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INTRODUCTION: Longitudinally extensive myelitis (LETM) has classically been grouped with the full or limited neuromyelitis optica spectrum disorders (NMOSD). However, differential diagnosis reveals a wide range of aetiologies. OBJECTIVE: To report on differential diagnosis and prognosis for LETM observed in a group of patients in Buenos Aires, Argentina. PATIENTS AND METHODS: Cross-sectional and retrospective multicentre study in two hospitals in Buenos Aires from June 2008 to June 2014. INCLUSION CRITERIA: medullary syndrome associated with spinal cord lesion extending for 3 or more contiguous spinal segments in magnetic resonance imaging (MRI). Clinical, radiological, and biochemical data were collected and subjects were rated on the Hughes functional disability scale (WHFDS) at 3 months. RESULTS: We evaluated 27 patients, 74% of whom were women; mean age was 35.22 years. The NMO-IgG antibody test was performed in 66.6% and oligoclonal band testing in 71%. NMO-IgG seropositivity was found exclusively in NMOSD patients (75%). Brain MRI was normal in 59.2% and revealed a mean of 7.9 affected spinal segments. Differential diagnoses revealed NMOSD (37%), idiopathic LETM (22.2%), lupus (11.1%), tumour (11.1%), dural fistula (7.4%), acute disseminated encephalomyelitis (7.4%), and a single case of multiple sclerosis (3.7%). Patients with lesions to ≥ 7 spinal segments showed poor recovery at 3 months (P<.001); these cases were associated with neoplastic, vascular, idiopathic, and lupus-related aetiologies. CONCLUSIONS: The most frequent causes of LETM in our cohort were NMOSD followed by idiopathic cases. Neoplastic, vascular, lupus-related, and idiopathic LETM may constitute a critical group with a distinct prognosis and other treatment needs.
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Diagnóstico Diferencial , Mielite Transversa/diagnóstico , Neuromielite Óptica/diagnóstico , Adulto , Argentina , Estudos Transversais , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Neuromielite Óptica/diagnóstico por imagem , Prognóstico , Estudos Retrospectivos , Medula Espinal/patologiaRESUMO
INTRODUCTION: Paroxysmal painful tonic spasms (PPTS) were initially described in multiple sclerosis (MS) but they are more frequent in neuromyelitis optica (NMO). The objective is to report their presence in a series of cases of NMO and NMO spectrum disorders (NMOSD), as well as to determine their frequency and clinical features. PATIENTS AND METHODS: We conducted a retrospective assessment of medical histories of NMO/NMOSD patients treated in 2 hospitals in Buenos Aires (Hospital Durand and Hospital Álvarez) between 2009 and 2013. RESULTS: Out of 15 patients with NMOSD (7 with definite NMO and 8 with limited NMO), 4 presented PPTS (26.66%). PPTS frequency in the definite NMO group was 57.14% (4/7). Of the 9 patients with longitudinally extensive transverse myelitis (LETM), 44.44% (9/15) presented PPTS. Mean age was 35 years (range, 22-38 years) and all patients were women. Mean time between NMO diagnosis and PPTS onset was 7 months (range, 1-29 months) and mean time from last relapse of LETM was 30 days (range 23-40 days). LETM (75% cervicothoracic and 25% thoracic) was observed by magnetic resonance imaging (MRI) in all patients. Control over spasms and pain was achieved in all patients with carbamazepine (associated with gabapentin in one case). No favourable responses to pregabalin, gabapentin, or phenytoin were reported. CONCLUSIONS: PPTS are frequent in NMO. Mean time of PPTS onset is approximately one month after an LETM relapse, with extensive cervicothoracic lesions appearing on the MRI scan. They show an excellent response to carbamazepine but little or no response to pregabalin and gabapentin. Prospective studies with larger numbers of patients are necessary in order to confirm these results.
