RESUMO
Human immunodeficiency virus (HIV-1) infection can be currently controlled by combined antiretroviral therapy, but a sterilizing cure is not achievable as this therapy does not target persistent HIV-1 in latent reservoirs. Therefore, different latency reversal agents are intensively explored in various models. We have previously observed that heme arginate, a drug approved for human use, reveals a strong synergism with PKC inducers in reactivation of the latent provirus. Heme is physiologically decomposed by heme oxygenases into 3 degradation products: iron (Fe2+), carbon monoxide (CO) and biliverdin which is further converted to bilirubin by biliverdin reductase. In this paper, we have studied the effects of individual heme-degradation products on latent HIV-1 reactivation in ACH-2 cells harboring integrated HIV-1 provirus and in H12 clone of Jurkat cells harboring HIV-minivirus expressing EGFP. We employed addition of ascorbate to generate Fe2+, resulting in increased expression of both HIV-1 p24 Ag and EGFP in PMA-stimulated ACH-2 and H12 cells, respectively, as characterized on RNA and protein levels. On the other hand, addition of a CO-donor or bilirubin decreased the p24 expression. The reactivation of latent HIV-1 by iron or heme arginate was inhibited by antioxidant N-acetyl cysteine, or by an iron chelator desferrioxamine, suggesting that the effects were mediated by iron- or heme-induced redox stress. Finally, we demonstrated the stimulatory effects of heme arginate and PMA on HIV-1 expression in peripheral blood mononuclear cells of HIV-infected patients cultured ex vivo. These results may constitute a new direction in the latent HIV-1 reactivation and therapy.
Assuntos
Arginina/farmacologia , Bilirrubina/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Heme/farmacologia , Latência Viral/fisiologia , Monóxido de Carbono , Linhagem Celular , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.
Assuntos
Caderinas/química , Sobrecarga de Ferro/fisiopatologia , Fígado/fisiopatologia , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Fígado/química , Camundongos , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Surgical antimicrobial prophylaxis (SAP) is one of the major purposes of antimicrobial use. AIM: To determine the adherence to the Japanese SAP guidelines in Japanese university hospitals. METHODS: This was a retrospective cohort study including 15 general hospitals and one dental university hospital. Up to three cases of 18 designated surgeries were evaluated regarding adherence to Japanese SAP guidelines: selection of antibiotics, timing of administration, re-dosing intervals, and duration of SAP. When all items were appropriate, surgery was defined as 'appropriate'. FINDINGS: In total, 688 cases (22-45 cases per surgery) were included. The overall appropriateness was 46.8% (322/688), and the appropriateness of each surgery ranged from 8.0% (2/25, cardiac implantable electronic device implantation) to 92.1% (35/38, distal gastrectomy). The appropriateness of each item was as follows: pre/intraoperative selections, 78.5% (540/688); timing of administrations, 96.0% (630/656); re-dosing intervals, 91.6% (601/656); postoperative selection, 78.9% (543/688); and duration of SAP, 61.4% (423/688). The overall appropriateness of hospitals ranged from 17.6% (9/51) to 73.3% (33/45). The common reasons for inappropriateness were the longer duration (38.5%, 265/688) and choice of antibiotics with a non-optimal antimicrobial spectrum before/during, and after surgery (19.0%, 131/688 and 16.9%, 116/688, respectively), compared to the guideline. CONCLUSIONS: Adherence to the guidelines differed greatly between the surgeries and hospitals. Large-scale multi-centre surveillance of SAP in Japanese hospitals is necessary to identify inappropriate surgeries, factors related to the appropriateness, and incidences of surgical site infections.
