RESUMO
The rectal mucosa is one of the routes of transmission of the HIV virus, although the mechanism of transmission is unknown. We carried out an immunohistological investigation of human rectal epithelium to detect CD4 glycoprotein and Fc receptors (FcR) for immunoglobulin G which may be involved in HIV infection. CD4 was not detected by monoclonal antibodies (MAb) in normal rectal epithelial cells, although CD4+ mononuclear cells were found in the lamina propria of the rectum. FcR3 and FcR2 were, however, detected in surface or crypt epithelial cells of rectal mucosa, using MAb to CD16 and CD32, respectively. In addition, CD16 messenger RNA (mRNA) was found in surface and crypt epithelial cells by in situ hybridization using an RNA probe. FcR3 and FcR2 were also detected in fetal recto-colonic tissue by immunohistology, suggesting that these are constitutive receptors. FcR3 and FcR2 gene transcripts were then demonstrated in fetal recto-colonic tissue using the polymerase chain reaction to amplify a portion of FcR3 and FcR2 coding sequences in complementary DNA (cDNA) prepared from fetal RNA. These findings suggest the possibility that rectal transmission of HIV-antibody complexes might be facilitated by the expression of FcR3 and FcR2 in rectal epithelial cells.
Assuntos
Antígenos CD4/análise , Receptores Fc/análise , Reto/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Anticorpos Monoclonais/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Sequência de Bases , Northern Blotting , Antígenos CD4/genética , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitélio/imunologia , Expressão Gênica/imunologia , HIV/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sondas RNA/genética , Receptores Fc/genética , Receptores Fc/imunologia , Receptores de IgG , Reto/embriologia , Reto/metabolismoRESUMO
This study used electron microscopy and confocal scanning laser microscopy interfaced with cytochemistry to study neuromuscular interrelationships in the ovijector of Ascaris suum. An extensive nerve plexus with both FaRPergic and non-FaRPergic components extends over the outer surface of the ovijector. The non-FaRPergic component is derived from nerve branches of the ventral nerve cord, whereas the FaRPergic component emanates from two large FMRFamide-immunoreactive neurons. In the vagina vera, most myofibrils are circular in orientation and a number of them divide and run for short distances in longitudinal and diagonal directions, their myofilaments are also orientated in a variety of directions. Parallel nerve fibres run in tracts along the length of the vagina vera with branches that penetrate the muscle layers. The vagina uteri possesses a thicker hypodermis than that of the vagina vera. It appears rich in secretory and phagocytic vesicles and the luminal side is invested with an electron-dense substance. The musculature of the vagina uteri is less well developed than that of the vagina vera, being restricted to circular myofibrils, with an apparent diagonal arrangement of myofilaments. Also, the innervation is less extensive in the vagina uteri with many fibres returning to the vagina vera to rejoin the nerve net and others continuing into the uteri.
Assuntos
Ascaris suum/anatomia & histologia , Ascaris suum/química , Genitália Feminina/química , Genitália Feminina/ultraestrutura , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/ultraestrutura , Neurônios/química , Neurônios/ultraestrutura , Animais , Ascaris suum/fisiologia , FMRFamida/análise , Feminino , Gânglios dos Invertebrados/química , Gânglios dos Invertebrados/fisiologia , Gânglios dos Invertebrados/ultraestrutura , Genitália Feminina/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Neurônios/fisiologia , Faloidina/análise , Serotonina/análise , Tubulina (Proteína)/análiseRESUMO
We have studied the primate cellular requirements in the antibody forming cell (AFC) assay, using a 185,000 molecular mass streptococcal antigen. Cultures of B cells alone stimulated with the antigen were unable to develop into AFC. Reconstitution of B cells with T cells resulted in a significant increase in the number of AFC. However, CD4 cells were more efficient than T cells in helping the antibody response and CD8 cells failed to induce B cells to synthesise antibody. A comparison between the specific IgG antibody synthesis and the number of AFC detected by the ELISPOT method showed a highly significant correlation between the number of AFC and the amount of specific IgG antibody. We suggest that the AFC (ELISPOT) assay can be readily used to investigate B cell interactions with T cell subsets.
