Low switched memory B cells are associated with no humoral response after SARS-CoV-2 vaccine boosters in kidney transplant recipients.

Introduction: The humoral response after SARS-CoV-2 vaccination and boosters in kidney transplant recipients (KTRs) is heterogeneous and depends on immunosuppression status. There is no validated immune measurement associated with serological response in clinical practice. Multicolor flow cytometric immunophenotyping could be useful for measuring immune response. This study aimed to study B- and T-cell compartments through Standardized EuroFlow PID Orientation after SARS-CoV-2 vaccination and their association with IgG SARS-CoV-2 seropositivity status after two doses or boosters. Methods: We conducted a multicenter prospective study to evaluate humoral response after SARS-CoV-2 vaccination in KTRs. Heterologous regimen: two doses of inactivated SARS-CoV-2 and two boosters of BNT162b2 mRNA (n=75). Homologous vaccination: two doses of BNT162b2 mRNA and one BNT162b2 mRNA booster (n=13). Booster doses were administrated to KTRs without taking into account their IgG SARS-CoV-2 seropositivity status. Peripheral blood samples were collected 30 days after the second dose and after the last heterologous or homologous booster. A standardized EuroFlow PID Orientation Tube (PIDOT) and a supervised automated analysis were used for immune monitoring cellular subsets after boosters. Results: A total of 88 KTRs were included and divided into three groups according to the time of the first detected IgG SARS-CoV-2 seropositivity: non-responders (NRs, n=23), booster responders (BRs, n=41), and two-dose responders (2DRs, n=24). The NR group was more frequent on mycophenolate than the responder groups (NRs, 96%; BRs, 80%; 2DRs, 42%; p=0.000). Switched memory B cells in the 2DR group were higher than those in the BR and NR groups (medians of 30, 17, and 10 cells/ul, respectively; p=0.017). Additionally, the absolute count of central memory/terminal memory CD8 T cells was higher in the 2DR group than in the BR and NR groups. (166, 98, and 93 cells/ul, respectively; p=0.041). The rest of the T-cell populations studied did not show a statistical difference. Conclusion: switched memory B cells and memory CD8 T-cell populations in peripheral blood were associated with the magnitude of the humoral response after SARS-CoV-2 vaccination. Boosters increased IgG anti-SARS-CoV-2 levels, CM/TM CD8 T cells, and switched MBCs in patients with seropositivity after two doses. Interestingly, no seropositivity after boosters was associated with the use of mycophenolate and a lower number of switched MBCs and CM/TM CD8 T cells in peripheral blood.

Case Report: Mycosis fungoides as an exclusive manifestation of common variable immunodeficiency in a family with a NFKB2 gene mutation.

Background: Common variable immunodeficiency disorders (CVIDs), which are primary immunodeficiencies characterized by the failure of primary antibody production, typically present with recurrent bacterial infections, decreased antibody levels, autoimmune features, and rare atypical manifestations that can complicate diagnosis and management. Although most cases are sporadic, approximately 10% of the patients may have a family history of immunodeficiency. Genetic causes involving genes related to B-cell development and survival have been identified in only a small percentage of cases. Case presentation: We present the case of a family with two brothers who presented with mycosis fungoides as an exclusive symptom of a common variable immunodeficiency disorder (CVID). Whole-exome sequencing of the index patient revealed a pathogenic variant of the NFKB2 gene. Based on this diagnosis and re-evaluation of other family members, the father and brother were diagnosed with this rare immune and preneoplastic syndrome. All CVID-affected family members presented with mycosis fungoides as their only symptom, which is, to the best of our knowledge, the first case to be reported. Conclusion: This case highlights the importance of high-throughput sequencing techniques for the proper diagnosis and treatment of hereditary hematological disorders.

Endothelium-biomarkers for postthrombotic syndrome: a case-control study.