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Neuromielite Óptica/complicações , Dor/etiologia , Espasmo/etiologia , Adulto , Analgésicos não Narcóticos/uso terapêutico , Carbamazepina/uso terapêutico , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Mielite Transversa/complicações , Neuromielite Óptica/diagnóstico por imagem , Neuromielite Óptica/tratamento farmacológico , Dor/tratamento farmacológico , Recidiva , Estudos Retrospectivos , Espasmo/diagnóstico por imagem , Espasmo/tratamento farmacológico , Adulto JovemRESUMO
Aims: The aim of this study was to establish consensus statements on medial patellofemoral ligament (MPFL) reconstruction, anteromedialization tibial tubercle osteotomy, trochleoplasty, and rehabilitation and return to sporting activity in patients with patellar instability, using the modified Delphi process. Methods: This was the second part of a study dealing with these aspects of management in these patients. As in part I, a total of 60 surgeons from 11 countries contributed to the development of consensus statements based on their expertise in this area. They were assigned to one of seven working groups defined by subtopics of interest. Consensus was defined as achieving between 80% and 89% agreement, strong consensus was defined as between 90% and 99% agreement, and 100% agreement was considered unanimous. Results: Of 41 questions and statements on patellar instability, none achieved unanimous consensus, 19 achieved strong consensus, 15 achieved consensus, and seven did not achieve consensus. Conclusion: Most statements reached some degree of consensus, without any achieving unanimous consensus. There was no consensus on the use of anchors in MPFL reconstruction, and the order of fixation of the graft (patella first versus femur first). There was also no consensus on the indications for trochleoplasty or its effect on the viability of the cartilage after elevation of the osteochondral flap. There was also no consensus on postoperative immobilization or weightbearing, or whether paediatric patients should avoid an early return to sport.
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Instabilidade Articular , Luxação Patelar , Articulação Patelofemoral , Humanos , Criança , Instabilidade Articular/cirurgia , Luxação Patelar/cirurgia , Articulação Patelofemoral/cirurgia , Técnica Delphi , Articulação do Joelho/cirurgia , Ligamentos Articulares/cirurgiaRESUMO
PURPOSE OF REVIEW: The indications for partial meniscectomy are becoming increasingly limited, and recent evidence suggests that the meniscus should be preserved whenever possible. Because of its many proposed advantages, all-inside meniscus repairs are becoming increasingly common. This review discusses the indications, advantages, disadvantages, and biomechanical and clinical outcomes of all-inside meniscus repair. RECENT FINDINGS: All-inside meniscus repair demonstrates equal functional outcomes, healing rates, and complications compared to inside-out repair of vertical longitudinal and bucket-handle tears with the advantages of decreased surgical time and faster post-operative recovery. In addition, return-to-sport and activity levels are high following all-inside repair regardless of whether concomitant anterior cruciate ligament reconstruction is performed. Biomechanical studies have demonstrated advantages of all-inside meniscal based repairs on radial and horizontal tears. All-inside meniscus repair compares favorably to inside-out repair of vertical longitudinal and bucket-handle tears and continues to increase in popularity. Both capsular based and meniscal based repairs can be used to repair a variety of tear patterns. While biomechanical results are encouraging, further research on the clinical outcomes of meniscal based repairs is needed to elucidate the role of these techniques in the future.
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PURPOSE: Clinical presentation of paediatric septic arthritis (SA) can be similar to other joint pathologies. Despite potential for infection in all major joints, most diagnostic criteria are based on values from the hip. This study identifies the best joint aspirate values in diagnosing SA in all joints. METHODS: In all, 166 patients who underwent 172 joint aspirations at the authors' institution between 01 September 2004 and 01 September 2014 were retrospectively identified. Recorded measures included age, sex, duration of symptoms, fever history, weight-bearing status, aspiration results, serum results and antibiotic administration. Patients were placed in the following four categories: 'culture confirmed SA' (C-SA), 'suspected SA' (S-SA), 'Other' and 'Other-rheumatologic' (Other-R), a subcategory of 'Other'. RESULTS: Most common sites of aspiration were the knee (55%) and hip (29%). Diagnostic grouping was as follows: C-SA = 44, S-SA = 45, Other = 83 (Other-R = 21). Fever and non-weight-bearing prior to admission were useful predictors of SA, though in C-SA patients, 21% did not have a fever and 23% could weight bear at the time of admission. Aspirate white blood cell (WBC) count was significantly greater in both C-SA (92 000 cells/hpf) and S-SA (54 000) than in Other (10 000) and Other-R (18 000) patients. The percentage of polymorphonuclear (%PMN) was also significantly greater in C-SA (81.1%) and S-SA (80.9%) than in Other (57.9%) and Other-R (63.3%). CONCLUSION: Joint aspirate values, especially %PMN, are valuable in diagnosing SA. Additionally, antibiotics pre-aspiration did not affect %PMN, facilitating subsequent diagnosis of infection. Lastly, while aspirate WBC count was a valuable indicator of SA, this finding is not as definitive as previous research suggests. LEVEL OF EVIDENCE: IV Case Series.
