Monitoring of HTLV-1-associated diseases by proviral load quantification using multiplex real-time PCR.

Proviral load (PVL) is one of the determining factors for the pathogenesis and clinical progression of the human T-lymphotropic virus type I (HTLV-1) infection. In the present study, we optimized a sensitive multiplex real-time PCR for the simultaneous detection and quantification of HTLV-1 proviral load and beta-globin gene as endogenous control. The values obtained for HTLV-1 PVL were used to monitor the clinical evolution in HTLV-1-infected individuals. A vector containing cloned DNA targets of the real-time PCR for the beta-globin gene and the HTLV-1pol region was constructed. For the reaction validation, we compared the amplification efficiency of the constructed vector and MT-2 cell line containing HTLV-1. The analytical sensitivity of the reaction was evaluated by the application of a standard curve with a high order of magnitude. PVL assay was evaluated on DNA samples of HTLV-1 seropositive individuals. The construct showed adequate amplification for the beta-globin and HTLV-1 pol genes when evaluated as multiplex real-time PCR (slope = 3.23/3.26, Y-intercept = 40.18/40.73, correlation coefficient r2 = 0.99/0.99, and efficiency = 103.98/102.78, respectively). The quantification of PVL using the MT-2 cell line was equivalent to the data obtained using the plasmidial curve (2.5 copies per cell). In HTLV-1-associatedmyelopathy/tropical spastic paraparesis patients, PVL was significantly higher (21315 ± 2154 copies/105 PBMC) compared to asymptomatic individuals (1253 ± 691 copies/105 PBMC). The obtained results indicate that the optimized HTLV-1 PVL assay using plasmidial curve can be applied for monitoring and follow-up of the progression of HTLV-1 disease. The use of a unique reference plasmid for both HTLV-1 and endogenous gene allows a robust and effective quantification of HTLV-1 PVL. In addition, the developed multiplex real-time PCR assay was efficient to be used as a tool to monitor HTLV-1-infected individuals.

Downregulation of histone methyltransferase EHMT2 in CD4+ T-cells may protect HTLV-1-infected individuals against HAM/TSP development.

Approximately 5% of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals will develop one of the HTLV-1-related diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T-cell leukemia. However, the mechanisms responsible for the appearance of symptoms have not been fully clarified. It is believed that viral factors, host genetic and epigenetic mechanisms are implicated in this process. Studies have shown the involvement of histone methyltransferases in retrovirus infection, but no study observed their expression in HTLV-1-infected patients. Among them, euchromatic histone-lysine N-methyltransferase (EHMT)-1 and EHMT-2 were related to retroviral latency in HIV-1 infection. We investigated whether histone methyltransferases EHMT1 and EHMT2 exert any influence on HAM/TSP development by assessing their expression levels in CD4+ T-cells from HTLV-1-infected patients. CD4+ T-cells were immunomagnetically isolated from peripheral blood mononuclear cells of HTLV-1-infected or non-infected individuals and the expression levels of EHMT1 and EHMT2 were determined by RT-qPCR. We observed that EHMT2 was negatively regulated in HTLV-1 asymptomatic carriers compared to non-infected individuals. No difference was observed for EHMT1. These results suggest that EHMT2 downregulation in CD4+ T-cells may be linked to a protection mechanism against the development of HAM/TSP.

HLA-G 3'-untranslated region polymorphisms are associated with HTLV-1 infection, proviral load and HTLV-associated myelopathy/tropical spastic paraparesis development.

