RESUMO
The Columbus steerable guidewire (Rapid Medical, Israel) is a 0.014 inch guidewire with a remotely controlled deflectable tip intended for neuronavigational purposes. 1 The tip can be shaped by pulling or pushing the handle. Pulling the handle decreases the radius (from 4 mm to 2 mm) and curves the tip, while pushing the handle increases the curvature radius and straightens the tip until it bends in the opposite direction. The amount of deflection is at the discretion of the operator. Video 1 The response of the Columbus guidewire to rotational movements is inferior to that of standard wires, and the tip is very soft and malleable but brings great support when bent. We present two cases where the Columbus guidewire was used. In the first case, the Columbus enabled us to probe a posterior cerebral artery arising from a giant basilar tip aneurysm without wall contact. In the second case, the Columbus was used as a secondary wire to help cannulate the pericallosal artery in a patient with a recurrent anterior complex aneurysm; this subsequently permitted successful stent-assisted coiling of the aneurysm. neurintsurg;14/10/1045/V1F1V1Video 1.
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Cateterismo , Stents , Humanos , Israel , Microcirurgia , Artéria Cerebral PosteriorRESUMO
The radiosynthesis of [(18)F]fluoropyruvate was investigated using numerous precursors were synthesized from ethyl 2,2-diethoxy-3-hydroxypropanoate (5) containing different leaving groups: mesylate, tosylate, triflate, and nonaflate. These precursors were evaluated for [(18)F]fluoride incorporation with triflate being superior. The subsequent hydrolysis step was investigated, and an acidic hydrolysis was optimized. After establishing suitable purification and formulation methods, the [(18)F]fluoropyruvate could be isolated in ca. 50% d.c. yield. The [(18)F]fluoropyruvate was evaluated in vitro for its uptake into tumor cells using adenocarcinomic human alveolar basal epithelial cells (A549) and unfortunately showed an uptake of approximately 0.1% of the applied dose per 100,000 cells after 30 min. Initial pharmacokinetic properties were assessed in vivo using nude mice showed a high degree of bone uptake from defluorination, which will limit its potential as an imaging agent for metabolic processes.
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Radioisótopos de Flúor , Piruvatos/química , Compostos Radiofarmacêuticos/química , Animais , Linhagem Celular Tumoral , Halogenação , Humanos , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons , Piruvatos/síntese química , Radioquímica , Compostos Radiofarmacêuticos/síntese química , Tomografia Computadorizada por Raios XRESUMO
OBJECTIVE: To investigate the suitability of blood granulocyte and monocyte sensitivity, as measured by the quantity of different agonists required to induce CD62L shedding, for assessment of perioperative immune changes in patients undergoing cardiac surgery with cardiopulmonary bypass. METHODS: Patients scheduled for aortocoronary bypass grafting or for valve surgery were included in this prospective observational study. Blood samples were drawn before anesthesia induction, directly after surgery and 48 hours after anesthesia induction. We determined the concentration of two different inflammatory stimuli--lipoteichoic acid (LTA) and tumor necrosis factor alpha (TNF)--required to induce shedding of 50% of surface CD62L from blood granulocytes and monocytes. In parallel monocyte surface human leukocyte antigen (HLA)-DR, and plasma interleukin (IL)-8, soluble (s)CD62L, soluble (s)Toll-like receptor (TLR)-2 and ADAM17 quantification were used to illustrate perioperative immunomodulation. RESULTS: 25 patients were enrolled. Blood granulocytes and monocytes showed decreased sensitivity to the TLR 2/6 agonist Staphylococcus aureus LTA immediately after surgery (p = 0.001 and p = 0.004 respectively). In contrast, granulocytes (p = 0.01), but not monocytes (p = 0.057) displayed a decreased postoperative sensitivity to TNF. We confirmed the presence of a systemic inflammatory response and a decreased immune sensitivity in the post-surgical period by measuring significant increases in the perioperative plasma concentration of IL-8 (p ≤ 0.001) and sTLR (p = 0.004), and decreases in monocyte HLA-DR (p<0.001), plasma sCD62L (p ≤ 0.001). In contrast, ADAM17 plasma levels did not show significant differences over the observation period (p = 0.401). CONCLUSIONS: Monitoring granulocyte and monocyte sensitivity using the "CD62L shedding assay" in the perioperative period in cardiac surgical patients treated with the use of cardiopulmonary bypass reveals common changes in sensitivity to TLR2/6 ligands and to TNF stimulus. Further long-term follow-up studies will address the predictive value of these observations for clinical purposes.
