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Introduction: DLBCL presents clinical, biological, and prognostic heterogeneity. The composition of the tumor TME strongly contributes for its biological diversity, being able to predict treatment response and determine prognosis. DLBCL with TME enriched in CD8+T-lymphocytes and NK cells demonstrate increased OS compared to those with TME enriched in histiocytes. Data from patients with solid tumors have established associations between absolute quantification of lymphocyte subpopulations (LS) in peripheral blood (PB) and prognosis. Therefore, it is hypothesized that the cellular constituents of PB may mirror the TME, and their quantification may constitute an easy and fast tool to access biological biomarkers predictive of therapeutic response and prognosis in malignancies. This study aims to determine the absolute quantification of LS in the PB of DLBCL patients, seeking associations with outcomes and specific clinical phenotypes. Methods: This study involved 54 newly-diagnosed DLBCL patients. PB collection was performed before starting any therapy, for blood count and immunophenotyping on a BD Facs Calibur flow cytometer. End points included OS and EFS. Survival curves were constructed using the KM method. The Mann-Whitney test and the Kruskal-Wallis test were used to establish the relationship between the lymphocyte profile, clinical variables, therapeutic response and outcomes. The Contal O'Quingley test was used to determine cut-offs for each LS capable of discriminating outcomes. Univariate analysis was performed using the Cox method and a p-value ≤ 0.05 was considered statistically significant. Results: The median age at diagnosis was 54 years and 55.6% were female. Clinical stage III/IV was observed in 77.8%, 57.4% had bulky ≥ 7 cm, 63.0% had B-symptoms, and 53.7% presented IPI ≥ 3. The ORR was 80.8%. With a median follow-up of 21.3 months, the estimated 2-year OS and EFS were 73.3% and 64.7%. The median absolute values obtained for the PB lymphocyte subpopulations were: 1026 cells for CD3+ T-lymphocytes, 591 cells for CD4+ T-helper lymphocytes, 412 cells for CD8+ T-cytotoxic lymphocytes, 1.6 for CD4/CD8 ratio, 240 cells for T-reg lymphocytes, 112 cells for LG T-lymphocytes, 173 cells for NK cells, and 70 cells for B-lymphocytes. In univariate analysis, using a cut-off value = 367 CD8+T-cells/mm3 and 141 NK-cells/mm3, cytotoxic T-lymphodepletion was associated with decreased OS (p = 0.005) and EFS (p < 0.001), as well as NK-lymphodepletion was associated with decreased EFS (p = 0.033), but not with shortened OS (p = 0.240). DLBCL patients presenting higher NK-cell counts had lower rates of chemoresistance to the R-CHOP (p = 0.024), and those with higher NK-cell counts (p = 0.033) and higher CD8+T-lymphocyte counts (p = 0.039) had lower probability to develop relapse, progression or death, confirming the central role of these PB cellular constituents in anti-tumor immunity. We observed that B-cell lymphodepletion was associated with an adverse clinical phenotype, including higher frequency of bulky disease (p = 0.009), B-symptoms (p < 0.001) and advanced clinical stage (p = 0.033). Conclusion: In newly-diagnosed DLBCL, the lymphodepletion of CD8+T-cells and NK cells quantified in PB were associated with decreased OS and EFS. Similarly, peripheral T-cytotoxic and NK lymphodepletion were associated with absence of response to the R-CHOP regimen, as well as higher probability of relapse, progression or death. Therefore, the absolute quantification of these LS in PB may be considered potential biomarkers capable of predicting response and prognosis in DLBCL. Also, we believe it may mirror the cellular composition of the immune TME.

Introduction: DLBCL commonly affects the older population. Elderly patients often present high-risk molecular profile, lower tolerability to chemotherapy, and poor outcomes. In this setting, attenuated therapeutic regimens, such as R-miniCHOP and R-miniCHOP of the elderly, have emerged for this fragile population. Albeit its relevance, response rates, clinical outcomes, and toxicities of these regimens remain poorly understood, because these individuals are usually excluded from controlled trials. Therefore, this study aims to assess the outcomes, determine survival predictors, and compare responses and toxicities between R-CHOP, R-miniCHOP, and R-miniCHOP of the elderly protocols in the management of elderly DLBCL patients. Methods: This retrospective, observational, and single-center study involved 185 DLBCL patients older than 70 years, treated at USP, from 2009 to 2020. Endpoints included ORR, OS, PFS. Survival curves were constructed using the KM method and the Log-Rank test was used to assess the relationship between variables and outcomes. The chi-square test and the Kruskal-Wallis test were applied to assess statistically significant differences in clinical characteristics, adverse event profile, and responses between different treatment modalities. Univariate analysis was performed using the Cox test and multivariate analysis by Cox regression method. The results were presented in HR and 95% CI, and a p-value ≤0.05 was considered significant. Results: The median age at diagnosis was 75 years, and 58.9% were female. Comorbidities were prevalent, including 19.5% with immobility, 28.1% with malnutrition and 24.8% with polypharmacy. Advanced clinical stage (III/IV) was observed in 72.4%, 48.6% had bulky ≥ 7 cm, 63.2% had B-symptoms, and 67.0% presented IPI ≥ 3. Among the 182 (98.4%) effectively treated cases, 57.1% received R-CHOP, 18.0% R-miniCHOP, 13.2% R-miniCHOP of the elderly, and 1.7% were palliated using R-CVP. The ORR for the whole cohort was 68.1%, with CR achieved in 65.9%. ORR was 72.1% for R-CHOP, 70.6% for R-miniCHOP, and 45.6% for R-miniCHOP of the elderly, p = 0.040. Although R-miniCHOP of the elderly regimen promoted lower ORR, patients in this group had higher rates of unfavorable clinical-laboratory findings, including hypoalbuminemia (p = 0.001), IPI ≥ 3 (p = 0.013), and NCCN-IPI ≥ 3 (p = 0.002). With a median follow-up of 6.3 years, the estimated 5-year OS and PFS were 50.2% and 44.6%, respectively. The estimated 2-year OS was 82.0% for R-CHOP, 67.5% for R-miniCHOP, and 48.1% for R-miniCHOP of the elderly, p = 0.003. The estimated 2-year PFS was 61.1% for R-CHOP, 56.4% for R-miniCHOP, and 20.5% for R-miniCHOP of the elderly, p = 0.005. Although correlated with increased OS and PFS in comparison to attenuated protocols, R-CHOP regimen was associated with higher rates of severe neutropenia (p = 0.003), but not translated into febrile neutropenia (p = 0.907), therapy interruption (p = 0.671), or higher early mortality rates (p = 0.681). In multivariate analysis, age≥75 years (HR: 2.08, p = 0.001), neutrophilia (HR: 2.18, p = 0.007), low lymphocyte/monocyte ratio (HR: 1.98, p = 0.010), and clinical stage III/IV (HR: 2.36, p = 0.003) were predictors of decreased OS. Conclusion: In this large and real-life LATAM cohort, we demonstrated that DLBCL patients older than 70 years still do not have satisfactory outcomes, with half of cases not reaching 5 years of life expectancy after diagnosis. Although a significant portion of older DLBCL patients is highly fragile and ineligible for enhanced regimens, attenuated anthracycle-based protocols promoted remarkably inferior outcomes compared to those achieved by the R-CHOP.