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HLA-G is an HLA class Ib gene that is highly expressed in human trophoblast cells. The single HLA-G mRNA is alternatively spliced to generate at least seven transcripts, three of which encode soluble isoforms. Many studies have shown that high levels of soluble antigens are associated with successful implantation and graft acceptance. To study expression, regulation and functions of two of the soluble isoforms, HLA-G5 and HLA-G6, we generated recombinant proteins in eukaryotic cells and developed monoclonal antibodies specific for each of the two proteins. In addition, we investigated the olive baboon Paan-AG gene as a potential functional correlate of HLA-G. Here, we present summaries of the studies that have been conducted in our laboratory using these tools and discuss the results within the context of the research on this topic that is ongoing in ours and other laboratories worldwide. Collectively, the data indicate that soluble HLA-G is a critical contributor to immune privilege in pregnancy and imply that this placenta-derived substance may impact other pathways leading to successful reproduction.
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Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Placenta/imunologia , Gravidez/imunologia , Receptores Imunológicos/imunologia , Blastocisto/imunologia , Feminino , Antígenos HLA/genética , Antígenos HLA/metabolismo , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , RNA Mensageiro/metabolismo , Receptores KIR2DL5 , Trofoblastos/imunologiaRESUMO
Several studies in multiple sclerosis (MS) suggest a trend of increasing disease frequency in women during the last decades. A direct comparison of gender ratio trends among MS populations from Argentina remains to be carried out. The objective of the study was to compare gender ratio trends, over a 50-year span in MS populations from Argentina. METHODS: multicenter study that included patients from 14 MS Centers of Argentina. Patients with definite MS with birth years ranging from 1940 to 1989 were included. Gender ratios were calculated by five decades based on year of birth and were adjusted for the F/M born-alive ratio derived from the Argentinean national registry of births. The F/M ratios were calculated using a multivariate logistic regression per five decades by the year of birth approach. Analyses were performed using Stata 10.1. RESULTS: 1069 patients were included. Gender ratios showed a significant increase from the first to the last decade in the whole MS sample (from 1.8 to 2.7; p value for trend=0.023). The Gender ratio did not show differences considering MS subtype. CONCLUSION: our study showed a modest increase of the F/M ratio (from 1.8 to 2.7) over time among patients affected by MS in Argentina.
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Esclerose Múltipla/diagnóstico , Esclerose Múltipla/epidemiologia , Razão de Masculinidade , Adulto , Argentina/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sistema de Registros , Estudos RetrospectivosRESUMO
UNLABELLED: The present study was carried out to assess if there is an anticipation of age at onset in younger generations of familial multiple sclerosis (FMS) vs. sporadic MS (SMS) in Argentina. METHODS: multicenter study that included patients from 14 MS Centers of Argentina. Patients were considered as FMS if they had in their family at least one relative of first or second degree diagnosed with MS; otherwise, patients were considered to have SMS. We compared the age at onset between familial and sporadic cases as well as the age at onset between relatives from different generations in FMS vs. SMS. RESULTS: 1333 patients were included, 97 of them were FMS (7.3%). A lower age at onset in the younger generations of FMS cases was found compared with older generations of FMS as well as. SMS cases (24.1±3.7 years vs. 30.3±5.7 years, and 32.4±9.4 respectively; p<0.001). No differences were observed between older generations of FMS vs. SMS cases (p=0.12). CONCLUSION: we observed an anticipation of age at onset of MS in younger generations of patients with FMS vs. older generations of FMS and SMS.
