RESUMO
Bone loss occurs frequently following allogeneic haematopoietic stem cell transplantation (alloSCT). The Australasian Leukaemia and Lymphoma Group conducted a prospective phase II study of pretransplant zoledronic acid (ZA) and individualised post-transplant ZA to prevent bone loss in alloSCT recipients. Patients received ZA 4 mg before conditioning. Administration of post-transplant ZA from days 100 to 365 post alloSCT was determined by a risk-adapted algorithm based on serial bone density assessments and glucocorticoid exposure. Of 82 patients enrolled, 70 were alive and without relapse at day 100. A single pretransplant dose of ZA prevented femoral neck bone loss at day 100 compared with baseline (mean change -2.6±4.6%). Using the risk-adapted protocol, 42 patients received ZA between days 100 and 365 post alloSCT, and this minimised bone loss at day 365 compared with pretransplant levels (mean change -2.9±5.3%). Femoral neck bone loss was significantly reduced in ZA-treated patients compared with historical untreated controls at days 100 and 365. This study demonstrates that a single dose of ZA pre-alloSCT prevents femoral neck bone loss at day 100 post alloSCT, and that a risk-adapted algorithm is able to guide ZA administration from days 100 to 365 post transplant and minimise further bone loss.
Assuntos
Densidade Óssea/fisiologia , Difosfonatos/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imidazóis/uso terapêutico , Condicionamento Pré-Transplante/efeitos adversos , Transplante Homólogo/efeitos adversos , Difosfonatos/farmacologia , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imidazóis/farmacologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Condicionamento Pré-Transplante/métodos , Transplante Homólogo/métodos , Ácido ZoledrônicoRESUMO
The atypical antipsychotic risperidone (RSP) is often associated with weight gain and cardiometabolic side effects. The mechanisms for these adverse events are poorly understood and, undoubtedly, multifactorial in etiology. In light of growing evidence implicating the gut microbiome in the host's energy regulation and in xenobiotic metabolism, we hypothesized that RSP treatment would be associated with changes in the gut microbiome in children and adolescents. Thus, the impact of chronic (>12 months) and short-term use of RSP on the gut microbiome of pediatric psychiatrically ill male participants was examined in a cross-sectional and prospective (up to 10 months) design, respectively. Chronic treatment with RSP was associated with an increase in body mass index (BMI) and a significantly lower ratio of Bacteroidetes:Firmicutes as compared with antipsychotic-naïve psychiatric controls (ratio=0.15 vs 1.24, respectively; P<0.05). Furthermore, a longitudinal observation, beginning shortly after onset of RSP treatment, revealed a gradual decrease in the Bacteroidetes:Firmicutes ratio over the ensuing months of treatment, in association with BMI gain. Lastly, metagenomic analyses were performed based on extrapolation from 16S ribosomal RNA data using the software package, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Those data indicate that gut microbiota dominating the RSP-treated participants are enriched for pathways that have been implicated in weight gain, such as short-chain fatty acid production.
Assuntos
Bacteroidetes , Firmicutes , Microbioma Gastrointestinal/efeitos dos fármacos , Transtornos Mentais , Risperidona , Aumento de Peso/efeitos dos fármacos , Adolescente , Antipsicóticos/administração & dosagem , Antipsicóticos/efeitos adversos , Bacteroidetes/efeitos dos fármacos , Bacteroidetes/isolamento & purificação , Criança , Estudos Transversais , Feminino , Firmicutes/efeitos dos fármacos , Firmicutes/isolamento & purificação , Humanos , Masculino , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/microbiologia , Risperidona/administração & dosagem , Risperidona/efeitos adversosRESUMO
AIM: To determine whether there is a sex difference in placebo and ibuprofen analgesia expectancy. METHODS: We measured detection and tolerance thresholds for electrically induced pain in the ear lobe in healthy subjects (10 male, 10 female) to study sex differences in expectancy following either ibuprofen 800 mg or placebo in four different expectancy states. Subjects took ibuprofen or placebo in a two-by-two factorial design (the balanced placebo design). We randomly assigned subjects to start in one of the four expectancy states. We analysed the results using analysis of variance for repeated measures with baseline pain as a covariate. RESULTS AND CONCLUSION: We found no sex difference in baseline pain threshold or tolerance levels. When partitioned by sex and expectancy state, analgesia only occurred in males during positive expectancy states at 2, 3 and 4 h post-placebo, and at 1 and 2 h post-ibuprofen. The time course of analgesic action in males was as expected considering the pharmacokinetic profile of ibuprofen. Our study found that dosages of 800 mg of ibuprofen are ineffective in producing analgesia in women regardless of their expectations. We hypothesize that ibuprofen analgesia is produced by a combination of specific pharmacological effects and a non-specific beta endorphin-mediated placebo effect. Whatever the mechanism responsible for the analgesic response seen in males, this research re-emphasizes the importance of psychological factors in determining drug response. It also shows that these factors can differ between men and women, and thus the contribution of psychological factors on analgesia needs to be seriously re-evaluated.
