RESUMO
Previous experiments observed a 4^{+} state in the N=28 nucleus ^{44}S and suggested that this state may exhibit a hindered E2-decay rate, inconsistent with being a member of the collective ground state band. We populate this state via two-proton knockout from a beam of exotic ^{46}Ar projectiles and measure its lifetime using the recoil distance method with the GRETINA γ-ray spectrometer. The result, 76(14)_{stat}(20)_{syst} ps, implies a hindered transition of B(E2;4^{+}â2_{1}^{+})=0.61(19) single-particle or Weisskopf units strength and supports the interpretation of the 4^{+} state as a K=4 isomer, the first example of a high-K isomer in a nucleus of such low mass.
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BACKGROUND: Traumatic brain injury (TBI) can result in significant morbidity and mortality if not diagnosed in a timely manner. Brain computed tomography (CT) is the diagnostic gold standard but is of limited availability in most South African public hospitals, resulting in transfer of TBI patients to tertiary hospitals. OBJECTIVE: To describe the referral patterns and outcomes of patients with TBI referred to Groote Schuur Hospital Trauma Centre. METHODS: This was a retrospective audit of all patients admitted to the Trauma Centre who had a brain CT scan for suspected TBI between 1 February 2022 and 31 March 2022. Demographic data (age, sex), mechanism of injury and Glasgow Coma Score were recorded. Referral pathways were determined, and final disposition of patients was recorded. RESULTS: A total of 522 patients had a brain CT for TBI. Of these, 314 (60.1%) were referred from other hospitals. CT scan was abnormal in 178 (34.1%) patients. Three hundred and two (58.6%) were discharged home within 24 hours. The mean time between referral and CT scan was 13 hours. CONCLUSION: More than half of patients referred for a CT scan were discharged from the Trauma Centre within 24 hours of admission, which indicates additional costs and inefficiencies in the health system. These data are useful to guide resource planning and allocation for district hospitals, since less expensive point-of-care modalities now exist to diagnose TBI, and which are useful in indicating the prognosis of patients.
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Lesões Encefálicas Traumáticas , Encaminhamento e Consulta , Tomografia Computadorizada por Raios X , Centros de Traumatologia , Humanos , Encaminhamento e Consulta/estatística & dados numéricos , Lesões Encefálicas Traumáticas/diagnóstico por imagem , Lesões Encefálicas Traumáticas/diagnóstico , Lesões Encefálicas Traumáticas/epidemiologia , Masculino , África do Sul/epidemiologia , Feminino , Estudos Retrospectivos , Adulto , Pessoa de Meia-Idade , Escala de Coma de Glasgow , Adolescente , Adulto Jovem , IdosoRESUMO
AIMS: To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants. METHODS AND RESULTS: Spores (≥ 7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2.7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid. CONCLUSIONS: Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials. SIGNIFICANCE AND IMPACT OF THE STUDY: A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials.
Assuntos
Bacillus anthracis/efeitos dos fármacos , Bacillus thuringiensis/efeitos dos fármacos , Descontaminação/métodos , Desinfetantes/farmacologia , Ácido Peracético/farmacologia , Bacillus anthracis/ultraestrutura , Bacillus thuringiensis/ultraestrutura , Desinfetantes/química , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/ultraestruturaRESUMO
AIMS: To develop test methods and evaluate the survival of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air. METHODS AND RESULTS: Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination. CONCLUSIONS: Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores with similar kinetics. SIGNIFICANCE AND IMPACT OF THE STUDY: Hot, humid air is a potential alternative to conventional chemical decontamination.
