RESUMO
Tobacco-specific nitrosamines (TSNAs) have been of concern to the public health community for decades and their reduction through agricultural practices, plant breeding, and tobacco processing has also been a decades-long industry effort. Despite those efforts, TSNAs, though lower, continue to be constituents of concern in tobacco products. This paper examines the TSNA levels of dark air-cured, dark fire-cured, and burley tobaccos purchased in the United States by U.S. Smokeless Tobacco Company LLC (USSTC) and of nine finished USSTC moist smokeless tobacco products. TSNA values of the incoming purchased tobaccos and the finished products showed considerable variability. For the incoming tobaccos, the coefficient of variation was generally more than 100 % for each tobacco type and for each of the measured TSNAs. The relative TSNA variability of the finished tobacco products was also considerable, averaging approximately 25 %. It was also found that the measured values for the finished products averaged well above the proposed FDA NNN proposed product standard of 1.0 µg/g dry weight. Because of the large variability in NNN values, products would have to average well below FDA's proposed product standard to be consistently compliant.
RESUMO
The present study reports novel aspects of the reproductive biology of the male common ringtail possum (Pseudocheirus peregrinus). Plasma testosterone was measured through a stimulation test using the gonadotrophin-releasing hormone agonist, buserelin. Following intra-muscular administration of buserelin, there was an increase (P<0.05) in testosterone concentration in the peripheral circulation 4 h later. Quantitative testicular histology of this species was described for the first time. Eight stages of the seminiferous epithelium cycle were identified in 10 possums and their relative frequency determined. Spermatozoa were recovered from the cauda epididymides of hemi-castrated possums and cryopreservation conducted in straws (6 degrees C min(-1)) using final glycerol concentrations ranging between 2 and 20% in Tris-citrate egg yolk extender (v/v). Frozen straws were thawed and post-thaw motility, rate of motility, the percentage of live-dead spermatozoa and the percentage of sperm with swollen decondensed nuclei recorded. Similar to other marsupial sperm, common ringtail possum cauda epididymidal spermatozoa required high levels of glycerol (10-16%) in order to maintain post-thaw viability.
Assuntos
Criopreservação , Espermatozoides/fisiologia , Testículo/anatomia & histologia , Testosterona/metabolismo , Animais , Busserrelina/farmacologia , Epididimo/citologia , Masculino , Phalangeridae , Motilidade dos Espermatozoides , Espermatozoides/efeitos dos fármacos , Temperatura , Testosterona/sangueRESUMO
The release of activin A in response to intravenous injection of the bacterial cell-wall component lipopolysaccharide (LPS) was investigated in an ovine model of acute inflammatory challenge in newborn and adult sheep, and in non-pregnant and pregnant ewes. Neonatal lambs (<20 days of age) showed a quantitatively similar response in terms of circulating concentrations of activin A, its binding protein follistatin and the cytokine interleukin-6 compared with adult ewes challenged with an equivalent dose (300 ng/kg bodyweight) of LPS. The fever response and plasma tumour necrosis factor-alpha release in response to LPS, however, were significantly (P < 0.01) less in lambs than in the adult group. Pregnant ewes in the last trimester of gestation had similar responses to LPS, in all aspects measured, compared with their non-pregnant counterparts, apart from an ablated fever response. Although the adult and neonatal sheep responded to LPS, a similar response was not apparent in the fetal circulation, possibly due to a protective effect of the placenta. A 10-fold increase in the dose of LPS (from 300 ng to 3 microg/kg bodyweight) given to neonatal lambs elicited an increase in several cytokine responses measured, with a significant (P< 0.05) increase in follistatin release. In contrast, the amount of activin released by the increased dose of LPS was similar to that invoked by the lower dose. The effect of tolerance to LPS was investigated by giving a second challenge of LPS 5 days after the initial injection. In all animals studied, there was an ablated (P < 0.05) response to the subsequent LPS injection, apart from a similar temperature-response profile. These data provide further evidence that activin A concentrations in the bloodstream are acutely responsive to inflammatory challenge in post-natal life and suggest that the response forms a significant component of the innate immune system.