Introduction: FL has heterogeneous biological behavior, variable clinical outcomes, and a natural history marked by histological transformation (HT). The management of FL and its outcomes are highly dependent on staging (CS) and tumor burden (TB). The identification of new molecular biomarkers is crucial, not only to understand FL's biology, but also, can guide better prognostic stratification, and determine new therapeutic targets. This study aims to outline the GEP in tissue samples from FL categorized by CS and TB using a large-scale gene microarray platform with subsequent expansion by RT-qPCR. Methods: This study involved 219 FL cases. FL patients were categorized into early-stage disease (I/II) (ES), advanced-stage (AS) (III/IV) with low tumor burden (LTB), and AS with high tumor burden (HTB) according to GELF criteria. The experimental phase involved 126 FFPE samples. Firstly, 42 samples (12 ES, 11 AS-LTV, 12 AS-HTV, 6 control lymph nodes and 1 control HELA) were submitted to the array using the Clariom D Array Human platform. The identification of differentially expressed genes (DEGs) between the 4 groups used the Transcriptome Analysis Console by the eBay ANOVA method. Bidimensional PCA sought to identify discriminatory DEGs clusters. A comparison between pairs was subsequently performed. The 7 most discriminatory DEGs were selected for a cohort expansion by RT-qPCR. Results: According to CS and TB, 14.6% of FL cases were categorized as ES, 17.8% as AS-LTB, and 67.6% as AS-HTB. With a median follow-up of 80.7 months, the median OS was 208.2, 130.5, and 126.3 months for ES, AS-LTB, and AS-HTB,p = 0.249. The median PFS was 113.3, 110.7, and 59.5 months, for the same groups, p = 0.018. The array identified 135,750 genes comparisons between the control, ES, AS-LTB, and AS-HTB. PCA analysis was not able to identify discriminatory DEGs clusters among the 4 groups. Pairwise comparative analysis identified 1913 DEGs among the 4 groups. Of these, 1759 (91.9%) were DEGs between FL and healthy controls, but only 218 (11.3%) were DEGs among the 3 different FL subgroups, evidencing a low molecular diversity in FL when categorized by CS and TB. In the cohort expansion by qRT-PCR, 7 pre-selected DEGs based on the microarray were tested, including the genes GNG7, ITGAM, AZN1, TIMD4, IL7R, PTGS2, and MTTL10. The comparison between the median expression of these genes among the 3 FL's groups showed that the expression of the ILR7 was downregulated with the progression of the disease, with marked hypoexpression in AS-HTB patients. These data may suggest downregulation of IL7R gene in the FLs natural history. We found that hypoexpression of the GNG7 (p = 0.024) and PTGS2 (p = 0.090) were associated with higher rates of disease progression/relapse, and hypoexpression of the GNG7 (p = 0.006) was associated with a higher probability of HT, highlighting the potential prognostic impact of these molecular biomarkers. Finally, we established phenotypic-genotypic associations, as hypoexpression of ITGAM was associated with higher rates of B-symptoms (p = 0.017) and bulky disease (p = 0.026), as well as hypoexpression of IL7R was correlated with a higher probability of bulky (p = 0.025) and extranodal involvement (p = 0.016). Conclusion: Although CS and TB are strong determinants of the prognostic heterogeneity of FL, this study did not find high molecular diversity by this categorization. We observed a relatively homogeneous GEP with low biological diversity in FL presenting different disease evolution phases. Moreover, new molecular biomarkers potentially implicated in FLs natural history, prognostication and specific clinical phenotypes were revealed.

Unsupervised machine learning techniques are employed to understand patterns and behaviors of variables in databases. Multiple Correspondence Analysis (MCA), an extension of correspondence analysis, can be used to verify the association of categorical variables and their categories. In this study, we evaluated the relationship between clinical-demographic variables of 154 patients with nodal peripheral T-cell lymphoma (PTCL) to understand how these variables relate to the unfavorable clinical outcome, death. Methodology: MCA was used to reduce the dimensionality of the real world database by creating two dimensions, 1 and 2, which were subsequently categorized into group 1 and group 2. Dimension 1, comprising ECOG, IPI, treatment, overall response, remission, and bone marrow transplant, represented about 30% of the total observed variance. Survival analysis was conducted to assess the association between these groups and overall survival (OS) and mortality rate. Results: The categories of dimension 1, group 1 and group 2, were associated with OS and mortality rate, with group 1 being associated with 5.5 months (95% CI: 2.70 – 8.40) of OS, while group 2 had a survival time of 277.20 months (95% CI: 91.21 – 463.12). Moreover, the mortality rate in group 1 was 87% (n = 67) and in group 2 it was 36% (n = 28) (p < 0.001). The risk of death for group 1 was 11.11 times (95% CI: 9.75-18.30; β= 2.41; p < 0.001). Discussion: These findings indicate a significant disparity in survival outcomes based on the clinical-demographic profiles of the patients. Group 1, characterized by poorer clinical indicators, exhibited substantially shorter OS and higher mortality rates. The ability of MCA to effectively reduce dimensionality while preserving the clinical relevance of the variables underscores its utility in identifying key prognostic factors. Conclusion: The MCA technique was effective in reducing the dimensionality of the database, maintaining the clinical characteristics of the variables robustly for use in Cox regression. This approach can aid in the identification of high-risk patient groups and inform treatment strategies aimed at improving clinical outcomes.