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Esclerose Múltipla/epidemiologia , Adulto , Idade de Início , Argentina/epidemiologia , Família , Seguimentos , Humanos , Masculino , Esclerose Múltipla/genética , Estudos Retrospectivos , Adulto JovemRESUMO
The ability of MuIFN-beta and MuIFN-gamma to potentiate the development of tumoricidal activity in proteose peptone-elicited murine peritoneal macrophages was investigated. Macrophages were stimulated with increasing concentrations of either MuIFN-beta or MuIFN-gamma, alone and in combination, in the presence of 1 ng/ml lipopolysaccharide (LPS). The priming activities attributable to the interferons (IFNs) were quantified using the dose-response curves obtained for these samples. The priming activity observed for mixtures of MuIFN-beta and MuIFN-gamma was greater than that expected if MuIFN-beta and MuIFN-gamma had acted in an additive manner. Isobologram analysis of data obtained when macrophages were stimulated with combinations of IFNs demonstrated that MuIFN-beta and MuIFN-gamma acted synergistically to prime macrophages for tumor cell killing. The greatest degree of synergy was observed when macrophages were stimulated with suboptimal and nearly equivalent concentrations of each class of IFN. Further studies demonstrated that macrophages stimulated with combinations of MuIFN-beta and MuIFN-gamma were more sensitive to the trigger signal provided by LPS than were cells primed with either IFN alone. Thus, the synergistic effects observed were quantitative in nature in that macrophages perceived combinations of MuIFN-beta and MuIFN-gamma as having higher priming activities than expected.
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Citotoxicidade Imunológica/efeitos dos fármacos , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Neoplasias Experimentais/imunologia , Animais , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C3HRESUMO
Macrophages activated for tumor cell killing by bacterial lipopolysaccharide (LPS) were shown to lose their cytolytic activity if exposed to physiological levels of prostaglandin E2 (PGE2). Increasing the LPS stimulus more than 100-fold over the amount needed to activate the cells did not substantially increase their resistance to the negative regulatory effect of PGE2. By contrast, killing mediated by macrophages activated by a mixture of LPS and gamma interferon was maintained. The degree of resistance conferred was directly related to the magnitude of the stimulus employed, reaching the point where not even 10(-5) M PGE2 would diminish killing. Killing by both activated resident and inflammatory peritoneal macrophages could be maintained, but it was easier to do so if the cells had been elicited by an inflammatory stimulus. A preparation of type I interferons produced by cells of the macrophage cell line J774A. 1 behaved similarly, but was over 500 times less efficient at helping to maintain killing than gamma (type II) interferon was. Alpha interferon alone, i.e., without LPS, was capable both of activating macrophages and of maintaining the activated state in the presence of PGE2. The capacity for both activation and maintenance could be strikingly enhanced, however, by mixing alpha and gamma interferons together under conditions that were free of detectable LPS. The data reported here collectively suggest that induction and maintenance of macrophage activation may be separable mechanistically, and that the interferons are important to host defense not only because they participate in the induction of macrophage activation for tumor cell killing but also because they help to maintain the activated state once it has been induced.
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Ativação de Macrófagos/efeitos dos fármacos , Prostaglandinas E/farmacologia , Animais , Dinoprostona , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peritônio/citologiaRESUMO
Investigators from two different laboratories have compared several variables in the short-term macrophage-mediated cytotoxicity assays used by each group to study the role of MuIFN-gamma in macrophage activation. The findings suggest that the capacity of MuIFN-gamma to activate macrophages without the need for a second triggering stimulus is related to assay conditions and, most especially, the strain of mouse used to provide the macrophages.
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Interferon gama/imunologia , Linfoma/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Sarcoma de Mastócitos/imunologia , Animais , Citotoxicidade Imunológica , Imunidade Celular , Camundongos , Projetos de Pesquisa , Especificidade da EspécieRESUMO
The suppressive effect of IFN-alpha and IFN-beta on the induction of tumoricidal activity in mouse bone marrow-derived macrophages was investigated. Macrophages incubated for 24 hr with IFN-beta developed lower levels of cytolytic activity when stimulated with IFN-gamma and LPS, in comparison with macrophages pretreated with medium. The suppressive effect was dependent on the pretreatment dose of IFN-beta over a concentration range of 1 to 1,000 U/ml. Analysis of IFN-gamma dose response curves of IFN-beta treated macrophages showed that these cells were less sensitive to IFN-gamma. The suppressive effects were fully neutralized by an antiserum to IFN-alpha/beta. Prostaglandins were apparently not involved in this process since the addition of indomethacin to IFN-beta treated macrophages did not prevent the loss of responsiveness to activating stimuli. In contrast to the results obtained with IFN-alpha and IFN-beta, macrophages pretreated with IFN-gamma did not develop lower levels of cytolytic activity when again stimulated with IFN-gamma and LPS. These observations provide evidence for a potentially important negative regulatory role for IFN-alpha and IFN-beta in macrophage activation for tumor cell killing.