Assuntos
Analgesia/psicologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Ibuprofeno/uso terapêutico , Dor/tratamento farmacológico , Caracteres Sexuais , Adolescente , Adulto , Anti-Inflamatórios não Esteroides/farmacologia , Estudos Cross-Over , Estimulação Elétrica , Feminino , Humanos , Ibuprofeno/farmacologia , Masculino , Pessoa de Meia-Idade , Dor/psicologia , Medição da Dor/efeitos dos fármacos , Medição da Dor/psicologia , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/psicologia , PlacebosRESUMO
The opportunistic protozoan parasite Toxoplasma gondii is well known as a strong inducer of cell-mediated immunity, largely as a result of proinflammatory cytokine induction during in vivo infection. Yet, during intracellular infection the parasite suppresses signal transduction pathways leading to these proinflammatory responses. The opposing responses are likely to reflect the parasite's need to stimulate immunity allowing host survival and parasite persistence, and at the same time avoiding excessive responses that could result in parasite elimination and host immunopathology. This Review summarizes past and present investigations into the effects of Toxoplasma on host cell signal transduction. These studies reveal insight into the profound suppression of proinflammatory cytokine responses that occurs when the parasite infects macrophages and other cells of innate immunity.
Assuntos
Interações Hospedeiro-Parasita/imunologia , Macrófagos/parasitologia , Transdução de Sinais/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Toxoplasmose/parasitologia , Animais , Citocinas/biossíntese , Citocinas/imunologia , Regulação para Baixo , Humanos , Imunidade Inata , Macrófagos/imunologiaRESUMO
BACKGROUND: Bortezomib has been shown to be a safe and efficacious for the treatment of relapsed and refractory multiple myeloma (MM). Here we report a subset analysis of Australian and New Zealand data from the International Extended Access Programme for bortezomib. METHODS: Patients with more than or equal to two prior lines of therapy were given bortezomib 1.3 mg/m(2) (i.v. bolus days 1, 4, 8, 11) for up to eight 21-day cycles (C). Dexamethasone, 20 mg/day p. o. on the day of, and day after, bortezomib was added after C2 for progressive disease or after C4 for stable disease. Efficacy was assessed using modified Southwest Oncology Group criteria in the intent-to-treat group. Results were compared between the Australian and New Zealand and international cohort. RESULTS: One hundred and eleven patients from 16 centres (55% men, median age 61.9 years) had a median of 5.2 +/- 2.8 treatment cycles of bortezomib. Among them, 82% had > or =3 prior therapies. Grade 3-4 treatment-related adverse events were reported in 57 patients (52%); the most common were thrombocytopenia (25.7%), anaemia (8.3%), peripheral neuropathy (7.3%) and diarrhoea (7.3%). Responses were evaluable in 106 patients: 22% achieved a best response of complete response/response and 20% partial response (overall response rate of 42%). Median times to first and best responses were 42 days and 69 days, respectively. Compared with the international cohort, the cohorts from Australian and New Zealand showed inferior overall response rates (54 vs 42%, P = 0.001), possibly due to heavier pretreatment (82% greater than or equal to three prior therapies vs 68%, P < 0.001). CONCLUSION: Our analysis confirms that bortezomib is safe and effective in relapsed and refractory MM in a real-life clinical setting.