Most human T-lymphotropic virus type 1 (HTLV-1)-infected patients remain asymptomatic throughout life. The factors associated with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development have not been fully elucidated; immunological and genetic factors may be involved. The association of 14 bp INS/DEL HLA-G polymorphism with HTLV-1 infection susceptibility has been reported previously. Here, other polymorphic sites at the HLA-G 3'-UTR (14-bp D/I, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142C/G, +3187A/G and +3196C/G) were evaluated in 37 HTLV-1-infected individuals exhibiting HAM/TSP, 45 HTLV-1 asymptomatic carriers (HAC) and 153 uninfected individuals, followed up at University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil. It was observed that: (i) 14bpDI genotype is a risk factor for HTLV-1 infection, while the 14bpDD and +3142CC genotypes were associated with protection against infection; (ii) the +3142C allele and the +3003CT and +3142CC genotypes were associated with susceptibility, while 14bpII and +3003TT genotypes were associated with protection against HAM/TSP development; and (iii) the 14bpII, +3010CC, +3142GG and +3187AA genotypes were associated with lower HTLV-1 proviral load compared to respective counterpart genotypes. Findings that HLA-G has a well-recognized immunomodulatory role and that the genetic variability at HLA-G 3'-UTR may post-transcriptionally modify HLA-G production indicate a differential genetic susceptibility to: (i) the development of HTLV-1 infection, (ii) the magnitude of HTLV-1 proviral load and (iii) HAM/TSP development.

Influence of verapamil on the pharmacokinetics of oxcarbazepine and of the enantiomers of its 10-hydroxy metabolite in healthy volunteers.

PURPOSE: Oxcarbazepine (OXC), a second-generation antiepileptic, and its chiral metabolite 10-hydroxycarbazepine (MHD) are substrates of P-glycoprotein, which can be inhibited by verapamil. This study evaluated the influence of verapamil on the pharmacokinetics of OXC and MHD enantiomers in healthy volunteers. METHODS: Healthy volunteers (n = 12) on occasion O (OXC monotherapy) received 300 mg OXC/12 h for 5 days, and on the O + V occasion (treatment with OXC + verapamil), they received 300 mg OXC/12 h and 80 mg verapamil/8 h for 5 days. Blood samples were collected over a period of 12 h. Total and free plasma concentrations of OXC and the MHD enantiomers were evaluated by LC-MS/MS. Noncompartmental pharmacokinetic analysis was performed using the WinNonlin program. RESULTS: The kinetic disposition of MHD was enantioselective with plasma accumulation (AUC(0-12) S-(+)/R-(-) ratio of 4.38) and lower fraction unbound (0.37 vs 0.42) of the S-(+)-MHD enantiomer. Treatment with verapamil reduced the OXC mean residence time (4.91 vs 4.20 h) and apparent volume of distribution (4.72 vs 3.15 L/kg). Verapamil also increased for both MHD enantiomers C max total [R-(-)-MHD: 2.65 vs 2.98 µg/mL and S-(+)-MHD: 10.15 vs 11.60 µg/mL], C average [R-(-)-MHD: 1.98 vs 2.18 µg/mL and S-(+)-MHD: 8.10 vs 8.83 µg/mL], and AUC(0-12) [R-(-)-MHD: 23.79 vs 26.19 µg h/mL and S-(+)-MHD: 97.87 vs 108.35 µg h/mL]. CONCLUSION: Verapamil increased the AUC values of both MDH enantiomers, which is probably related to the inhibition of intestinal P-glycoprotein. Considering that the exposure of both MHD enantiomers was increased in only 10 %, no OXC dose adjustment could be recommended in the situation of verapamil coadministration.

Analysis of oxcarbazepine and the 10-hydroxycarbazepine enantiomers in plasma by LC-MS/MS: application in a pharmacokinetic study.

Oxcarbazepine is a second-generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic-clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10-hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)-(+)- and R-(-)-MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Aliquots of 100 µL of plasma were extracted with a mixture of methyl tert-butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD-H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC-MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S-(+)-MHD enantiomer compared to R-(-)-MHD and an AUC(0-12) S-(+)/R-(-) ratio of 5.44.

Upregulation of hsa-miR-125b in HTLV-1 asymptomatic carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis patients.