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Citotoxicidade Imunológica , Interferon Tipo I/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Animais , Tolerância Imunológica/efeitos dos fármacos , Imunidade Celular , Indometacina/farmacologia , Interferon gama/antagonistas & inibidores , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Neoplasias Experimentais/imunologiaRESUMO
Macrophages and natural killer (NK)-like cells are the major hematopoietic cell populations in the cycling and pregnant mouse uterus and are also found in the embryo. In order to evaluate potential receptivity of these cells to interferon-gamma (IFN-gamma), tissues taken from cycling and pregnant mice were tested for IFN-gamma receptor (IFN-gamma R) mRNA and protein. Macrophages were identified immunohistochemically by using the specific monoclonal antibody F4/80. NK cells were identified by their large size, distinctive intracellular granules, and binding of a monoclonal antibody to the common leukocyte antigen. In cycling uteri, the abundance of IFN-gamma R mRNA relative to an invariant message (glyceraldehyde-3-phosphate dehydrogenase) increased during progression of the hormonally regulated estrous cycle. IFN-gamma R mRNA in situ hybridization signals were slightly higher in macrophage-like than in other types of endometrial stromal cells. In pregnant uteri, the highest proportions of IFN-gamma R mRNA were observed at gestation day (g.d.) 16. Specific message and protein were present in uterine macrophages by g.d. 7 and in NK cells by g.d. 9. IFN-gamma R expression in both lineages remained stable through the balance of pregnancy. In embryos, IFN-gamma R mRNA increased between g.d. 14 and 16. Specific transcripts were present in many cells at g.d. 14, but none were detected in embryonic liver macrophages until g.d. 16. The results of this study suggest relationships between IFN-gamma R expression and ovarian hormones as well as cell maturation and support the postulate that IFN-gamma receptor-ligand interactions may improve the ability of uterine and embryonic hematopoietic cells to perform specific tasks during gestation.
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Embrião de Mamíferos/citologia , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/fisiologia , Prenhez/genética , Receptores de Interferon/genética , Útero/citologia , Animais , Anticorpos Monoclonais , Northern Blotting , Linhagem Celular , Embrião de Mamíferos/química , Embrião de Mamíferos/ultraestrutura , Feminino , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/ultraestrutura , Imuno-Histoquímica , Hibridização In Situ , Células Matadoras Naturais/química , Células Matadoras Naturais/fisiologia , Células Matadoras Naturais/ultraestrutura , Macrófagos/química , Macrófagos/fisiologia , Macrófagos/ultraestrutura , Camundongos , Gravidez , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Interferon/análise , Transcrição Gênica , Útero/química , Útero/ultraestrutura , Receptor de Interferon gamaRESUMO
Nitric oxide (NO), a potent and versatile free radical, is synthesized in macrophages and mast cells as well as in other types of cells by the inducible form of nitric oxide synthase (iNOS). In this study, cells containing iNOS were identified in the uteri of cycling mice by using a rabbit antibody generated to an iNOS-specific peptide. Macrophages were identified in semiserial sections of the same tissues with the monoclonal antibody, F4/80, and mast cells were identified by toluidine blue staining. In tissue sections of uteri obtained from mice in the four stages of the estrous cycle (8 to 11 mice per stage), iNOS immunoreactivity was strongest in diestrus-I uteri and weakest in diestrus-II uteri. Myometrial mast cells and endometrial epithelial cells were prominent locations of iNOS, and specific protein was also present in myometrial smooth muscle and macrophage-like cells in the endometrial stroma. Because cyclic variations suggested regulation of iNOS expression by ovarian steroid hormones, studies were done using ovariectomized mice. Seven days after ovariectomy, immunoreactive iNOS was low but detectable in mast cells and luminal epithelial cells. In the uteri of ovariectomized, estradiol-17 beta (E2)-treated mice, mast cells were iNOS+ after 24 h whereas epithelial cells were negative; the reverse was observed in progesterone (P4)-treated mice. Both mast cells and epithelial cells were iNOS+ in the uteri of mice that had received a combination of E2 + P4. These results indicate that several types of uterine cells produce iNOS and that expression of this enzyme in specific cell lineages is governed by ovarian steroid hormones. The data are consistent with the postulate that NO derived from uterine leukocytes and other types of cells plays a role in uterine cyclicity and preparation for pregnancy.