Assuntos
Ativinas/sangue , Envelhecimento/imunologia , Subunidades beta de Inibinas/sangue , Lipopolissacarídeos/farmacologia , Prenhez/imunologia , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Feminino , Feto/efeitos dos fármacos , Exposição Materna , Troca Materno-Fetal , Gravidez , Prenhez/sangue , OvinosRESUMO
In several biological systems, the inhibin beta(A) homodimer activin A is stimulated by, and in turn, inhibits the action of interleukin (IL)-1 (both IL-1alpha and IL-1beta) and IL-6. The possibility that a similar regulatory relationship operates within the testis was investigated. Sertoli cells from immature (20-day-old) rats were cultured with human IL-1alpha or IL-1beta, human IL-6 and/or ovine FSH or dibutyryl cAMP. Activin A and the inhibin dimers, inhibin A and inhibin B, were measured by specific ELISA. Immunoreactive inhibin (ir-inhibin) was measured by RIA. Activin/inhibin subunit mRNA expression was measured by quantitative real-time PCR. Both IL-1 isoforms, but not IL-6, stimulated activin A secretion through increased synthesis of beta(A)-subunit mRNA. IL-1 also stimulated activin A secretion by testicular peritubular cells. In contrast to the effect on activin A, IL-1 suppressed inhibin beta(B)-subunit and, to a lesser extent, alpha-subunit mRNA expression, thereby reducing basal and FSH-stimulated inhibin B secretion by the Sertoli cells. Conversely, FSH inhibited basal activin A secretion and antagonised the stimulatory effects of IL-1. Dibutyryl cAMP partially inhibited the action of IL-1 on activin A secretion, but had no significant effect on basal activin A secretion. Secretion of inhibin A was low in all treatment groups. These data demonstrate that IL-1 and FSH/cAMP exert a reciprocal regulation of activin A and inhibin B synthesis and release by the Sertoli cell, and suggest a role for activin A as a potential feedback regulator of IL-1 and IL-6 activity in the testis during normal spermatogenesis and in inflammation.
Assuntos
Ativinas/farmacologia , Hormônio Foliculoestimulante/farmacologia , Subunidades beta de Inibinas/farmacologia , Inibinas/farmacologia , Interleucina-1/farmacologia , Células de Sertoli/metabolismo , Animais , Bucladesina/farmacologia , Técnicas de Cultura de Células , Ensaio de Imunoadsorção Enzimática/métodos , Retroalimentação Fisiológica , Interleucina-6/farmacologia , Masculino , RNA Mensageiro/análise , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Sertoli/efeitos dos fármacos , Estimulação QuímicaRESUMO
While it is well known that serious illness and inflammation reduce male fertility, the mechanisms involved are poorly understood. In adult male rats, a single injection of lipopolysaccharide at doses that induced either mild or severe inflammation, caused a biphasic decline in Leydig cell testosterone production and gonadotropin responsiveness. In the high dose group only, serum LH levels also were reduced; however, intratesticular testosterone concentrations remained at a level adequate to support qualitatively normal spermatogenesis in both treatment groups. Testicular interstitial fluid formation also declined in a dose-dependent fashion after lipopolysaccharide treatment. In the high dose group only, these hormonal and vascular changes were accompanied by an increase in endothelial permeability, microhemorrhage, and inflammatory cells in the testis, followed by vacuolization of round spermatid nuclei, disruption of Sertoli-germ cell contacts at stages I-IV of the cycle of the seminiferous epithelium, and subsequently apoptosis of spermatocytes at stages II-V. These data indicate that mild inflammation causes local inhibition of Leydig cell function with relatively little spermatogenic damage. The pathological changes in spermatogenic function during severe inflammation are most likely due to direct effects of inflammatory mediators on the seminiferous epithelium or testicular vasculature, rather than inhibition of the brain-pituitary-Leydig cell axis.
