RESUMO
Immune thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare disease that seldom occurs in the elderly. Few reports have studied the clinical course of iTTP in older patients. In this study, we have analysed the clinical characteristics at presentation and response to therapy in a series of 44 patients with iTTP ≥60 years at diagnosis from the Spanish TTP Registry and compared them with 209 patients with <60 years at diagnosis from the same Registry. Similar symptoms and laboratory results were described in both groups, except for a higher incidence of renal dysfunction among older patients (23% vs. 43.1%; p = 0.008). Front-line treatment in patients ≥60 years was like that administered in younger patients. Also, no evidence of a difference in clinical response and overall survival was seen in both groups. Of note, 14 and 25 patients ≥60 years received treatment with caplacizumab and rituximab, respectively, showing a favourable safety and efficacy profile, like that observed in patients <60 years.
Assuntos
Púrpura Trombocitopênica Idiopática , Púrpura Trombocitopênica Trombótica , Trombose , Humanos , Idoso , Púrpura Trombocitopênica Trombótica/diagnóstico , Púrpura Trombocitopênica Trombótica/epidemiologia , Púrpura Trombocitopênica Trombótica/terapia , Púrpura Trombocitopênica Idiopática/terapia , Rituximab/uso terapêutico , Trombose/terapia , Troca Plasmática , Sistema de Registros , Proteína ADAMTS13RESUMO
BACKGROUND: Transversus abdominis release (TAR) is a relatively recent surgical technique for ventral hernia repair which allows placement of a large prosthesis in the retro-muscular plane with considerable myofascial medialization. A retrospective review of 100 cases who underwent TAR for complex ventral hernias was performed to evaluate the safety and efficacy of TAR in a series of large ventral hernias. METHODS: Between March 2016 and May 2019, 100 consecutive patients who underwent open TAR were identified from our prospectively maintained database. A retrospective review was performed to analyze patient demographics, peri-operative events, adverse outcomes and recurrence. RESULTS: 12 primary and 88 incisional hernia cases underwent TAR with prosthetic mesh repair during the study period. Mean age was 52.5 years, mean BMI was 30.87 kgs/m2, mean ASA class 1.95. In our series, 41% were diabetic, 11% had COPD. All patients underwent preoperative CT scans. The mean defect was 140.18 cm2. Average mesh area was 1344 cm2. Average blood loss was 245 mL. Defects were bridged in 19% cases despite bilateral component separation. Readmission rate at 1 month was 3%, for wound complications. We recorded 9 surgical site infections, 17 surgical site occurrences, 10 of which needed procedural interventions. We recorded no recurrences at a mean follow-up duration of 20.2 months. CONCLUSIONS: Our early results with TAR are encouraging. We have demonstrated that the repair allows anatomical reconstruction with a large sublay mesh while inflicting minimal morbidity. TAR can be a valuable tool in complex ventral hernia repair.
Assuntos
Parede Abdominal , Hérnia Ventral , Músculos Abdominais/cirurgia , Parede Abdominal/cirurgia , Hérnia Ventral/cirurgia , Herniorrafia/efeitos adversos , Humanos , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Telas Cirúrgicas , Resultado do TratamentoRESUMO
Although many experts position statements on autologous stem cell mobilization have been published, there are some aspects that are still under discussion. A Spanish Hematologist expert group was summoned to settle on agreements and uncertainties on PBSCs mobilization, including factors not always considered; as apheresis and cytometry key factors that determine a successful PBSC collection. This document reviews critical factors that define poor mobilizer patients and the tools to better collect the desired stem cells for a successful autologous haematopoietic stem cell transplant.