Assuntos
Bacillus anthracis/isolamento & purificação , Bacillus thuringiensis/isolamento & purificação , Descontaminação/métodos , Temperatura Alta , Umidade , Ar , Microscopia Eletrônica de Transmissão , Esporos Bacterianos/isolamento & purificação , Esporos Bacterianos/ultraestrutura , Estatística como AssuntoRESUMO
BACKGROUND: Damage control laparotomy (DCL) is associated with high mortality. The purpose of this study was to review the outcomes of DCL. METHODS: All patients undergoing DCL for penetrating trauma from May 2015 to July 2017 were reviewed. Data retrieved were demographics, mechanism of injury, vitals, and biochemical parameters. Injury severity was described by the revised trauma score (RTS), penetrating abdominal trauma index (PATI), injury severity score (ISS) and trauma and injury severity score (TRISS). Indications for DCL, length of ICU stay, number of procedures and primary abdominal closure rates, complications and mortality were recorded. RESULTS: Fifty-one patients underwent DCL and 47 patients sustained gunshot injuries. Indications for laparotomy were haemodynamic instability (n = 27) and peritonism in stable patients (n = 22). The medians for the different severity scores were RTS 7.36, ISS 20, and PATI 30. The organs most commonly injured, in decreasing frequency, were small bowel (33), large bowel (25), abdominal vasculature (22), liver (18), stomach (14), kidney (10), diaphragm (10), spleen (9) and pancreas (8). DCL procedures performed were abdominal packing (36), temporary bowel ligation (30), vascular (5) and ureteric (1) shunting. The median number of laparotomies performed per patient was three, with a primary fascial closure rate of 69%. The mortality rate was 29%. CONCLUSION: DCL in our centre is associated with a 29% mortality rate. Severe acidosis, massive blood transfusion in first 24 hours and median PATI score more than 47 are independent risk factors associated with increased mortality.
Assuntos
Traumatismos Abdominais , Laparotomia , Traumatismos Abdominais/cirurgia , Humanos , Escala de Gravidade do Ferimento , Laparotomia/efeitos adversos , Centros de Traumatologia , Resultado do TratamentoRESUMO
BACKGROUND: Management of colon injuries has significantly evolved in the recent decades resulting in considerably decreased morbidity and mortality. We set out to investigate penetrating colon injuries in a high-volume urban academic trauma center in South Africa. METHODS: All patients with penetrating colon injuries admitted between 1/2015 and 1/2018 were prospectively enrolled. Data collection included demographics, injury profile and outcomes. Primary outcome was in-hospital mortality. Secondary outcome was morbidity. RESULTS: Two-hundred and five patients were included in the analysis. Stab and gunshot wounds constituted 18% and 82% of the cases, respectively. Mean age was 28.9 (10.2) years and 96.1% were male. Median injury severity score (ISS) and penetrating abdominal trauma index (PATI) were 16 (9-25) and 19 (10-26), respectively. A total of 47.8% of the patients had a complication per Clavien-Dindo classification. Colon leak rate was 2.4%. Wound and abdominal organ/space infection rate was 15.1 and 6.3%, respectively. Overall in-hospital mortality was 9.3%. Risk factors for mortality were higher ISS and PATI, shock on admission, need for blood transfusion, intra-abdominal vascular injury, damage control surgery, and extra-abdominal severe injuries. CONCLUSIONS: Contemporary overall complication rate remains high in penetrating colon injuries, however, anastomotic leak rate is decreasing. Colon injury associated mortality is related to overall injury burden and hemorrhage rather than to colon injuries.
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Traumatismos Abdominais , Ferimentos por Arma de Fogo , Ferimentos Penetrantes , Traumatismos Abdominais/epidemiologia , Traumatismos Abdominais/cirurgia , Adulto , Colo/lesões , Colo/cirurgia , Feminino , Humanos , Escala de Gravidade do Ferimento , Masculino , Estudos Prospectivos , Estudos Retrospectivos , África do Sul/epidemiologia , Ferimentos por Arma de Fogo/epidemiologia , Ferimentos por Arma de Fogo/cirurgia , Ferimentos Penetrantes/epidemiologia , Ferimentos Penetrantes/cirurgiaRESUMO
AIMS: To compare physical properties of spores that were produced in broth sporulation media at greater than 10(8) spores ml(-1). METHODS AND RESULTS: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0.68 +/- 0.11 microm wide and 1.21 +/- 0.18 microm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0.48 +/- 0.38 microm(3) and 0.97 +/- 0.07 microm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l(-1) glutamate and antifoam. Spores were 0.95 +/- 0.11 microm wide and 1.31 +/- 0.17 microm long. Spore volumes were 0.78 +/- 0.61 microm(3) and volume-equivalent spherical diameters were 1.14 +/- 0.11 microm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. CONCLUSIONS: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. SIGNIFICANCE AND IMPACT OF THE STUDY: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.