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Aminoácido Oxirredutases/análise , Aminoácido Oxirredutases/biossíntese , Estradiol/farmacologia , Progesterona/farmacologia , Útero/enzimologia , Animais , Western Blotting , Endométrio/citologia , Endométrio/enzimologia , Indução Enzimática/efeitos dos fármacos , Estro/fisiologia , Feminino , Histocitoquímica , Macrófagos/citologia , Macrófagos/enzimologia , Mastócitos/citologia , Mastócitos/enzimologia , Camundongos , Miométrio/citologia , Miométrio/enzimologia , NADPH Desidrogenase/análise , Óxido Nítrico Sintase , Útero/citologia , Útero/fisiologiaRESUMO
Five different short term assays (less than 48 h) used to measure macrophage-mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: three different populations of macrophages; four different kinds of target cells; two types of radioisotopes; and two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were apparent in assays of less than 24 h duration, disappeared when the same kinds of targets were compared in assays of greater than 40 h duration. The results of this study are an important first step toward standardizing the way in which macrophage-mediated, nonspecific cytotoxicity is measured in short-term assays, laboratory to laboratory.
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Testes Imunológicos de Citotoxicidade , Macrófagos/imunologia , Linhagem Celular , Cromo , Relação Dose-Resposta Imunológica , Técnicas In Vitro , Índio , Interferon gama/farmacologia , Ativação de Macrófagos , Fatores de TempoRESUMO
To facilitate investigation of its physical and functional properties, 11 monoclonal antibodies (mAbs) and a goat polyclonal IgG specific for the mouse interferon- (IFN-gamma) receptor were characterized and their potential uses studied. Eight of the mAbs interacted with epitopes on the extracellular domain of the receptor, two interacted with epitopes on the intracellular domain, and one interacted with an epitope that could not be localized definitively to either region. Of the 11 mAbs, the majority (8) were IgGs, 2 were IgMs, and 1 was an IgA. Relative avidities of the seven that could be determined ranged from 333 to 0.002 microM-1. Both the polyclonal goat IgG and mAb GR-20 (the latter specific for an epitope in the binding site for IFN-gamma) blocked binding of the ligand and, as expected, prevented induction by IFN-gamma of priming of macrophages for tumor cell killing. None of the other mAbs had an effect despite the fact that GR-22 partially (greater than 50%) blocked binding of IFN-gamma. Neither the polyclonal IgG nor any of the mAbs had an agonist effect. The relative usefulness of the antibodies for immunoprecipitation, immunoblotting, immunoassay, and cell staining with and without prior fixation is described. The results of immunocytochemical staining directly confirmed that the majority of immunologically reactive receptor protein expressed by cells is intracellular. To facilitate use by other investigators, the hybridomas that produce these mAbs will be offered to the American Type Culture Collection.