Introduction: Peripheral T-cell lymphomas (PTCL) are a rare group of malignancies derived from mature T-cells or natural killer cells, characterized by significant clinical-biological heterogeneity despite being grouped together. These entities have unique clinical, morphological, phenotypic, and molecular signatures. The predominantly nodal presentation group includes the histological variants ALK-positive anaplastic large cell lymphoma (ALK1+ ALCL), ALK-negative anaplastic large cell lymphoma (ALK1- ALCL), angioimmunoblastic T-cell lymphoma (AITL), and PTCL, not otherwise specified (PTCL-NOS). The central pillar of treatment for this subgroup of lymphomas is based on anthracycline-containing polychemotherapy regimens and upfront consolidation with autologous hematopoietic stem cell transplantation, although unfavorable outcomes such as primary refractory disease and early relapses are still common, except for the ALK1+ ALCL subgroup. Methodology: Hence, a multicenter, real-world, ambispective study was conducted, involving reference institutions in Brazil and two in the USA, to evaluate the main clinical-demographic characteristics associated with the outcomes of 179 patients diagnosed with nodal PTCL. Results and discussion: Individuals with nodal PTCL often presented at diagnosis with unfavorable clinical features, including a high frequency of advanced disease (stages III and IV), high tumor burden, poor performance status (ECOG ≥ 2 points), and frequent allocation in high-risk categories of the IPI and PIT prognostic indices. Patients with nodal PTCL included in this study globally exhibited low Complete Response rates, as well as shortened Overall Survival (OS) and Progression-Free Survival (PFS). In this cohort, we did not identify a robust survival benefit from the intensification of the CHOP regimen with the addition of etoposide (CHOEP protocol) or with BV-CHP. Age ≥ 60 years, ECOG ≥ 2, and lack of complete remission after primary therapy centered on CHOP-like chemotherapy were identified as independent prognostic factors associated with worse survival in this cohort represented by the four main histopathological subtypes of nodal PTCL. Conclusion: This analysis demonstrated a high frequency of unfavorable clinical features and poor OS and PFS outcomes in patients with nodal PTCL, regardless of the frontline chemotherapy regimen.

Introduction: Circulating free DNA (cfDNA), particularly circulating tumor DNA (ctDNA), has emerged as a pivotal tool for diagnosis, response assessment, and monitoring of various malignancies. However, in most cancers, it has yet to be incorporated into clinical practice due to the lack of reproducible and widely accessible methodological standardization. In this context, numerous studies have been conducted to achieve such standardization. The purpose of this study was to evaluate the quantification of cfDNA and the identification of plasma ctDNA using the Z-scan technique in patients with diffuse large B-cell lymphoma (DLBCL). Methods: Peripheral blood samples were prospectively collected from DLBCL patients at diagnosis, after the fourth treatment cycle, and at the end of R-CHOP-like treatment. Simultaneously, samples from healthy individuals matched by sex and age were collected. After obtaining cfDNA, its concentration was assessed using Nanodrop, Qubit, Bioanalyzer, and Z-scan equipment. The results were compared and correlated with the clinical characteristics of the patients. Results: From January 2018 to December 2022, 54 DLBCL patients were recruited, with a median age of 58 years (ranging from 21 to 91 years), 33 (62.9%) of whom were female. The overall response rate of the cohort was 68.52%, with a median follow-up of 24.6 months, showing an overall survival of 32 months and event-free survival of six months. The concentrations of cfDNA varied significantly among the different reading methodologies used. The peak-to-valley distance (Theta values - Θ) of cfDNA from patients at diagnosis (mean of 0.438) obtained in the Z-scan was statistically distinct between the control group (background) and patients at different collection points. The peak-to-valley distance (Θ) of cfDNA samples collected at diagnosis was higher than that of the control group (background), p = 0.068, those collected after cycle 4 (mean 0.33), p = 0.016, and after treatment completion (mean 0.33), p = 0.0005. Several cut-off points for “Θ” were tested to identify the best discriminatory value between DLBCL and healthy control, with a sensitivity of 64.71% (95% CI: 50.99% to 76.37%) and specificity of 86.61% (95% CI: 62.86% to 93.02%) for Θ > 0.406. No correlations were found between “Θ” values and the clinical and demographic characteristics of the patients. Conclusion: The Z-Scan technique was able to distinguish DLBCL patients from healthy controls, as well as samples obtained during and after treatment from those collected before the start of anti-lymphoma therapy. We hypothesize that this discrimination was possible because the Z-scan technique may have been capable of distinguishing ctDNA from cfDNA.

Introduction: Infection with Human T-Lymphotropic Virus Type 1 (HTLV-1) is endemic in various regions worldwide. Brazil has an estimated 800,000 to 2.5 million infected individuals. This virus is associated with a broad spectrum of clinical manifestations, ranging from asymptomatic infection to severe diseases such as tropical spastic paraparesis/HTLV-1-associated myelopathy (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL). Both conditions are linked to high morbidity and mortality. HTLV-1 can infect and alter the function of immune system cells, particularly CD4+ T lymphocytes, causing abnormalities in genes involved in cellular senescence, proliferation, and apoptosis. This study aimed to evaluate the differential expression of genes regulating senescence and cellular proliferation in asymptomatic HTLV-1 carriers. Methods: Peripheral blood samples were collected from healthy individuals (controls), asymptomatic HTLV-1 carriers, and ATLL patients. Gene expression analysis was performed using RNA extracted from CD4+ T lymphocytes separated by magnetic microbeads. The extracted RNA was converted into cDNA for quantitative real-time polymerase chain reaction (qRT-PCR). Results: A total of 32 asymptomatic HTLV-1 carriers, 30 healthy controls, and 7 ATLL cases were analyzed (N = 69). Of the 32 asymptomatic HTLV-1 carriers, 22 (68.8%) were female; in the control group, 16 (53.3%) were female, and in the ATLL group, 5 (71.4%) were female. The age of asymptomatic HTLV-1 carriers ranged from 20 to 87 years (median 58 years), from 31 to 66 years (median 52 years) in the control group, and from 43 to 79 years (median 64 years) in the ATLL group. Three (43%) patients were classified as having smouldering ATLL, and 1 (14.3%) as acute ATLL. The genes CDKN1A, PIK3CA, and RBL2 were underexpressed in asymptomatic HTLV-1 carriers. ATLL patients exhibited underexpression of CDKN1A and overexpression of COL3A1. Conclusion: Asymptomatic HTLV-1 carriers showed underexpression of CDKN1A, PIK3CA, and RBL2 in CD4+ T lymphocytes. CDKN1A was also significantly underexpressed in ATLL. Asymptomatic carriers and ATLL patients did not show differential expression of the genes AKT1, ID1, IFNG, NFKB1, PTEN, TERT, TWIST1, and SPARC. COL3A1 was overexpressed in ATLL patients.