Assuntos
Bacillus/fisiologia , Meios de Cultura/química , Bacillus/ultraestrutura , Ácido Glutâmico , Microscopia Eletrônica , Sais , Esporos Bacterianos/fisiologia , Esporos Bacterianos/ultraestruturaRESUMO
Ultrasound is both a valuable diagnostic tool and a promoter of beneficial tissue bioeffects for the treatment of cardiovascular disease. Vascular effects can be mediated by mechanical oscillations of circulating microbubbles that may also encapsulate and shield therapeutic agents in the bloodstream. Here, the effect of color-Doppler ultrasound exposure on bevacizumab-loaded liposome delivery into the vascular bed was assessed in atheromatous porcine carotids. Bevacizumab, an anti-angiogenic antibody to vascular endothelial growth factor (VEGF-A), was loaded into echogenic liposomes (BEV-ELIP) and confirmed to be immunoreactive. BEV-ELIP flowing within the lumen were exposed to color-Doppler ultrasound at three acoustic pressures for 3.5 min during treatment at physiologic temperature and fluid pressure. To confirm the presence of bubble activity, cavitation was detected within the lumen by a single-element passive cavitation detector. After treatment, the artery was fixed at physiologic pressure and subjected to immunohistochemical analysis to assess the penetration of bevacizumab within the carotid wall. The results suggest that other factors may more strongly influence the deposition of bevacizumab into carotid tissue than color-Doppler ultrasound and cavitation. In both sets of arteries, preferential accumulation of bevacizumab occurred in locations associated with atheroma progression and neointimal thickening: fibrous tissue, necrotic plaque and areas near macrophage infiltration. The delivery of bevacizumab to carotid vascular tissue correlated with the properties of the tissue bed, such as permeability, or affinity for growth-factor binding. Future investigations using this novel therapeutic strategy may focus on characterizing the spatial extent of delivery and bevacizumab colocalization with biochemical markers of atheroma.
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Bevacizumab/administração & dosagem , Bevacizumab/química , Lipossomos/química , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/química , Animais , Suínos , Ultrassom/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
A case of interatrial septal aneurysm discovered by two-dimensional echocardiography and confirmed by ultrasound contrast technique is described. Previous reports are reviewed, emphasizing the differential diagnosis, prognosis and recommendations for follow-up.
Assuntos
Ecocardiografia/métodos , Aneurisma Cardíaco/diagnóstico , Adulto , Meios de Contraste , Feminino , Septos Cardíacos , HumanosRESUMO
Much effort has recently been directed toward ultrasound characterization of normal and abnormal left ventricular myocardium. The purpose of this study was to evaluate the normal acoustic properties of all four cardiac chambers as a first step toward tissue characterization of the atria and ventricles. The hypothesis was that integrated ultrasound backscatter would follow the pattern of collagen concentration in the cardiac chambers, being higher in the right side of the heart than in the left and in the atria compared with the ventricles. Seven normal canine hearts, perfusion-fixed in 10% formalin, were examined. Sections of the free walls of right and left ventricles and atria were studied in vitro with a 5 MHz transducer positioned at the focal distance from the epicardium. The radio frequency ultrasound signal energy from each specimen was derived, corrected for sample thickness and expressed as integrated backscatter, in decibel units less than the reflected energy from a stainless steel block. The backscatter was higher from the right ventricle than from the left ventricle (-64.5 +/- 1.25 [mean +/- SEM] [n = 7] versus -73.6 +/- 1.32; p less than 0.05), higher from the right atrium than from the right ventricle (-58.5 +/- 0.83 versus -64.5 +/- 1.25; p less than 0.05) and higher from the left atrium than from the left ventricle (-62.8 +/- 1.14 versus -73.6 +/- 1.32; p less than 0.05). These data show that backscatter is higher in the right ventricle than in the left ventricle and in the atria compared with the ventricles.(ABSTRACT TRUNCATED AT 250 WORDS)
