RESUMO
Schizophrenia (SZ) is a mental illness characterized by psychosis, negative symptoms, and cognitive deficits. The anterior cingulate cortex (ACC), a structurally and functionally diverse region, is one of several brain regions that is abnormal in SZ. The present study compared synaptic organization and mitochondrial number and morphology in postmortem ACC in SZ versus normal control (NC). Total synaptic density in the combined ACC was decreased in SZ, to 72% of normal controls (NCs), due to selective decreases in axospinous synapses, both asymmetric (excitatory) and symmetric (inhibitory). These changes were present in layers 3 and 5/6. The density of mitochondria in all axon terminals combined in SZ was decreased to 64% of NC. In layer 3, mitochondrial density was decreased only in terminals forming asymmetric synapses with spines, while in layers 5/6 mitochondrial density was decreased in terminals forming symmetric synapses with spines and dendrites. The proportion of terminals making symmetric synapses that contained mitochondria was significantly lower in SZ than in NCs, especially for symmetric axospinous synapses. The number of mitochondria per neuronal somata was decreased in the ACC in SZ compared to NCs; this finding was present in layers 5-6. The size of mitochondria in neuronal somata and throughout the neuropil was similar in SZ and NCs. Our results, though preliminary, are well supported by the literature, and support an anatomical substrate for some of the altered executive functions found in SZ.
Assuntos
Giro do Cíngulo/ultraestrutura , Mitocôndrias/ultraestrutura , Esquizofrenia/patologia , Sinapses/ultraestrutura , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-IdadeRESUMO
The process of glutamate release, activity, and reuptake involves the astrocyte, the presynaptic and postsynaptic neurons. Glutamate is released into the synapse and may occupy and activate receptors on both neurons and astrocytes. Glutamate is rapidly removed from the synapse by a family of plasma membrane excitatory amino acid transporters (EAATs), also localized to neurons and astrocytes. The purpose of the present study was to examine EAAT labeling in the postmortem human cortex at the light and electron microscopic (EM) levels. The postmortem prefrontal cortex was processed for EAAT1 and EAAT2 immunohistochemistry. At the light microscopic level, EAAT1 and EAAT2 labeling was found in both gray and white matter. Most cellular labeling was in small cells which were morphologically similar to glia. In addition, EAAT1-labeled neurons were scattered throughout, some of which were pyramidal in shape. At the EM level, EAAT1 and EAAT2 labeling was found in astrocytic soma and processes surrounding capillaries. EAAT labeling was also found in small astrocytic processes adjacent to axon terminals forming asymmetric (glutamatergic) synapses. While EAAT2 labeling was most prevalent in astrocytic processes, EAAT1 labeling was also present in neuronal processes including the soma, axons, and dendritic spines. Expression of EAAT1 protein on neurons may be due to the hypoxia associated with the postmortem interval, and requires further confirmation. The localization of EAATs on the astrocytic plasma membrane and adjacent to excitatory synapses is consistent with the function of facilitating glutamate reuptake and limiting glutamate spillover. Establishment that EAAT1 and EAAT2 can be measured at the EM level in human postmortem tissues will permit testing of hypotheses related to these molecules in diseases lacking analogous animal models.
Assuntos
Transportador 1 de Aminoácido Excitatório/metabolismo , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/ultraestrutura , Adulto , Idoso , Animais , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Transportador 1 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório , Feminino , Humanos , Masculino , Camundongos Knockout , Microscopia Eletrônica , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/ultraestrutura , Córtex Pré-Frontal/irrigação sanguínea , Especificidade da EspécieRESUMO
Recent evidence suggests that prostanoids are an important participant in the pathobiology of gastric adenocarcinoma, but the location and identity of cells in tumor-adjacent gastric mucosa able to synthesize and/or bind specific prostanoids is not clear. Using probes for cyclooxygenase 1 and 2 mRNA and protein as well as for the EP family of PGE(2) receptors, we sought to define the biology of prostanoids in adjacent human gastric mucosa at the site of tumor invasion. In mucosa adjacent to an invasive gastric adenocarcinoma, expression of cyclooxygenase was prominent, with COX 1 primarily in mucosal T lymphocytes surrounding nests of tumor cells. Densitometry showed these tumor-adjacent cells had substantial levels of COX 1 immunoreactive protein (relative intensity, 3.2). Cyclooxygenase 2 was newly expressed among these cells as well, but was limited in number (<25% of cyclooxygenase-positive T lymphocytes) in tumor-adjacent mucosa. Further, CD3(+) mononuclear cells, adjacent to tumor, strongly expressed prostanoid receptor EP(4) (relative intensity, 8.0), but cells with this receptor were not evident in the tumor itself. In contrast, normal gastric mucosa showed a consistent and structured expression of cyclooxygenase and PGE(2) receptor immunoreactive protein among mucosal cells. Cyclooxygenase 1 and PGE(2) receptor EP(4) were expressed on mucosal CD3(+) T lymphocytes in the lumenal (upper) third of gastric mucosa; and prostanoid receptors EP(2), EP(3) and EP(4), on gastric epithelia lining gastric pits. In situ hybridization with COX cDNAs confirmed these findings, and neither COX 2-specific mRNA nor protein was detected in normal gastric tissue. Our studies suggest that synthetic machinery and receptors for PGE(2), prominently expressed by T lymphocytes in gastric mucosa at the boundary of normal mucosa with tumor cells, may play a central role in prostanoid-driven tumorigenesis of this tissue.