BACKGROUND: The postthrombotic syndrome (PTS) is a long-term complication of deep venous thrombosis (DVT). Increase knowledge on the PTS pathophysiology and novel biomarkers are needed in order to predict PTS development and to improve treatment results. The aim of this study was to analyze novel endothelium-biomarkers for PTS in patients with DVT out of the acute phase. METHODS: A case-control study was conducted. Inclusion criteria were symptomatic and confirmed DVT patients treated with anticoagulants for at least 3 months. Villalta score was performed at the time of inclusion and used to diagnose and classify the severity of PTS. Plasma inter-cellular adhesion molecule 1 (ICAM-1), P-selectin, fractalkine and vascular endothelial growth factor (VEGF) were quantified using cytometric bead array. Endothelial progenitor cells (EPCs) and circulating endothelial cells (CEC) level were quantified by flow cytometry. RESULTS: Thirty two patients and 61 controls were included. PTS patients showed higher levels of CEC (0.56/µl (0.34-1.5) vs. 0.20/µl (0.11-0.77); P  = 0.04) and EPC (0.75/µl (0.38-1.52) vs. 0.09/µl (0.05-0.82); P  = 0.0021) compared to no PTS patients. Patients with PTS had significantly higher levels of fractalkine (387.60 pg/ml (222.30-597.90) vs. 98.00 pg/ml (82.30-193.02); P  = 0.044) than patients without PTS. Fracktalkine levels showed a strong linear correlation with Villalta score, r  = 0.86, P  < 0.0001. No differences were observed in P-selectin, ICAM-1 and VEGF between studied groups. CONCLUSIONS: The formation and early resolution of DVT are characterized by inflammation and endothelial/platelet activation. We have identified possible novel biomarkers such as CEC, EPC and fractalkine for the development of PTS. These results suggest a possible role of these mediators in the maintenance and worsening of PTS turning them into potential therapeutic targets.

Circulating T regulatory cell subsets in patients with untreated lung cancer

IntroductionRegulatory T Cells (Tregs) play an important role in carcinogenesis and tumor immunoediting by preventing the development of effective antitumor immunity. Several reports showed that circulating Tregs are increased in patients with solid tumors, including lung cancer. Treg population could be categorized into “naive,” “effector,” and “memory” subtypes, bearing potential unique functions. However, the data regarding the prognostic impact of these Tregs subtypes is limited in lung cancer. The aim of this study was to investigate the frequency of different circulating Tregs subtypes in lung cancer and their correlation with clinical outcomes.MethodsWe analyzed the frequency of circulating CD4, CD8 and, Tregs lymphocytes in 66 patients with lung cancer and 32 healthy controls using flow cytometry. Circulating Tregs subtypes: naïve (CD3+ , CD4+ , CCR4+ , CD25+ and CD127low, CD45RO−), memory (CD3+ , CD4+ , CCR4+ , CD25+ and CD127low, CD45RO+) and the expression of the activation marker HLA-DR were correlated with overall survival.ResultsThe percentage and the absolute number of total, memory and activated Tregs was significantly higher in lung cancer patients than healthy controls. Patients with a Tregs percentage higher than 5.4% and higher than 20% of HLA-DR + Tregs had worse overall survival than those with lower levels.ConclusionsCirculating Tregs and activated Tregs are a potential prognostic factor in patients with lung cancer treated with conventional therapy and could be considered a predictive biomarker in patients not eligible for immune blockade treatments. Additionally, it will be interesting to study these Tregs subsets for immune treatments in future clinical trials. (AU)

Circulating T regulatory cell subsets in patients with untreated lung cancer.

INTRODUCTION: Regulatory T Cells (Tregs) play an important role in carcinogenesis and tumor immunoediting by preventing the development of effective antitumor immunity. Several reports showed that circulating Tregs are increased in patients with solid tumors, including lung cancer. Treg population could be categorized into "naive," "effector," and "memory" subtypes, bearing potential unique functions. However, the data regarding the prognostic impact of these Tregs subtypes is limited in lung cancer. The aim of this study was to investigate the frequency of different circulating Tregs subtypes in lung cancer and their correlation with clinical outcomes. METHODS: We analyzed the frequency of circulating CD4, CD8 and, Tregs lymphocytes in 66 patients with lung cancer and 32 healthy controls using flow cytometry. Circulating Tregs subtypes: naïve (CD3+ , CD4+ , CCR4+ , CD25+ and CD127low, CD45RO-), memory (CD3+ , CD4+ , CCR4+ , CD25+ and CD127low, CD45RO+) and the expression of the activation marker HLA-DR were correlated with overall survival. RESULTS: The percentage and the absolute number of total, memory and activated Tregs was significantly higher in lung cancer patients than healthy controls. Patients with a Tregs percentage higher than 5.4% and higher than 20% of HLA-DR + Tregs had worse overall survival than those with lower levels. CONCLUSIONS: Circulating Tregs and activated Tregs are a potential prognostic factor in patients with lung cancer treated with conventional therapy and could be considered a predictive biomarker in patients not eligible for immune blockade treatments. Additionally, it will be interesting to study these Tregs subsets for immune treatments in future clinical trials.