Assuntos
Endotoxinas/farmacologia , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Testículo/patologia , Animais , Líquidos Corporais/fisiologia , Gonadotropina Coriônica/farmacologia , Escherichia coli , Humanos , Marcação In Situ das Extremidades Cortadas , Inflamação/induzido quimicamente , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/patologia , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Testosterona/sangue , Fatores de TempoRESUMO
Recent evidence suggests a role for activin A, and its binding protein, follistatin, in inflammatory pathways. However, whether activin is released systemically during inflammation is not known. In this study, a release of activin A into the circulation occurred in sheep within 1 hour of injection of lipopolysaccharide. This rapid peak in activin A preceded the release of the key inflammatory cytokines, tumor necrosis factor-alpha and interleukin-6. Follistatin release into the circulation occurred some 4 hours after the peak of activin A and continued out to 24 hours from lipopolysaccharide treatment. These data are the first to document a circulatory response of activin A to an inflammatory stimulus, and together with previous findings, suggest that activin A may have both pro- and anti-inflammatory actions in regulating cytokine-driven pathways.
Assuntos
Glicoproteínas/sangue , Inflamação/sangue , Inibinas/sangue , Ativinas , Animais , Ensaio de Imunoadsorção Enzimática , Folistatina , Inflamação/induzido quimicamente , Interleucina-6/sangue , Lipopolissacarídeos , Ovinos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Activins are pluripotent growth factors that have recently been shown to be present in placental and fetal membrane preparations. Our previous studies have identified and purified activin A from ovine amniotic and allantoic fluids. In this study, ligand blots of side fractions from the isolation of activin A from allantoic fluid suggested the presence of activin-binding proteins other than follistatin. Further purification of one of these fractions involved two sequential reverse phase HPLC steps and a Superose 12HR fractionation. SDS-PAGE revealed a single protein band of 55 kDa, which was identified by NH2-terminal sequencing as ovine uterine milk protein (UTMP), a member of the serine protease inhibitor (serpin) superfamily of proteins. Further binding studies, using ligand blot techniques and Superose 12HR fractionation in the presence of [125I]activin, demonstrated UTMP to be an activin-binding protein with a lower affinity for activin than that of follistatin. A study of the specific binding behavior of UTMP to activin, using surface plasmon resonance, revealed an apparent equilibrium dissociation constant (Kd) of 49 +/- 25 nM, compared with the follistatin-activin Kd of 379 +/- 51 pM. Similar to another activin-binding protein, alpha2-macroglobulin, UTMP was unable to neutralize the bioactivity of activin in a bioassay based on the capacity of activin to inhibit the proliferation of an MPC-11 plasmacytoma cell line. The high concentrations of this protein in uterine fluid during pregnancy and its ability to bind activin suggest that UTMP may act as a low affinity, high capacity binding protein to sequester activin in the local uterine environment.
Assuntos
Alantoide/metabolismo , Líquidos Corporais/metabolismo , Glicoproteínas/metabolismo , Serpinas , Ovinos/metabolismo , Ativinas , Animais , Feminino , Folistatina , Inibinas/metabolismo , GravidezRESUMO
Recent clinical studies have questioned whether there is a qualitative change in the circulating isoforms of LH and FSH after stimulation by GnRH. The present study investigated the median charge of serum gonadotropin isoforms before and after an exogenous challenge of 100 micrograms GnRH in 10 girls and 10 boys undergoing pubertal development. All of the children had basal serum levels of LH and FSH and responses 30 and 60 min after GnRH treatment that were considered normal for puberty. LH and FSH in serum and eluates after electrophoresis in 0.10% agarose suspension were measured with sandwich fluoroimmunoassays. The increases in serum gonadotropin concentrations were generally similar for both sexes, but girls had significantly (P < 0.05) higher LH levels at 30 and 60 min than boys and a larger (P < 0.05) relative increase in serum FSH levels after GnRH treatment. In terms of the median charge of serum isoforms, the girls had significantly less negative (i.e. more basic) isoforms of LH (P < 0.05) and FSH (P < 0.001) than the boys. There was a change to more basic isoforms of both LH and FSH in all children 30 min after GnRH administration. For LH, the charge had returned to pretreatment values by 90 min after GnRH, but for FSH, the charge was still significantly (P < 0.05) more basic at this time (n = 4/sex). When the LH isoforms were more acidic before GnRH treatment, the change in median charge was larger than when the isoforms were more basic beforehand. A similar relationship was not found for FSH. Conversely, there was for FSH, but not for LH, a significant (P < 0.001) relationship between the relative increase in serum concentrations and the change in charge of the isoforms 60 min after GnRH treatment. These findings show that the circulating forms of LH and FSH become more basic after an exogenous challenge of GnRH in children undergoing pubertal development and suggest that the differences in the responses of LH and FSH isoforms may be due to differing degrees of selective secretion and/or survival.