Assuntos
Ácidos Borônicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Pirazinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Austrália/epidemiologia , Ácidos Borônicos/efeitos adversos , Bortezomib , Estudos de Coortes , Feminino , Gastroenteropatias/induzido quimicamente , Gastroenteropatias/epidemiologia , Humanos , Cooperação Internacional , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/epidemiologia , Mieloma Múltiplo/prevenção & controle , Nova Zelândia/epidemiologia , Pirazinas/efeitos adversos , Recidiva , Resultado do TratamentoRESUMO
Toll-like receptor (TLR)/MyD88 signalling has emerged as a major pathway of pathogen recognition in the innate immune system. Here, we review recent data that begin to show how this pathway controls the immune response to protozoan infection, with particular emphasis on the opportunistic pathogen Toxoplasma gondii. The various ways that the parasite activates and suppresses TLR/MyD88 signalling defines several key principals that illuminate the complexities of the host-pathogen interaction. We also speculate how TLR/MyD88 signalling might be exploited to provide protection against Toxoplasma, as well as other protozoa and infection in general.
Assuntos
Receptores Toll-Like/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Interações Hospedeiro-Parasita , Humanos , Imunidade Celular , Imunidade nas Mucosas , Ligantes , Transdução de Sinais/imunologiaRESUMO
Numerous lines of preclinical and clinical evidence support the existence of a graft-versus-leukemia effect, but less evidence supporting a comparable graft-versus-lymphoma effect exists. We review here current clinical data addressing the graft-versus-lymphoma effect, including comparisons of autologous, syngeneic, and allogeneic transplantation; responses to immunomodulation; and responses to nonmyeloablative stem cell transplantation. Despite several limitations of the data, we believe that there is sufficient evidence suggesting a significant graft-versus-lymphoma effect. In addition, we discuss approaches for clinical management of lymphoma patients, opportunities for mechanistic studies afforded by donor leukocyte infusions and nonmyeloablative transplantation, and suggestions for clinical studies to further define the magnitude and applicability of the graft-versus-lymphoma effect.
Assuntos
Efeito Enxerto vs Tumor , Transplante de Células-Tronco Hematopoéticas/métodos , Linfoma/terapia , Humanos , Transfusão de Leucócitos , Condicionamento Pré-Transplante/métodos , Imunologia de TransplantesRESUMO
Control of microbial infection requires regulated induction of NF-kappaB-dependent proinflammatory cytokines such as IL-12 and TNF-alpha. Activation of this important transcription factor is driven by phosphorylation-dependent degradation of the inhibitory IkappaB molecule, an event which enables NF-kappaB translocation from the cytoplasm to the nucleus. In this study, we show that intracellular infection of macrophages with the protozoan parasite Toxoplasma gondii induces rapid IkappaB phosphorylation and degradation. Nevertheless, NF-kappaB failed to translocate to the nucleus, enabling the parasite to invade cells without triggering proinflammatory cytokine induction. Infected cells subsequently subjected to LPS triggering were severely crippled in IL-12 and TNF-alpha production, a result of tachyzoite-induced blockade of NF-kappaB nuclear translocation. Our results are the first to demonstrate the ability of an intracellular protozoan to actively interfere with the NF-kappaB activation pathway in macrophages, an activity that may enable parasite survival within the host.
Assuntos
Núcleo Celular/metabolismo , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Proteínas I-kappa B , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , NF-kappa B/antagonistas & inibidores , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/imunologia , Transporte Ativo do Núcleo Celular/imunologia , Animais , Linhagem Celular , Núcleo Celular/imunologia , Núcleo Celular/parasitologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Inflamação/imunologia , Inflamação/parasitologia , Inflamação/prevenção & controle , Interleucina-12/antagonistas & inibidores , Interleucina-12/biossíntese , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Fosforilação , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Ubiquitina/metabolismoRESUMO
Neutrophils are well known to rapidly migrate to foci of infection, where they exert microbicidal functions. We sought to determine whether neutrophils responding to in vivo infection with the protozoan pathogen Toxoplasma gondii were capable of IL-12 production as suggested by recent in vitro studies. Intraperitoneal infection induced a neutrophil influx by 4 h, accompanied by ex vivo IL-12 p40 and p70 release. Approximately 85% of the neutrophils displayed intracellular stores of IL-12, as determined by flow cytometry and confocal fluorescence microscopy. Neutrophils from IFN-gamma knockout mice also expressed IL-12, ruling out an IFN-gamma-priming requirement. Neither infected nor uninfected peritoneal macrophages displayed intracellular IL-12, but these cells were strongly IL-10(+). Infection per se was unnecessary for IL-12 production because peritoneal and peripheral blood neutrophils from uninfected animals contained IL-12(+) populations. Expression of the granulocyte maturation marker Gr-1 (Ly-6G) was correlated with IL-12 production. Mice depleted of their granulocytes by mAb administration at the time of infection had decreased serum levels of IL-12 p40. These results suggest a model in which neutrophils with prestored IL-12 are rapidly mobilized to an infection site where they are triggered by the parasite to release cytokine. Our findings place neutrophils prominently in the cascade of early events leading to IL-12-dependent immunity to T. gondii.