Mobilized and apheresis-collected endothelial progenitor cells with plerixafor.

BACKGROUND: Endothelial progenitor cells (EPCs) are immature cells able to proliferate and contribute to endothelial repair, vascular homeostasis, neovascularization, and angiogenesis. It therefore seems likely that circulating EPCs have therapeutic potential in ischemic and vascular diseases. In this study we evaluated the efficiency of EPC mobilization and collection by large volume leukapheresis in subjects with hematological diseases, treated with plerixafor in association with G-CSF. METHODS: Twenty-two patients with lymphoid malignancies underwent rHuG-CSF and plerixafor treatment followed by leukapheresis. Blood samples before and after treatment and apheresis liquid sample were taken and analyzed by flow cytometry in order to quantified EPC. RESULTS: The percentage of CD34+ cells and EPCs among circulating total nuclear cells (TNCs) increased significantly by approximately 2-fold and 3-fold, respectively, after plerixafor treatment. Consequently, the absolute number of CD34+ cells and EPCs were increased 4-fold after plerixafor treatment. The median PB concentration of EPCs before and after treatment were 0.77/µL (0.31-2.15) and 3.41/µL (1.78-4.54), respectively, P < .0001. The total EPCs collected per patient were 3.3×107 (0.8×107 -6.8×107 ). CONCLUSION: We have shown that plerixafor in combination with G-CSF allows the mobilization and collection of large amounts of EPCs along with CD34+ cells in lymphoid neoplasm patients. The possibility to collect and to store these cells could represent a promising therapeutic tool for the treatment of ischemic complications without the need of in vitro expansion.

Germline CEBPA mutation in familial acute myeloid leukemia.

Myeloid Neoplasms with germline predisposition become part of 2016 World Health Organization (WHO) classification of hematological malignancies since 2016. CCAAT/enhancer binding protein-alpha (CEBPA) is a myeloid transcription factor located in chromosome 19q. Acute myeloid leukemia (AML) with biallelic mutations of CEBPA AML with recurrent genetic abnormalities according to WHO classification. The inheritance of a germline CEBPA mutation predisposes to the development of AML with autosomal dominant inheritance. Familial CEBPA AML share characteristics with somatic CEBPA AML. However, a higher relapse incidence is reported. We present the case of a 46-years-old male with family history of acute leukemia who was diagnosed with single mutated CEBPA acute myeloid leukemia. The same mutation was found in two of his siblings. The clinical suspicion and proper diagnosis of familial cases is necessary, especially when a related allogenic transplant is indicated in order to select an adequate donor.

Blood cell respiration rates and mtDNA copy number: A promising tool for the diagnosis of mitochondrial disease.

Human mitochondrial diseases are a group of heterogeneous diseases caused by defects in oxidative phosphorylation, due to mutations in mitochondrial (mtDNA) or nuclear DNA. The diagnosis of mitochondrial disease is challenging since mutations in multiple genes can affect mitochondrial function, there is considerable clinical variability and a poor correlation between genotype and phenotype. Herein we assessed mitochondrial function in peripheral blood mononuclear cells (PBMCs) and platelets from volunteers without known metabolic pathology and patients with mitochondrial disease. Oxygen consumption rates were evaluated and respiratory parameters indicative of mitochondrial function were obtained. A negative correlation between age and respiratory parameters of PBMCs from control individuals was observed. Surprisingly, respiratory parameters of PBMCs normalized by cell number were similar in patients and young controls. Considering possible compensatory mechanisms, mtDNA copy number in PBMCs was quantified and an increase was found in patients with respect to controls. Hence, respiratory parameters normalized by mtDNA copy number were determined, and in these conditions a decrease in maximum respiration rate and spare respiratory capacity was observed in patients relative to control individuals. In platelets no decay was seen in mitochondrial function with age, while a reduction in basal, ATP-independent and ATP-dependent respiration normalized by cell number was detected in patients compared to control subjects. In summary, our results offer promising perspectives regarding the assessment of mitochondrial function in blood cells for the diagnosis of mitochondrial disease, minimizing the need for invasive procedures such as muscle biopsies, and for following disease progression and response to treatments.