Assuntos
Anti-Infecciosos , Antibioticoprofilaxia , Humanos , Estudos Retrospectivos , Hospitais Universitários , Japão , Fidelidade a Diretrizes , Antibacterianos/uso terapêutico , Infecção da Ferida Cirúrgica/epidemiologia , Infecção da Ferida Cirúrgica/prevenção & controle , Infecção da Ferida Cirúrgica/tratamento farmacológico , Anti-Infecciosos/uso terapêuticoRESUMO
Hepcidin is a key regulator of iron homeostasis, while hemojuvelin is an important component of the hepcidin regulation pathway. It has been recently proposed that soluble hemojuvelin, produced from hemojuvelin by the protease furin, decreases hepcidin expression. The aim of the presented study was to examine the downregulation of hepcidin by chronic bleeding in hemojuvelin-mutant mice. Male mice with targeted disruption of the hemojuvelin gene (Hjv-/- mice) and wild-type littermates were maintained on an iron-deficient diet and subjected to weekly phlebotomies for 7 weeks. Gene expression was examined by real-time PCR. In wild type mice, repeated bleeding decreased hepcidin mRNA by two orders of magnitude. In Hjv-/- mice, basal hepcidin expression was low; however, repeated bleeding also decreased hepcidin mRNA content by an order of magnitude. Phlebotomies reduced hepatic iron overload in Hjv-/- mice by 80 %. Liver and muscle furin mRNA content was not significantly changed. No effect on hepatic Tmprss6 mRNA content was observed. Results from the study indicate that soluble hemojuvelin is not the sole factor responsible for hepcidin downregulation. In addition, the presented data suggest that, under in vivo conditions, tissue hypoxia does not transcriptionally regulate the activity of furin or TMPRSS6 proteases.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Eritropoese , Hemorragia/metabolismo , Proteínas de Membrana/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Hipóxia Celular , Modelos Animais de Doenças , Regulação para Baixo , Furina/metabolismo , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Hemorragia/etiologia , Hemorragia/genética , Hepcidinas , Deficiências de Ferro , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/prevenção & controle , Ferro da Dieta/administração & dosagem , Fígado/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Flebotomia , RNA Mensageiro/metabolismo , Serina Endopeptidases/metabolismo , Fatores de TempoRESUMO
The purpose of this study was to evaluate the topographic anatomy of the vertebral vein (VV) in the lower neck and thoracic inlet using CT scans. Enhanced CT scans using 32-MDCT were obtained for 199 consecutive patients. Reconstructed images with 1-mm section thickness/intervals were evaluated by two radiologists examining the drainage point, number, and route of VVs using frame forwarding and the rewind function on the DICOM viewer. The VV was classified into four types as follows: Type A (80.6%), a VV that descended ventral to the subclavian artery (SA) and drained into the upper portion of the brachiocephalic vein (BCV); Type B (5.8%), a VV that descended dorsal to the SA and drained into the upper portion or the lower portion of the BCV; Type C (8.3%), a doubled VVs that crossed both sides of the SA and drained into the upper portion of the BCV and formed a common trunk; Type D (5.3%), a VV ventral to the SA that drained into the upper portion of the BCV and another VV dorsal to the SA drained into the upper portion or the lower portion of the BCV. Some variations were observed in regard to the drainage point, number, and route of the VVs. Classification of the VV may be useful for interpreting chest CT scans and in better understanding the embryologic development of the vertebral vein.