Assuntos
Movimento Celular/imunologia , Interleucina-12/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Doença Aguda , Animais , Animais não Endogâmicos , Antígenos Ly/biossíntese , Regulação para Baixo/imunologia , Feminino , Granulócitos/imunologia , Granulócitos/metabolismo , Humanos , Injeções Intraperitoneais , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Interleucina-12/sangue , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutropenia/imunologia , Neutropenia/parasitologia , Neutrófilos/parasitologia , Neutrófilos/patologia , Peritonite/imunologia , Peritonite/parasitologia , Peritonite/patologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose Animal/patologiaRESUMO
The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC, arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) and arsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidans is gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functional trxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR and arsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.
Assuntos
Acidithiobacillus thiooxidans/genética , Adenosina Trifosfatases/genética , Antimônio/farmacologia , Arsênio/farmacologia , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Bombas de Íon , Complexos Multienzimáticos , Acidithiobacillus thiooxidans/classificação , Acidithiobacillus thiooxidans/efeitos dos fármacos , ATPases Transportadoras de Arsenito , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Cinética , Proteínas de Membrana/genética , Filogenia , Plasmídeos , Mapeamento por Restrição , Tiorredoxinas/metabolismoRESUMO
Trichinella spiralis L1 larvae infect susceptible hosts by invading epithelial cells that line the small intestine. Invasion also occurs in vitro when larvae are inoculated into cultures of epithelial cells from several different animal species. To further investigate the mechanism of invasion, we studied the interaction of larvae with the rat epithelial cell line IEC-6. Larvae did not invade IEC-6 cells, but did cause the cells to take up parasite glycoproteins. Glycoprotein bearing cells remained viable and were detectable in monolayers for as long as 24 h, suggesting that the glycoproteins were not toxic for cells. Immunofluorescence revealed that parasite glycoproteins localized in the nuclei, mitochondria and cytoplasm and we found evidence for selection of certain molecules between nuclear and cytoplasmic compartments. Using fluorescent dextrans as fluid phase markers we found 17-38% of the cells in inoculated monolayers were engorged with dextran and that dextran was free in the cytoplasm. Increased dextran uptake was not lethal, required the presence of activated larvae, and was often associated with uptake of parasite glycoproteins. These observations suggest that larvae caused plasma membrane wounds. Our results showed that neither delivery of glycoproteins nor mechanical wounding is sufficient to allow entry of the parasite into resistant epithelial cells. Because both invasion-resistant and susceptible epithelial cells undergo non-lethal wounding, we propose that larvae-induced injury to epithelial cells may result in release of cell-specific mediators that signal larva to invade a particular cell line or, alternatively, to ignore it.