Lymphotropic Viruses EBV, KSHV and HTLV in Latin America: Epidemiology and Associated Malignancies. A Literature-Based Study by the RIAL-CYTED.

The Epstein-Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV) and human T-lymphotropic virus (HTLV-1) are lymphomagenic viruses with region-specific induced morbidity. The RIAL-CYTED aims to increase the knowledge of lymphoma in Latin America (LA), and, as such, we systematically analyzed the literature to better understand our risk for virus-induced lymphoma. We observed that high endemicity regions for certain lymphomas, e.g., Mexico and Peru, have a high incidence of EBV-positive lymphomas of T/NK cell origin. Peru also carries the highest frequency of EBV-positive classical Hodgkin lymphoma (HL) and EBV-positive diffuse large B cell lymphoma, not otherwise specified (NOS), than any other LA country. Adult T cell lymphoma is endemic to the North of Brazil and Chile. While only few cases of KSHV-positive lymphomas were found, in spite of the close correlation of Kaposi sarcoma and the prevalence of pathogenic types of KSHV. Both EBV-associated HL and Burkitt lymphoma mainly affect young children, unlike in developed countries, in which adolescents and young adults are the most affected, correlating with an early EBV seroconversion for LA population despite of lack of infectious mononucleosis symptoms. High endemicity of KSHV and HTLV infection was observed among Amerindian populations, with differences between Amazonian and Andean populations.

TH17/Treg imbalance and IL-17A increase after severe aneurysmal subarachnoid hemorrhage.

To evaluate cerebrospinal fluid (CSF) and peripheral blood (PB) Treg, TH17 cells, TH1, TH2 and related cytokines in the acute phase of aSAH we assessed TH17, TH1, TH2, T regulatory cells and neutrophils in 39 aneurysmal subarachnoid hemorrhage (aSAH) patients and 56 controls. PB TH17 cells and TH17/Treg ratio were higher in CSF and PB of aSAH patients. Serum and CSF IL-17A levels were increased in aSAH. Serum IL-17A levels were associated with vasospasm and ICU mortality. Study results support the role of TH17/IL17 axis in aSAH pathogenesis, turning it into a potential clinical biomarker and a novel target for research.

Flow cytometry "Ogata score" for the diagnosis of myelodysplastic syndromes in a real-life setting. A Latin American experience.

INTRODUCTION: Flow cytometry (FC) is a helpful tool for the diagnosis of myelodysplastic syndrome (MDS). Different FC score systems have been developed. The "Ogata score" is a simple diagnostic score that has been validated having a sensitivity of 69% and a specificity of 92% in low-risk MDS. We aimed to study the feasibility and the utility of the "Ogata score" for the diagnosis of MDS among Latin America (LA) Laboratories. METHODS: This is a case and control study conducted in LA institutions members of Grupo Latinoamericano de Mielodisplasia (GLAM). A total of 146 MDS patients and 57 control patients were included. "Ogata score" was calculated. RESULTS: The sensitivity of "Ogata score" was 75.6% (95% CI, 66.8-81.3), specificity was 91.2% (95% CI, 79.7-96.7), PPV was 95.6% (95% CI, 88.5-98.3), and NPV was 65.4% (95% CI, 49.1-71.9). In low/intermediate-1 IPSS patients group, the sensitivity was 70.1% (95% CI, 60.2-78.2), specificity was 91.2% (CI-95%, 79.7-96.7), PPV was 94.2% (95% CI, 86.4-97.8), and NPV was 62.1% (95% CI, 53.0-78.7). In the group of patients "without MDS specific markers" (patients without ring sideroblasts, blast excess, or chromosomal abnormalities), the sensitivity was 66.7% (CI-95%, 55.8-76.0), specificity was 91.2% (95% CI, 79.7-96.7), PPV was 92.3% (95% CI, 82.2-97.1), and NPV was 63.5% (95% CI, 51.9-73.5). CONCLUSIONS: The diagnostic power found in this study was similar to the reported by Della-Porta et al. Also in LA, the analysis was made in modern equipment with acquisition of at least 100 000 events which permits a good reproducibility of the results.

Activity of the Calcineurin Pathway in Patients on the Liver Transplantation Waiting List: Factors of Variability and Response to Tacrolimus Inhibition.