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Ponte Cardiopulmonar/métodos , Cardiopatias/sangue , Cardiopatias/imunologia , Selectina L/sangue , Proteínas ADAM/metabolismo , Proteína ADAM17 , Idoso , Ensaio de Imunoadsorção Enzimática , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Inflamação , Interleucina-8/metabolismo , Ligantes , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
INTRODUCTION: Independent measurement of the levels of both the estrogen receptors, ERα and ERß, in breast cancer could improve prediction of benefit from endocrine therapies. While ERα levels can be measured by positron emission tomography (PET) using 16α-[(18)F]fluoroestradiol (FES), no effective agent for imaging ERß by PET has yet been reported. METHODS: We have prepared the fluorine-18 labeled form of 8ß-(2-fluoroethyl)estradiol (8BFEE(2)), an analog of an ERß-selective steroidal estrogen, 8ß-vinylestradiol; efficient incorporation of fluorine-18 was achieved, but required very vigorous conditions. We have examined the biodistribution of this compound, as well as of Br-041, an analog of a known non-steroidal ERß-selective ligand (ERB-041), labeled with bromine-76. Studies were done in immature female rodents, with various pharmacological and endocrine perturbations to assess ERß selectivity of uptake. RESULTS: Little evidence of ERß-mediated uptake was observed with either [(18)F]8BFEE(2) or [(76)Br]Br-041. Attempts to increase the ERß content of target tissues were not effective and failed to improve biodistribution selectivity. CONCLUSIONS: Because on an absolute basis level, ERß levels are low in all target tissues, these studies have highlighted the need to develop improved in vivo models for evaluating ERß-selective radiopharmaceuticals for use in PET imaging. Genetically engineered breast cancer cells that are being developed to express either ERα or ERß in a regulated manner, grown as xenografts in immune-compromised mice, could prove useful for future studies to develop ER subtype-selective radiopharmaceuticals.
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Estradiol/síntese química , Receptor beta de Estrogênio/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Animais , Técnicas de Química Sintética , Estradiol/metabolismo , Estradiol/farmacocinética , Receptor alfa de Estrogênio/metabolismo , Feminino , Letrozol , Ligantes , Camundongos , Nitrilas/farmacologia , Ratos , Especificidade por Substrato , Triazóis/farmacologiaRESUMO
UNLABELLED: Bombesin is a peptide exhibiting high affinity for the gastrin-releasing peptide receptor (GRPr), which is highly overexpressed on prostate cancer cells. In the present study, we developed an (18)F-labeled bombesin analog, (18)F-BAY 86-4367, which is currently being clinically tested for use in PET of prostate cancer. METHODS: In vitro pharmacologic studies were performed to characterize the nonradioactive ((19)F) standard of the bombesin analog for binding affinity and selectivity for GRPr. The stability of (18)F-BAY 86-4367 was determined in murine and human plasma. In vivo, the tumor-targeting potential and pharmacokinetic profile of the (18)F tracer were analyzed with biodistribution experiments and PET studies of prostate tumor-bearing mice. RESULTS: The nonradioactive ((19)F) standard of the bombesin analog showed subnanomolar and GRPr-selective binding affinity. The stability of the tracer in murine and human plasma was found to be high. In 2 prostate cancer xenograft models (PC-3 and LNCaP), (18)F-BAY 86-4367 showed more specific and effective GRPr-based targeting in vivo than the benchmark radiotracers (18)F-fluoroethylcholine and (18)F-FDG. In addition, rapid tumor targeting and fast renal excretion (â¼70%) and hepatobiliary excretion (â¼10%) were identified in both xenograft models. Furthermore, PET studies provided clear and specific visualization of PC-3 tumors in mice. CONCLUSION: Favorable preclinical data showing specific and effective tumor targeting by (18)F-BAY 86-4367 suggest that a clinical trial be undertaken to test its diagnostic utility in PET for prostate carcinoma patients.