Assuntos
Formação de Anticorpos , Células Produtoras de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/biossíntese , Subpopulações de Linfócitos , Macaca mulatta , Streptococcus/imunologiaRESUMO
A streptococcal antigen (SA) of 185 kDa was isolated from Streptococcus mutans and this antigen induced in vitro helper, suppressor and contrasuppressor activities with primate peripheral blood lymphocytes. The 185-kDa SA was then treated by sodium dodecyl sulfate and yielded a 4-kDa SA which was capable of eliciting only helper activity. We have now cleaved the 185-kDa SA with cyanogen bromide, in an attempt to identify suppressor and contrasuppressor determinants. A 14-kDa SA was separated from the cyanogen bromide digest and its ability to elicit T cell and B cell functional activities was tested in rhesus monkeys. Whereas the 185-kDa SA (and 4-kDa SA) elicited high serum anti-SA antibodies and the CD4 cells showed an increase in DNA synthesis, this was not demonstrable with the 14-kDa SA. However, the 14-kDa SA, unlike the 185-kDa SA, activated a significant proportion of CD4 and CD8 cells to bind the Vicia villosa lectin (VV) and this is a characteristic feature of contrasuppressor cells. We then studied the effect of sequential immunization of monkeys with the 14-kDa SA, followed by the 185-kDa SA. The results of this showed suppression of the CD4 proliferative response, in the presence of a normal antibody production. We suggest that the split tolerance between the T cell proliferative and B cell differentiating functions might be interpreted on the basis of suppressor CD8 cells inhibiting the CD4 proliferative phase and the VV-adherent CD8 cells contrasuppressing B cell antibody formation.
Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Linfócitos B/imunologia , Lectinas de Plantas , Streptococcus mutans/imunologia , Linfócitos T/imunologia , Animais , Formação de Anticorpos , Diferenciação Celular , Tolerância Imunológica , Imunização , Lectinas , Ativação Linfocitária , Macaca mulattaRESUMO
Human serum amyloid A (SAA) proteins are a group of 12-14 kDa apolipoproteins found predominantly in the high-density lipoprotein (HDL) fraction of plasma. Several functions have been proposed for SAA, but its primary physiological function remains elusive. In this report, we used the monocytic cell line THP-1 to investigate whether recombinant SAA1 (rSAA) or the HDL-rSAA protein complex can affect the capacity of these cells to produce inflammatory cytokines in vitro. Incubation of rSAA, plasma HDL (which contains < or = 30 microg/ml of SAA) or HDL-rSAA complex with THP-1 cells induced synthesis of IL-1beta, IL-1ra and sTNFR-II protein and mRNA. The induction of cytokine synthesis was not due to endotoxin contamination since the effect was abrogated by protein denaturation. The rSAA and HDL-rSAA complex did not induce detectable levels of IL-6 or TNFalpha protein or mRNA. In contrast 10 microg/ml LPS stimulated secretion of the inflammatory cytokines, IL-1beta, IL-6 and TNFalpha, as well as IL-1ra and sTNFR-II from THP-1 cells. We confirmed that rSAA has chemoattractant properties in vivo, by subcutaneous injections into mice and examined the histology of the injection site at 72 h, however, the HDL-rSAA complex has a substantially reduced effect.