BACKGROUND: We sought to evaluate, in patients on a liver transplantation waiting list, potential biomarkers of the base calcineurin pathway activity with use of a new model of nonstimulated peripheral blood mononuclear cells (PBMC) and ex vivo response to tacrolimus (TAC). METHODS: The calcineurin pathway activity was explored ex vivo in stimulated and nonstimulated PBMC from 19 patients. The inhibition of NFAT1 translocation to PBMC nuclei, expression of intracellular IL-2, and membrane CD25 in different T-cell subsets were measured by multiparametric flow cytometry before and after exposure to TAC. We also studied the influence on the individual response of polymorphisms in 3 key genes of the calcineurin pathway: PPIA, PPP3CA, and IL2RA. RESULTS: All pharmacodynamics profiles closely fitted an I/Imax sigmoid model. Interindividual variability was higher in nonstimulated than in stimulated conditions, as well as in the presence of TAC. IL-2+CD8+ cells at TAC Imax showed the highest interindividual variability, suggesting its usefulness as a biomarker of individual TAC effects integrating many different sources of regulation and variability. Moreover, in the absence of TAC, patients with end-stage liver disease exhibited lower NFAT1 translocation and T-cell activation than healthy volunteers from a previous study under similar conditions. Multivariate statistical analysis showed strong and significant associations between TAC pharmacodynamic parameters and 2 polymorphisms in the gene-coding cyclophilin A (rs8177826 and rs6850). CONCLUSIONS: We show the feasibility of using nonstimulated PBMCs to explore the calcineurin pathway under more physiologic conditions and point toward potential biomarkers for TAC pharmacodynamic monitoring. ClinicalTrials.gov Identifier: NCT01760356.

[Adult T-cell leukemia/lymphoma. Report of a case in Uruguay]. / Un caso de leucemia/linfoma T del adulto en Uruguay.

Adult T-cell leukemia/lymphoma belongs to the group of mature T-cell malignancies according to the WHO classification. It constitutes a rare entity and has a strong association with infection by human T-lymphotropic virus 1. In Uruguay, this viral infection is very infrequent and, to our knowledge, no case of adult T-cell leukemia/lymphoma has been previously reported. We describe the case of a woman, immigrant from Peru, who presented with persistent lymphocytosis, intestinal parasitic diseases, and skin involvement. The diagnosis was delayed and the patient died before initiating oncological treatment. We therefore emphasize the relevance of an early clinical suspicion and serology for this virus, especially in patients coming from endemic countries like Peru.

Un caso de leucemia/linfoma T del adulto en Uruguay / Adult T-cell leukemia/lymphoma. Report of a case in Uruguay

La leucemia/linfoma T del adulto pertenece al grupo de neoplasias T maduras y constituye una entidad clínica de baja incidencia. Su etiopatogenia se asocia a la infección por el virus linfotrópico humano 1. En Uruguay se registra una incidencia muy baja de infección por este virus y no se ha comunicado a la fecha ningún caso de leucemia/linfoma T del adulto. Presentamos el caso de una inmigrante de Perú, quien se presentó con linfocitosis sostenida, múltiples parasitosis intestinales y compromiso cutáneo. El diagnóstico fue tardío y la paciente falleció antes de iniciar tratamiento oncoespecífico. Destacamos la importancia de la sospecha clínica de esta entidad y el estudio de la serología para el virus, en particular en casos, como el nuestro, procedentes de área endémica.

Adult T-cell leukemia/lymphoma belongs to the group of mature T-cell malignancies according to the WHO classification. It constitutes a rare entity and has a strong association with infection by human T-lymphotropic virus 1. In Uruguay, this viral infection is very infrequent and, to our knowledge, no case of adult T-cell leukemia/lymphoma has been previously reported. We describe the case of a woman, immigrant from Peru, who presented with persistent lymphocytosis, intestinal parasitic diseases, and skin involvement. The diagnosis was delayed and the patient died before initiating oncological treatment. We therefore emphasize the relevance of an early clinical suspicion and serology for this virus, especially in patients coming from endemic countries like Peru.

