+ cells and lower frequencies of plasmacytoid dendritic cells (pDC) than AC. Our findings are consistent with a model in which HTLV‐1, along with the host genetic background, drives quantitative and qualitative changes in pDC and CD4+ Foxp3+ cells that lead to a predominance of inflammatory responses over lytic responses in the CD8+ T‐cell response of individuals predisposed to develop HAM/TSP.

Evaluation of Host Genetic and Viral Factors as Surrogate Markers for HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis in Peruvian HTLV-1-Infected Patients

Human T‐lymphotropic virus 1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a complication that affects up to 5% of HTLV‐1‐infected individuals. Several host genetic and viral factors have been associated with the risk of HAM/TSP. The aim of this study was to evaluate the performance of a prognostic model for HAM/TSP developed in Japan in a Peruvian population of 71 HAM/TSP patients and 94 asymptomatic carriers (ACs). This model included age, proviral load (PVL), the presence of HLA‐A*02 and HLA‐Cw*08 alleles, SDF‐1 +801, and TNF‐α −863 polymorphisms, and viral subgroup. We describe frequencies for the four host genetic markers and demonstrate the presence of the HTLV‐1 tax B subgroup in Peru. Using cross‐validation, we show that the predictive ability of the prognostic model, as characterized by the area under the receiver‐operating characteristic curve (AUC), does not differ from a model containing PVL only (both AUC = 0.74). We found some suggestive evidence of a protective effect of the HLA‐A*02 allele but failed to replicate the associations with the other three genetic markers and with viral subgroup. A logistic model containing PVL, age, gender, and HLA‐A*02 provided the best predictive ability in the Peruvian cohort (AUC = 0.79). J. Med. Virol. 82:460–466, 2010.

Immunological response in cases of complicated and uncomplicated bartonellosis during pregnancy

Bartonellosis (Carrions Disease) during pregnancy is associated with high rates of maternal and perinatal mortality. We report the immunological patterns in two cases of human bartonellosis during pregnancy. One patient had an uncomplicated course while the second patient developed life threatening anasarca and cardiac tamponade. The patient with a complicated course had a Th1 response with a higher elevation of IL-10. This elevation has been associated with poor outcome pregnancies during bacterial infections.

Cytokines and T-Lymphocute count in patients in the acute and chronic phases of Bartonella bacilliformis infection in an endemic area in peru: a pilot study

Human Bartonellosis has an acute phase characterized by fever and hemolytic anemia, and a chronic phase with bacillary angiomatosis-like lesions. This cross-sectional pilot study evaluated the immunology patterns using pre- and post-treatment samples in patients with Human Bartonellosis. Patients between five and 60 years of age, from endemic areas in Peru, in the acute or chronic phases were included. In patients in the acute phase of Bartonellosis a state of immune peripheral tolerance should be established for persistence of the infection. Our findings were that elevation of the anti-inflammatory cytokine IL-10 and numeric abnormalities of CD4(+) and CD8(+) T-Lymphocyte counts correlated significantly with an unfavorable immune state. During the chronic phase, the elevated levels of IFN-γ and IL-4 observed in our series correlated with previous findings of endothelial invasion of B. henselae in animal models.

Genome sequence and comparative analysis of Avibacterium paragallinarum.

Background: Avibacterium paragallinarum, the causative agent of infectious coryza, is a highly contagious respiratory acute disease of poultry, which affects commercial chickens, laying hens and broilers worldwide. Methodology: In this study, we performed the whole genome sequencing, assembly and annotation of a Peruvian isolate of A. paragallinarum. Genome was sequenced in a 454 GS FLX Titanium system. De novo assembly was performed and annotation was completed with GS De Novo Assembler 2.6 using the H. influenzae str. F3031 gene model. Manual curation of the genome was performed with Artemis. Putative function of genes was predicted with Blast2GO. Virulence factors were identified by comparison with the Virulence Factor Database. Results: The genome obtained has a length of 2.47 Mb with 40.66% of GC content. Seventy five large contigs (>500 nt) were obtained, which comprised 1,204 predicted genes. All the contigs are available in Genbank [GenBank: PRJNA64665]. A total of 103 virulence factors, reported in the Virulence Factor Database, were found in A. paragallinarum. Forty four of them are present in 7 species of Haemophilus, which are related with pathogenesis, virulence and host immune system evasion. A tetracycline-resistance associated transposon (Tn10), was found in A. paragallinarum, possibly acting as a defense mechanism. Discussion and conclusion: The availability of A. paragallinarum genome represents an important source of information for the development of diagnostic tests, genotyping, and novel antigens for potential vaccines against infectious coryza. Identification of virulence factors contributes to better understanding the pathogenesis, and planning efforts for prevention and control of the disease.