The retrovirus human T lymphotropic virus type 1 (HTLV-1) promotes spastic paraparesis, adult T cell leukaemia and other diseases. Recently, some human microRNAs (miRNAs) have been described as important factors in host-virus interactions. This study compared miRNA expression in control individuals, asymptomatic HTLV-1 carriers and HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis patients. The proviral load and Tax protein expression were measured in order to characterize the patients. hsa-miR-125b expression was significantly higher in patients than in controls (p = 0.0285) or in the HAM group (p = 0.0312). Therefore, our findings suggest that miR-125b expression can be used to elucidate the mechanisms of viral replication and pathogenic processes.

Update on the diagnosis and management of neurocysticercosis.

BACKGROUND: Neurocysticercosis (NCC) is a serious public health problem in several developing countries, including those in Latin America, Asia, and Africa. NCC is considered to be the main cause of late-onset epilepsy in endemic areas. OBJECTIVE: This review summarizes recent advances in diagnosis and therapy of NCC. Methods: Relevant articles and books were reviewed and used as a source of information for this review. RESULTS: The diagnosis of NCC is based upon neuroimaging studies (MRI and computed tomography) and laboratory analysis of the cerebrospinal fluid (CSF). Praziquantel and albendazole are considered parasiticidal drugs against NCC, but there is an intense debate over the value and safety of these drugs. CONCLUSION: Given the relative scarcity of clinical trials, more comparative interventional studies, especially randomized controlled trials in long-term clinical evolution, are required in order to clarify the controversy over the validity of parasitic therapy in patients with NCC.

The Expression of Tax and HBZ Genes in Serum-Derived Extracellular Vesicles From HTLV-1 Carriers Correlates to Proviral Load and Inflammatory Markers.

Human T-lymphotropic virus 1 (HTLV-1) is the etiologic agent of adult cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the major questions in HTLV-1 studies is related to the understanding of causes that lead to different clinical manifestations. However, it is well known that the viral genes tax and HTLV-1 basic leucine zipper factor (HBZ) are related to viral infectivity and the development of neurological and hematological diseases. Currently, there is evidence that HTLV-1 infected cells can release small extracellular vesicles (sEVs) involved in the mechanisms of viral particles spreading. Therefore, we evaluated the expression levels of tax and HBZ viral transcripts in serum-derived sEVs from HTLV-1 carriers, as well as the role of these vesicles in the modulation of the immune response. Three HAM/TSP carriers presented detectable levels of tax and HBZ transcripts in sEVs and were positively correlated to the proviral load (PVL) in peripheral blood mononuclear cells (PBMCs). The viral transcripts were only detectable in individuals with a PVL higher than 6,000/105 PBMCs. Additionally, it was observed that HBZ presented a 2-12-folds increase over tax expression units. Gene expression and secretory protein analysis indicated that PBMCs from blood donors and HTLV-1 carriers exposed to increasing doses of tax+ HBZ+ sEVs showed a dose-dependent increase in interferon (IFN)-γ and interleukin (IL)-8 transcripts and proteins. Interestingly, the increase in IL-8 levels was close to those seen in HTLV-1-infected PBMCs with high PVL. Taken together, these findings indicate that the expression of viral transcripts in serum-derived sEVs of HTLV-1 carriers is related to the PVL presented by the infected individual. Additionally, tax+ HBZ+ sEVs can induce the production of inflammatory cytokines in patients with low PVL, which may be related to the development of symptoms in HTLV-1 infection.

Albendazole-praziquantel interaction in healthy volunteers: kinetic disposition, metabolism and enantioselectivity.