Estudio del ratio de FLT3-ITD como factor pronóstico en leucemias agudas mieloides: primeros casos estudiados en Uruguay / Analysis of the FLT3-ITD ratio as a prognostic factor in acute myeloid leukemias. First cases studied in Uruguay

Introducción: en los últimos años ha existido un avance significativo en el conocimiento biológico de la leucemia aguda mieloide (LAM) que se ha traducido en que el tratamiento de los pacientes afectados se realice guiado por el perfil citogenético y molecular. Las duplicaciones internas en tándem del gen FLT3 (FLT3-ITD) representan las mutaciones más frecuentes en LAM y confieren un mal pronóstico en pacientes con riesgo citogenético intermedio. Se ha reportado que la presencia de un ratio FLT3-ITD elevado (relación entre cantidad de alelo portador de ITD y de alelo salvaje) confiere un mayor pronóstico adverso. Objetivo: estandarizar una técnica, no disponible en Uruguay, para determinar el ratio de FLT3-ITD en pacientes portadores de LAM de riesgo citogenético intermedio. Discutir los primeros casos de LAM FLT3+ a los que se realizó el ratio. Material y método: para la detección de FLT3-ITD se amplificó un fragmento correspondiente a los exones 14 y 15 del gen en muestras de médula ósea al debut de la enfermedad. En los casos positivos se determinó el ratio de FLT3-ITD mediante análisis de fragmentos por electroforesis capilar. Resultados: en este trabajo mostramos la estandarización de un método para la determinación del ratio de FLT3-ITD y los primeros casos analizados en nuestro país. Se estudiaron 12 pacientes y se detectó la presencia de FLT3-ITD en tres de ellos. El ratio de FLT3-ITD encontrado fue en dos casos menor a 0,8 y en un caso mayor o igual a 0,8. Conclusiones: disponemos de una técnica de determinación del ratio de FLT3-ITD con importante valor pronóstico para pacientes portadores de LAM.

Abstract Introduction: In recent years, significant progress has been made in the biological knowledge of acute myeloid leukemia (AML), which has been reflected on treatment of affected patients being guided by cytogenetics and molecular profiling. FLT3 internal tandem duplications (FLT3/ITDs) represent the most frequent mutations in AML and confer a bad prognosis in patients with intermediate cytogenetic risk. It has been reported that the presence of a high FLT3-ITD ratio (relationship between number of ITD carrier allele and wild type allele). Objective: To standardize a technique, still not available in Uruguay, to determine the FLT3-ITD ratio in patients carriers of AML of intermediate cytogenetic risk. To discuss the first cases of AML FLT3+ who underwent ratio analysis. Methods: In order to identify FLT3-ITD, the fragment corresponding to exons 14 and 15 of the gene was amplified in bone marrow samples upon debut of the disease. In the cases it was positive, the FLT3-ITD ratio was determined by the analysis of fragments with capillary electrophoresis. Results: This study presented the standardization of a method to determine the FLT3-ITD ratio and the first cases analysed in our country. Twelve patients were studied and the presence of FLT3-ITD was detected in three of them. In two cases, the FLT3-ITD ratio found was below 0.8 and in one case it was greater than or equal to 0.8. Conclusions: We have a technique to determine the FLT3-ITD ratio with an important prognostic value for patients carriers of AML.

Resumo Introdução: nos últimos anos observou-se um avanço significativo do conhecimento biológico da leucemia aguda mieloide (LAM) que fez com que o tratamento destes pacientes seja orientado por seus perfis citogenético e molecular. As duplicações internas no tandem do gen FLT3 (FLT3-ITD) são as mutações mais frequentes na LAM e conferem um mal prognóstico em pacientes com risco citogenético intermediário. Foi descrito que uma proporção de FLT3-ITD elevada (relação entre a quantidade do alelo portador de ITD e do alelo selvagem) está vinculada com um maior prognóstico adverso. Objetivo: padronizar uma técnica, não disponível no Uruguai, para determinar a proporção de FLT3-ITD em pacientes portadores de LAM com risco citogenético intermediário. Discutir os primeiros casos de LAM FLT3+ cuja proporção foi calculada. Materiais e métodos: para a detecção de FLT3-ITD, foi realizada a ampliação de um fragmento correspondente aos exons 14 e 15 do gen em amostras de medula óssea no inicio da doença. Nos casos positivos, a proporção de FLT3-ITD foi determinada usando análise de fragmentos por eletroforese capilar. Resultados: neste trabalho mostramos a padronização de um método para a determinação da proporção de FLT3-ITD e os primeiros casos estudados no nosso país. Foram estudados 12 pacientes e a presença de FLT3-ITD foi determinada em 3. Em dois casos a proporção de FLT3-ITD era menor que 0,8 e em 1 caso maior ou igual a 0,8. Conclusões: contamos com uma técnica de determinação da proporção de FLT3-ITD com importante valor prognóstico para pacientes portadores de LAM.