Assuntos
Hormônio Foliculoestimulante/sangue , Hormônio Liberador de Gonadotropina , Hormônio Luteinizante/sangue , Puberdade/fisiologia , Adolescente , Criança , Feminino , Humanos , Cinética , Masculino , Caracteres SexuaisRESUMO
An acute challenge of exogenous GnRH elicits rapidly increased serum gonadotropin levels with qualitative changes to more basic isoforms of both FSH and LH. Chronic GnRH agonist therapy suppresses endogenous gonadotropins, and the serum levels of FSH and LH are low and fairly constant. A possible qualitative change in the gonadotropins during GnRH agonist therapy was investigated by determination of the median charge of the gonadotropin isoforms before and during therapy in 18 pubertal children. Two different GnRH agonists were studied: buserelin, given intranasally or as a sc implant for 1.5-34 months to five girls, aged 7-10 yr, and for 5-6 months to two boys, aged 11-13 yr; and triptorelin, administered as a depot preparation for 3-6 months to four girls, aged 9-12.5 yr, and for 1-24 months to seven boys, aged 10.5-12 yr. FSH and LH in serum and eluates after electrophoresis in 0.10% agarose suspension were measured with sandwich fluoroimmunoassays. The mean serum FSH and LH levels decreased significantly (P < 0.05) in girls during triptorelin therapy, whereas only the FSH level decreased (P < 0.05) in the boys. There were no significant (P > 0.05) changes in serum gonadotropin levels during buserelin therapy. All of the children had more basic serum isoforms of LH, and all but one had more basic forms of FSH during the GnRH agonist treatments. In a girl who had more basic gonadotropin isoforms after treatment with triptorelin for 2 and 6 months, a GnRH challenge elicited the release of still more basic isoforms. The changes in mean median charge to more basic gonadotropin isoforms were highly significant for both busereline (P < 0.01) and triptorelin (P < 0.001) treatment. An increased (P < 0.001) degree of charge heterogeneity was observed for FSH after triptorelin therapy. These findings show that there is a qualitative change in the isoforms of both FSH and LH in serum during GnRH agonist therapy in pubertal children. The changes in charge to more basic gonadotropin isoforms most likely reflect a direct effect at the pituitary level, leading to the synthesis and/or selective release of less sialylated and sulfated isoforms of the gonadotropins. The observed qualitative changes in the gonadotropin isoforms in these pubertal children may be part of the clinical effects of GnRH agonist therapy, leading to an arrest or regression of puberty.
Assuntos
Busserrelina/uso terapêutico , Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Puberdade/sangue , Pamoato de Triptorrelina/uso terapêutico , Adolescente , Criança , Feminino , Humanos , MasculinoRESUMO
Concentrations of LH and FSH are known to increase during normal pubertal development, but changes in the isoforms of the gonadotropins at this time have not been investigated in depth. We examined the median charge of serum LH and FSH using agarose suspension electrophoresis in 81 normal children at pubertal stages I-V. In pubertal girls there were no significant (P > 0.05) differences in the median charge of LH, but there was a small (P = 0.05) shift to more acidic FSH isoforms between pubertal stages I and IV. In boys there was a significant (P < 0.01) shift to more acidic isoforms for both LH and FSH by pubertal stage II. Further changes were not found later in puberty. Except for LH at pubertal stage I, where the median charge was similar (P > 0.05) for both sexes, the median charge was more basic (P < 0.001) for both LH and FSH in girls compared with boys at all five pubertal stages. The degree of charge heterogeneity of FSH, estimated as the peak width at half the peak height, was significantly (P < 0.01) larger at pubertal stage I than at pubertal stages III-V in both boys and girls. The charge heterogeneity of LH was similar for all pubertal stages in both sexes. In conclusion, there were few qualitative changes in the gonadotropins during normal female puberty, whereas in the male there was a dramatic shift to more acidic isoforms of LH and FSH early in puberty. This information may assist our understanding of normal and pathological processes during puberty and may be of clinical relevance in detecting the initiation of puberty in boys.