AIM: This study investigated the kinetic disposition, metabolism and enantioselectivity of albendazole (ABZ) and praziquantel (PZQ) administered alone and in combination to healthy volunteers. METHODS: A randomized crossover study was carried out in three phases (n= 9), in which some volunteers started in phase 1 (400 mg ABZ), others in phase 2 (1500 mg PZQ), and the remaining volunteers in phase 3 (400 mg ABZ + 1500 mg PZQ). Serial blood samples were collected from 0-48 h after drug administration. Pharmacokinetic parameters were calculated using a monocompartmental model with lag time and were analyzed using the Wilcoxon test; P ≤ 0.05. RESULTS: The administration of PZQ increased the plasma concentrations of (+)-ASOX (albendazole sulphoxide) by 264% (AUC 0.99 vs. 2.59 µg ml(-1) h), (-)-ASOX by 358% (0.14 vs. 0.50 µg ml(-1) h) and albendazole sulfone (ASON) by 187% (0.17 vs. 0.32 µg ml(-1) h). The administration of ABZ did not change the kinetic disposition of (+)-(S)-PZQ (-)-(R)-4-OHPZQ or (+)-(S)-4-OHPZQ, but increased the plasma concentration of (-)-(R)-PZQ by 64.77% (AUC 0.52 vs. 0.86 µg ml(-1) h). CONCLUSIONS: The pharmacokinetic interaction between ABZ and PZQ in healthy volunteers was demonstrated by the observation of increased plasma concentrations of ASON, both ASOX enantiomers and (-)-(R)-PZQ. Clinically, the combination of ABZ and PZQ may improve the therapeutic efficacy as a consequence of higher concentration of both active drugs. On the other hand, the magnitude of this elevation may represent an increased risk of side effects, requiring, certainly, reduction of the dosage. However, further studies are necessary to evaluate the efficacy and safety of this combination.

Polymorphisms at GLUT1 gene are not associated with the development of TSP/HAM in Brazilian HTLV-1 infected individuals and the discovery of a new polymorphism at GLUT1 gene.

The development of HTLV-1 associated clinical manifestations, such as TSP/HAM and ATLL, occur in 2-4% of the infected population and it is still unclear why this infection remains asymptomatic in most infected carriers. Recently, it has been demonstrated that HTLV uses the Glucose transporter type 1 (GLUT1) to infect T-CD4(+) lymphocytes and that single nucleotide polymorphisms (SNP) in the GLUT1 gene are associated with diabetic nephropathy in patients with diabetes mellitus in different populations. These polymorphisms could contribute to a higher GLUT1 protein expression on cellular membrane, facilitating the entry of HTLV and its transmission cell by cell. This could result in a higher provirus load and consequently in the development of TSP/HAM. To evaluate the role of GLUT1 gene polymorphisms in the development of TSP/HAM in HTLV-1 infected individuals, the g.22999G > T, g.15339T > C and c.-2841A > T sites were analyzed by PCR/RFLP or sequencing in 244 infected individuals and 102 normal controls. The proviral load of the HTLV-1 infected patients was also analyzed using Real Time Quantitative PCR. Genotypic and allelic frequencies of the three sites did not differ significantly between controls and HTLV-1 infected individuals. There was no difference in genotypic and allelic distributions among patients as to the presence or absence of HTLV-1 associated clinic manifestations. As regards the quantification of the provirus load, we observed a significant reduction in the asymptomatic individuals compared with the oligosymptomatic and TSP/HAM individuals. These results suggest that g.22999G > T, g.15339T > C, and c.-2841A > T SNP do not contribute to HTLV-1 infection nor to the genetic susceptibility of TSP/HAM in Brazilian HTLV-1 infected individuals.

Neurosyphilis in the age of AIDS: clinical and laboratory features.

Neurosyphilis remains to be a challenging diagnostic possibility worldwide. The aim of our study was to identify and report the clinical and laboratory profile of neurosyphilis, comparing features of HIV-infected and HIV-negative patients. A retrospective investigation of all cases of neurosyphilis, defined as positive VDRL test on cerebrospinal fluid, diagnosed at Hospital das Clínicas, Ribeirão Preto School of Medicine between January 1988 and December 2005, was carried out. We identified 35 patients with a mean age of 42.1 years, 28.6% of them HIV infected and 74.3% of them were male. HIV-infected patients were younger (34.6 years), presented with a higher frequency of the early forms of neurosyphilis, higher titers of serum VDRL and higher mean proteinorrachia at the suboccipital level. Neurosyphilis is still characterized by clinical polymorphism and there are significant differences in its epidemiological, clinical and laboratory profile when HIV-infected patients are compared with HIV-negative patients.