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Bombesina/análogos & derivados , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismo , Compostos Radiofarmacêuticos , Receptores da Bombesina/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Bombesina/química , Bombesina/farmacocinética , Linhagem Celular , Ácido Cisteico/química , Estabilidade de Medicamentos , Radioisótopos de Flúor , Humanos , Indicadores e Reagentes , Marcação por Isótopo , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Receptores da Bombesina/genética , Distribuição TecidualRESUMO
There is a high demand for tumor specific PET tracers in oncology imaging. Besides glucose, certain amino acids also serve as energy sources and anabolic precursors for tumors. Therefore, (18)F-labeled amino acids are interesting probes for tumor specific PET imaging. As glutamine and glutamate play a key role in the adapted intermediary metabolism of tumors, the radiosynthesis of 4-[(18)F]fluoro l-glutamic acid (BAY 85-8050) as a new specific PET tracer was established. Cell-uptake studies revealed specific tumor cell accumulation.
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Glutamatos/síntese química , Compostos Radiofarmacêuticos/síntese química , Linhagem Celular Tumoral , Radioisótopos de Flúor , Glutamatos/química , Glutamatos/metabolismo , Humanos , Marcação por Isótopo , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , EstereoisomerismoRESUMO
The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with (18)F, and characterized in vitro and in vivo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K(i) = 0.7 nM) and 7b (K(i) = 0.1 nM) were chosen for further in vivo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [(18)F]6b, compound [(18)F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In vivo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [(18)F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.
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Bombesina/análogos & derivados , Bombesina/metabolismo , Radioisótopos de Flúor , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores da Bombesina/metabolismo , Animais , Bombesina/química , Bombesina/farmacocinética , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Evaluation of the technical and diagnostic feasibility of commercial multiplex real-time polymerase chain reaction (PCR) for detection of blood stream infections in a cohort of intensive care unit (ICU) patients with severe sepsis, performed in addition to conventional blood cultures. DESIGN: Dual-center cohort study. SETTING: Surgical ICU of two university hospitals. PATIENTS AND PARTICIPANTS: One hundred eight critically ill patients fulfilling the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) severe sepsis criteria were included. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: PCR results obtained in 453 blood samples from 108 patients were compared with corresponding blood culture results. PCR resulted in a twofold higher positivity rate when compared with conventional blood culture (BC) testing (114 versus 58 positive samples). In 40 out of 58 PCR positive assays the results of the corresponding blood cultures were identical to microorganisms detected by PCR. In 18 samples PCR and BC yielded discrepant results. Compared with conventional blood culture the sensitivity and specificity of PCR was 0.69 and 0.81, respectively. Further evaluation of PCR results against a constructed gold standard including conventional microbiological test results from other significant patient specimen (such as bronchio-alveolar lavage fluid, urine, swabs) and additionally generated clinical and laboratory information yielded sensitivity of 0.83 and specificity of 0.93. CONCLUSIONS: Our cohort study demonstrates improved pathogen detection using PCR findings in addition to conventional blood culture testing. PCR testing provides increased sensitivity of blood stream infection. Studies addressing utility including therapeutic decision-making, outcome, and cost-benefit following diagnostic application of PCR tests are needed to further assess its value in the clinical setting.