ABCs and LRP transport proteins are involved in several metabolic processes, among which the transport of lipids and xenobiotics can be highlighted. Thus, the overexpression of some of these proteins, such as ABC-G2 and the LRP protein, are associated with the phenotype of resistance to multiple drugs, one of the main causes of relapses and relapses in patients with acute myeloid leukemia. Mesenchymal stem cells (MSC) are multipotent cells present in the bone marrow and are part of the hematopoietic niche. Phenotypic changes in MSCs are related to AML progression. The oxysterols, 7-KC and Triol, are derived from the auto-oxidation of the cholesterol molecule; and have functions in cell signaling, survival and death. Therefore, the objective of this work was to evaluate the action of oxysterols, in subtoxic concentrations, of 7-KC (30 μM, 50 μM and 70 μM) and Triol (20 μM, 30 μM and 40 μM) on the ABCs and LRP transport proteins of MSCs derived from bone marrow from patients with AML (MSC-D), being used as a comparator MSC of bone marrow from healthy individuals (MSC-S). Using the indirect immunofluorescence technique, it was observed that the amount of ABC-A1 protein was higher in MSC-S strains and that treatment with 30 μM of 7-KC increased ABC-A1 only in MSC-S strains. On the other hand, treatment with Triol decreased the concentration of ABC-A1 in MSC-S strains, and this decrease in ABC-A1 was associated with a decrease in the amount of the LXR-β receptor. Furthermore, treatment with 70 μM of 7-KC decreased LRP protein expression in MSC-S strains, while in MSC-D strains there was no difference. Treatment with Triol showed no significant effect on LRP protein expression in MSC-S, only in MSC-D strains, where there was an increase in the concentration of 20 μM, followed by a decrease in the concentrations of 30 μM and 40 μM of Triol. Both 7-KC and Triol treatment increased the concentration of SMO protein in the cytoplasm and its translocation to the nucleus in MSC-S cell lines. This effect was not observed in MSC-D strains. Treatment with Triol increased the concentration of ABC-D4 protein in the cytoplasm. Furthermore, the presence of ABC-D4 protein was observed in the nucleus of MSC-S and MSC-D strains. Our results demonstrated that in the MSCs lineages the effect of 7-KC and Triol is mainly metabolic, in which an increase in the oxidized glutathione content, alteration in the processes of mitochondrial dynamics with induction of genes related to the fission and fusion process in both 7-KC and Triol-treated MSC lines. Increased expression of the BCL2 gene and cytoplasmic survivin was also observed in MSCs treated with 7-KC. In conclusion, it is possible to observe differences in the expression of ABC transporters and LRP between CTM-S and CTM-D strains. Furthermore, at the same concentrations of 7-KC and Triol used, the observed effect was different between MSC-S and MSC-D, which demonstrates that there are different mechanisms between mesenchymal stem cell lines. In summary, our results are characteristic of the metabolic action of the oxysterols 7-KC and Triol on MSCs.

Introduction: Mantle cell lymphoma (MCL) is a rare malignancy with heterogeneous behavior. Despite the therapeutic advances recently achieved, MCL remains incurable. Currently, the standard of care for young and fit patients involves induction immunochemotherapy followed by up-front ASCT. However, the role of more intensive induction regimens, such as those based on high-doses of cytarabine (HDAC) remains controversial in the management of ASCT-eligible patients. Based on this premise, the current study aims to describe clinical and laboratory characteristics, assess outcomes, determine survival predictors, and compare responses between different primary therapeutic strategies, with focus on assessing the impact of HDAC-based regimens on outcomes in ASCT-eligible patients. Methods: This retrospective, observational, and single-center study involved 165 MCL patients, diagnosed and treated at the University of São Paulo, Brazil, from 2010 to 2022. Endpoints included overall OS, EFS, and POD-24 from up-front treatment. Survival curves were constructed using the Kaplan-Meier method and the Log-Rank test was used to assess the relationship between variables and outcomes. Univariate analysis was performed using the semi-parametric Cox test and multivariate analysis by Cox regression method or proportional ratios model. The results were presented in HR and 95% CI, and a p-value ≤ 0.05 was considered statistically significant. Results: The median age at diagnosis was 65 years and 73.9% were male. More than 90% of cases had a classic nodal variant (cnMCL), 76.4% had bone marrow infiltration, and 56.4% presented splenomegaly. Bulky ≥ 7 cm, B-symptoms, ECOG ≥ 2, and advanced-stage III/IV were observed in 32.7%, 64.8%, 32.1%, and 95.8%, respectively. Sixty-four percent of patients were categorized as high-risk according to MIPI score. With a median follow-up of 71.1 months (95% CI: 61.8-80.4) the estimated 2-year OS and EFS were 64.1% (95% CI: 56.2-71.9%) and 31.8% (95% CI: 23.0-40.6%), respectively. The ORR for the whole cohort was 64.7% (95% CI: 57.1-71.7%), with CR achieved in 41.5% (95% CI: 34.1-49.1%). POD-24 was observed in 46.7% (95% CI: 39.1-54.3%), and the overall mortality rate during all follow-up was 58.2% (95% CI: 50.6-65.5%), with disease progression being the main cause of death. Patients treated with R-HDAC-based regimens (43.4%-66/152) had higher ORR (85.9% vs 65.7%, p = 0.007) compared to those receiving R-CHOP (46.0%-70/152), as well as lower POD-24 rates (61.9% vs 80.4%, p = 0.043) and lower mortality (43.9% vs 68.6%, p = 0.004). However, intensified induction regimens using R-HDAC were not associated with a real OS benefit in MCL patients undergoing up-front consolidation with ASCT (2-year OS: 88.7% for R-HDAC plus ASCT vs 78.8% for R-CHOP plus ASCT, p = 0.289). Furthermore, up-front ASCT was independently associated with increased OS (p < 0.001), EFS (p = 0.005), and lower POD-24 rates (p < 0.001) in MCL. Additionally, CNS involvement (HR: 3.12, p = 0.004), tumor lysis syndrome (HR: 2.79, p = 0.026), albumin < 3.5 g/dL (HR: 2.69, p < 0.001), and failure to achieve remission after induction therapy (HR: 3.71, p < 0.001) were predictors of poor OS. Conclusion: In the largest Latin American cohort of MCL reported to date, we confirmed the OS benefit promoted by up-front consolidation with ASCT in young anf fit patients, regardless of the intensity of the immunochemotherapy regimen used in the pre-ASCT induction. Although HDAC-based regimens were not associated with an unequivocal increase in OS, it was associated with higher ORR and lower rates of early-relapses.