Assuntos
Remoção de Componentes Sanguíneos , Células-Tronco de Sangue Periférico , Consenso , Mobilização de Células-Tronco Hematopoéticas , Humanos , Transplante AutólogoRESUMO
BACKGROUND: The optimal blood management after cardiac surgery remains controversial. Moreover, blood transfusions may have an impact on long-term outcomes. OBJECTIVE: The aim of this study is to characterize the impact of liberal red blood cell transfusions on Health-Related Quality of life (HRQoL) after cardiac surgery. METHODS: We studied a cohort of 205 consecutive patients after ICU discharge. Baseline characteristics and clinical data were recorded, and HRQoL was assessed using the EuroQoL-5D instrument, applied 6 months after ICU discharge. A specific question regarding the improvement in the quality of life after the surgical intervention was added to the HRQoL questionnaire. Risk factors related to impaired quality of life were identified using univariate comparisons and multivariate regression techniques. RESULTS: The median (interquartile range, IQR) of transfused red blood cells was 3 (1-4). Among 205 patients, 178 were studied 6 months after discharge. Impairment in at least one dimension of the EuroQoL-5D questionnaire was observed in 120 patients, with an overall score of 0.8 (IQR 0.61-1). The number of red blood cell transfusions was related to an impaired HRQoL (OR 1.17 per additional unit, 95% confidence interval 1.03-1.36, p=0.03), a trend to lower visual analog scale score (coefficient -0.75 per additional unit, 95% confidence interval -1.61 to 0.1, p=0.09) and an absence of improvement in HRQoL after surgery compared to the previous status (OR 1.13, 95% confidence interval 1.03-1.25, p=0.01). CONCLUSIONS: Liberal red blood cell transfusions increase the risk of impaired HRQoL after cardiac surgery.
Assuntos
Procedimentos Cirúrgicos Cardíacos , Transfusão de Eritrócitos/efeitos adversos , Qualidade de Vida , Idoso , Transfusão de Eritrócitos/métodos , Transfusão de Eritrócitos/estatística & dados numéricos , Feminino , Humanos , Masculino , Período Pós-Operatório , Estudos Prospectivos , Análise de Regressão , Fatores de Risco , Inquéritos e Questionários , Fatores de TempoRESUMO
BACKGROUND: Numerous studies have demonstrated significant differences in the expression level across continental human populations. Most of published results were performed on B-cell lines materials examined under specific laboratory conditions, without further validation in a primary biological material. The goal of our study was to identify mRNA markers characterized by a significant and stable difference in the gene expression profile in Caucasian and Chinese populations, both in the commercially available B-lymphocyte cell lines and in the primary samples of the peripheral blood. RESULTS: The preliminary selection of population-differentiating transcripts was based on Illumina expression microarray analysis of the representative group of ethnically-specified B-lymphocyte cell lines. Twenty genes with the inter-population difference in the mean expression characterized by the at least 1.5-fold change and FDR < 0.05 were identified. Subsequently, a two-step validation procedure was carried out. In the first step, a subset of selected population- differentiating transcripts was tested in the independent set of B-lymphocyte cell lines, using TLDA cards. Based on TLDA analysis, three transcripts representing Fch > 2 were chosen for validation. The differentiating status was confirmed for all of them: UTS2, UGT2B17 and SLC7A7. The mean expression of UTS2 was higher in CHB (25.8-fold change compared to CEU), while the expression of UGT2B17 and SLC7A7 was higher in CEU (3.2- and 2.2-fold change, respectively). In the next validation step, two transcripts were verified in the primary biological material. As an ultimate result of our study, two mRNA markers (UTS2 and UGT2B17) exhibiting population differences in the expression level in both B-cell line and in the blood were identified. Further statistical analysis confirmed the discriminatory potential of these two markers. CONCLUSIONS: An inter-population differences on the level of gene expression were identified in both B-cell lines and peripheral blood samples. These findings may have a practical application in the field of forensic science. In particular, these transcripts, targeted by specific probes, may be used as population-specific targets in the efforts aiming to separate mixture of blood from individuals of different populations. Notwithstanding, these results have to be confirmed on extended population group.