Short Communication: Human Bone Marrow Stromal Cells Exhibit Immunosuppressive Effects on Human T Lymphotropic Virus Type 1 T Lymphocyte from Infected Individuals.

Human multipotent mesenchymal stromal cells (MSCs) display immunoregulatory functions that can modulate innate and adaptive cellular immune responses. The suppressive and immunomodulatory activities of MSCs occur through the action of soluble factors that are constitutively produced and released by these cells or, alternatively, after MSC induction by stimuli of inflammatory microenvironments. However, to date the contribution of MSCs in the inflammatory microenvironment resulting from viral infection is unknown. In our study, we evaluated the MSC immunosuppressive effect on human T lymphotropic virus type 1 (HTLV-1) infected T lymphocytes. To evaluate if MSC immunoregulation can influence the proliferation of HTLV-1 infected T lymphocytes, we compared the proliferation of lymphocytes obtained from HTLV-1 infected and healthy individuals cocultured in the presence of MSCs. It was observed that the lymphoproliferative inhibition by MSCs on infected lymphocytes was similar compared to the cells obtained from healthy individuals. In addition, this suppressive effect was related to a significant increase of indoleamine-2,3-dioxygenase and prostaglandin E2 gene expression (p ≤ .05). Furthermore, the HTLV-1 pol gene was less expressed after coculturing with MSCs, suggesting that the MSC immunoregulation can have effective suppression on HTLV-1 infected T cells. In conclusion, this study suggests that MSCs could be involved in the immunomodulation of the HTLV-1 infected T lymphocytes.

Analysis of unbound plasma concentration of oxcarbazepine and the 10-hydroxycarbazepine enantiomers by liquid chromatography with tandem mass spectrometry in healthy volunteers.

This study describes the development and validation of a method for the analysis of unbound plasma concentrations of oxcarbazepine (OXC) and of the enantiomers of its active metabolite 10-hydroxycarbazepine (MHD) [S-(+)- and R-(-)-MHD] using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, the free fraction of the drug is described in healthy volunteers (n=12) after the oral administration of 300mg OXC/12h for 5days. Plasma aliquots of 200µL were submitted to ultrafiltration procedure and 50µL of the ultrafiltrate were extracted with a mixture of tert-butyl methyl ether:dichloromethane (2:1, v/v). OXC and the MHD enantiomers were separated on a OD-H chiral phase column. The method was linear in the range of 4.0-2.0µg/mL for OXC and of 20.0-6.0µg/mL plasma for the MHD enantiomers. The limit of quantification was 4ng for OXC and 20ng for each MHD enantiomer/mL plasma. The intra- and inter-day precision and inaccuracy were less than 15%. The free fraction at the time of peak plasma concentration of OXC was 0.27 for OXC, 0.37 for S-(+)-MHD and 0.42 for R-(-)-MHD. Enantioselectivity in the free fraction of MHD was observed, with a higher proportion of R-(-)-MHD compared to S-(+)-MHD.

Fluconazole levels in serum and cerebrospinal fluid according to daily dosage in patients with cryptococcosis and other fungal infections.

Fluconazole is extensively used for the treatment of candidiasis and cryptococcosis. Among other factors, successful treatment is related to appropriate fluconazole levels in blood and cerebrospinal fluid. In the present study, fluconazole levels were determined in 15 patients, 14 of whom had AIDS and 13 had neurocryptococcosis. The only selection criterion was treatment with fluconazole, which was performed with a generic or similar form of the drug. Fluconazole level was determined by high performance liquid chromatography and the susceptibility profile of Cryptococcus spp. isolated from the patients was assessed by broth microdilution. Blood and cerebrospinal fluid fluconazole levels were found to be related to the fluconazole daily dose, and exceeded the minimum inhibitory concentration of this antifungal for the Cryptococcus spp. isolates. A good correlation was observed between serum and cerebrospinal fluid drug concentration. In conclusion, treatment with non-original fluconazole under usual medical practice conditions results in appropriate blood and cerebrospinal fluid levels of the drug for inhibiting Cryptococcus spp. susceptible to this antifungal drug. The relatively common failures of neurocryptococcosis treatment appear not to be due to insufficient fluconazole levels in the cerebrospinal fluid, especially with the use of daily doses of 400-800mg.