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Ecocardiografia , Coração/anatomia & histologia , Acústica , Animais , Colágeno/metabolismo , Cães , Miocárdio/metabolismoRESUMO
Coronary arterial remodeling is a compensatory mechanism that may limit the adverse effects of coronary obstructive lesions by expansion of the entire vascular segment. To determine if this compensatory anatomic change occurs in patients, high-frequency epicardial echocardiography using a 12 MHz transducer was performed during open heart surgery in 33 patients (10 with normal coronary arteries undergoing valvular surgery and 23 with coronary atherosclerosis). From stop-frame videotape high-frequency epicardial echocardiographic images, cross-sectional measurements of luminal area and total arterial area (lumen, intima, media and dense adventitia) were made in the patients with atherosclerosis at the site of arterial lesions and from the most proximal portion of the same artery. Remodeling was defined as enlargement of the total arterial area. In normal arteries measurements were made from proximal and midarterial locations. In the patients with normal coronary arteries, total arterial area, as determined by high-frequency echocardiography, decreased from the proximal site to the midportion of the artery (from 10.4 +/- 0.9 to 8.4 +/- 1.0 mm2, p less than 0.05); luminal area also decreased (from 6.0 +/- 0.6 to 4.5 +/- 0.7 mm2, p less than 0.05). In patients with coronary arterial lesions, luminal area also decreased from the proximal site to the arterial lesion site (from 5.3 +/- 0.6 to 2.3 +/- 0.3 mm2, p less than 0.05), but total arterial area increased (from 11.6 +/- 1.0 to 13.0 +/- 1.0 mm2, p less than 0.05). Of the 25 coronary arteries evaluated, only 4 had angiographic evidence of coronary collateral formation. These data indicate that coronary arterial remodeling is an important compensatory mechanism in obstructive coronary disease.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Doença da Artéria Coronariana/diagnóstico por imagem , Vasos Coronários/diagnóstico por imagem , Ecocardiografia/métodos , Procedimentos Cirúrgicos Cardíacos , Circulação Colateral/fisiologia , Doença da Artéria Coronariana/fisiopatologia , Vasos Coronários/patologia , Feminino , Humanos , Cuidados Intraoperatórios , Masculino , Pessoa de Meia-IdadeRESUMO
To further define the capacity for recovery after acute phase electrical and mechanical injury in patients with Q wave myocardial infarction who were treated with standard measures, 120 lead body surface potential maps and radionuclide angiograms were recorded at day 5 before discharge and month 6 after infarction in 23 patients with a first infarction (12 anterior and 11 inferior by standard 12 lead electrocardiographic criteria). In addition to assessment of spatial changes in electrocardiographic and wall motion patterns, five quantitative variables were evaluated: minimal Q zone integral, sigma Q wave integral, maximal ST integral, left ventricular ejection fraction and left ventricular wall motion abnormality score. From day 5 to month 6 after infarction, the only change in the inferior infarction group was a gain in sigma Q wave (-91 +/- 40 mu V X s X 10(2) to -68 +/- 24 mu V X s X 10(2); p less than 0.05). In contrast, all variables improved over the same time period in the anterior infarction group: Q zone minimum, -34 +/- 20 to -24 +/- 13 mu V X s (p less than 0.05); sigma Q wave, -160 +/- 122 X 10(2) to -120 +/- 90 mu V X s X 10(2) (p less than 0.05); ST maximum, 44 +/- 19 to 18 +/- 9 mu V X s (p less than 0.01); ejection fraction, 54 +/- 7 to 63 +/- 17% (p less than 0.05); and wall motion score, 6 +/- 3 to 3 +/- 3 (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Eletrocardiografia , Coração/diagnóstico por imagem , Infarto do Miocárdio/fisiopatologia , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Idoso , Eletrocardiografia/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/tratamento farmacológico , Nitroglicerina/uso terapêutico , Cintilografia , Volume SistólicoRESUMO