Assuntos
Membrana Celular/parasitologia , Células Epiteliais/parasitologia , Glicoproteínas/metabolismo , Trichinella spiralis/patogenicidade , Animais , Linhagem Celular , Membrana Celular/ultraestrutura , Dextranos/metabolismo , Endocitose , Citometria de Fluxo , Fluorescência , Proteínas de Helminto/metabolismo , Hexoses/metabolismo , Larva/metabolismo , Larva/patogenicidade , Microscopia Confocal , Ratos , Frações Subcelulares , Trichinella spiralis/crescimento & desenvolvimentoRESUMO
OBJECTIVE: To determine the performance characteristics of enzyme-based immunoassay (EIA) kits for the detection of antinuclear and other autoantibodies of defined specificities. METHODS: Nine manufacturers of EIA kits to detect antibodies of defined specificities participated in a study in which they received coded sera from the Centers for Disease Control and Prevention. These coded sera contained different dilutions of antibody of one specificity mixed with sera containing antibodies of other specificities. The manufacturers were asked to use their standard technology to determine antibody content and send the data to a committee of the International Union of Immunological Societies for analysis. The data were analyzed for sensitivity and specificity in the detection of anti-double-stranded DNA (anti-dsDNA), anti-single-stranded DNA, antihistone, anti-Sm, anti-U1 RNP, anti-SSA/Ro, anti-SSB/La, anti-Scl-70 (DNA topoisomerase I), anticentromere, and anti-Jo-1 antibodies. In addition, replicate samples were included in the coded sera to evaluate the precision of each EIA method. RESULTS: Lack of sensitivity and specificity was most evident in the anti-dsDNA and anti-Sm kits, although 2 kits for anti-dsDNA achieved acceptable sensitivity and specificity. Generally, anti-SSA/Ro, anti-SSB/La, anti-Scl-70, anticentromere, and anti-Jo-1 kits performed well. Many false-positive results were obtained with a multiple myeloma serum containing cryoprecipitates, but multiple myeloma sera without cryoprecipitates presented no problem in the EIA system. Precision, based on evaluation of replicate samples, varied from very good to poor. CONCLUSION: No single manufacturer was clearly superior to others in terms of their products' overall sensitivity, specificity, and precision. Areas that needed improvement were in kits for the detection of antibodies to dsDNA and to Sm antigen. Some EIA kits achieved good sensitivity and specificity. Individual manufacturers were informed of the performance of their respective kits so they could take measures to correct perceived deficiencies and thus improve the reliability of a group of important diagnostic assays used in the evaluation of systemic rheumatic diseases.
Assuntos
Anticorpos Antinucleares/análise , Anticorpos Antinucleares/imunologia , Especificidade de Anticorpos , Doenças Autoimunes/diagnóstico , Técnicas Imunoenzimáticas/métodos , RNA Citoplasmático Pequeno , Autoantígenos/análise , Autoantígenos/genética , Autoantígenos/imunologia , Doenças Autoimunes/genética , DNA/imunologia , DNA Topoisomerases Tipo I , DNA de Cadeia Simples/imunologia , Humanos , Técnicas Imunoenzimáticas/normas , Proteínas Nucleares/análise , Proteínas Nucleares/imunologia , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Ribonucleoproteína Nuclear Pequena U1/análise , Ribonucleoproteína Nuclear Pequena U1/imunologia , Ribonucleoproteínas/análise , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Sensibilidade e Especificidade , Antígeno SS-BRESUMO
Intracellular staining for cytokines and parasites, combined with two-color flow cytometric analyses, were used to examine the frequencies of IL-12-, TNF-alpha- and IL-6-producing macrophages in response to Leishmania major infection and/or activation with IFN-gamma/lipopolysaccharide (LPS). Inflammatory macrophages were obtained from nonimmune granulomas, initiated by the injection of polyacrylamide microbeads (Bio-gel P-100) into subcutaneous pouches of different mouse strains. Infection of inflammatory macrophages in vitro using metacyclic promastigotes produced identical effects on cytokine responses regardless of whether cells from genetically resistant or susceptible mouse strains were used: IL-12 was not produced in response to infection itself, virtually every infected cell lost its ability to produce IL-12 in response to IFN-gamma/LPS, and the IL-6 response was partially inhibited, while the TNF-alpha response of infected cells was unimpaired. Low-multiplicity infection of inflammatory macrophages in vivo using either metacyclic promastigotes or tissue amastigotes also resulted in the complete and selective inhibition of IL-12 responses in infected cells. These data establish the physiologic relevance of prior observations regarding the selective impairment of IL-12 induction pathways in infected macrophages, and suggest a mechanisms for the delayed onset of cell-mediated control mechanisms that is typical of even self-limiting forms of leishmanial disease.