Assuntos
Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Puberdade/sangue , Adolescente , Criança , Pré-Escolar , Eletroforese em Gel de Ágar , Feminino , Humanos , Isomerismo , Masculino , Valores de Referência , Caracteres SexuaisRESUMO
Recent studies suggest that an age-related decline in ovarian inhibin B may play a role in the increase in follicular phase FSH in menstrual cycles of older women. Considering that the peripheral feedback regulation of FSH is dictated by the overall tone of inhibins, activins, and follistatins as well as estradiol, it is essential to determine the relative inputs of all of these regulators in assessing whether the collective peripheral input to FSH is one of inhibition or stimulation. To test the hypothesis that changes in the overall tone of peripheral feedback may contribute to this hallmark sign of aging, we compared the concentrations of dimeric inhibin A, inhibin B, activin A, and total and free follistatin in 7 young (mean age, 27.9 +/- 2.6 yr) and 10 older (mean age, 43.6 +/- 0.9 yr) cycling women during the follicular (FOLL; cycle day 6) and midluteal (ML; 7 days post-LH surge) phases of the menstrual cycle. Subjects were preselected on the basis of FOLL phase FSH levels (older, > or = 8.0 mIU/mL; younger, < 8 mIU/mL). Circulating FSH regulatory peptide concentrations were determined from samples pooled from blood drawn every 10 min for 8 daytime h using specific 2-site assays. In the older group, cycle length was shorter (29.1 +/- 0.5 vs. 26.1 +/- 0.5, young vs. older; P < 0.001), mean LH levels during the follicular phase were higher (LH, 5.6 +/- 0.8 vs. 8.8 +/- 1.1 mIU/mL, young vs. older; P < 0.001). Mean FSH levels for the older and younger groups averaged 10.8 +/- 0.8 and 6.2 +/- 0.3 mIU/mL, respectively. Estradiol levels were higher, but not statistically different, than those in the younger group (99 +/- 13 vs. 169 +/- 25 pmol/L, young vs. older; P = 0.06). In both age groups, inhibin B levels were higher in the FOLL vs. ML phase, inhibin A levels were higher in the ML vs. FOLL phase, but total activin A and total and free follistatin did not differ across cycle days. FOLL phase inhibin A levels were higher in the older group (16.3 +/- 2.4 vs. 26.4 +/- 3.4 pg/mL, young vs. older; P = 0.024), but levels of inhibin B were lower (323 +/- 80 vs. 163 +/- 24 pg/mL, young vs. older; P = 0.03). Overall, the estimated total inhibin activity (inhibin A plus inhibin B) was lower in older cycling than in younger women (339 +/- 82 and 189 +/- 24 pg/mL, young vs. older). Total and free follistatin levels were not different among the 2 groups of women. In contrast, total activin A levels were higher in the older cycling group (0.51 +/- 0.05 and 0.68 +/- 0.05 ng/mL, young vs. older; P = 0.02). No differences in age groups were observed during the ML phase for any of the variables measured. These data suggest that a net increase in stimulatory input resulting from a decrease in inhibin B and an increase in activin A may contribute in part to the monotropic FSH increase in aging women.