Assuntos
Citocinas/biossíntese , Monócitos/metabolismo , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/farmacologia , Animais , Células Cultivadas , Citocinas/efeitos dos fármacos , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacologia , Camundongos , Monócitos/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
T cell clones were generated from the peripheral blood of rhesus monkeys that had been immunized with a soluble Mr 185,000 Ag (SAI/II) derived from Streptococcus mutans. The clones were CD3+ CD8+ CD4- alpha beta TCR+ and were specifically stimulated to proliferate by SAI/II. The proliferative responses of the cloned cells were class I restricted, as demonstrated by reconstitution of the cloned T cells with APC matched at various MHC class I and II loci, as well as by inhibition with anti-class I and not anti-class II mAb. The function of the CD8+ cloned cells was examined in vitro for their effect on antibody synthesis by Ag-stimulated CD4+ cells and B cells from immunized animals. Indeed, four of the five clones suppressed SAI/II-specific IgG antibody synthesis when activated with SAI/II and the appropriate MHC-matched APC. Although activation of the suppressor clones was Ag specific, the effector function of the suppression of antibody synthesis was Ag nonspecific. The latter was probably mediated by lymphokines and, indeed, the culture supernatant generated by stimulating the cloned CD8+ cells with anti-CD3 mAb suppressed both the specific and nonspecific antibody synthesis. Cytotoxicity studies showed that all five CD8+ clones showed a low level of lectin-dependent cytotoxicity. However, because four of the five clones expressed significant suppression of antibody synthesis, the suppressor activity was unlikely to be a function of the weak cytotoxicity. The results suggest that immunization of rhesus monkeys with a soluble streptococcal Ag induced CD8+ alpha beta TCR+ T cell clones that show SAI/II-specific, MHC class I-restricted proliferative responses and nonspecific down-regulatory function of in vitro antibody synthesis.
Assuntos
Antígenos de Bactérias/imunologia , Streptococcus mutans/imunologia , Linfócitos T/imunologia , Animais , Formação de Anticorpos , Células Apresentadoras de Antígenos , Antígenos de Diferenciação de Linfócitos T/análise , Northern Blotting , Antígenos CD8/imunologia , Células Clonais , Citotoxicidade Imunológica , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária , Macaca mulatta/imunologia , Complexo Principal de Histocompatibilidade , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Reguladores/imunologia , Toxoide Tetânico/imunologiaRESUMO
The percentages of various leucocyte subsets in the blood, spleen and popliteal lymph nodes of animals with experimental allergic neuritis (EAN) were determined at various times between the initiation of the disease and recovery. The total cell yield from these tissues and the weights of the spleens and lymph nodes were recorded. Clinical and histological signs of disease were assessed at the same time points. Normal and adjuvant control animals were also studied. The most marked change observed was that the percentage of MRC OX 8+ ('suppressor/cytotoxic') cells in the blood of animals with EAN was significantly below the normal value during the most severe clinical disease, and then returned to normal during recovery. Adjuvant controls did not show this change. No significant differences were observed in the other lymphocyte subsets studied (surface Ig+, Ia+, W3.13+ and W3.25+ cells) or in the total white cell count in the blood. There were no changes from normal values in any of the parameters examined in the spleen. The popliteal lymph nodes were enlarged in both adjuvant controls and animals with EAN and both showed a decrease in the percentage of W3.25+ lymphocytes from normal unenlarged nodes.