Assuntos
Veias Braquiocefálicas/anatomia & histologia , Vértebras Cervicais/irrigação sanguínea , Tomografia Computadorizada por Raios X/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Veias Braquiocefálicas/diagnóstico por imagem , Vértebras Cervicais/diagnóstico por imagem , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pescoço/irrigação sanguínea , Pescoço/diagnóstico por imagem , Radiografia Torácica , Estudos Retrospectivos , Tórax/irrigação sanguínea , Tomógrafos Computadorizados , Tomografia Computadorizada por Raios X/instrumentação , Adulto JovemRESUMO
BACKGROUND: Outbreaks of vancomycin-resistant enterococcus (VRE) are a serious problem in hospitals. Inferring the transmission route is an important factor to institute appropriate infection control measures; however, the methodology has not been fully established. AIM: To reconstruct and evaluate the transmission model using sequence variants extracted from whole genome sequencing (WGS) data and epidemiological information from patients involved in a VRE outbreak. METHODS: During a VRE outbreak in our hospital, 23 samples were collected from patients and environmental surfaces and analysed using WGS. By combining genome alignment information with patient epidemiological data, the VRE transmission route was reconstructed using a Bayesian approach. With the transmission model, evaluation and further analyses were performed to identify risk factors that contributed to the outbreak. FINDINGS: All VREs were identified as Enterococcus faecium belonging to sequence type 17, which consisted of two VRE genotypes: vanA (N = 8, including one environmental sample) and vanB (N = 15). The reconstruction model using the Bayesian approach showed the transmission direction with posterior probability and revealed transmission through an environmental surface. In addition, some cases acting as VRE spreaders were identified, which can interfere with appropriate infection control. Vancomycin administration was identified as a significant risk factor for spreaders. CONCLUSION: A Bayesian approach for transmission route reconstruction using epidemiologic data and genomic variants from WGS can be applied in actual VRE outbreaks. This may contribute to the design and implementation of effective infection control measures.
Assuntos
Transmissão de Doença Infecciosa , Enterococcus faecium/isolamento & purificação , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções por Bactérias Gram-Positivas/transmissão , Epidemiologia Molecular , Enterococos Resistentes à Vancomicina/isolamento & purificação , Sequenciamento Completo do Genoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Teorema de Bayes , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/transmissão , Surtos de Doenças , Enterococcus faecium/classificação , Enterococcus faecium/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Fatores de Risco , Enterococos Resistentes à Vancomicina/classificação , Enterococos Resistentes à Vancomicina/genéticaRESUMO
Leishmania exist as extracellular promastigotes which multiply in the gut of the sandfly insect vector and as intracellular amastigotes which divide in the phagolysosome of mononuclear phagocytic cells of the mammalian host. Promastigotes express a major surface glycoprotein of 63 kDa, referred to as GP63. The expression of GP63 in both Leishmania life stages was studied using rabbit antibodies against native GP63 as well as rabbit antibodies against recombinant GP63 that was synthesized in an Escherichia coli expression system. Immunofluorescence staining detected GP63 in intracellular amastigotes contained within a macrophage cell line and within freshly isolated lesion amastigotes. Western blot analysis using anti-recombinant GP63 antibodies also demonstrated that amastigotes synthesize GP63 which may undergo differential post-translational processing as compared to promastigote GP63.
Assuntos
Leishmania/crescimento & desenvolvimento , Glicoproteínas de Membrana/biossíntese , Metaloendopeptidases , Proteínas de Protozoários/biossíntese , Animais , Antígenos de Protozoários , Western Blotting , Imunofluorescência , Leishmania/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Leishmania tropica/crescimento & desenvolvimento , Proteínas Recombinantes/biossínteseRESUMO
New derivatives of 4-homoisotwistane (tricyclo[5.3.1.0(3,8)]undecane) (3), the olefin (8), bromide (4), alcohols (7,9,11, and 14), ketone (10), acid (12), esters (13, 18a, and 18b), amides (5, 16, and 19a-d), nitrile (20), and amines and their hydrochlorides (6, 17, and 21), were prepared, and antiviral activities of these compounds were determined in vitro on a monolayer culture of chick embryo fibroblasts against Newcastle disease virus. 4-Homoisotwist-3-ylamine hydrochloride (6) and 4-homoisotwist-3-ylmethylamine hydrochloride (21) were found 30-50 times more potent than amantadine hydrochloride in this assay. Methods of preparing the test compounds included those functionalization reactions of 3 which revealed many interesting features of the reactivity of the recently discovered polycyclic hydrocarbon 3.