Assuntos
Tipagem Molecular/métodos , Transcriptoma , Povo Asiático/genética , Biomarcadores , Linhagem Celular , Genética Forense/métodos , Perfilação da Expressão Gênica , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , População Branca/genéticaRESUMO
Assays that allow analysis of the biogeographic origin of biological samples in a standard forensic laboratory have to target a small number of highly differentiating markers. Such markers should be easy to multiplex and the assay must perform well in the degraded and scarce biological material. SNPs localized in the genome regions, which in the past were subjected to differential selective pressure in various populations, are the most widely used markers in the studies of biogeographic affiliation. SNPs reflecting biogeographic differences not related to any phenotypic traits are not sufficiently explored. The goal of our study was to identify a small set of SNPs not related to any known pigmentation/phenotype-specific genes, which would allow efficient discrimination between populations of Europe and East Asia. The selection of SNPs was based on the comparative analysis of representative European and Chinese/Japanese samples (B-lymphocyte cell lines), genotyped using the Infinium HumanOmniExpressExome microarray (Illumina). The classifier, consisting of 24 unlinked SNPs (24-SNP classifier), was selected. The performance of a 14-SNP subset of this classifier (14-SNP subclassifier) was tested using genotype data from several populations. The 14-SNP subclassifier differentiated East Asians, Europeans and Africans with â¼100% accuracy; Palestinians, representative of the Middle East, clustered with Europeans, while Amerindians and Pakistani were placed between East Asian and European populations. Based on these results, we have developed a SNaPshot assay (EurEAs_Gplex) for genotyping SNPs from the 14-SNP subclassifier, combined with an additional marker for gender identification. Forensic utility of the EurEAs_Gplex was verified using degraded and low quantity DNA samples. The performance of the EurEAs_Gplex was satisfactory when using degraded DNA; tests using low quantity DNA samples revealed a previously not described source of genotyping errors, potentially important for any SNaPshot-based assays.
Assuntos
Povo Asiático/genética , Genética Forense/métodos , Grupos Raciais/genética , População Branca/genética , DNA/análise , DNA/sangue , DNA/genética , Primers do DNA , Frequência do Gene , Genética Populacional , Genótipo , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex/métodos , Fenótipo , Filogeografia , Polimorfismo de Nucleotídeo Único , Grupos Raciais/classificação , Análise para Determinação do Sexo/métodosRESUMO
Glioblastomas (GBM) are some bad prognosis brain tumors despite a conventional treatment associating surgical resection and subsequent radio-chemotherapy. Among these heterogeneous tumors, a subpopulation of chemo- and radioresistant GBM stem-like cells appears to be involved in the systematic GBM recurrence. Moreover, recent studies showed that differentiated tumor cells may have the ability to dedifferentiate and acquire a stem-like phenotype, a phenomenon also called plasticity, in response to microenvironment stresses such as hypoxia. We hypothesized that GBM cells could be subjected to a similar dedifferentiation process after ionizing radiations (IRs), then supporting the GBM rapid recurrence after radiotherapy. In the present study we demonstrated that subtoxic IR exposure of differentiated GBM cells isolated from patient resections potentiated the long-term reacquisition of stem-associated properties such as the ability to generate primary and secondary neurospheres, the expression of stemness markers and an increased tumorigenicity. We also identified during this process an upregulation of the anti-apoptotic protein survivin and we showed that its specific downregulation led to the blockade of the IR-induced plasticity. Altogether, these results demonstrated that irradiation could regulate GBM cell dedifferentiation via a survivin-dependent pathway. Targeting the mechanisms associated with IR-induced plasticity will likely contribute to the development of some innovating pharmacological strategies for an improved radiosensitization of these aggressive brain cancers.