Introduction: FL is the most common low-grade B-cell NHL. Although indolent, it has heterogeneous biological behavior and variable clinical outcomes. Histologic transformation (HT) to high-grade B-cell NHL is associated with dismal survival. The therapeutic management of FL and its prognosis are highly dependent on its staging and tumor burden. Here we aimed to describe clinical characteristics, assess outcomes, determine predictors of survival and HT, and compare responses between different therapies applied in a large cohort of FL patients. Methods: This retrospective study involved 223 patients with FL G1-3A, diagnosed at USP, Brazil, from 2006 to 2022. FL patients were categorized into early-stage (I/II) (ES), advanced-stage (AS) (III/IV) with low tumor burden (LTV), and AS with high tumor burden (HTV) according to the GELF criteria. Endpoints were OS, PFS, early-relapse (< 24 months) and HT rates. Survival curves were constructed using the KM method and the Log-Rank test was used to assess the relationship between variables and outcomes. Univariate and multivariate analysis were performed. The results were presented in HR and 95% CI and p ≤ 0.05 was considered significant. Results: The median age at diagnosis was 60 years and 54.8% were female. 15% of cases had ES, 18.4% had AS-LTV, and 66.8% had AS-HTV. BM involvement, leukemic presentation, splenomegaly, and serous effusions occurred in 48.9%, 11.7%, 12.6%, and 21.1%, respectively. Bulky ≥ 7 cm, B-symptoms, ECOG ≥ 2, and involvement of ≥ 4 nodal areas were observed in 44.4%, 51.6%, 15.2%, and 56%, respectively. 49% of patients were categorized as high-risk according to the FLIPI score. With a median follow-up of 79.5 months (95% CI 67.0-92.0) the OS medians were 18 years (95% CI 14.9-21.1), 11 years (95% CI 9.2-12.9), and 10.5 years (95% CI 9.3-11.8) for ES, AS-LTV, and AS-HTV, respectively, p = 0.138. Similarly, the PFS medians were 6.7 years (95% CI 3.6-9.8), 8.4 years (95% CI 2.0-14.8), and 3.5 years (95 CI 1.8-5.3) for ES, AS-LTV, and AS-HTV, respectively, p = 0.171. Early-relapses occurred in 36.3% of cases, being 18.2%, 31.7%, and 41.0% for ES, AS-LTV, and AS-HTV, respectively, p = 0.032. The overall mortality rate was 29.1% for the whole cohort. HT was documented in 16.7% of cases, being 3.0%, 14.6% and 20.1% for ES, AS-LTV, and AS-HTV, respectively, p = 0.053. Among patients with AS-HTV, the R-CHOP regimen did not promote increased OS compared to the R-CVP regimen (p = 0.415), however it was associated with a substantial increase in PFS (p = 0.005). Age ≥ 60 years (HR 1.06, p < 0.001), ≥ 2 comorbidities (HR 3.85, p = 0.014), B-symptoms (HR 2.35, p = 0.015), HT (HR 2.77, p = 0.002) and thrombocytopenia (HR 2.96, p = 0.028) were predictors of poor OS. Similarly, age ≥ 60 years (HR 1.08, p < 0.001), involvelment of ≥ 4 nodal areas (HR 1.27, p < 0.001), and high LDH (HR 1.65, p = 0.002) predicted decreased PFS. Serous effusions (HR 2.09, p = 0.05), albumin < 3.5 g/dL (HR 2.82, p = 0.05), B-symptoms (HR 2.00, p = 0.006), involvement of ≥ 4 nodal areas (HR 1.21, p = 0.014), and BM infiltration (HR 2.11, p = 0.05) were the main predictors for HT. Conclusion: Although it is characteristically an indolent disease, we demonstrated that a significant portion of FL patients have shortened survival. We confirmed this prognostic heterogeneity, particularly considering the clinical staging and tumor burden. Therefore, FL patients with AS-HTV had higher HT rates and early-relapses, both classic adverse prognostic markers, as well as a tendency to higher mortality. Additionally, we identified clinical and laboratory predictors for HT, which may in a near future direct adapted-risk therapeutic strategies.