Assuntos
Antivirais/síntese química , Hidrocarbonetos Aromáticos com Pontes/síntese química , Compostos Policíclicos/síntese química , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Células Cultivadas , Embrião de Galinha , Fibroblastos/metabolismo , Conformação Molecular , Vírus da Doença de Newcastle/efeitos dos fármacos , Compostos Policíclicos/farmacologia , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Functionalization reactions via cationic intermediates of tricyclo[4.3.1.1 2,5]undecane (2) were investigated to prepare derivatives with potential antiviral activities. Bromination of 2 took place regiospecifically at C-1, and the resulted bromide 5 was converted into the hydroxide 9, the carboxylic acid 12, and the amine 22, from which were synthesized a variety of secondary derivatives, including homologous esters 10 and 20, amides 14 and 19, carbamates 24, and ureas 17 and 25. The hydroxide 9, the acid 12, and the acetamide 21 were also obtainable directly from tricyclo[5.2.1.0 2,6]dec-endo-2-ylcarbinol (1), the precursor for the synthesis of the hydrocarbon 2. Success in these functionalization-rearrangements was attributed to the inability of the intermediate 2-1-yl cation (2+) for further skeletal isomerizations. Among the 1-substituted derivatives of 2 prepared, the amine hydrochlorides (16 and 23), a few esters (20b and 20d), and some N-alkylamides (19c, 19d, and 19e) exhibited marked antiviral activities as compared to amantadine hydrochloride, when tested in vitro on a monolayer culture of chick embryo fibroblasts against Newcastle disease virus.
Assuntos
Antivirais/síntese química , Compostos Policíclicos/síntese química , Animais , Sobrevivência Celular/efeitos dos fármacos , Fenômenos Químicos , Química , Embrião de Galinha , Vírus da Doença de Newcastle/efeitos dos fármacos , Compostos Policíclicos/farmacologiaRESUMO
For peripheral nerve allografts, the conditions for successful nerve regeneration and the possibility of transplanting Schwann cells were examined using a model of pretreated rats. Incomplete immunosuppression was achieved in recipient rats with donor red blood cell infusions given before allogeneic nerve grafting (RBC group). The origin of Schwann cells in the grafts was assessed by immunohistochemical staining from 1 week to 12 weeks after transplantation. In the RBC group, the replacement of donor Schwann cells by recipient cells was detected at 3-8 weeks, with the graft being filled with many of these cells at all times, and successful nerve regeneration was seen after 12 weeks on morphometric and electrophysiologic evaluations. In peripheral nerve allografts, pretreatment with donor-specific blood transfusion did not induce significant immunosuppression compared with allotransplantations of some tissues and organs. Clinically, if the state of immunosuppression can be controlled by RBC transfusion, it is possible that donor tissues may be replaced by recipient tissues and that nerves will regenerate successfully.
Assuntos
Regeneração Nervosa , Nervos Periféricos/transplante , Células de Schwann/imunologia , Condicionamento Pré-Transplante , Animais , Doadores de Sangue , Transfusão de Sangue , Eritrócitos/imunologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Isoanticorpos/análise , Linfonodos/imunologia , Masculino , RatosRESUMO
BACKGROUND: The origin of Schwann cells and effect of a limited course of immunosuppression using cyclosporine (CsA) were examined in rat peripheral nerve allotransplants. METHODS: Phenotypes of Schwann cells in groups without, with continuing, and with limited (12 weeks) CsA treatment were examined immunohistochemically in allogeneically and syngeneically transplanted animals from 4 to 36 weeks after transplantation. RESULTS: In the group receiving no CsA, little nerve regeneration was obtained; donor Schwann cells were rejected and replaced by recipient cells. In continuing and limited-course CsA groups, successful nerve regeneration was achieved at postoperative week 36, as was also observed in the syngeneic group. Schwann cells in the continuing CsA group remained donor-derived. In the limited-course CsA group, graft rejection and loss of function occurred after the withdrawal of CsA, and donor Schwann cells were replaced by recipient cells in the part of the graft where rejection had been complete. However, many donor Schwann cells remained at week 36, when the rejection response subsided. CONCLUSION: Possible clinical use of a limited course of immunosuppression was supported by this demonstration of long term persistence of donor Schwann cells.
Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Células de Schwann/citologia , Nervo Isquiático/transplante , Animais , Eletrofisiologia , Rejeição de Enxerto/tratamento farmacológico , Antígenos de Histocompatibilidade Classe I/imunologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Endogâmicos Lew , Células de Schwann/efeitos dos fármacos , Células de Schwann/imunologia , Nervo Isquiático/citologia , Nervo Isquiático/fisiologiaRESUMO
The effects of glucocorticoid (GC) on thymocytes have been utilized to investigate the maturation and differentiation of thymocytes, but these experiments have mainly been performed on mouse thymocytes. We investigated the cell surface antigens expressed by LEW rat thymocytes during thymic reconstitution after GC treatment. Three-color flow cytofluorometric analysis of CD4, CD8 and the T cell antigen receptor (TCR alpha beta) clearly demonstrated that normal rat thymocytes contain CD4-8+ TCR alpha beta- and CD4+8- TCR alpha beta- cells. After GC treatment, we observed significant increases in the percentages of CD4-8+ TCR alpha beta- and CD4+8- TCR alpha beta- cells. The extent of the increase in the percentage of CD4-8+ TCR alpha beta- cells was greater than that of CD4+8- TCR alpha beta- thymocytes. Two-color analysis of TCR alpha beta and major histocompatibility complex (MHC) class I antigen showed that GC treatment significantly increased the percentage of TCR alpha beta- MHC class Ihi cells. Three-color analysis of CD4, CD8 and MHC class I demonstrated that normal rat thymocytes contain CD4-8- MHC class Ihi cells, which increased in number after GC treatment. These results indicate that rat thymocytes contain no fewer CD4-8+ TCR alpha beta- and CD4+8- TCR alpha beta+ cells than do mouse thymocytes, and that CD4-8+ TCR alpha beta- cells predominate over CD4+8- TCR alpha beta- cells in LEW rat thymus. Rat CD4-8- cells seemed to be divided into two subsets of TCR alpha beta- MHC class Ihi and TCR alpha beta- MHC class I- cells.
Assuntos
Antígenos de Superfície/efeitos dos fármacos , Dexametasona/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Timo/citologia , Animais , Anticorpos Monoclonais , Antígenos CD4/efeitos dos fármacos , Antígenos CD8/efeitos dos fármacos , Diferenciação Celular/imunologia , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T alfa-beta/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Timo/efeitos dos fármacosRESUMO
The expression of cell surface antigens recognized by UB-12 monoclonal antibody against rat hemopoietic cells was examined in fetal liver, and adult bone marrow using immunohistochemistry, radioimmunoassay, and flow cytofluorometry. Comparisons were made with the expression of the antigens recognized by OX-7 (anti-Thy-1) or W3/13 (anti-leukocyte sialoglycoprotein) antibodies. UB-12 positive cells appeared at earlier stages of gestation and in higher cell frequency than OX-7 or W3/13 positive cells in fetal liver. The antigen detected with UB-12 antibody was positive in fetal liver at day 12 and increased at day 15, reaching about 90% of the positive cells. In bone marrows, UB-12 positive cells started to appear around birth and were about 30-40% of total nucleated cells during adult periods. Thus, the antigen recognized by UB-12 was found only during early stages of blood cells. UB-12 monoclonal antibody recognized a B-cell lymphoma, Y-3, but did not react with a rat T lymphoma cell line. This monoclonal antibody may be important for distinguishing normal and neoplastic B cells and their precursors.