The purpose of this study was to determine the accuracy of a new high frequency echocardiographic technique for the quantitative assessment of coronary artery luminal and wall dimensions. In 32 open chest animals, high frequency echocardiographic measurements (echo) of luminal diameter correlated well with in vitro histologic measurements (Histo) (r = 0.86; high frequency echo = 0.89 Histo + 0.79) (range 1.7 to 5.8 mm). Similar results were found in the evaluation of five human autopsy hearts studied in vitro. Coronary artery wall thickness measurements in human autopsy hearts showed a good correlation with high frequency echocardiographic measurements (r = 0.86; high frequency echo = 0.65 Histo + 0.24) (range 0.3 to 0.8 mm). In eight open chest calves, high frequency echocardiographic measurements of total vessel diameter correlated well with sonomicrometer measurements (Sono) (r = 0.94; high frequency echo = 1.03 Sono + 0.4) (range 2.1 to 5.3 mm). Inter- and intraobserver variability measurements of high frequency echocardiographic measurements demonstrated excellent reproducibility (r = 0.95, interobserver variability for wall thickness; r = 0.97, interobserver variability for luminal diameter; n = 10 postmortem human coronary arteries). In conclusion, high frequency echocardiography is an accurate and reproducible method of measuring coronary luminal and wall geometry and may be a potentially useful tool for in vivo coronary artery evaluation in patients.
Assuntos
Vasos Coronários/anatomia & histologia , Ecocardiografia , Animais , Doença das Coronárias/diagnóstico , Cães , Humanos , Especificidade da EspécieRESUMO
OBJECTIVES: The purpose of this study was to target acoustically reflective liposomes to atherosclerotic plaques in vivo for ultrasound image enhancement. BACKGROUND: We have previously demonstrated the development of acoustically reflective liposomes that can be conjugated for site-specific acoustic enhancement. This study evaluates the ability of liposomes coupled to antibodies specific for different components of atherosclerotic plaques and thrombi to target and enhance ultrasonic images in vivo. METHODS: Liposomes were prepared with phospholipids and cholesterol using a dehydration/ rehydration method. Antibodies were thiolated for liposome conjugation with N-succinimidyl 3-(2-pyridyldithio) propionate resulting in a thioether linkage between the protein and the phospholipid. Liposomes were conjugated to antifibrinogen or anti-intercellular adhesion molecule-1 (anti-ICAM-1). In a Yucatan miniswine model, atherosclerosis was developed by crush injury of one carotid and one femoral artery and ingestion of a hypercholesterolemic diet. After full plaque development the arteries were imaged (20-MHz intravascular ultrasound catheter and 7.5-MHz transvascular linear probe) after injection of saline, unconjugated liposomes and antibody conjugated liposomes. RESULTS: Conjugated liposomes retained their acoustically reflective properties and provided ultrasonic image enhancement of their targeted structures. Liposomes conjugated to antifibrinogen attached to thrombi and fibrous portions of the atheroma, whereas liposomes conjugated to anti-ICAM-1 attached to early atheroma. CONCLUSIONS: Our data demonstrate that this novel acoustic agent can provide varying targeting with different antibodies with retention of intravascular and transvascular acoustic properties.