Assuntos
Envelhecimento/sangue , Hormônio Foliculoestimulante/sangue , Fase Folicular/fisiologia , Inibinas/sangue , Ciclo Menstrual/fisiologia , Ativinas , Adulto , Dimerização , Retroalimentação , Feminino , Folistatina , Glicoproteínas/sangue , Humanos , Hormônio Luteinizante/sangue , Pessoa de Meia-IdadeRESUMO
Recent evidence has suggested that activin A complexed to its binding protein, follistatin, may be present on the surface of cells through their interaction with heparan sulfate proteoglycans. As heparin is used routinely in many cardiovascular procedures for its anticoagulation properties, it may also cause the release of heparin-binding growth factors, including activin and follistatin, from the vascular endothelium. We examined the effect of two cardiovascular procedures and the use of heparin directly on the circulating concentrations of activin A and follistatin. A rapid and robust release of activin A and follistatin occurred in the circulation of patients undergoing abdominal aortic aneurysm repair or carotid endarterectomy at the time of vessel clamping and administration of heparin (5000 IU). This release pattern was dissimilar to that of the inflammatory marker, interleukin-1beta. However, administering heparin (2500 IU) to coronary angiography patients produced a similar activin and follistatin response, whereas placebo-treated angiography patients had no response. These findings illustrate that the routine use of heparin in surgical procedures elicits a rapid and robust release of activin and follistatin. This has direct clinical relevance by potentially activating heparin-binding growth factors that are important in injury, hyperplasia, and restenosis of vessels.
Assuntos
Anticoagulantes/efeitos adversos , Procedimentos Cirúrgicos Cardiovasculares/efeitos adversos , Glicoproteínas/metabolismo , Substâncias de Crescimento/metabolismo , Heparina/efeitos adversos , Inibinas/metabolismo , Ativinas , Idoso , Idoso de 80 Anos ou mais , Aneurisma da Aorta Abdominal/cirurgia , Angiografia Coronária , Endarterectomia das Carótidas , Feminino , Folistatina , Humanos , Interleucina-1/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
An immunofluorescent assay was used to characterize precisely the potency and specificity of fluorescein-conjugated immunoglobulin class-specific and polyvalent anti-sera. Stable antigen standards consisted of highly purified immunoglobulin antigens covalently bound to polyaminostyrene beads. Linear regressions of weighted logit transformations of relative fluorescent intensities versus the logarithm of relative conjugate concentrations were determined. The potency of conjugates was compared using two different methods derived from the logit transformation. Inappropriate specificities were measured in some conjugates described as immunoglobulin 'class-specific'.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Especificidade de Anticorpos , Imunofluorescência , Imunodifusão , Imunoglobulinas/classificação , MétodosRESUMO
Seventeen patients with multiple myeloma were given intravenous immunoglobulin at doses ranging from 150 mg/kg to 500 mg/kg in a phase I study. The intravenous immunoglobulin was well tolerated with only three transient episodes of mild clinical toxicity during 27 infusions. In no instance was hepatic or renal toxicity seen. Marked biologic variability over the one month study period in total IgG levels in patients with non-IgG myeloma and IgG subclasses in many of the patients was observed, making intravenous immunoglobulin half-life determinations based on IgG or IgG subclass levels problematical. The decay of functional antibody to hepatitis B surface antigen was determined. Analysis of the hepatitis antibody data suggested that intravenous immunoglobulin half-life was in the range of seven to 20 days for the entire study group and was not related to the isotype of the myeloma paraprotein or to the baseline levels of IgG. No infections were observed in the study group during the study period, but the potential for infection prophylaxis by intravenous immunoglobulin in myeloma patients must be evaluated in a randomized, prospective, controlled phase III study.
Assuntos
Agamaglobulinemia/terapia , Imunoglobulina G/análogos & derivados , Mieloma Múltiplo/complicações , Adulto , Agamaglobulinemia/complicações , Agamaglobulinemia/imunologia , Idoso , Ensaios Clínicos como Assunto , Feminino , Meia-Vida , Anticorpos Anti-Hepatite B/análise , Humanos , Imunização Passiva , Imunoglobulina G/administração & dosagem , Imunoglobulina G/classificação , Imunoglobulina G/metabolismo , Imunoglobulinas Intravenosas , Infusões Parenterais/efeitos adversos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologiaRESUMO
We have recently demonstrated that the proinflammatory cytokine, interleukin-6 (IL-6), could upregulate the production of protein S in the human hepatoma cell line, HepG-2, but not in endothelial cells. In this study, we have demonstrated that the combination of exogenous IL-6 and soluble IL-6 receptor (sIL-6R) could significantly upregulate protein S production in both primary human umbilical vein endothelial cells (HUVEC) and in the immortalized human microvascular endothelial cell line, HMEC-1. The IL-6/sIL-6R complex was also able to rapidly induce tyrosine phosphorylation of the IL-6 transducer, gp130. Neutralizing antibodies directed against either IL-6 or gp130 blocked protein S upregulation by the IL-6/sIL-6R complex. It was also observed that exogenous sIL-6R could also upregulate protein S by forming a complex with IL-6 constitutively produced by the endothelial cell. Two other cytokines which also utilize the gp130 receptor, oncostatin M (OSM) and leukemia inhibitory factor (LIF), were also able to upregulate endothelial cell protein S. This study demonstrates a mechanism that allows endothelial cells to respond to IL-6 and also illustrates the potential importance of circulating soluble receptors in the regulation of the anticoagulation pathway.