Assuntos
Desdiferenciação Celular/efeitos da radiação , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas Inibidoras de Apoptose/metabolismo , Células-Tronco Neoplásicas/patologia , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Humanos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos da radiação , Fenótipo , SurvivinaRESUMO
Cryobanking and transplantation of ovarian tissue is a promising approach to restore fertility in cancer patients. However, ischemic stress following avascular ovarian cortex grafting is known to induce stromal tissue fibrosis and alterations in follicular development. The aim of the study was to analyze the impact of freeze-thawing and grafting procedures on gene expression in human ovarian tissue. Frozen-thawed ovarian tissue from 14 patients was xenografted for 7 days to nude mice and one ungrafted fragment was used as a control. Immediately after recovery, grafts were processed for RNA extraction and histological analysis. Their expression profile was screened by whole-genome oligonucleotide array (n = 4) and validated by reverse-transcriptase polymerase chain analysis (n = 10). After data filtering, the Limma package was used to build a linear regression model for each gene and to compute its fold change between tissues on Days 0 and 7. After adjusting the P-value by the Sidak method, 84 of the transcripts were significantly altered after 7 days of grafting, including matrix metalloproteinase-9 and -14 and angiogenic factors such as placental growth factor and C-X-C chemokine receptor type 4 (CXCR4). Major biological processes were related to tissue remodeling, including secretory processes, cellular adhesion and response to chemical and hormonal stimuli. Angiopoietin signaling, the interleukin-8 pathway and peroxisome proliferator-activated receptor activation were shown to be differentially regulated. On Day 7, overexpression was confirmed by PCR for interleukin-8, transforming growth factor-beta 1, matrix metalloproteinase-14 and CXCR4, compared with ungrafted controls. In conclusion, new as well as known genes involved in tissue restructuring and angiogenesis were identified and found to play a key role during the first days after human ovarian tissue transplantation. This will facilitate the development of strategies to optimize grafting techniques.
Assuntos
Expressão Gênica , Redes e Vias Metabólicas/genética , Ovário/metabolismo , RNA Mensageiro/genética , Adulto , Animais , Criopreservação , Feminino , Perfilação da Expressão Gênica , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Modelos Lineares , Camundongos , Camundongos Nus , Ovário/transplante , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , RNA Mensageiro/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Transplante HeterólogoRESUMO
PURPOSE: To assess the safety of reimplantation of cryopreserved ovarian tissue from advanced-stage breast cancer patients. METHODS: Cryopreserved ovarian cortical fragments were obtained from 13 advanced-stage breast cancer patients aged 17-35 years. After thawing, part of the ovarian cortical tissue was grafted to severe combined immunodeficient mice for 6 months. The presence of malignant mammary cells in ovarian tissue was evaluated after thawing as well as after grafting by 1) histology and immunohistochemistry (epithelial membrane antigen, Her2/neu and gross cystic disease fluid protein 15 identification), and 2) detection of the MGB2 gene by qPCR. RESULTS: No malignant cells were evidenced by histology and immunohistochemistry. None of the mice died during the 6-month grafting period, nor developed macroscopically visible masses. MGB2 gene expression was detected by qPCR and confirmed by sequencing in frozen-thawed ovarian tissue in 4 cases and in grafts in 1 case. CONCLUSIONS: This pilot study is the first to evaluate the risk of contamination of cryopreserved ovarian tissue from advanced-stage breast cancer patients by xenotransplantation for 6 months to immunodeficient mice, associated with more conventional screening methods. Our xenografting results are reassuring, but caution needs to be exercised, as MGB2 gene expression was detected in some cases. Larger numbers of ovarian tissue samples from patients with advanced-stage breast cancer are required to confirm our findings before ovarian tissue transplantation can be contemplated in these patients.
Assuntos
Neoplasias da Mama/patologia , Preservação da Fertilidade/métodos , Folículo Ovariano/transplante , Adolescente , Adulto , Idoso de 80 Anos ou mais , Animais , Proteínas de Transporte/metabolismo , Criopreservação , Feminino , Glicoproteínas/metabolismo , Humanos , Mamoglobina B/biossíntese , Mamoglobina B/genética , Proteínas de Membrana Transportadoras , Camundongos , Camundongos SCID , Projetos Piloto , Receptor ErbB-2/metabolismo , Transplante Heterólogo , Adulto JovemRESUMO
AIMS: The use of bone marrow mononuclear cells (BMMNCs) as a source of mesenchymal stem cells (MSCs) for therapy has recently attracted the attention of researchers because BMMNCs can be easily obtained and do not require in vitro expansion before their use. This study was designed to quantify the MSC population in bone marrow (BM) samples obtained for cell therapy using flow cytometry to detect the CD271 antigen. MATERIAL AND METHODS: Autologous BM was obtained by posterior superior iliac crest aspiration under topical anesthesia. Mononuclear cells isolated from the BM aspirate on a Ficoll density gradient were used to treat patients with pressure ulcer (n = 13) bone nonunions (n = 3) or diabetic foot ulcers (n = 5). RESULTS: Our flow cytometry data revealed a low percentage as well as a high variability among patients of CD271(+)CD45(-) cells (range, 0.0017 to 0.0201%). All cultured MSC adhered to plastic dishes showing a capacity to differentiate into adipogenic and osteogenic lineages. CONCLUSIONS: Our findings suggested that the success of cell therapy was independent of the number of MSCs present in the BM aspirate used for autologous cell therapy.