Lymphocyte subpopulations in myocardial infarction: a comparison between peripheral and intracoronary blood.

The frequency and profile of lymphocyte subsets within the culprit coronary artery were investigated in 33 patients with myocardial infarction and compared to their systemic circulating counterparts. T cell subsets including CD4(+)CD28null, activated and regulatory T-cells, TH1/TH2/TH17 phenotypes, NK and B-cells were studied in intracoronary (IC) and arterial peripheral blood (PB) samples. CD4(+)CD28null T-lymphocytes were significantly increased in IC compared to PB (3.7 vs. 2.9 %, p < 0.0001). Moreover, patients with more than 6 h of evolution of STEMI exhibited higher levels of CD4(+)CD28null T-cells suggesting that this subset may be associated with more intense myocardial damage. The rare NK subpopulation CD3(-)CD16(+)CD56(-) was also increased in IC samples (5.6 vs. 3.9 %, p = 0.006). CD4(+)CD28null T-cells and CD3(-)CD16(+)CD56(-) NK subpopulations were also associated with higher CK levels. Additionally, IFN-γ and IL10 were significantly higher in IC CD4(+) lymphocytes. Particular immune cell populations with a pro-inflammatory profile at the site of onset were increased relative to their circulating counterparts suggesting a pathophysiological role of these cells in plaque instability, thrombi and myocardial damage.

Immune cells subpopulations in cerebrospinal fluid and peripheral blood of patients with Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: There is growing evidence supporting the role of inflammation in aneurysmal subarachnoid hemorrhage (aSAH) pathophysiology and it is of great interest to elucidate which immune mechanisms are involved. METHODS: 12 aSAH patients and 28 healthy controls were enrolled prospectively. We assessed leukocytes subpopulations and their activation status by flow cytometry in cerebrospinal fluid (CSF) and peripheral blood (PB) of SAH patients at the same time and in PB of controls. RESULTS: Monocytes and neutrophils were activated in CSF of aSAH patients. The percentage of CD14(++)CD16(+) monocytes were higher in CSF than in PB of aSAH patients, and were also increased in PB of aSAH patients compared with controls. An enhanced expression of CD69 was shown in CSF neutrophils compared with PB in aSAH patients. PB of aSAH patients showed lower percentage of total lymphocytes compared with controls PB. Additionally, lymphocytes were activated in CSF and PB of aSAH patients. CD4(+) and CD8(+) T cells had a decreased expression on CD3 and higher levels of CD69 in CSF compared with PB in aSAH patients. Moreover, PB CD4(+) and CD8(+) T cells of aSAH patients were activated compared with controls. Additionally, CD28 expression was decreased on CSF T lymphocytes. CONCLUSIONS: Our data suggest an important recruitment of leukocytes to the site of injury in aSAH as well as an increased activation at this level. Overall, these results indicate that aSAH probably stimulates both the innate and adaptive immune responses.

Salmonella enterica serovar Typhimurium immunotherapy for B-cell lymphoma induces broad anti-tumour immunity with therapeutic effect.

Despite the efficacy of current immune-chemotherapy for treatment of B-cell non-Hodgkin lymphoma, a substantial proportion of patients relapse, highlighting the need for new therapeutic modalities. The use of live microorganisms to develop anti-tumoural therapies has evolved since Coley's toxin and is now receiving renewed attention. Salmonella Typhimurium has been shown to be highly effective as an anti-tumour agent in many solid cancer models, but it has not been used in haemato-oncology. Here, we report that intra-tumoural administration of LVR01 (attenuated S. Typhimurium strain with safety profile) elicits local and systemic anti-tumour immunity, resulting in extended survival in a lymphoma model. LVR01 induces intra-tumoural recruitment of neutrophils and activated CD8(+) T cells, as well as increasing the natural killer cell activation status. Furthermore, a systemic specific anti-tumour response with a clear T helper type 1 profile was observed. This approach is an alternative therapeutic strategy for lymphoma patients that could be easily moved into clinical trials.