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Anticorpos Monoclonais , Anticorpos , Receptores Imunológicos/química , Animais , Anticorpos/classificação , Anticorpos Monoclonais/classificação , Especificidade de Anticorpos , Western Blotting , Células Cultivadas , Epitopos/análise , Citometria de Fluxo , Imuno-Histoquímica , Interferon gama/análise , Ligantes , Camundongos , Camundongos Endogâmicos C3H , Testes de Precipitina , Ratos , Receptores Imunológicos/fisiologia , Receptores de Interferon , Coloração e Rotulagem , Células Tumorais CultivadasRESUMO
The inducible protein p71/73 marks the response of mouse macrophages to one of several stimuli (e.g., bacterial lipopolysaccharide or poly I:C) that trigger the expression of cytolytic activity when these cells have previously been primed for tumor cell killing by interferon-gamma (IFN-gamma). The results reported here identify this marker protein as the inducible prostaglandin endoperoxide synthase (PES), TIS10/PES-2. Identification was based on four findings: (1) p71/73, like the TIS10/PES-2 protein, was associated with cellular membranes; (2) the sequence of amino acids in the NH2 terminus of both p71 and p73 was 96% identical to the predicted NH2-terminal sequence of the TIS10/PES-2 protein; (3) a polyclonal antiserum raised against the COOH-terminal region of the TIS10/PES-2 gene product recognized p71/73 in immunoblots; and (4) dexamethasone, which blocks induction of TIS10/PES-2 expression, inhibited the induction of both p71/73 synthesis and tumoricidal activity in macrophage. Several regulatory roles for this protein in the activation process are possible.
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Ativação de Macrófagos , Macrófagos/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Sequência de Aminoácidos , Animais , Anticorpos , Biomarcadores , Células da Medula Óssea , Células Cultivadas , Galinhas , Grânulos Citoplasmáticos/enzimologia , Citosol/enzimologia , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Indução Enzimática , Humanos , Immunoblotting , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Prostaglandina-Endoperóxido Sintases/química , Prostaglandina-Endoperóxido Sintases/isolamento & purificação , Homologia de Sequência de Aminoácidos , OvinosRESUMO
Activation of mouse macrophages for tumor cell killing is negatively regulated by prostaglandin E2 (PGE2). The effect of this hormone is to shut off cytolytic activity that is expressed as a consequence of activation. A lymphokine in the culture supernates of concanavalin A stimulated spleen cells has been shown to change the sensitivity of activated macrophages to the negative regulatory effects of PGE2, thereby maintaining activation, as manifested by the continued expression of tumor cell killing by these cells. Using a highly specific polyclonal antiserum and gamma interferon produced either by a T-cell hybridoma or by recombinant DNA technology we show here that one lymphokine responsible for mediating the maintenance effect is gamma interferon.
Assuntos
Interferon gama/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Prostaglandinas E/antagonistas & inibidores , Animais , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo , Células Cultivadas , Cromatografia em Gel , Citotoxicidade Imunológica , Dinoprostona , Hibridomas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Testes de Neutralização , Recombinação GenéticaRESUMO
Enriched fractions of spermatogenic cells were isolated by unit gravity sedimentation and analyzed both for the presence of secreted tumor necrosis factor-alpha (TNF alpha) in vitro by bioassay and for the presence of TNF alpha mRNA by Northern blot analysis. Small quantities of bioactive TNF alpha were consistently detected in medium conditioned by round spermatid fractions. Both pachytene spermatocyte and round spermatid fractions contained RNA that hybridized with murine cDNA probes for TNF alpha, with pachytene spermatocytes containing a normal 1.9-kilobase (kb) transcript, while round spermatids contained principally an approximately 2.8-kb transcript. Both the normal size transcript and the larger haploid-specific transcript were enriched when total RNA from pachytene spermatocyte and round spermatid fractions was passed through an oligo(dT) column. The normal 1.9-kb transcript within pachytene spermatocytes could be induced by exposing the spermatogenic cells to lipopolysaccharides in vitro, yet the approximately 2.8-kb transcript within round spermatids appeared uninduced by LPS treatment. In situ hybridization for the TNF alpha message by using digoxigenin label antisense TNF alpha riboprobe labeled pachytene spermatocytes, round spermatids, and presumptive interstitial macrophages. Spermatogonia and elongating spermatids as well as other interstitial cells were unlabeled or very lightly labeled. Hybridization of 16-day-old prepuberal testis resulted in the labeling of spermatocytes and presumptive interstitial macrophages. RNA from Sertoli cells, but not pachytene spermatocytes or round spermatids, hybridized with human TNF alpha receptor p60 probe in Northern blot analysis. These results are consistent with the working hypothesis that spermatids release TNF alpha, which is detected by Sertoli cells and may serve as a paracrine factor, regulating an as yet unidentified process in spermatogenesis.