Assuntos
Células da Medula Óssea/citologia , Leucócitos Mononucleares/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Adapaleno , Adipócitos/citologia , Adulto , Idoso , Diferenciação Celular , Feminino , Citometria de Fluxo , Humanos , Antígenos Comuns de Leucócito/metabolismo , Masculino , Pessoa de Meia-Idade , Naftalenos/metabolismo , Úlcera por PressãoRESUMO
CONTEXT: Pressure ulcers are especially difficult to treat in patients with spinal cord injury (SCI) and recurrence rates are high. Prompted by encouraging results obtained using bone marrow stem cells to treat several diseases including chronic wounds, this study examines the use of autologous stem cells from bone marrow to promote the healing of pressure ulcers in patients with SCI. OBJECTIVE: To obtain preliminary data on the use of bone marrow mononuclear cells (BM-MNCs) to treat pressure ulcers in terms of clinical outcome, procedure safety, and treatment time. PARTICIPANTS: Twenty-two patients with SCI (19 men, 3 women; mean age 56.41 years) with single type IV pressure ulcers of more than 4 months duration. INTERVENTIONS: By minimally invasive surgery, the ulcers were debrided and treated with BM-MNCs obtained by Ficoll density gradient separation of autologous bone marrow aspirates drawn from the iliac crest. RESULTS: In 19 patients (86.36%), the pressure ulcers treated with BM-MNCs had fully healed after a mean time of 21 days. The number of MNCs isolated was patient dependent, although similar clinical outcomes were observed in each case. Compared to conventional surgical treatment, mean intra-hospital stay was reduced from 85.16 to 43.06 days. Following treatment, 5 minutes of daily wound care was required per patient compared to 20 minutes for conventional surgery. During a mean follow-up of 19 months, none of the resolved ulcers recurred. CONCLUSIONS: Our data indicate that cell therapy using autologous BM-MNCs could be an option to treat type IV pressure ulcers in patients with SCI, avoiding major surgical intervention.
Assuntos
Células da Medula Óssea/fisiologia , Transplante de Medula Óssea/métodos , Úlcera por Pressão/cirurgia , Adulto , Idoso , Diagnóstico por Imagem/métodos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Úlcera por Pressão/etiologia , Traumatismos da Medula Espinal/complicações , Transplante Autólogo/métodos , Resultado do TratamentoRESUMO
BACKGROUND AND AIMS: TPMT deficiency is associated with azathioprine (AZA)-induced myelosuppression (MS). However, in one previous study, only about » of MS episodes in Crohn's Disease patients under AZA can be attributed to TPMT deficiency. Recently, new TPMT mutations have been described and our aim is to investigate their clinical relevance before and after a first MS episode on thiopurine therapy. METHODS: Clinical data from 61 IBD patients having developed MS during AZA therapy were collected. Sequencing analysis was carried out on TPMT cDNA for the presence of all currently known mutations. RESULTS: Only TPMT *2, *3A and *3C mutations were found in this cohort. TPMT mutations were observed in 15 out of 61 patients (25%). Four out of 15 were homozygous for a TPMT mutation (low methylator, LM genotype) and 11 were heterozygous (intermediate methylator, IM genotype). Median delays of MS onset were 2, 2.75 and 6months in the LM, IM and HM (high methylator, wild type TPMT) groups, respectively. After the first MS episode, 36 patients resumed thiopurine treatment of which 13 experienced a second MS episode. This second episode was also rarely associated with TPMT mutations. CONCLUSIONS: One quarter of MS episodes during AZA were associated with TPMT deficient genotype. After a first leucopenia episode, thiopurine therapy may be resumed in a majority of patients independently of their TPMT genotype.