Introduction: DLBCL is a malignancy with marked clinical, biological and prognostic heterogeneity. Its different outcomes may be predicted by the identification of molecular biomarkers. Therefore, quantification of circulating cell-free DNA (ccfDNA) and circulating tumor DNA (ctDNA) have emerged as minimally invasive methods able to predict its prognosis. However, such measurements are based on the use of costly technologies and with restricted access in clinical practice. Here, we aimed to quantify ccfDNA in DLBCL using capillary electrophoresis (CEF), a low-cost and widely available methodology, in order to establish prognostic associations between its serum concentrations, clinical characteristics and outcomes. Methods: This prospective study involved 54 DLBCL patients, diagnosed at USP, from 2019 to 2020, and 23 healthy-controls. All cases were treated with 6-8 cycles of R-CHOP. Plasma samples were collected from DLBCL at three moments: at diagnosis, after the 4th CT cycle and at the end of treatment. ccfDNA was extracted using the QIAamp MinElute ccfDNA kit and quantified by CEF (Agilent 2100 Bioanalyzer). Comparison between two groups was performed using the Mann-Whitney test, and between three groups using the Kruskall-Wallis test. The cut-off for [ccfDNA] (1147 ng/μl) was determined by ROC methodology. Survival curves were constructed using the KM method and Log-Rank test was used to assess the relationship between variables and outcomes, and p≤0.05 was considered significant. Results: The median age at diagnosis was 57 years and 61.1% were female. Advanced-stage was observed in 59.3%, 29.6% had ECOG ≥ 2, 52.6% had IPI ≥ 3, and 44.4% had bulky ≥ 10 cm. BM infiltration and involvement of ≥ 2 EN sites occurred in 5.6% and 16.8%, respectively. With a median follow-up of 32 months, the medians OS and EFS were 32 months (95% CI 28-35) and 31 months (95% CI 28-34), respectively. The ORR was 76% (95% CI 63.5-86). The overall mortality rate was 20.4% (95% CI 11.1-32.3). ccfDNA concentrations were significantly higher in DLBCL compared to controls (p < 0.001), however, it did not differ significantly between samples from DLBCL collected at the 3 different time points (p = 0.602). We observed positive associations between ccfDNA [ ] in diagnostic DLBCL samples and biomarkers of high tumor burden, including higher LDH levels (p = 0.003), bulky (p = 0.011), ECOG ≥ 2 (p < 0.001), and IPI ≥ 3 (p = 0.069). However, ccfDNA[ ] at diagnosis were not statistically associated with ORR (p = 0.264), nor with clinical outcomes, including OS (1-year OS: 92% for [ccfDNA] < 1147 ng/μl vs 76% for [ccfDNA] ≥ 1147 ng/μl, p = 0.217) and EFS (1-year EFS: 92% for [ccfDNA] < 1147 ng/μl vs 69% for [ccfDNA] ≥ 1147 ng/μl, p = 0.270). We believe that the short follow-up time and sample number restriction may have contributed to the lack of statistically significant association between ccfDNA [ ] and responses/outcomes observed. In this sense, cohort expansion and NGS techniques for ctDNA identification are ongoing to assess its potential impact as a prognostic biomarker in our cohort. Conclusion: We demonstrated that ccfDNA [ ] measured by CEF were able to accurately discriminate healthy individuals from DLBCL, highlighting its potential role as a minimally invasive diagnostic biomarker. Furthermore, ccfDNA [ ] at diagnosis in DLBCL patients were positively associated with markers of high tumor burden and adverse prognosis. Our results suggest that measurement of ccfDNA by CEF may be a highly cost-effective methodology to identify high-grade B-cell NHLs and recognize DLBCL subgroups with adverse clinical-prognostic characteristics.

Extensive research in the last few decades has conclusively demonstrated the significant influence of experimental conditions, surfactants, and synthesis methods on semiconductors' properties in technological applications. Therefore, in this study, the synthesis of molybdenum oxide (MoO 3 ) was reported by the addition of 2.5 (MoO 3 &#95;2.5), 5 (MoO 3 &#95;5), 7.5 (MoO 3 &#95;7.5), and 10 mL (MoO 3 &#95;10) of nitric acid, obtaining the respective concentrations of 0.6, 1.10, 1.6, and 0.6 mol L -1 . In this study, all samples were synthesized by the hydrothermal method at 160 °C for 6 h. The materials obtained were structurally characterized by X-ray diffraction (XRD) and structural Rietveld refinement, Raman spectroscopy, and infrared spectroscopy (FTIR), confirming the presence of all crystallographic planes and bands associated with active modes for the pure hexagonal phase ( h -MoO 3 ) when the solution's concentration was 0.6 mol L -1 of nitric acid. For concentrations of 1.10, 1.60, and 2.10 mol L -1 , the presence of crystallographic planes and active modes associated with the formation of mixtures of molybdenum oxide polymorphs was confirmed, in this case, the orthorhombic, monoclinic, and hexagonal phases. X-ray photoelectron spectroscopy reveals the occurrence of the states Mo 4+ , Mo 5+ , and Mo 6+ , which confirm the predominance of the acid Lewis sites, corroborating the analysis by adsorption of pyridine followed by characterization by infrared spectroscopy. The images collected by scanning electron microscopy confirmed the information presented in the structural characterization, where microcrystals with hexagonal morphology were obtained for the MoO 3 &#95;2.5 sample. In contrast, the MoO 3 &#95;5, MoO 3 &#95;7.5, and MoO 3 &#95;10 samples exhibited hexagonal and rod-shaped microcrystals, where the latter morphology is characteristic of the orthorhombic phase. The catalytic tests carried out in the conversion of oleic acid into methyl oleate, using the synthesized samples as a heterogeneous catalyst, resulted in conversion percentages of 52.5, 58.6, 69.1, and 97.2% applying the samples MoO 3 &#95;2.5, MoO 3 &#95;5, MoO 3 &#95;7.5, and MoO 3 &#95;10, respectively. The optimization of the catalytic tests with the MoO 3 &#95;10 sample revealed that the conversion of oleic acid into methyl oleate is a thermodynamically favorable process, with a variation in the Gibbs free energy between -67.3 kJ mol -1 and 83.4 kJ mol -1 as also, the energy value of activation of 24.6 kJ mol -1 , for the temperature range from 80 to 140 °C, that is, from 353.15 to 413.15 K, respectively. Meanwhile, the catalyst reuse tests resulted in percentages greater than 85%, even after the ninth catalytic cycle. Therefore, the expressive catalytic performance of the mixture of h -MoO 3 and α-MoO 3 (MoO 3 &#95;10) phases is confirmed, associated with the synergistic effect, mainly due to the increase in the surface area and available Lewis sites of these phases.

Gait variability is related to functional decline in the elderly. The dual-task Timed Up and Go Test (TUG-DT) reflects the performance in daily activities. Objective To evaluate the differences in time to perform the TUG with and without DT in elderly women with different ages and levels of education and physical activity. Method Ninety-two elderly women perfomed the TUG at usual and fast speeds, with and without motor and cognitive DT. Results Increases in the time to perform the TUG-DT were observed at older ages and lower educational levels, but not at different levels of physical activity. More educated women performed the test faster with and without DT at both speeds. When age was considered, significant differences were found only for the TUG-DT at both speeds. Conclusion Younger women with higher education levels demonstrated better performances on the TUG-DT.