Design of a predicted MHC restricted short peptide immunodiagnostic and vaccine candidate for Fowl adenovirus C in chicken infection

Fowl adenoviruses (FAdVs) are the ethiologic agents of multiple pathologies in chicken. There are five different species of FAdVs grouped as FAdV-A, FAdV-B, FAdV-C, FAdV-D, and FAdV-E. It is of interest to develop immunodiagnostics and vaccine candidate for Peruvian FAdV-C in chicken infection using MHC restricted short peptide candidates. We sequenced the complete genome of one FAdV strain isolated from a chicken of a local farm. A total of 44 protein coding genes were identified in each genome. We sequenced twelve Cobb chicken MHC alleles from animals of different farms in the central coast of Peru, and subsequently determined three optimal human MHC-I and four optimal human MHC-II substitute alleles for MHC-peptide prediction. The potential MHC restricted short peptide epitope-like candidates were predicted using human specific (with determined suitable chicken substitutes) NetMHC MHC-peptide prediction model with web server features from all the FAdV genomes available. FAdV specific peptides with calculated binding values to known substituted chicken MHC-I and MHC-II were further filtered for diagnostics and potential vaccine epitopes. Promiscuity to the 3/4 optimal human MHC-I/II alleles and conservation among the available FAdV genomes was considered in this analysis. The localization on the surface of the protein was considered for class II predicted peptides. Thus, a set of class I and class II specific peptides from FAdV were reported in this study. Hence, a multiepitopic protein was built with these peptides, and subsequently tested to confirm the production of specific antibodies in chicken.

IFN-γ Response Is Associated to Time Exposure Among Asymptomatic Immune Responders That Visited American Tegumentary Leishmaniasis Endemic Areas in Peru

Clinical manifestations of American Tegumentary Leishmaniasis (ATL) include cutaneous (CL) and mucous forms (ML); however, there are asymptomatic individuals who despite being infected do not present any clinical manifestations. This study characterized the cell-mediated immunity of travelers who lived in the Andean highlands of Cusco, free of leishmaniasis transmission, which eventually visited leishmaniasis endemic in the Amazonian basin and returned home without any clinical signs of the disease. Their immune response was compared with CL and ML patients who acquired the disease during their stage in the same region. Fifty-four human subjects from the highlands of Cusco (Peru), who have visited an endemic area, were enrolled: 28 of them did not show any symptoms, 12 showed CL and 14 showed ML. Ten healthy subjects from a non-endemic area (HS) were included as controls. T-cell proliferation was evaluated using peripheral blood mononuclear cells (PBMC) stimulated for 5 days with a total soluble leishmanial antigen (TSLA) of L. (V.) braziliensis. Th1/Th2/Th17 cytokines were also quantified in the supernatants by a flow cytometry multiplex assay. T-cell proliferation was expressed as stimulation index (SI) and the cut off was fixed at SI >2.47. Fifteen out of 28 subjects did not show any signs of disease (54%); subjects with an SI above the cut off. They were defined as asymptomatic immune responders (AIR). CL and ML patients presented a higher SI than HS and AIR. Among the latter group, the exposure time to Leishmania was clearly associated with the IFN-γ response. Increased levels of this cytokine were observed in individuals who remained <90 days in an endemic area of leishmaniasis. Our results evidenced two sub-populations among asymptomatic individuals, one AIR who did not develop clinical disease manifestations when they were exposed to Leishmania in endemic areas. Exposure time to Leishmania in the wild was associated with the IFN-γ response.