Assuntos
Azatioprina/efeitos adversos , Hipersensibilidade a Drogas/complicações , Doenças Inflamatórias Intestinais/complicações , Metiltransferases/genética , Erros Inatos do Metabolismo da Purina-Pirimidina/complicações , Adolescente , Adulto , Idoso , Azatioprina/uso terapêutico , Análise Mutacional de DNA , Hipersensibilidade a Drogas/etiologia , Hipersensibilidade a Drogas/genética , Feminino , Genótipo , Humanos , Imunossupressores , Doenças Inflamatórias Intestinais/tratamento farmacológico , Leucopenia , Masculino , Pessoa de Meia-Idade , Mutação , Pancitopenia/induzido quimicamente , Pancitopenia/genética , Erros Inatos do Metabolismo da Purina-Pirimidina/etiologia , Erros Inatos do Metabolismo da Purina-Pirimidina/genética , Estudos Retrospectivos , Adulto JovemAssuntos
Anemia Hemolítica Autoimune/induzido quimicamente , Antibacterianos/efeitos adversos , Anemia Hemolítica Autoimune/diagnóstico , Antibacterianos/imunologia , Teste de Coombs , Humanos , Masculino , Pessoa de Meia-Idade , Ácido Penicilânico/efeitos adversos , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/imunologia , Piperacilina/efeitos adversos , Piperacilina/imunologia , Combinação Piperacilina e TazobactamRESUMO
In clinically organ-confined prostate cancer patients, bloodstream tumour cell dissemination generally occurs, and may be enhanced by surgical prostate manipulation. To evaluate cancer-cell seeding impact upon patient recurrence-free survival, 155 patients were prospectively enrolled then followed. Here, 57 patients presented blood prostate cell shedding preoperatively and intraoperatively (group I). Of the 98 preoperatively negative patients, 53 (54%) remained negative (group II) and 45 (46%) became intraoperatively positive (group III). Median biological and clinical recurrence-free time was far shorter in group I (36.2 months, P<0.0001) than in group II (69.6 months) but did not significantly differ in group II and III (69.6 months vs 65.0). Such 5-year follow-up data show that preoperative circulating prostate cells are an independent prognosis factor of recurrence. Moreover, tumour handling induces cancer-cell seeding but surgical blood dissemination does not accelerate cancer evolution.
Assuntos
Células Neoplásicas Circulantes , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia , Idoso , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/sangue , Estadiamento de Neoplasias , Prognóstico , Estudos Prospectivos , Neoplasias da Próstata/cirurgiaRESUMO
Human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) is a key enzyme in the detoxification of thiopurine drugs widely used in the treatment of various diseases, such as inflammatory bowel diseases, acute lymphoblastic leukaemia and rheumatic diseases. The TPMT gene is genetically polymorphic and the inverse relationship between TPMT activity and the risk of developing severe hematopoietic toxicity is well known. In this study, the entire coding sequence of TPMT, together with its 5'-flanking promoter region, was analysed in patients with an intermediate phenotype for thiopurine drug methylation. Four polymorphisms were identified, two previously described, c.356A>C (p.Lys(119)Thr, TPMT*9) and c.205C>G (p.Leu(69)Val, TPMT*21), and two novel missense mutations, c.537G>T (p.Gln(179)His, TPMT*24) and c.634T>C (p.Cys(212)Arg, TPMT*25). Structural investigations, using molecular modeling, were undertaken in an attempt to explain the potential impact of the amino acid substitutions on the structure and activity of the variant proteins. Additionally, in order to determine kinetic parameters (K(m) and V(max)) of 6-thioguanine (6-TG) methylation, the four variants were expressed in a recombinant yeast expression system. Assays were performed by HPLC and the results were compared with those of wild-type TPMT. The p.Leu(69)Val and the p.Cys(212)Arg substitutions encode recombinant enzymes with a significantly decreased intrinsic clearance compared to that of the wild-type protein, and, consequently, characterise non-functional alleles of TPMT. The p.Lys(119)Thr and the p.Gln(179)His substitutions do not affect significantly the catalytic activity of the corresponding variant proteins, which prevents to unambiguously describe these latter alleles as defective TPMT variants.