Estudio de ploidía de células plasmáticas por citometría de flujo en pacientes conmieloma múltiple: primeros casos estudiados en Uruguay / Flow cytometry for plasma cells ploidy analysis in patients with multiple myeloma: first cases studied in Uruguay

Introducción: la historia natural del mieloma múltiple (MM) es heterogénea con sobrevidas que van desde pocas semanas a más de 20 años. El análisis de los factores pronósticos es esencial para establecer una terapéutica adaptada al riesgo. El estudio de la ploidía de las células plasmáticas es un factor que ha demostrado tener un importante valor pronóstico. Objetivo: estandarizar una técnica, no disponible en Uruguay, para determinar la ploidía de las células plasmáticas por citometría de flujo. Material y método: el estudio de ploidía se realizó en médula ósea utilizando para la marcación de las células plasmáticas los anticuerpos monoclonales anti CD38 y CD138. Para el estudio del contenido del ácido desoxirribonucleico (ADN) se utilizó ioduro de propidio. En el análisis se calculó el índice de ADN (cociente entre la moda del pico correspondiente a la cantidad de ADN de las células plasmáticas en fase Go/G1 y la moda del pico Go/G1 de las células normales residuales). Resultados: en este trabajo mostramos la estandarización de la determinación de ploidía por citometría de flujo y los primeros casos analizados en nuestro país. Se estudiaron nueve pacientes con diagnóstico de MM, hallándose dos casos hipoploides (no hiperploide), un caso diploide (no hiperploide) y seis casos hiperploides. Conclusiones: disponemos de una técnica de determinación de ploidía de células plasmáticas que es sencilla, rápida de realizar y con importante valor pronóstico para pacientes portadores de MM.

Introduction: the natural history of multiple myeloma (MM) is heterogeneous, survival rates ranging from a few weeks to over 20 years. Analysis of prognostic factors is essential to decide on a therapy that is adapted to specific risks. Plasma cells ploidy analysis has proved to be a high prognostic value factor.Objective: to standardize a technique, unavailable in Uruguay, consisting in flow cytometry for plasma cells ploidy analysis in order to determine ploidy values in plasma cells.Method: ploidy analysis was performed in the bone marrow, and monoclonal anti-CD38 and CD-138 antibodies were used to mark plasma cells. Propidium iodide was used to study the content of deoxyribonucleic acid (DNA). The DNA was calculated in the analysis (the ratio of the peak mode corresponding to the DNA present in the plasma cells during the Go/G1 phase and the Go/G1 peak mode of residual normal cells). Results:the study presented the standardization of flow cytometry ploidy analysis and the first cases analysed in our country. Nine patients with a diagnosis of MMwere studied, having found two hypoploid cases (non-hyperploid), one diploid case (non-hyperploid),and six hyperploid cases.Conclusions: there is a technique for ploidy determination of plasma cells that is simple, fast to perform and has an important prognostic value for patients with MM.

Introdução: a historia natural do mieloma múltiplo (MM) é heterogênea com sobrevidas que variam de poucas semanas a mais de 20 anos. A análise dos fatores prognósticos é fundamental para estabelecer uma terapêutica adaptada ao risco. O estudo da ploidia das células plasmáticas é um fator que mostrou ter valor prognóstico importante. Objetivo: padronizar uma técnica, não disponível no Uruguai, para determinar a ploidia das células plasmáticas por citometria de fluxo. Material e método: o estudo da ploidia foi reão da determinação da ploidia por citometria de fluxo e os primeiros casos analisados no nosso país. Nove pacientes com diagnóstico de MM foram estudados, sendo encontrados dois casos hipoploides (não hiperploide), um caso diploide (não hiperploide) e seis casos hiperploides. Conclusões: dispomos de uma técnica de determinação de ploidía de células plasmáticas que é simples, rápida de realizar e com valor prognóstico importante para pacientes portadores de MM.

A therapeutic vaccine using Salmonella-modified tumor cells combined with interleukin-2 induces enhanced antitumor immunity in B-cell lymphoma.

Therapeutic vaccination holds potential as complementary treatment for non-Hodgkin's lymphoma (NHL). B-NHL cells are antigen-presenting cells, but they cannot elicit proper antitumor responses because they lack expression of co-stimulatory molecules. Here, we report a novel approach to design improved whole tumor cell vaccines for B-NHL. We demonstrated that Salmonella infection significantly up-regulates CD80, CD86, CD40 and MHC II expression in lymphoma cells, and that therapeutic vaccination with infected and then irradiated lymphoma cells combined with IL-2 elicits strong anti-tumor specific immunity and extended survival in lymphoma-bearing mice. This may represent the basis of an effective immunotherapy against B-NHL that could be easily translated into the clinics.