Assuntos
Antígenos CD/fisiologia , Citocinas/farmacologia , Endotélio Vascular/metabolismo , Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Proteína S/biossíntese , Receptores de Interleucina/fisiologia , Células Cultivadas , Dexametasona/farmacologia , Endotélio Vascular/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Humanos , Interleucina-1/farmacologia , Interleucina-11/farmacologia , Cinética , Fator Inibidor de Leucemia , Linfocinas/farmacologia , Oncostatina M , Peptídeos/farmacologia , Receptores de Interleucina-6 , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Veias UmbilicaisRESUMO
Several pro-inflammatory cytokines have been shown to be important in the modulation of the procoagulant response. However, what role these cytokines may have in the regulation of coagulation inhibitors is poorly understood. While the hepatocyte is a primary site of synthesis for the anticoagulant protein C and S, it is also a major target cell for the proinflammatory cytokine, IL-6. We have found that stimulation of HepG-2 hepatoma cells with IL-6 (5 ng/ ml) significantly increased the production of the anticoagulant cofactor, protein S, in both a time and dose dependent fashion. This increase was seen at both the RNA and protein level. A mouse monoclonal neutralizing antibody to human IL-6 suppressed the IL-6 effect in a concentration dependent fashion. IL-6 also increased the release of the C4b-binding protein but had no effect on protein C production. When combined with either dexamethasone or soluble IL-6 receptor, the IL-6 response was significantly enhanced. Oncostatin M, a functionally related cytokine, had a similar effect while other related cytokines, IL-11 and leukemia inhibitory factor, only had a marginal effect. IL-1, TGF-beta, and TNF-alpha had no significant effect on protein S production. These results indicate that IL-6 may play an important regulatory role in the anti-coagulant pathway.
Assuntos
Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/biossíntese , Proteína S/biossíntese , Animais , Anticorpos Monoclonais/farmacologia , Sequência de Bases , Citocinas/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Humanos , Interleucina-6/antagonistas & inibidores , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteína S/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Estimulação Química , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Despite thromboprophylaxis, deep vein thrombosis is a common complication of major orthopedic surgery. Predisposing genetic risk factors are unknown. In this case-control study, we investigated the association of the insertion (I)/deletion (D) angiotensin converting enzyme (ACE) gene polymorphism, Factor V Leiden (R506Q) mutation, and 5,10 methylenetetrahydrofolate reductase (MTHFR) gene polymorphism with post-operative venous thrombosis in 85 patients who underwent elective total hip arthroplasty. The odds of a thrombotic event following hip surgery among subjects with the DD genotype of the ACE gene was increased more than 10-fold compared to subjects with the II genotype (odds ratio 11.7 [95% confidence interval 2.3-84.5]); it was increased 5-fold in subjects with the ID genotype compared to the II genotype (odds ratio 5.0 [95% confidence interval 1.1-34.9]). Mean plasma ACE level in control subjects not on ACE inhibitors at the time of study (n=43) was lowest in persons homozygous for the I allele (18.9+/-7.95 U/l), intermediate in patients with the ID genotype (31.6+/-10.8 U/l) and highest in subjects homozygous for the D allele (44.0+/-7.14 U/l). Mean plasma ACE level among cases was higher (33.0 U/l, n=25) than among controls (29.4 U/l, n=43) but this difference was not statistically significant. Neither the Factor V Leiden mutation nor MTHFR gene polymorphism increased the risk of thrombosis following hip replacement. These results demonstrate that the I/D ACE gene polymorphism is a potent risk factor for thrombosis in subjects undergoing total hip arthroplasty.