Assuntos
Anticorpos , Arteriosclerose/diagnóstico por imagem , Aumento da Imagem , Lipossomos , Ultrassonografia de Intervenção/métodos , Animais , Anticorpos/administração & dosagem , Arteriosclerose/complicações , Artérias Carótidas/diagnóstico por imagem , Portadores de Fármacos , Endotélio Vascular/diagnóstico por imagem , Endotélio Vascular/metabolismo , Artéria Femoral/diagnóstico por imagem , Fibrinogênio/imunologia , Injeções Intra-Arteriais , Molécula 1 de Adesão Intercelular/imunologia , Lipossomos/administração & dosagem , Lipossomos/química , Suínos , Porco Miniatura , Tromboembolia/diagnóstico por imagem , Tromboembolia/etiologia , Gravação em VídeoRESUMO
The purpose of this study was to evaluate the in vivo characteristics of coronary atherosclerosis by using high frequency epicardial echocardiography. High frequency epicardial echocardiography was used to evaluate residual lumen and wall morphology at the sites of maximal coronary atherosclerosis in 26 patients undergoing coronary artery bypass grafting. The maximal/minimal wall thickness ratio was 3.1 +/- 0.2 (mean +/- SEM) with a large range (1.3 to 7.5). Portions of the wall were normal in 16 of 31 lesions; the percent normal circumference ranged from 9% to 85%. Maximal/minimal lumen diameter ratio was 1.5 +/- 0.1 (range 1.1 to 2.9). The shape of the residual coronary lumen was noncircular in 16 lesions: oval in 13 and complex in 3. The residual coronary lumen was eccentrically placed within six arteries. These data emphasize the variability of residual lumen and wall geometry in atherosclerosis.
Assuntos
Doença da Artéria Coronariana/patologia , Vasos Coronários/patologia , Ponte de Artéria Coronária , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/cirurgia , Vasos Coronários/diagnóstico por imagem , Ecocardiografia/métodos , Feminino , Humanos , Período Intraoperatório , MasculinoRESUMO
Selective transport of polymeric (p) immunoglobulins (Ig) of IgA and IgM isotypes into external secretions by pIg receptor-mediated mechanism depends on the incorporation of joining (J) chain into the polymers. Until now, availability of a free J chain for immunological and biophysical studies has been limited to preparations of denatured J chain forms with moderate yield. Here we report that a recombinant J chain (rJ) can be over-expressed as a soluble fusion protein with thioredoxin using a modified vector pET32 in Escherichia coli. An intact J chain was released by digestion with IgA1 protease from Neisseria gonorrhoeae and isolated in a good yield with immunological and biochemical properties similar to those of J chain obtained by chemical cleavage from pIgA.
Assuntos
Cadeias J de Imunoglobulina/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Humanos , Imunoglobulina A , Cadeias J de Imunoglobulina/genética , Imunoglobulina M , Dados de Sequência Molecular , Serina Endopeptidases/metabolismo , Tiorredoxinas/biossíntese , Tiorredoxinas/genéticaRESUMO
In order to assess cytokine-producing cells at the single cell level, the cytokine-specific ELISPOT assay has proven to be an important and sensitive method. The purpose of this study was to adapt this method to elucidate individual cells producing murine IL-2, IL-4 or IL-6. In order to establish these cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific cDNA transfected myeloma cell lines, e.g., X63-Ag8-653 X2, X63-Ag8-653 X4 and X63-Ag8-653 X6, respectively, were used as specific cytokine-producing cells. In the IL-2 ELISPOT assay, the coating reagent, monoclonal antibody (mAb) rat IgG2a anti-mouse IL-2 (CR #40014) was used while rabbit IgG polyclonal anti-mouse IL-2 was employed for detection of IL-2 spot forming cells (SFC). The mAbs anti-mouse IL-4, BVD4-1D11 and BVD6-24G2 were selected as capture and detection antibodies for enumeration of IL-4 SFC. For the IL-6 ELISPOT assay, anti-mouse IL-6 (MP5-20F3) mAb was used for coating and MP5-32C11 mAb was used for detection of IL-6 SFC. When IL-2 producing X63-Ag8-653 X2 cells were subjected to these three different ELISPOT assays, IL-2-specific SFC were only noted with the IL-2 ELISPOT system. In the case of IL-4 SFC, only X63-Ag8-653 X4 cells formed specific spots using the tandem of BVD4-1D11 and BVD6-24G2 mAbs. IL-6-specific spots developed in MP5-20F3 mAb pre-coated wells containing X63-Ag8-653 X6 cells, when developed with mAb anti-IL-6 (MP5-32C11). Addition of cycloheximide (50 micrograms/ml) inhibited formation of IL-2, IL-4 and IL-6 SFC by approximately 90%. When an unrelated mAb was used as detection antibody in these three different cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific SFC were not detected. Further, when concanavalin A stimulated T cells from Peyer's patch of normal mice were subjected to the respective cytokine-specific ELISPOT assay, IL-2, IL-4 and IL-6 SFC were enumerated. These results have shown that cytokine-specific IL-2, IL-4 and IL-6 ELISPOT assays have now been established and will allow analysis of the frequency of cytokine-secreting cells at the single cell level.