Assuntos
Linfócitos/fisiologia , Neurite Autoimune Experimental/imunologia , Animais , Anticorpos Monoclonais/imunologia , Doenças Autoimunes/imunologia , Imunofluorescência , Contagem de Leucócitos , Linfonodos/citologia , Linfonodos/fisiopatologia , Linfócitos/imunologia , Masculino , Camundongos/imunologia , Tamanho do Órgão , Ratos , Ratos Endogâmicos Lew , Baço/citologia , Baço/fisiopatologiaRESUMO
The mechanism of transmission of HIV from the male to the female genital tract or in the reverse order is not clear. CD4 glycoprotein is the receptor for HIV and Langerhans cells and the related dendritic cells could play a role in the initial transmission of HIV. Fc receptors (FcR) for IgG might be involved in antibody-mediated binding of HIV. We carried out an immunohistological study of normal human cervical and vaginal epithelia for the presence of CD4 glycoprotein, Langerhans cells and FcR to IgG. CD4+ glycoprotein was not found in the vaginal or cervical epithelium, with the exception of a few endocervical epithelial cells. A small number of CD4+ mononuclear cells were found in the endocervical epithelium of a third of the specimens but a large number of CD4+ cells was found in the submucosa of most of the cervical and vaginal specimens. Langerhans cells expressing CD4, HLA class II, Fc gamma R2 and Fc gamma R3 were detected in most vaginal, ectocervical and transformation zone epithelia and in 9/14 endocervical tissues. Fc gamma R3 was detected in about two-thirds of the columnar endocervical epithelium and the transformation zone. A smaller number of specimens expressed Fc gamma R2 in these epithelia, but Fc gamma R1 was not detected. We then demonstrated mRNA for Fc gamma R3 in the columnar endocervical epithelial cells and transformation zone by in situ hybridization, using a CD16-RNA probe. Fc gamma R3 and Fc gamma R2 gene transcripts were also found in fetal cervical tissue by applying the polymerase chain reaction to amplify portions of the Fc gamma R3 and Fc gamma R2 coding sequences in cDNA prepared from fetal RNA. HLA-DR was found in the endocervical cells, transformation zone and in Langerhans cells of all specimens. The presence of Langerhans cells, Fc gamma receptors and HLA class II antigen offers three potential mechanisms for cervico-vaginal HIV transmission: (i) direct HIV infection of Langerhans cells, (ii) binding of HIV antibody complexes to cervical epithelial Fc gamma receptors and (iii) binding of HIV infected CD4+ cells to cervical HLA class II antigen which may infect these or the adjacent CD4+ cells.
Assuntos
Colo do Útero/ultraestrutura , Antígenos de Histocompatibilidade Classe II/análise , Células de Langerhans/citologia , Receptores Fc/análise , Receptores Fc/genética , Vagina/ultraestrutura , Adulto , Sequência de Bases , Linfócitos T CD4-Positivos/citologia , Colo do Útero/química , Colo do Útero/imunologia , Epitélio/química , Epitélio/ultraestrutura , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Células de Langerhans/química , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Transcrição Gênica , Vagina/química , Vagina/imunologiaRESUMO
A novel formulation of cationic liposomes containing the novel cytofectin ACHx was used for delivery of an anti-inflammatory cytokine gene, IL-10, to mice with established collagen induced arthritis. A single intraperitoneal injection of human IL-10 expression plasmid complexed with liposomes 2 to 4 days after the onset of arthritis was sufficient to give significant and prolonged amelioration of arthritis for 30 days. Preliminary experiments suggested that the therapeutic effect was IL-10 dose-dependent. The distribution of the human IL-10 DNA after injection was widespread, including the inflamed paws. Human IL-10 mRNA was also detected in the paws 24 h after injection. IL-10 protein was below the level of detection in paws and serum but was detected in some tissues up to 10 days after injection. The target cell of transfection was demonstrated to be the macrophage. These results suggest that systemic therapy with plasmid DNA complexed with cationic liposomes merits further development as an alternative method for anti-inflammatory treatment of arthritis.
Assuntos
Artrite Experimental/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Interleucina-10/genética , Transfecção/métodos , Animais , Cátions , Colágeno , Relação Dose-Resposta a Droga , Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Injeções Intraperitoneais , Interleucina-10/análise , Lipossomos/administração & dosagem , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos DBA , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
Ascaris suum possesses a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8/PF3) have been shown to modulate the intrinsic, rhythmic activity of the vagina vera of A. suum in vitro. In the present study, the effects of the nematode FaRPs, SDPNFLRFamide (PF1), SADPNFLREamide (PF2) and KPNFIRFamide (PF4) (from Panagrellus redivivus) and AVPGVLRFamide (AF3) and GDVPGVLRFamide (AF4) (from A. suum) on the in vitro activity of the vagina vera were examined. The effects of each of the peptides were qualitatively and quantitatively distinct. All 3 FaRPs from P. redivivus were inhibitory, causing a cessation of contractions. PF2 was 3 times more potent than PF1, with a threshold of 1 nM. Although PF4 was the least potent (threshold, 10 nM), its effects at > or = 10 nM were quantitatively the greatest. Both AF3 and AF4 (1 microM) induced complex, multiphasic responses consisting of an initial contraction and spastic paralysis followed by a return of contractile activity of increased amplitude. AF3 was 3 times more potent than AF4. The effects of these peptides had some similarities to those observed on A. suum somatic body wall muscle in vitro, with PF1, PF2 and PF4 being inhibitory and AF3 and AF4 being excitatory.