Detection of HTLV-1 proviral DNA in BM mononuclear cells and cultured mesenchymal stromal cells isolated from patients with HTLV-1 infection.

The bone marrow (BM) biology during HTLV-1 infection is obscure. In this study, we investigated BM mononuclear cells and mesenchymal stromal cells (MSC) from HTLV-1 asymptomatic and symptomatic individuals. An infiltration of CD4+ T-cell lymphocytes in the BM of HTLV-1-infected individuals was observed when compared to healthy controls. The provirus detection in the BM CD4+ T cells confirmed the presence of integrated HTLV DNA. In regard to MSC, we observed that the number of fibroblast progenitor cells was lower in HTLV-1 infected individuals than in healthy controls. Isolated HTLV-1 infected BM-MSC demonstrated surface expression markers and in vitro differentiation potential similar to uninfected individuals. The presence of HTLV-1 proviral DNA in the BM-MSC of HTLV-1-infected patients was demonstrated but no p19 antigen was detected in supernatant from cultured MSC. We suppose that HTLV-1 infects human MSC probably by cell-to-cell contact from the infected CD4+ T-lymphocytes infiltrated into the bone marrow.

Simultaneous determination of albendazole metabolites, praziquantel and its metabolite in plasma by high-performance liquid chromatography-electrospray mass spectrometry.

The analysis of albendazole sulfoxide, albendazole sulfone, praziquantel and trans-4-hydroxypraziquantel in plasma was carried out by high-performance liquid chromatography-mass spectrometry ((LC-MS-MS). The plasma samples were prepared by liquid-liquid extraction using dichloromethane as extracting solvent. The partial HPLC resolution of drug and metabolites was obtained using a cyanopropyl column and a mobile phase consisting of methanol:water (3:7, v/v) plus 0.5% of acetic acid, at a flow rate of 1.0 mL/min. Multi reaction monitoring detection was performed by electrospray ionization in the positive ion mode, conferring additional selectivity to the method. Method validation showed relative standard deviation (precision) and relative errors (accuracy) lower than 15% for all analytes evaluated. The quantification limit was 5 ng/mL and the linear range was 5-2500 ng/mL for all analytes. The method was used for the determination of drug and metabolites in swine plasma samples and proved to be suitable for pharmacokinetic studies.

Update on the diagnosis and management of neurocysticercosis / Atualização no diagnóstico e manejo da neurocisticercose

ABSTRACT Background: Neurocysticercosis (NCC) is a serious public health problem in several developing countries, including those in Latin America, Asia, and Africa. NCC is considered to be the main cause of late-onset epilepsy in endemic areas. Objective: This review summarizes recent advances in diagnosis and therapy of NCC. Methods: Relevant articles and books were reviewed and used as a source of information for this review. Results: The diagnosis of NCC is based upon neuroimaging studies (MRI and computed tomography) and laboratory analysis of the cerebrospinal fluid (CSF). Praziquantel and albendazole are considered parasiticidal drugs against NCC, but there is an intense debate over the value and safety of these drugs. Conclusion: Given the relative scarcity of clinical trials, more comparative interventional studies, especially randomized controlled trials in long-term clinical evolution, are required in order to clarify the controversy over the validity of parasitic therapy in patients with NCC.