Assuntos
Anticorpos Monoclonais/imunologia , Células da Medula Óssea , Células-Tronco Hematopoéticas/imunologia , Fígado/citologia , Animais , Separação Celular , Reações Cruzadas , Citometria de Fluxo , Hematopoese , Fígado/embriologia , Linfoma/patologia , Ratos , Ratos Endogâmicos/crescimento & desenvolvimento , Ratos Endogâmicos/imunologia , Células Tumorais Cultivadas/imunologiaRESUMO
An alternative complement pathway-inhibiting protein (ACPIP), which inhibits the activation of the alternative complement pathway (ACP) of the human serum, was isolated from larval hemolymph of the silkworm, Bombyx mori, by using ammonium sulfate fractionation and column chromatographies to homogeneity. About 400microg of ACPIP was routinely obtained from 20ml hemolymph. The purified ACPIP preparation consisted of two distinct polypeptides (34 and 32kDa) on SDS-PAGE. The amino acid compositions of the two polypeptides were nearly identical; 21% of the amino acid residues were acidic. The amino terminal amino acid sequences up to 20 residues in these two polypeptides were also identical. Addition of the ACPIP to human serum resulted in a dose-dependent inhibition of the hemolysis of intact rabbit erythrocytes via the ACP, whereas in no inhibition of hemolysis of sensitized-sheep erythrocytes (EA) via the classical pathway.
Assuntos
Bombyx/química , Proteínas Inativadoras do Complemento/isolamento & purificação , Hemolinfa/química , Proteínas de Insetos/isolamento & purificação , Sequência de Aminoácidos , Animais , Composição de Bases , Proteínas Inativadoras do Complemento/química , Proteínas Inativadoras do Complemento/imunologia , Via Alternativa do Complemento/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Hemólise/efeitos dos fármacos , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/imunologia , Larva/química , Dados de Sequência Molecular , Peso Molecular , Coelhos , OvinosRESUMO
The monoclonal antibody Kp62 recognized surface antigenic determinants of some strains of Klebsiella pneumoniae. The antigen recognized by Kp62 was demonstrated on the bacterial surface using immunoelectron microscopy. Kp62 reacted with K. pneumoniae No. 1 or K. pneumoniae B 5055 and lipopolysaccharide (LPS) from the same bacteria. However, Kp62 was not inhibited by the LPS from Escherichia coli (E. coli) O111:B4 and E. coli O55:B5. Thus, Kp62 might be a useful monoclonal antibody to detect K. pneumoniae and LPS from K. pneumoniae. The possibility to visualize the localization of K. pneumoniae LPS injected into animals using immunohistochemical methods with this monoclonal antibody was examined. It was possible to detect the injected LPS in the spleen of mouse and rat with the monoclonal antibody to K. pneumoniae. In order to detect the early events taking place in the spleen after intravenous injection of LPS, time course of LPS distribution in mice and rats was studied. After 30 min, 2, 4, 8 and 24 h LPS localized in the marginal zone (MZ) in mice and rats, although the degree of LPS positive cells varied. The cells responsible for trapping the injected LPS appeared to be marginal zone macrophages. The early trapping of LPS by marginal zone macrophages was thought to be important for the following immune responses to the injected LPS. Interestingly the antigenic determinant on the injected LPS appeared to last long on or within the cells in the spleen from the injected animals. Such a remaining antigen might be important for the continuous stimulation of B cells by the LPS. With respect to the distribution of red pulp (RP) and white pulp (WP), we found the varied distribution of LPS between mouse and rat, and SPF and conventionally fed (Conv) animals. For example, LPS-positive cells in RP of rat were scarce, while significant degree of LPS-positive cells were observed in mice. And in WP, LPS-positive cells were observed in Conv DA rats, but not in mice or SPF-fed Wistar rats. These results may suggest that the mode of antigen processing may be different in the spleen of rat and mouse or even among the different strain of rats and previous sensitization to the LPS (or the similar antigenic determinants) may lead to the different distribution of LPS in the spleen. The monoclonal antibody specifically raised against K. pneumoniae was shown to be very useful to follow the fate of LPS derived from K. pneumoniae using immunohistochemical method.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Anticorpos Antibacterianos/análise , Klebsiella pneumoniae/imunologia , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Antígenos de Bactérias/análise , Ligação Competitiva , Imunofluorescência , Imuno-Histoquímica/métodos , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Radioimunoensaio , Ratos , Ratos EndogâmicosRESUMO