Assuntos
Alelos , Metiltransferases/genética , Mutação de Sentido Incorreto , Polimorfismo Genético , Região 5'-Flanqueadora/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Cristalografia por Raios X , DNA/genética , Genótipo , Humanos , Inativação Metabólica/genética , Leucócitos/enzimologia , Leucócitos/metabolismo , Metiltransferases/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Purinas/farmacocinética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , População Branca/genéticaRESUMO
The aim of this study was to be able to amplify and to detect on one array 27 different etiologic agents found in nosocomial pneumonia, some being phylogenetically closely related and others very distant. The assay is based on the use of consensus primers combined with the identification of the resulting amplicons by hybridization on specific capture probes present on an array. Three genes were necessary in order to cover the different pathogens. We took a redundancy of at least two positive spots to confirm the identity of each species. Each probe was present in triplicate on the array. The detection limit was between 10 and 1,000 DNA copies in the assay depending on the bacteria and the probe. The assay was also specific when tested both on reference collection strains corresponding to the 27 species of interest and on 57 other bacterial species of the normal human flora. Accuracy of the assay was assessed on 200 clinical isolates and some polymorphisms were indeed observed for 5 species. Effectiveness of the assay was preliminarily validated on 25 endotracheal aspirates and sputum samples, and the results were in accordance either with the cell culture or with the sequencing. Polybacterial infections were well detected in three samples. The results show that a combination of appropriate polymerase chain reaction (PCR) and redundancy of signals on the array allows specific screening of bacteria belonging to different species and genus and even fungi. The results open the way for a possible molecular detection of bacteria in the clinical diagnostic setting.
Assuntos
Bactérias/isolamento & purificação , Infecção Hospitalar/microbiologia , Fungos/isolamento & purificação , Análise em Microsséries/métodos , Pneumonia/microbiologia , Bactérias/genética , Sequência de Bases , Primers do DNA , Fungos/genética , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: Individuals with Type 2 diabetes are at increased risk of stroke. Plasma homocysteine (tHcy) is an independent risk factor for cardiovascular (CV) disease. The methylene-tetrahydrofolate reductase (MTHFR) gene polymorphism (thermolabile variant C(677)T) is associated with CV risk, partly as a result of increased Hcy, especially in homozygous subjects. AIM: To relate the occurrence of the MTHFR polymorphism with stroke prevalence by examining allelic frequency and genotype distribution in 165 subjects with Type 2 diabetes studied for the presence of thermolabile C(677)T MTHFR mutation. RESULTS: Mean age was 67.7 years, and tHcy 18.2 micromol/l. T allele frequency was 38.5%. MTHFR genotypes were: normal (CC) 40%; heterozygous (CT) 43%; homozygous (TT) 17%. Serum levels of folic acid and B12 vitamin were within normal limits. Stroke prevalence was 14%. Sixty-four per cent of stroke-free subjects had the normal C allele vs. 46% in stroke subjects. The frequencies of genotypes (CC-CT-TT) were (%): 44-41-15 in stroke-free vs. 17-57-26 in stroke patients. Coronary (CAD) and peripheral artery disease (PAD) were common in all groups, with no differences according to genotypes. Stroke prevalence was markedly higher in genotypes CT and TT (18 and 21%) compared with CC (6%). Mean tHcy levels were higher in TT subjects. CONCLUSION: The allelic frequency of C(677)T MTHFR mutation in Type 2 diabetes subjects with stroke is markedly different from that of subjects without stroke. Genotypic characteristics suggest that C(677)T MTHFR mutation confers a higher risk for stroke to both homozygous and heterozygous T allele carriers that cannot be ascribed solely to raised tHcy and/or lower folate status in CT subjects, nor to phenotypic expression of conventional risk factors for stroke. The impact of the MTHFR polymorphism on stroke may result from T allele-linked deleterious effects, or C allele-linked protection. Confirmatory studies are warranted, as this cohort was not randomly selected, and a type 1 error cannot be ruled out.