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Infecções Bacterianas/diagnóstico , Sepse/microbiologia , Adolescente , Adulto , Idoso , Infecções Bacterianas/complicações , Infecções Bacterianas/epidemiologia , Calcitonina/sangue , Estudos de Coortes , Comorbidade , Estado Terminal , Estudos de Viabilidade , Feminino , Humanos , Unidades de Terapia Intensiva , Interleucina-6/sangue , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Precursores de Proteínas/sangue , Sepse/sangue , Sepse/mortalidade , Índice de Gravidade de Doença , Taxa de Sobrevida , Adulto JovemRESUMO
Methods for the radiolabeling molecules of interest with [18F]-fluoride need to be rapid, convenient, and efficient. Numerous [18F]-labeled prosthetic groups, e.g., N-succinimidyl 4 [18F]-fluorobenzoate ([18F]-SFB), 4-azidophenacyl-[18F]-fluoride ([18F]-APF), and 1-(3-(2-[18F]fluoropyridin-3-yloxy)propyl)pyrrole-2,5-dione ([18F]-FpyMe), for conjugating to biomolecules have been developed. As the synthesis of these prosthetic groups usually requires multistep procedures, there is still a need for direct methods for the nucleophilic [18F]-fluorination of biomolecules. We report here on the development of a procedure based on the trimethylammonium (TMA) leaving group attached to an aromatic ring and activated with different electron-withdrawing groups (EWGs). A series of model compounds containing different electron-withdrawing substituents, a trimethylammonium leaving group, and carboxylic functionality for subsequent coupling to peptides were designed and synthesized. The optimal model compound, 2-cyano-4-(methoxycarbonyl)-N,N,N-trimethylbenzenaminium trifluoromethanesulfonate, was converted to carboxylic acid and coupled to peptides. The results of the one-step [18F]-fluorination of tetrapeptides and bombesin peptides show that the direct 18F-labeling of peptides is feasible under mild conditions and in good radiochemical yields.
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Derivados de Benzeno/química , Peptídeos/química , Compostos de Trimetil Amônio/química , Radioisótopos de Flúor/química , Estrutura Molecular , EstereoisomerismoRESUMO
Infection is the main treatment-related cause of mortality in cancer patients. Rapid and accurate diagnosis to facilitate specific therapy of febrile neutropenia is therefore urgently warranted. Here, we evaluated a commercial PCR-based kit to detect the DNA of 20 different pathogens (SeptiFast) in the setting of febrile neutropenia after chemotherapy. Seven hundred eighty-four serum samples of 119 febrile neutropenic episodes (FNEs) in 70 patients with hematological malignancies were analyzed and compared with clinical, microbiological, and biochemical findings. In the antibiotic-naïve setting, bacteremia was diagnosed in 34 FNEs and 11 of them yielded the same result in the PCR. Seventy-three FNEs were negative in both systems, leading to an overall agreement in 84 of 119 FNEs (71%). During antibiotic therapy, positivity in blood culture occurred only in 3% of cases, but the PCR yielded a positive result in 15% of cases. In six cases the PCR during antibiotic treatment detected a new pathogen repetitively; this was accompanied by a significant rise in procalcitonin levels, suggestive of a true detection of infection. All patients with probable invasive fungal infection (IFI; n = 3) according to the standards of the European Organization for Research and Treatment of Cancer had a positive PCR result for Aspergillus fumigatus; in contrast there was only one positive result for Aspergillus fumigatus in an episode without signs and symptoms of IFI. Our results demonstrate that the SeptiFast kit cannot replace blood cultures in the diagnostic workup of FNEs. However, it might be helpful in situations where blood cultures remain negative (e.g., during antimicrobial therapy or in IFI).
Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Febre/etiologia , Fungos/isolamento & purificação , Micoses/diagnóstico , Neutropenia/etiologia , Reação em Cadeia da Polimerase/métodos , Idoso , Bactérias/genética , Infecções Bacterianas/microbiologia , Sangue/microbiologia , DNA Bacteriano/genética , DNA Fúngico/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Fungos/genética , Humanos , Hospedeiro Imunocomprometido , Masculino , Pessoa de Meia-Idade , Micoses/microbiologia , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Kit de Reagentes para Diagnóstico , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: In transgenic animal models of sepsis, members of the Bcl-2 family of proteins regulate lymphocyte apoptosis and survival of sepsis. This study investigates the gene regulation of pro-apoptotic and anti-apoptotic members of the Bcl-2 family of proteins in patients with early stage severe sepsis. METHODS: In this prospective case-control study, patients were recruited from three intensive care units (ICUs) in a university hospital. Sixteen patients were enrolled when they fulfilled the criteria of severe sepsis. Ten critically ill but non-septic patients and 11 healthy volunteers served as controls. Blood samples were immediately obtained at inclusion. To confirm the presence of accelerated apoptosis in the patient groups, caspase-3 activation and phosphatidylserine externalisation in CD4+, CD8+ and CD19+ lymphocyte subsets were assessed using flow cytometry. Specific mRNAs of Bcl-2 family members were quantified from whole blood by real-time PCR. To test for statistical significance, Kruskal-Wallis testing with Dunn's multiple comparison test for post hoc analysis was performed. RESULTS: In all lymphocyte populations caspase-3 (p < 0.05) was activated, which was reflected in an increased phosphatidylserine externalisation (p < 0.05). Accordingly, lymphocyte counts were decreased in early severe sepsis. In CD4+ T-cells (p < 0.05) and B-cells (p < 0.001) the Bcl-2 protein was decreased in severe sepsis. Gene expression of the BH3-only Bim was massively upregulated as compared with critically ill patients (p < 0.001) and 51.6-fold as compared with healthy controls (p < 0.05). Bid was increased 12.9-fold compared with critically ill patients (p < 0.001). In the group of mitochondrial apoptosis inducers, Bak was upregulated 5.6-fold, while the expression of Bax showed no significant variations. By contrast, the pro-survival members Bcl-2 and Bcl-xl were both downregulated in severe sepsis (p < 0.001 and p < 0.05, respectively). CONCLUSIONS: In early severe sepsis a gene expression pattern with induction of the pro-apoptotic Bcl-2 family members Bim, Bid and Bak and a downregulation of the anti-apoptotic Bcl-2 and Bcl-xl proteins was observed in peripheral blood. This constellation may affect cellular susceptibility to apoptosis and complex immune dysfunction in sepsis.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Sepse/sangue , Adulto , Idoso , Apoptose/genética , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/sangue , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/biossíntese , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/sangue , Proteína 11 Semelhante a Bcl-2 , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Estudos Prospectivos , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/sangue , Sepse/genética , Sepse/patologia , Índice de Gravidade de DoençaRESUMO
The successful use of peptides as potential radiopharmaceuticals essentially requires the modification of the bioactive peptide hormones to introduce chelators for radiolabeling. In this study, four Y 1/Y 2 receptor-selective NPY analogues with different receptor subtype specificities have been investigated. For in vitro studies, the cold metal surrogate was used. Gallium and indium complexes were introduced by using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid as bifunctional chelator. The peptides were synthesized by solid-phase peptide synthesis (SPPS), the chelator was coupled either at the N-terminus or at the N(epsilon) side chain of Lys(4) of the resin-bound peptide, and the labeling was performed in solution after cleavage. Competitive binding assays showed high binding affinity of the receptor-selective analogues at NPY receptor expressing cells. To test internalization of the novel peptide analogues and the metabolic stability in human blood plasma, the corresponding 5(6)-carboxyfluorescein (CF) analogues were prepared and investigated. One of the most promising analogues, the Y 1-receptor selective [Lys(DOTA)(4), Phe(7), Pro(34)]NPY was labeled with (111)In and injected into nude mice that bear MCF-7 breast cancer xenografts, and biodistribution studies were performed. In vitro and in vivo studies suggest that receptor-selective analogues of NPY have promising characteristics for future applications in nuclear medicine for breast tumor diagnosis and therapy.
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Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Neoplasias/metabolismo , Neuropeptídeo Y/síntese química , Neuropeptídeo Y/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Bovinos , Linhagem Celular , Linhagem Celular Tumoral , Quelantes/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Humanos , Radioisótopos de Índio , Ligantes , Camundongos , Neoplasias/radioterapia , Neuropeptídeo Y/química , Neuropeptídeo Y/farmacocinética , Receptores de Neuropeptídeo Y/metabolismo , Coloração e Rotulagem , Distribuição TecidualRESUMO
The oxidoreductase Macrophage Migration Inhibitory Factor (MIF) is discussed as a promising target for immunomodulatory therapy in patients with severe sepsis. Moreover, MIF expresses tautomerase as well as thiol-protein oxidoreductase activities and has a potential role in cellular redox homeostasis, apoptosis inhibition, endotoxin responsiveness as well as regulation of nuclear transcription factors. To further elucidate a potential role of intracellular MIF in severe sepsis, we assessed alterations of intracellular MIF content in peripheral blood leukocytes of patients with severe sepsis in comparison to healthy controls and non-septic patients after major surgery. Intracellular MIF was significantly elevated simultaneously in lymphocytes, B-cells, macrophages and granulocytes of patients with severe sepsis when compared to healthy control individuals (p < 0.05) and increased when compared to non-septic patients after major surgery. In parallel, plasma MIF levels were elevated in severe sepsis (p < 0.05). There was no difference of intracellular MIF in lymphocytes, B-cells, macrophages or granulocytes between surviving and non-surviving patients with severe sepsis (p > 0.05). However, in survivors LPS ex vivo stimulation increased MIF secretion but not in non-survivors of sepsis (p < 0.05). This finding underlines the role of intracellular MIF in inflammatory diseases. It suggests monitoring of intracellular MIF in further clinical and non-clinical research valuable.