RESUMO Antecedentes: A neurocisticercose (NCC) é grave problema de saúde pública nos países em desenvolvimento, especialmente na América Latina, Ásia e África. A NCC é considerada a principal causa de epilepsia de início tardio nas regiões endêmicas. Objetivo: Este artigo pretende discutir os recentes avanços no diagnóstico e tratamento da NCC. Métodos: Artigos científicos e livros relevantes serviram de fonte de informação para esta revisão. Resultados: O diagnóstico da NCC é fundamentado nos exames de neuroimagem (ressonância magnética e tomografia computadorizada) e do líquido cefalorraquiano (LCR). Atualmente, praziquantel e albendazole são considerados eficazes na terapêutica etiológica da NCC, mas há intenso debate quanto à validade e segurança desses medicamentos. Conclusão: Pela relativa carência de ensaios clínicos, são necessários novos estudos particularmente randomizados, controlados e com análise de desfechos clínicos a longo prazo para o esclarecimento da polêmica envolvendo a validade da terapêutica parasiticida na NCC.

Population pharmacokinetics of oxcarbazepine and its metabolite 10-hydroxycarbazepine in healthy subjects.

Oxcarbazepine is indicated for the treatment of partial or generalised tonic-clonic seizures. Most of the absorbed oxcarbazepine is converted into its active metabolite, 10-hydroxycarbazepine (MHD), which can exist as R-(-)- and S-(+)-MHD enantiomers. Here we describe the influence of the P-glycoprotein (P-gp) inhibitor verapamil, on the disposition of oxcarbazepine and MHD enantiomers, both of which are P-gp substrates. Healthy subjects (n=12) were randomised to oxcarbazepine or oxcarbazepine combined with verapamil at doses of 300mg b.i.d. and 80mg t.i.d., respectively. Blood samples (n=185) were collected over a period of 12h post oxcarbazepine dose. An integrated PK model was developed using nonlinear mixed effects modelling using a meta-analytical approach. The pharmacokinetics of oxcarbazepine was described by a two-compartment model with absorption transit compartments and first-order elimination. The concentration-time profiles of both MHD enantiomers were characterised by a one-compartment distribution model. Clearance estimates (95% CI) were 84.9L/h (69.5-100.3) for oxcarbazepine and 2.0L/h (1.9-2.1) for both MHD enantiomers. The volume of distribution was much larger for oxcarbazepine (131L (97-165)) as compared to R-(-)- and S-(+)-MHD (23.6L (14.4-32.8) vs. 31.7L (22.5-40.9), respectively). Co-administration of verapamil resulted in a modest increase of the apparent bioavailability of oxcarbazepine by 12% (10-28), but did not affect parent or metabolite clearances. Despite the evidence of comparable systemic levels of OXC and MHD following administration of verapamil, differences in brain exposure to both moieties cannot be excluded after P-glycoprotein inhibition.

Distribution of human T cell lymphotropic virus type 1 (HTLV-1) subtypes in Brazil: genetic characterization of LTR and tax region.

We report the molecular and epidemiological characterization of 128 human T cell lymphotropic virus type 1 (HTLV-1) isolates from Brazilian patients with different clinical manifestations of the infection. Thirty-two percent of the patients were asymptomatic, 44% had HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and 23% had adult T cell leukemia/lymphoma (ATLL). Phylogenetic analysis performed using part of the LTR region of the viral genome revealed that all Brazilian isolates belonged to the Cosmopolitan subtype, with the following distribution within the Transcontinental subgroup: 81.6% within the Latin American cluster and 15.8% outside the Latin American cluster. Two isolates belonged to the Japanese subgroup. Molecular analysis of the tax region showed a high nucleotide similarity ( approximately 99%) with 41 prototype sequences, including the ATK-1 isolate. The mean number of nucleotide substitutions ranged from 1 to 8. Five specific nucleotide substitutions, C7401T, T7914C, C7920T, C7982T, and G8231A, were highly conserved among the Brazilian isolates (79.6%), with a frequency ranging from 81.6% to 100% in the sample group and from 18.4% to 24.1% in the prototypes used, suggesting the existence of a molecular signature. These changes were not correlated with a specific clinical status of the patients and could be a molecular characteristic of the HTLV-1 strains that circulate in Brazil.

[Cloning and transmembrane glycoprotein expression of the retrovirus HTLV-1 in mammals' cells]. / Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos.

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.