FF-6 is a transplantable squamous cell carcinoma which originally arose in the facial skin of a DA rat. It was established after maintaining the tumor in the subcutaneous tissue or peritoneal cavity of DA rats conventionally for over 30 generations. When the soybean-sized original FF-6 tumor was transplanted subcutaneously, it became an oval, hard, whitish, solitary and thumb-head-sized nodule within one month. After intraperitoneal transplantation of FF-6, it formed many nodules ranging from miliary to thumb-head size, which adhered and/or metastasized to many abdominal organs. When FF-6, cut into small pieces, was injected into the lower lip, the tumor grew bigger in situ, and metastasized to regional lymph nodes. Histologically, FF-6 was characterized as a well-differentiated squamous cell carcinoma, showing positive staining with anti-keratin, anti-laminin, anti-collagen type IV, anti-fibronectin and UB-14 antibodies. This transplantable tumor may be useful for analyzing the mechanisms of proliferation and metastasis of squamous cell carcinoma in vivo, and the host defence mechanism in rats, as well as being a suitable model of human squamous cell carcinoma.
Assuntos
Carcinoma de Células Escamosas/patologia , Colágeno/análise , Queratinas/análise , Laminina/análise , Neoplasias Cutâneas/patologia , Neoplasias do Timo/patologia , Animais , Divisão Celular , Linhagem Celular , Fibronectinas/análise , Humanos , Imuno-Histoquímica/métodos , Metástase Linfática , Metástase Neoplásica , Transplante de Neoplasias , Ratos , Ratos EndogâmicosRESUMO
The effects of allogeneic lymphocytes on the rat thymus following sublethal irradiation were investigated using immunofluorescence. The recovery of thymus weight following irradiation was delayed in rats 6 days after receiving lymphocytes compared to controls. Allogeneic cells forming colonies were detected by immunofluorescence in both the cortex and medulla of the host thymus, most frequently on day 15 when an appropriate number (3 x 10(6)) was injected. The allogeneic cells detected in the host thymus, presumably T lymphocytes, appeared to disturb thymic reconstitution following irradiation. However, double-immunofluorescence staining revealed that allogeneic cells did not affect the thymic stromal microenvironment. Allogeneic cells may have subsequently affected thymic tissue via cytokines. It is important to investigate not only the character of allogeneic cells in the host thymus but also the interactions of donor allogeneic cells, host immature lymphocytes and thymic epithelial cells because of the possibility that these allogeneic cells in the host thymus could prevent the rejection of allogeneic transplants.
Assuntos
Transplante de Células/fisiologia , Linfócitos T/fisiologia , Timo/citologia , Timo/efeitos da radiação , Animais , Anticorpos Monoclonais , Diferenciação Celular/fisiologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Doença Enxerto-Hospedeiro/patologia , Técnicas Imunoenzimáticas , Tamanho do Órgão , Ratos , Ratos Endogâmicos LewRESUMO
Immunohistochemical examination of rat skeletal muscle during graft-versus-host disease (GVHD), a systemic immune reaction, was performed to investigate specific immune reactivities focusing on major histocompatibility complex (MHC) expression and inflammatory cell infiltration of skeletal muscle during a systemic immune reaction. MHC class II expression and inflammatory cell infiltration did not increase. MHC class I was expressed along the contour of muscle fibres, and most strongly expressed by the cells which were distributed throughout the endomysium and perimysium. Seventy-six percent of these MHC class I+ cells carried endothelial cell-markers, while 24% of them did not. The latter cells were revealed not to be inflammatory cells such as lymphocytes, granulocytes or macrophages when examined by immunostaining using several exudate-cell markers. Neither were they myosatellite cells because they were located outside the basement membrane. These results may be useful for considering animal models of inflammatory myopathies such as polymyositis and dermatomyositis.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Músculos/imunologia , Animais , Peso Corporal/fisiologia , Transplante de Células , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica/fisiologia , Genes MHC Classe I/imunologia , Genes MHC da Classe II/imunologia , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Linfócitos/imunologia , Músculos/transplante , Inclusão em Parafina , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos , Pele/imunologiaRESUMO