Occupational exposure to organic solvents present in paints is responsible for an increased production of reactive species, thus enabling the development of several diseases. Besides, both exo- and endogenous antioxidant defense systems are necessary to avoid oxidative tissue damage. This study investigated possible protective effects of the exo- and endogenous antioxidants on oxidative damage in painters occupationally exposed to organic solvents (n = 42) and controls (n = 28). Retinol, lycopene and β-carotene were significantly lower in the exposed group. Despite the fact that blood toluene was below the biological exposure limits, malondialdehyde levels and antioxidant enzyme activities were increased, whereas reduced glutathione levels were decreased in painters, compared to nonexposed subjects. Moreover, multivariate regression models showed that reduced glutathione and carotenoids (mainly β-carotene) have the major influence on lipid peroxidation (LPO). The present work suggests that the exogenous antioxidants, such as carotenoids, could protect occupationally exposed subjects to xenobiotics from LPO.

Owing to concerns over the effects of the physicochemical properties of nanoparticles and their interaction with biological systems, further investigation is required. We investigated, for the first time, the toxicity of lipid-core nanocapsules (LNCs) containing a polymeric wall of poly(ε-caprolactone) and a coating of polysorbate 80 used as drug delivery devices (~245nm) in Wistar rats after single- and repeated-dose treatments. The suspensions were prepared by interfacial deposition of the polymer and were physicochemically characterized. Toxicological effects were determined after single doses of 18.03, 36.06, and 72.12 × 10(12) LNC/kg and repeated doses of 6.01, 12.02, and 18.03 × 10(12) LNC/kg for 28 days by ip administration. The results for both the treatments showed no mortality or permanent body weight changes during the experiments. A granulomatous foreign body reaction was observed in the liver and spleen of higher dose groups in acute and subchronic treatments. Most of the hepatotoxicity and nephrotoxicity markers were within the reference values and/or were similar to the control group. However, a slight alteration in the hematologic parameters was observed in both the studies. Thus, to verify a possible methodological influence, we performed an in vitro test to confirm such influence. These findings are in agreement with earlier reports regarding no appreciable toxicity of biodegradable polymeric nanoparticles, indicating that LNC might be a safe candidate for drug delivery system. Furthermore, the results presented in this study are important for health risk assessment and to implement strategies for testing biodegradable polymeric nanoparticles.

Background: Ischemia-reperfusion injury is believed to be a major cause of transferred skin flap failure. Cigarette smoking is known to be associated with endogenous antioxidant depletion, hypercoagulability, and cutaneous vasoconstriction. This investigation was carried out to study possible effects of pentoxyfilline or heparin on rat skin reperfusion injury under tobacco exposure., Materials and Methods: Thirty-six rats were randomized into two major groups: 18 were exposed to cigarette smoke during a 4 wk period prior to surgery; the remaining 18 underwent a sham smoking procedure. Each group was further divided into three equal subgroups: heparin, pentoxyfilline, and saline solution. One identical skin flap was raised in each animal. The vasculature of the flap was clamped for 3 h and reperfused for 5 min. A venous blood sample was obtained from the flap after reperfusion for serum malondialdehyde (MDA) and myeloperoxidase (MPO) analysis. Flap survival was assessed 7 d after the procedure., Results: The lipid peroxidation levels and flap necrosis were significantly higher in the cigarette-smoking group skin flaps. There was also a decrease of MPO activity in this group compared with the non-smoking group. Heparin-treated rats had significantly lower MDA levels and showed the most viable percent area among smoking rats., Conclusions: These data suggest that heparin had a significant beneficial effect both on flap survival and on the lipid peroxidation reduction after smoke exposure in the rat axial-pattern skin flap subjected to ischemia and reperfusion injury. Pharmacologic therapy may represent an alternative way to counteract tobacco effects in flap surgery in emergency situations., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Aim: This study aims to describe the incidence of complications on scalp from which a thin split-skin graft was harvested (0.005-0.007 in.) of the donor site in children and adult burn victims., Methods: We reviewed the medical records of 295 burn patients admitted in the Burn Unit of the Clinical Hospital of the Faculty of Medicine of Ribeirão Preto, from January 1998 to December 2007, whose scalps were used as donor site for grafts. Skin-graft thickness varied from 0.005 in. to 0.007 in. The occurrence of pathological healing was evaluated clinically and the time of epithelisation by the main surgeon and a plastic surgeon or a staff nurse., Results: Of the 295 patients whose scalps were used as donor site, 274 were followed from 6 months to 10 years after the procedure (median 18.2 months). Twenty-one patients were lost to follow-up in the first 6 months. No hypertrophic scarring or keloids on the donor site was observed. Five patients (1.82%) presented with folliculitis and two of them were evaluated with small areas of alopecia (0.7%), treated with resection of these areas and primary suture. The average time of epithelisation of the donor site was 7 days., Conclusion: The harvest of thinner split graft from the scalp is a safe procedure., (2009 Elsevier Ltd and ISBI. All rights reserved.)

Objective: To analyze and compare the mutations in two different regions of the katG gene, which is responsible for isoniazid (INH) resistance., Methods: We analyzed 97 multidrug-resistant Mycobacterium tuberculosis strains isolated in cultures of sputum samples obtained from the Professor Hélio Fraga Referral Center, in Brasília, Brazil. Another 6 INH-sensitive strains did not present mutations and were included as controls. We used PCR to amplify two regions of the katG gene (GenBank accession no. U06258)-region 1, (from codon 1 to codon 119) and region 2 (from codon 267 to codon 504)-which were then sequenced in order to identify mutations., Results: Seven strains were resistant to INH and did not contain mutations in either region. Thirty strains carried mutations in region 1, which was characterized by a high number of deletions, especially at codon 4 (24 strains). Region 2 carried 83 point mutations, especially at codon 315, and there was a serine-to-threonine (AGC-to-ACC) substitution in 73 of those cases. The analysis of region 2 allowed INH resistance to be diagnosed in 81.4% of the strains. Nine strains had mutations exclusively in region 1, which allowed the proportion of INH-resistant strains identified to be increased to 90.6%., Conclusions: The number of mutations at codon 315 was high, which is consistent with cases described in Brazil and in other countries, and the analysis of region 1 resulted in a 9.2% increase in the rate at which mutations were identified.