Assuntos
Inflamação/imunologia , Interleucina-12/biossíntese , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Macrófagos/imunologia , Animais , Feminino , Inflamação/patologia , Interleucina-12/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
OBJECTIVE: To determine the range of antinuclear antibodies (ANA) in "healthy" individuals compared with that in patients with systemic lupus erythematosus (SLE), systemic sclerosis (SSc; scleroderma), Sjögren's syndrome (SS), rheumatoid arthritis (RA), or soft tissue rheumatism (STR). METHODS: Fifteen international laboratories experienced in performing tests for ANA by indirect immunofluorescence participated in analyzing coded sera from healthy individuals and from patients in the 5 different disease groups described above. Except for the stipulation that HEp-2 cells should be used as substrate, each laboratory used its own in-house methodology so that the data might be expected to reflect the output of a cross-section of worldwide ANA reference laboratories. The sera were analyzed at 4 dilutions: 1:40, 1:80, 1:160, and 1:320. RESULTS: In healthy individuals, the frequency of ANA did not differ significantly across the 4 age subgroups spanning 20-60 years of age. This putatively normal population was ANA positive in 31.7% of individuals at 1:40 serum dilution, 13.3% at 1:80, 5.0% at 1:160, and 3.3% at 1:320. In comparison with the findings among the disease groups, a low cutoff point at 1:40 serum dilution (high sensitivity, low specificity) could have diagnostic value, since it would classify virtually all patients with SLE, SSc, or SS as ANA positive. Conversely, a high positive cutoff at 1:160 serum dilution (high specificity, low sensitivity) would be useful to confirm the presence of disease in only a portion of cases, but would be likely to exclude 95% of normal individuals. CONCLUSION: It is recommended that laboratories performing immunofluorescent ANA tests should report results at both the 1:40 and 1:160 dilutions, and should supply information on the percentage of normal individuals who are positive at these dilutions. A low-titer ANA is not necessarily insignificant and might depend on at least 4 specific factors. ANA assays can be a useful discriminant in recognizing certain disease conditions, but can create misunderstanding when the limitations are not fully appreciated.
Assuntos
Anticorpos Antinucleares/análise , Doenças Reumáticas/imunologia , Adulto , Artrite Reumatoide/imunologia , Feminino , Fibromialgia/imunologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , Valores de Referência , Escleroderma Sistêmico/imunologia , Síndrome de Sjogren/imunologia , Células Tumorais CultivadasRESUMO
Resistance of perforin knockout (PKO) mice to infection with Toxoplasma gondii was assessed in models of acute infection and during chronic disease. PKO mice vaccinated with the attenuated mutant, ts-4, displayed severely defective CTL responses against tachyzoite-infected targets. Lysis of the NK target, YAC-1, was also severely impaired in PKO mice following ts-4 vaccination. In contrast, wild-type mice developed high levels of CTL and NK lytic activity after ts-4 vaccination. Despite severely defective lytic activity, vaccinated PKO animals were completely resistant to challenge with the virulent strain RH, which normally causes a lethal acute infection. Resistance was attributable to production of IFN-gamma, which remained unimpaired in the PKO animals. In contrast, when PKO mice were infected with low virulence parasite strain ME49, which progresses to the cyst-forming stage after passage through an acute phase, accelerated mortality was observed beginning at 75 days postinfection. A three- to fourfold increase in brain cyst numbers was also found by day 30 in infected PKO animals. Nevertheless, the PKO strain produced normal levels of IFN-gamma after ME49 infection, ruling out impaired production of the latter cytokine as a cause of increased susceptibility. Together, these results show that perforin-dependent cytolytic function is not required for host resistance to lethal acute infection in preimmunized animals, but that the latter activity contributes to the control of infection during the chronic stage.