Assuntos
Artroplastia de Quadril , Peptidil Dipeptidase A/genética , Polimorfismo Genético , Complicações Pós-Operatórias/epidemiologia , Embolia Pulmonar/epidemiologia , Deleção de Sequência , Trombofilia/epidemiologia , Trombose Venosa/epidemiologia , Resistência à Proteína C Ativada/epidemiologia , Resistência à Proteína C Ativada/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Anticoagulantes/uso terapêutico , Doenças Cardiovasculares/epidemiologia , Estudos de Casos e Controles , Comorbidade , Fator V/análise , Fator V/genética , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2) , Pessoa de Meia-Idade , New Jersey/epidemiologia , Obesidade/epidemiologia , Razão de Chances , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Peptidil Dipeptidase A/sangue , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Prevalência , Embolia Pulmonar/etiologia , Grupos Raciais/genética , Fatores de Risco , Fumar/epidemiologia , Trombofilia/genética , Trombose Venosa/etiologia , Trombose Venosa/prevenção & controleRESUMO
We measured the association constant (Ka) for 42 murine monoclonal antibodies (MAbs) to human IgG epitopes. Included are antibodies, previously evaluated in an IUIS/WHO collaborative study, to various epitopes on the four subclasses of human IgG - IgG Fc, IgG Fab, kappa, and lambda - and to selected IgG allotopes. We used a sequential-saturation immunofluorescent assay and interactive computer program to determine the Ka by Scatchard analysis. Kas ranged from unmeasurably low by this method (approximately 10(6) L/M) to 3.8 X 10(9) L/M. Some class specific MAbs had large Ka differences for different subclasses and some subclass specific MAbs had large Ka differences for molecules of the same subclass but of different light-chain types.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Afinidade de Anticorpos , Epitopos/imunologia , Humanos , Alótipos de Imunoglobulina/imunologia , Imunoglobulina G/classificação , Cadeias Leves de Imunoglobulina/imunologia , CinéticaRESUMO
We used an immunofluorescent sequential-saturation-of-antibody assay and an interactive computer program for Scatchard analysis to determine association constants (Ka) of 33 murine monoclonal antibodies (Mabs) specific for human IgA epitopes. Ka ranged from 0.37 to 690 x 10(7) liters per mole (an approximate 1900-fold difference). Specificity was validated with a panel of 18 highly purified IgA1 and IgA2 myeloma proteins and secretory IgA using an immunofluorometric assay. Western blots of bacterial IgA protease digests were used to locate the epitopes of IgA specific Mabs in either the Fab, Fc, or hinge region. Mabs specific for unique epitopes on secretory IgA or free secretory component (FSC) were produced and evaluated.
Assuntos
Anticorpos Monoclonais/análise , Imunoglobulina A/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sítios de Ligação , Western Blotting , Eletroforese em Gel de Poliacrilamida , Epitopos , Imunofluorescência , Humanos , Imunoglobulina A/classificação , CamundongosRESUMO
Seventy-four monoclonal antibodies (McAb) of putative specificity for human IgG (11), the IgG sub-classes (59) or Gm allotypes (4) have been evaluated for reactivity and specificity in eight laboratories employing different assay techniques or protocols. For the IgG, IgG3, IgG4, G1m(f) and G3m(u) specificities McAb have been produced that can be satisfactorily applied in most methodologies employed and have potential as reference reagents. The IgG1 and particularly IgG2 specificities proved problematical with all McAb evaluated demonstrating apparent assay restriction and whilst performing well in some assays proved to be poor or inactive reagents in others. However, the study identifies McAb individually suited to application within most commonly employed methodologies. Epitope display is the probable variability rather than capricious behaviour by the McAb. IgG1 and IgG2 were the least immunogenic of the sub-class proteins and there is evidence that epitope display is influenced by the physical and chemical procedures used to immobilize or fix antigen - a common requirement in the assay systems studied.