The expression of membrane antigens on rat hepatocytes transplanted syngeneically and heterotopically was analyzed immunohistochemically using monoclonal antibodies against rat hepatocytes. Isolated adult and fetal hepatocytes were able to survive in the spleen, salivary gland, thymus, or subcapsular region of the kidney for various periods after transplantation. Fairly clear expression of HAM2, 4, and 8 antigens was observed on hepatocytes transplanted into syngeneic spleen, suggesting that the cells might be functionally equivalent to hepatocytes in situ. HAM4 antigen was localized specifically on the newly formed bile-canalicular faces of hepatocytes. The expression of HAM2 (MHC class I) antigen on the transplanted hepatocytes appeared much stronger on the side facing lymphoid tissues, than on the other faces, suggesting that some immunological reactions may take place between hepatocytes and lymphoid tissue. HAM8 antigen, which is localized on gap junctions between neighboring hepatocytes in rat liver, was also recognized between transplanted hepatocytes. In salivary glands where hepatocytes were transplanted, bile-canaliculus-like structures were observed not only between neighboring hepatocytes but also between hepatocytes and salivary acinar cells, suggesting good interaction between the two different epithelial cell types. Hepatocytes transplanted into thymus appeared viable, but most showed fatty degeneration. Some healthy hepatocytes survived in the interlobular connective tissue and the thymic cortical tissue. When fetal hepatocytes were transplanted heterotopically, they formed a mass consisting of hepatocytes and bile duct-like structures 7 wk after transplantation. The inoculated hepatocytes possessed HAM4 antigen, which was not recognized on fetal hepatocytes at day 14 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antígenos de Superfície/biossíntese , Transplante de Fígado/fisiologia , Transplante Heterotópico/fisiologia , Transplante Isogênico/fisiologia , Animais , Antígenos de Superfície/análise , Células Cultivadas , Feminino , Transplante de Tecido Fetal/fisiologia , Sobrevivência de Enxerto , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/biossíntese , Rim , Gravidez , Ratos , Ratos Endogâmicos , Glândulas Salivares , Baço , Timo , Fatores de TempoRESUMO
To investigate the mechanism of interstitial inflammation in diabetic nephropathy, we used spontaneously diabetic KKAy mice. Twelve KKAy mice were divided into two groups; six mice were fed standard mouse chow ad libitum and six mice were placed on a diet (i.e. they received the same amount of chow as six control C57BL mice). Diabetic KKAy mice developed hypercholesterolemia and albuminuria. Animals were killed at 16 weeks of age and renal tissues were immunostained for vascular cell adhesion molecule-1 (VCAM-1). In diabetic KKAy mice, the renal interstitium was infiltrated by monocytes, lymphocytes, plasma cells, and other cells. The walls of venules near the infiltrating cells were more intensely stained for VCAM-1 when compared with other sites. In contrast, the VCAM-1 staining of arterioles and peritubular capillaries was not significantly increased. There was weak VCAM-1 staining of the infiltrating cells, including lymphocytes, monocytes, and other cells. Electron microscopy demonstrated immunolabeling for VCAM-1 on the cell surface and in the cytoplasm of both infiltrating cells and vascular endothelial cells. In KKAy mice placed on a diet, there was less staining for VCAM-1 and cellular infiltration was also decreased. Thus, increased expression of VCAM-1 by the endothelial cells of venules and VCAM-1 expression by infiltrating cells were demonstrated in the interstitium of kidneys from diabetic mice. These results suggest that increased expression of VCAM-1 by endothelial cells and infiltrating cells contributes to interstitial inflammation in diabetic nephropathy.