Assuntos
Glicoproteínas de Membrana/fisiologia , Linfócitos T Citotóxicos/fisiologia , Toxoplasmose Animal/imunologia , Animais , Citotoxicidade Imunológica , Feminino , Interferon gama/fisiologia , Células Matadoras Naturais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Perforina , Proteínas Citotóxicas Formadoras de PorosRESUMO
OBJECTIVE: To define the fine specificity of the 10 reference sera used for determination of antinuclear antibodies (ANA) and ANA subsets which are available from the Arthritis Foundation (AF) and from the Centers for Disease Control and Prevention (CDC). METHODS: AF/CDC sera were assessed by experienced laboratory staff, using indirect immunofluorescence and Western blotting. RESULTS: The original assignment of fluorescence patterns to 4 reference sera was confirmed, and the fluorescence intensities were determined using reference fluorescent beads. On Western blots, sera AF/CDC2 (anti-SS-B/La) and AF/CDC7 (anti-SS-A/Ro) did not detect Ro antigens, sera AF/CDC9 and AF/CDC10 appeared to be monospecific anti-Scl-70 and anti-Jo-1 sera, respectively, serum AF/CDC4 (anti-U1 small nuclear RNP) recognized the 70-kd band, and serum AF/CDC5 recognized the Sm antigen with its multiple bands. Semiquantitative analyses revealed that AF/CDC5, AF/CDC2, and AF/CDC10 were strongly reactive sera, whereas AF/CDC4 and AF/CDC9 were much weaker and should be used at lower dilutions on Western blots. CONCLUSION: The AF/CDC ANA reference sera, originally described as reference reagents for indirect immunofluorescence and double immunodiffusion techniques, are also useful for Western blotting. The data presented herein further support the use of these sera for reference purposes.
Assuntos
Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/imunologia , Especificidade de Anticorpos , Autoantígenos/imunologia , Western Blotting , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Estudos Multicêntricos como Assunto , Padrões de Referência , Ribonucleoproteína Nuclear Pequena U1/imunologia , Ribonucleoproteínas Nucleares Pequenas/imunologia , Proteínas Centrais de snRNPRESUMO
To study the function of side chain oligosaccharides of the cell-surface lipophosphoglycan (LPG), mutagenized Leishmania major defective in side chain biosynthesis were negatively selected by agglutination with the monoclonal antibody WIC79.3, which recognizes the galactose-containing side chains of L. major LPG. One such mutant, called Spock, lacked the ability to bind significantly to midguts of the natural L. major vector, Phlebotomus papatasi, and to maintain infection in the sand fly after excretion of the digested bloodmeal. Biochemical characterization of Spock LPG revealed its structural similarity to the LPG of Leishmania donovani, a species whose inability to bind to and maintain infections in P. papatasi midguts has been strongly correlated with the expression of a surface LPG lacking galactose-terminated oligosaccharide side chains. An in vitro galactosyltransferase assay using wild-type or Spock membranes was used to determine that the defect in Spock LPG biosynthesis is a result of defective beta1,3-galactosyltransferase activity as opposed to a modification of LPG, which would prevent it from serving as a competent substrate for galactose addition. The results of these experiments show that Spock lacks the beta1, 3-galactosyltransferase for side chain addition and that the LPG side chains are required for L. major to bind to and to produce transmissible infection in P. papatasi.
Assuntos
Galactosiltransferases/fisiologia , Glicoesfingolipídeos/fisiologia , Insetos Vetores/parasitologia , Leishmania major/patogenicidade , Phlebotomus/parasitologia , Animais , Anticorpos Monoclonais/imunologia , Sistema Livre de Células , Intestinos/parasitologia , Mutação , Oligossacarídeos/química , Sequências Repetitivas de Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.
Assuntos
Moléculas de Adesão Celular/metabolismo , Endotélio Vascular/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Cálcio/metabolismo , Adesão Celular , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , Toxina Pertussis , Receptores de Formil Peptídeo , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The activities of three lysosomal hydrolases and creatinine levels were measured in the plasma and urine of 11 adults (mean age, 28.1 years) with insulin-dependent diabetes mellitus and 14 non-diabetic controls (mean age, 27.9 years). All of the patients were free of diabetic complications and non exhibited microalbuminuria. Fractional enzyme excretion (FEE) values between the two groups of subjects were calculated and compared for the following enzymes: beta-hexosaminidase (N-acetyl-glucosaminidase), beta-glucuronidase and alpha-galactosidase. The FEE value was calculated as the ratio of enzyme clearance to creatinine clearance. Relative to the non-diabetic control group, the FEE value for beta-hexosaminidase was approximately 2-fold lower (P = 0.02) in the diabetic subjects (means, 0.424 vs. 0.242, respectively). The FEE values for beta-glucuronidase and alpha-galactosidase were not significantly different (P > 0.4) between the diabetic and control groups. These easily measured biochemical parameters in blood and urine and the resultant FEE value for beta-hexosaminidase may provide a means of assessing subtle deteriorative changes in renal function which occur in the early stage of diabetes before the onset of clinically evident complications.