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Técnicas Imunológicas , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Interleucina-6/biossíntese , Animais , Células Cultivadas , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C3H , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , TransfecçãoRESUMO
1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)- alpha-phenylacetate (IQNP, 3), an analogue of QNB in which a phenyl ring has been replaced with an iodopropenyl substituent, was prepared and evaluated in vitro and in vivo for m-AChR selectivity and specificity. High specific activity [125]IQNP ([125I]-3) was synthesized in greater than 60% yield utilizing an electrophilic iododestannylation reaction with hydrogen peroxide for the oxidation of iodide. In in vitro receptor binding studies, 3 demonstrated high affinity for M1 (Ki = 0.78 nM), M2 (Ki = 1.06 nM), and M3 (Ki = 0.27 nM) subtypes. In vivo biodistribution studies in female rats [125I]-3 demonstrated high uptake in areas rich in muscarinic receptors such as the brain (cortex and striatum) and the heart. Blocking studies were performed with a series of receptor specific agents and demonstrated that the uptake of [125I]-3 was selective and specific for cerebral muscarinic receptor rich areas and that the binding to m-AChR is reversible. The high-yield preparation and specificity and selectivity of high specific activity [125I]IQNP for muscarinic receptors suggest that this is an attractive new agent for potential imaging of cerebral receptors using single photon tomographic imaging (SPECT).
Assuntos
Quinuclidinas/síntese química , Quinuclidinil Benzilato/análogos & derivados , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Radioisótopos do Iodo , Ligantes , Quinuclidinas/metabolismo , Quinuclidinas/farmacocinética , Quinuclidinil Benzilato/metabolismo , Ratos , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/metabolismo , Relação Estrutura-Atividade , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
A new class of substituted 1-phenyl-3-piperazinyl-2-propanones with antimuscarinic activity is reported. As part of a structure-activity relationship study of this class, various structural modifications, particularly ones involving substitution of position 1 and the terminal piperazine nitrogen, were investigated. The objective of this study was to derive new antimuscarinic agents with potential utility in treating urinary incontinence associated with bladder muscle instability. These compounds were examined for M1, M2, and M3 muscarinic receptor selectivity in isolated tissue assays and for in vivo effects on urinary bladder contraction, mydriasis, and salivation in guinea pigs. Potency and selectivity in these assays were influenced most notably by the nature of the substituent group on the terminal nitrogen of the piperazine moiety. Benzyl substitution was particularly advantageous in producing compounds with functional M3 receptor (smooth muscle) and bladder selectivity; it provided several candidates for clinical study. In vivo, 3-(4-benzyl-piperazinyl)-1-cyclobutyl-1-hydroxy-1-phenyl-2-propanone (24) demonstrated 11- and 37-fold separations in its effect on bladder function versus mydriatic and salivation responses, respectively. The corresponding 2-chlorobenzyl derivative 25 was more than 178-fold selective for M3 versus M1 and M2 muscarinic receptors. 3-(4-Benzylpiperazinyl)-1,1-diphenyl-1-hydroxy-2-propanone (51) was 18-fold selective for M3 versus M1 and 242-fold selective for M3 versus M2 receptors. It was also selective in guinea pigs, where it displayed 20- and 41-fold separations between bladder function and effect on mydriasis and salivation, respectively. In general, the results of this study are consistent with the proposition that the described piperazinylpropanones interact with muscarcinic receptors in a hydrogen-bonded form that presents a conformation similar to that apparently adopted by classical antimuscarinic agents.