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Leucócitos/enzimologia , Fatores Inibidores da Migração de Macrófagos/sangue , Oxirredutases/sangue , Sepse/sangue , Antígenos CD/sangue , Antígenos CD19/sangue , Complexo CD3/sangue , Antígenos CD4/sangue , Antígenos CD8/sangue , Moléculas de Adesão Celular/sangue , Feminino , Proteínas Ligadas por GPI , Humanos , Receptores de Lipopolissacarídeos/sangue , Subpopulações de Linfócitos/enzimologia , Masculino , Sepse/enzimologia , Sepse/mortalidadeRESUMO
During sepsis, a severe systemic disorder, micronutrients often are decreased. Apoptosis is regarded as an important mechanism in the development of often significant immunosuppression in the course of the disease. This study aimed to investigate alpha-tocopherol and selenium in reference to apoptosis in patients with sepsis. 16 patients were enrolled as soon as they fulfilled the criteria of severe sepsis. 10 intensive care patients without sepsis and 11 healthy volunteers served as controls. alpha-Tocopherol, selenium and nucleosomes were measured in serum. Phosphatidylserine externalization and Bcl-2 expression were analyzed in T-cells by flow cytometry. Serum alpha-tocopherol and selenium were decreased in severe sepsis but not in non-septic critically ill patients (p < 0.05). Conversely, markers of apoptosis were increased in sepsis but not in critically ill control patients: Nucleosomes were found to be elevated 3 fold in serum (p < 0.05) and phosphatidylserine was externalized on an expanded subpopulation of T-cells (p < 0.05) while Bcl-2 was expressed at lower levels (p < 0.05). The decrease of micronutrients correlated with markers of accelerated apoptosis. Accelerated apoptosis in sepsis is associated with low alpha-tocopherol and selenium. The results support the investigation of micronutrient supplementation strategies in severe sepsis.
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Apoptose , Selênio/sangue , Sepse/sangue , alfa-Tocoferol/sangue , Adulto , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Fosfatidilserinas/análise , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sepse/metabolismo , Linfócitos T/metabolismoRESUMO
INTRODUCTION: The potent endogenous antimicrobial peptide human beta-defensin 2 (hBD2) is a crucial mediator of innate immunity. In addition to direct antimicrobial properties, different effects on immune cells have been described. In contrast to the well-documented epithelial beta-defensin actions in local infections, little is known about the leukocyte-released hBD2 in systemic infectious disorders. This study investigated the basic expression levels and the ex vivo inducibility of hBD2 mRNA in peripheral whole blood cells from patients with severe sepsis in comparison to non-septic critically ill patients and healthy individuals. METHODS: This investigation was a prospective case-control study performed at a surgical intensive care unit at a university hospital. A total of 34 individuals were tested: 16 patients with severe sepsis, 9 critically ill but non-septic patients, and 9 healthy individuals. Serial blood samples were drawn from septic patients, and singular samples were obtained from critically ill non-septic patients and healthy controls. hBD2 mRNA levels in peripheral white blood cells were quantified by real-time polymerase chain reaction in native peripheral blood cells and following ex vivo endotoxin stimulation. Defensin plasma levels were quantified by enzyme-linked immunosorbent assay. RESULTS: Endotoxin-inducible hBD2 mRNA expression was significantly decreased in patients with severe sepsis compared to healthy controls and non-septic critically ill patients (0.02 versus 0.95 versus 0.52, p < 0.05, arbitrary units). hBD2 plasma levels in septic patients were significantly higher compared to healthy controls and critically ill non-septic patients (541 versus 339 versus 295 pg/ml, p < 0.05). CONCLUSION: In contrast to healthy individuals and critically ill non-septic patients, ex vivo inducibility of hBD2 in peripheral blood cells from septic patients is reduced. Impaired hBD2 inducibility may contribute to the complex immunological dysfunction in patients with severe sepsis.