Assuntos
Diabetes Mellitus Tipo 2/genética , Angiopatias Diabéticas/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo Genético/genética , Acidente Vascular Cerebral/genética , Idoso , Estudos de Coortes , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/genética , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Feminino , Ácido Fólico/sangue , Frequência do Gene , Heterozigoto , Homozigoto , Humanos , Masculino , Mutação/genética , Doenças Vasculares Periféricas/complicações , Doenças Vasculares Periféricas/genética , Fatores de Risco , Acidente Vascular Cerebral/complicações , Vitamina B 12/sangueRESUMO
The pharmacokinetics (PK) of isepamicin were studied in 8 febrile neutropenic patients with hematologic malignancy and in 20 young women with acute pelvic inflammatory disease (PID). Isepamicin was given as a slow intravenous infusion over 30 min at a dose of 15 mg/kg once daily (OD). Serum levels of isepamicin were determined by fluorescence polarization immunoassay, and PK analyses were obtained based on a one-compartment open model after 24 hours (steady state) and after 7 days. On day 1, the volume of distribution (Vd) of isepamicin, for both populations, appeared about 30% higher than classically reported in healthy individuals: 0.31 and 0.36 L/kg for neutropenic and PID patients respectively. However on day 7, Vd displayed significant reduction (0.28 and 0.27 L/kg, respectively for neutropenic and PID patients). A reduction of isepamicin clearance was also observed between day 1 and day 7 (137 vs 120 mL/min and 130 vs 101 mL/min for neutropenic and PID populations, respectively). Such changes are consistent with a significant increase in the Cmax concentrations (45 vs 50 mg/L, and 38 vs 49 mg/L) and in the AUC (136 vs 158 and 137 vs 162 mg/L.h) observed after a week of treatment in neutropenic and PID patients, respectively. In conclusion, taking into account the importance of reaching early active concentrations, we recommend the use of higher loading dose of isepamicin (>15 mg/kg) in neutropenic cancer patients and in women with PID, particularly in case of a combination with a possibly ineffective antibacterial agent, in case of infection with bacteria at upper limit of susceptibility, in the presence of high infectious inoculum or in the presence of sequestered sites of infection.
Assuntos
Antibacterianos/farmacocinética , Neoplasias Hematológicas/complicações , Neutropenia/tratamento farmacológico , Doença Inflamatória Pélvica/complicações , Doença Aguda , Adulto , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Área Sob a Curva , Feminino , Febre/etiologia , Gentamicinas/administração & dosagem , Gentamicinas/farmacocinética , Gentamicinas/uso terapêutico , Humanos , Infusões Intravenosas , Pessoa de Meia-Idade , Neutropenia/etiologiaRESUMO
OBJECTIVE: The aim of the study was to determine the prevalence of the C677T mutation in a cohort of type 2 diabetic patients with and without elevated total plasma homocysteine (tHcy). METHODS: 80 type 2 diabetic patients with hyperhomocysteinaemia (group 1, tHcy: 21.3 +/- 6.7 micromol/L) and 50 subjects with normal levels (group 2, tHcy 11.2 +/- 2.3 micromol/L) were studied. C677T mutation was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Homozygosity was present in 23% of patients in group 1 and 8% in group 2 (P<0.02). No significant difference in heterozygosity frequency was observed between patients with and without hyperhomocysteinaemia. T allele frequency was 0.43 in group 1 and 0.35 in group 2. CONCLUSION: C677T mutation is frequent in diabetic patients with hyperhomocysteinaemia and could contribute, besides non genetic factors, to increased levels of tHcy.