Assuntos
Ascaris suum/fisiologia , FMRFamida/análogos & derivados , FMRFamida/fisiologia , Animais , Ascaris suum/química , Relação Dose-Resposta a Droga , Feminino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Vagina/fisiologiaRESUMO
Ascaris suum possesses a well-developed nervous system which is regulated by a number of classical neurotransmitters including acetylcholine (ACh), gamma-aminobutyric acid (GABA), glutamate and serotonin. The vagina vera, the distal part of the ovijector, displays intrinsic, rhythmic activity which has been shown to be modulated by FMRFamide-related peptides (FaRPs) in vitro. Confocal scanning laser microscopy coupled with immunocytochemistry, and histochemical studies, revealed that the nerve plexus of the ovijector contains GABAergic and glutamatergic innervation. Although no distinctive cholinergic or serotoninergic innervation was apparent, cholinesterase activity was localized to discrete areas of the musculature of the vagina vera. The effects of classical transmitters on the activity of the vagina vera in vitro were examined. ACh was excitatory, stimulating a brief but powerful contraction of the vagina vera with a threshold for activity of 1 microM. Both GABA and glutamate were inhibitory, causing a cessation of contractile activity at high concentrations (> 10 microM). Although less potent than glutamate, GABA had more profound effects and induced longer-lasting paralysis of the tissue. The threshold concentrations for activity were 5 microM for glutamate and 10 microM for GABA. Serotonin had no consistent effect on the vagina vera. This study demonstrates that classical transmitters modulate the activity of the ovijector of A. suum.
Assuntos
Ascaris suum/fisiologia , Neurotransmissores/fisiologia , Acetilcolina/fisiologia , Animais , Ascaris suum/química , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Ácido Glutâmico/fisiologia , Imuno-Histoquímica , Microscopia Confocal , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculos/lesões , Músculos/fisiologia , Serotonina/fisiologia , Transdutores , Ácido gama-Aminobutírico/fisiologiaRESUMO
Ascaris suum contains a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8, also called PF3) have been extensively studied and are known to exert actions on somatic muscle strips of the worm. In the present study, the effects of AF1, AF2 and AF8 on the activity of the vagina vera of female A. suum have been examined in vitro. The vagina vera is a muscular tube connecting the uterus and vagina uteri to the gonopore and is probably involved in regulating egg output. The tissue exhibited spontaneous, rhythmic contractions in vitro, which were modulated by each of the FaRPs tested. The effects of each of the peptides were qualitatively and quantitatively different, and in each case were reversible. AF1 (1 microM) caused a biphasic response in the form of a transient lengthening of the preparation, followed by a shortening; contractions were initially inhibited but resumed 5 min post-addition of the peptide. Lower concentrations (< or = 0.1 microM) induced a less marked effect, with rhythmic contractions returning 5 min post-addition. AF2 and AF8 reduced contraction frequency at concentrations > or = 0.1 microM. Both peptides also caused the tissue to shorten, although the effects of AF8 on baseline tension were inconsistent. The apparent potencies of AF1 and AF8 on contraction frequency of the vagina vera were 10-fold greater than AF2 and, unlike their actions on A. suum somatic body wall muscles, the actions of AF1 and AF2 were qualitatively different. Indeed, the effects of each of these FaRPs on the vagina vera were markedly different from those observed on the somatic muscle.