Mining of metallic sulfide ore produces acidic water with high metal concentrations that have harmful consequences for aquatic life. To understand the composition and structure of microbial communities in acid mine drainage (AMD) waters associated with Zn mine tailings, molecular diversity of 16S genes was examined using a PCR, cloning, and sequencing approach. A total of 78 operational taxonomic units (OTUs) were obtained from samples collected at five different sites in and around mining residues in Sepetiba Bay, Brazil. We analyzed metal concentration, physical, chemical, and microbiological parameters related to prokaryotic diversity in low metal impacted compared to highly polluted environments with Zn at level of gram per liter and Cd-Pb at level of microgram per liter. Application of molecular methods for community structure analyses showed that Archaea and Bacteria groups present a phylogenetic relationship with uncultured environmental organisms. Phylogenetic analysis revealed that bacteria present at the five sites fell into seven known divisions, alpha-Proteobacteria (13.4%), beta-Proteobacteria (16.3%), gamma-Proteobacteria (4.3%), Sphingobacteriales (4.3%), Actinobacteria (3.2%) Acidobacteria (2.1%), Cyanobacteria (11.9%), and unclassified bacteria (44.5%). Almost all archaeal clones were related to uncultivated Crenarchaeota species, which were shared between high impacted and low impacted waters. Rarefaction curves showed that bacterial groups are more diverse than archaeal groups while the overall prokaryotic biodiversity is lower in high metal impacted environments than in less polluted habitats. Knowledge of this microbial community structure will help in understanding prokaryotic diversity, biogeography, and the role of microorganisms in zinc smelting AMD generation and perhaps it may be exploited for environmental remediation procedures in this area.

Objective: To analyze the clinical characteristics and evolution of a group of patients with positive sputum cultures for multidrug-resistant (MDR) Mycobacterium tuberculosis and treated at a referral center in the city of Rio de Janeiro, Brazil., Methods: Based on the positive results in sputum cultures for MDR M. tuberculosis, 50 patients were selected, and their clinical data were obtained from the Brazilian Ministry of Health MDR-TB Database. The frequencies of noncompliance, relapses, failures and previous treatments for TB up to diagnosis of MDR-TB were compiled. The radiological patterns were classified as unilateral or bilateral, and with or without cavitation. Two years after the end of the standard treatment for MDR-TB, the outcome (cure, failure, noncompliance or death) for each patient was evaluated and reassessed every two years. The post-treatment follow-up period was eight years., Results: The mean number of previous treatments was 2.3 +/- 0.9. The mean interval between the initial diagnosis and the development of MDR-TB was 2.0 +/- 1.7 years. Two years after the initial treatment for MDR-TB, 2 patients had abandoned treatment, 8 had died, 18 had been cured, and 22 had presented treatment failure. The bivariate analysis showed that bilateral pulmonary involvement and cavitary pattern markedly reduced the chances for cure, with a relative risk of 1-0.6 (40%) and 1-0.7 (30%), respectively. At the end of the follow-up period, 2 patients had abandoned treatment, 9 had presented treatment failure, 17 had been cured, and 22 had died., Conclusions: Bilateral pulmonary involvement and cavity pattern greatly reduced the chances for cure of the patients with MDR-TB. Most patients who presented treatment failure died within the 8-year follow-up period.

Here we investigated the effect of lifelong supplementation of the diet with coconut fat (CO, rich in saturated fatty acids) or fish oil (FO, rich in n-3 polyunsaturated fatty acids) on tumor growth and lactate production from glucose in Walker 256 tumor cells, peritoneal macrophages, spleen, and gut-associated lymphocytes. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) and CO supplemented was approximately 30 g. Supplementation of the diet with FO significantly reduced tumor growth by 76%. Lactate production (nmol h(-1) mg(-1) protein) from glucose by Walker 256 cells in the group fed regular chow (W) was 381.8 +/- 14.9. Supplementation with coconut fat (WCO) caused a significant reduction in lactate production by 1.6-fold and with fish oil (WFO) by 3.8-fold. Spleen lymphocytes obtained from W and WCO groups had markedly increased lactate production (553 +/- 70 and 635 +/- 150) when compared to non-tumor-bearing rats ( approximately 260 +/- 30). FO supplementation reduced significantly the lactate production (297 +/- 50). Gut-associated lymphocytes obtained from W and WCO groups increased lactate production markedly (280 +/- 31 and 276 +/- 25) when compared to non-tumor-bearing rats ( approximately 90 +/- 18). FO supplementation reduced significantly the lactate production (168 +/- 14). Lactate production by peritoneal macrophages was increased by tumor burden but there was no difference between the groups fed the various diets. Lifelong consumption of FO protects against tumor growth and modifies glucose metabolism in Walker tumor cells and lymphocytes but not in macrophages.

In this study we investigated the effect of lifelong supplementation of the diet with coconut oil (CO, rich in saturated fatty acids) or fish oil (FO, rich in n-3 polyunsaturated fatty acids, PUFAs) on tumor growth, animal survival, and metabolic indicators of cachexia in adult rats. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation, and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) was approximately 20 g. These animals displayed cancer cachexia, which was characterized by loss of weight, hypoglycemia, hyperlacticidemia, hypertriacylglycerolemia, and depletion of glycogen stores. Supplementation of the diet with CO did not change these parameters, except that there was a smaller decrease in serum triacylglycerol concentration. Supplementation of the diet with FO significantly decreased tumor growth (by approximately 60%), increased survival (50% at 30 days postinoculation vs. 30% in the controls and 13.5% in the CO group), and prevented the fall in body weight. Furthermore, FO supplementation partly abolished the fall in serum glucose, totally prevented the elevation in serum lactate concentrations, partly prevented the hypertriacylgylcerolemia, and preserved tissue glycogen stores. Lifelong consumption of FO, rich in n-3 PUFAs, protects against tumor growth and cancer cachexia and improves survival.

Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.