Assuntos
Sepse/sangue , beta-Defensinas/sangue , Adulto , Estudos de Casos e Controles , Estado Terminal , Expressão Gênica , Humanos , Interleucina-6/sangue , Leucócitos/metabolismo , Estudos Prospectivos , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Valores de Referência , beta-Defensinas/biossíntese , beta-Defensinas/genéticaRESUMO
OBJECTIVE: Cardiac surgery causes induction and release of inflammatory mediators that may be regulated by genetic background. Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator that is known to be up-regulated in patients undergoing cardiac operations. Here we analyzed genotype distribution and allele frequency of the MIF-173*G/C single nucleotide polymorphism (SNP) and MIF plasma levels in patients undergoing surgical revascularization with (on-pump, n=45) and without (off-pump, n=34) cardiopulmonary bypass (CPB). METHODS: Genotyping was performed using a real-time PCR-based system with a hybridization probe system specific for the MIF-173*G/C SNP. In on-pump patients, blood samples were drawn before start of CPB, after termination of CPB and 12h postoperatively. In off-pump patients, blood samples were collected before stabilizer placement, after removal of the stabilizer and 12h postoperatively. MIF levels were measured using ELISA technique. RESULTS: Genotype distribution and allele frequencies were comparable between on-pump and off-pump patients. When comparing patients according to MIF genotype, a significant increase of MIF plasma levels after completed coronary bypass grafting using CPB was found in patients heterozygous for the MIF-173*G/C SNP (p<0.05). Moreover, on-pump patients showed significantly decreased MIF plasma levels after 12h postoperatively (p<0.05). In off-pump patients, MIF plasma levels were not significantly different over the time-course and were independent of the genotype. CONCLUSIONS: Patients carrying the C-allele showed significantly increased levels of the proinflammatory cytokine MIF compared to G/G homozygous when revascularization was carried out using CPB. The G/C genotype may be associated with a severe inflammatory reaction and therefore preoperative screening could be beneficial for patients undergoing cardiac surgery using CPB.
Assuntos
Ponte Cardiopulmonar/efeitos adversos , Ponte de Artéria Coronária/métodos , Inflamação/genética , Fatores Inibidores da Migração de Macrófagos/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Ponte de Artéria Coronária sem Circulação Extracorpórea , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Inflamação/sangue , Inflamação/etiologia , Fatores Inibidores da Migração de Macrófagos/sangue , Pessoa de Meia-Idade , Período Pós-OperatórioRESUMO
Contamination of polymerase chain reaction (PCR) reagents continues to be a major problem when consensus primers are used for detection of low concentrations of bacterial DNA. We designed a real-time polymerase chain reaction (PCR) for quantification of bacterial DNA by using consensus primers that bind specifically to the 16S region of bacterial DNA. We have tested four different methods of decontamination of PCR reagents in a project aimed at detecting bacterial DNA at low concentrations: deoxyribonuclease (DNAse) treatment, restriction endonuclease digestion, UV irradiation, and 8-methoxypsoralen in combination with long-wave UV light to intercalate contaminating DNA into double-stranded DNA. All four methods result in inhibition of the PCR reaction, and most of the decontamination procedures failed to eliminate the contaminating bacterial DNA. Only the DNAse decontamination proved to be efficient in eliminating contaminating DNA while conserving PCR efficiency. All four decontamination methods are time consuming and have the possibility of carrying new contamination into the reaction mixture. However, decontamination with DNAse may help, together with the use of highly purified PCR reagents, in detecting small amounts of bacterial DNA in clinical specimens.