Assuntos
Dinoprostona/biossíntese , Mucosa Gástrica/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Prostaglandina E/metabolismo , Neoplasias Gástricas/metabolismo , Anticorpos , Mucosa Gástrica/enzimologia , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Gástricas/enzimologiaRESUMO
Chronic ingestion of non-steroidal anti-inflammatory medication is reported to delay or, in part, reverse development of polyps in the colon, but the mechanism for this effect is unknown. Using mRNA and immunoglobulin probes, specific for prostanoid receptors and for prostaglandin endoperoxide synthase (COX 1 and 2), we sought to define, by in situ and in vitro techniques, changes in PGE2 receptors and synthesis in cell populations of precancerous familial adenomatous polyposis (FAP) colonic mucosa. In FAP, expression of prostanoid receptors EP3 and EP4 among colonic lamina propria mononuclear and lateral crypt epithelial cells was robust, with 53.9+/-5.3% of mononuclear cells staining EP4+. When sections of normal colonic mucosa were examined by similar techniques, prostanoid receptor EP4 was expressed on only 21.3+/-1.2% of lamina propria mononuclear cells (including CD4+ T lymphocytes), as well as on surface and lateral crypt epithelium, and this distribution was found at the mRNA level as well. When receptor expression was quantitated by densitometry, immunoreactive EP3 protein on deep basolateral (but not other) FAP crypt epithelium was enhanced 2.8-fold over normal, and the number of prostanoid receptor EP4+ mononuclear cells by 2.5-fold. On the other hand, while COX 1 expression in mononuclear cells was prominent in normal and FAP mucosa, densitometric analysis showed immunoreactive prostaglandin endoperoxide synthase levels were further increased in FAP, due to a greater than fourfold elevation of COX 2 expression among mononuclear cells and epithelia. Our data suggest enhanced cell-specific prostanoid receptor expression and increased prostanoid synthesis in precancerous FAP mucosa.
Assuntos
Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/fisiopatologia , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/fisiologia , Receptores de Prostaglandina E/genética , Sequência de Aminoácidos , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Dinoprostona/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/química , Isoenzimas/análise , Isoenzimas/genética , Proteínas de Membrana , Dados de Sequência Molecular , Prostaglandina-Endoperóxido Sintases/análise , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/análise , Receptores de Prostaglandina E/análise , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4RESUMO
While in situ techniques have been valuable in identifying the presence and localization of cytoplasmic and membrane components in tissue (1), there is often a need to study directly one or more cell types, free from its own microenvironment. For the human colon, isolation techniques to allow direct study have been described for mononuclear cells in the lamina propria, smooth muscle cells at or below the muscularis mucosae, and cells of the enteric nervous system, located between the subserosa and the lamina propria (2-4). More recently, interest has risen to isolate populations of intestinal epithelial cells, for investigations of human colonic adenocarcinoma-which originates from colonic epithelia; as well as for study of the epithelial response to infection and inflammation. The technique for isolating epithelial cells from the human colon involves mechanical dissection to separate mucosa from the muscle layers which are discarded; and enzymatic digestion of collagen, followed by discontinuous gradient centrifugation in Percoll. The goal is to isolate>90% pure epithelial cells. Although the cells appear intact under the microscope, viability is variable from 50-80%. The yield depends on the size of the available tissue.
RESUMO
The mucosa of the colon occupies 25% of the intestinal wall, from the muscularis mucosa to the lumen-lining epithelium, and contains a variety of cell types, predominantly B and T lymphocytes, but also monocytes, mast cells, and macrophages (1,2). These cells secrete a variety of cytokines, including interleukins, leakotrienes, and prostaglandins. Most of these are locally active, diffuse throughout the mucosa, and do not substantially contribute to the concentration of cytokines in the blood.
RESUMO
The tissue concentration of PGE(2)is heightened during mucosal inflammation. Nevertheless, the cellular targets of this prostanoid and its effects on epithelial cell physiology are incompletely understood. We used a panel of specific immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGE(2)on cells of histologically normal and inflamed human colonic mucosa, and then examined the physiological consequences for the epithelial component of intestine, with special attention to its barrier function. Prostanoid receptors were selectively expressed on a limited number of human colonic mucosal cells, and differed markedly between normal and inflamed tissue. In non-inflamed mucosa, EP(2)and EP(3)were expressed on epithelia at the apex of crypts; while EP(4)was expressed on surface and lateral crypt epithelia. Dual immunostaining and in situ hybridization with digoxygenin-labelled RNA probes largely confirmed the epithelial localization of EP(4). On the other hand, during inflammation, lateral crypt (non-surface) epithelial cells newly and significantly expressed prostanoid receptors EP(2)and EP(3)(p<0.05, by computer-assisted densitometry). Functionally, exogenous E series prostanoids applied to epithelial monolayers in nM concentrations brought about a 24% increase in the level of barrier function; an associated rise in intracellular cAMP (EC(50)of 281); and protection of epithelium from the effects of T cell cytokines. A major perturbation in the number and distribution of functional eicosonoid receptors on epithelia occurs in chronic inflammation of human colonic mucosa.
Assuntos
Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Receptores de Prostaglandina E/metabolismo , Alprostadil/farmacologia , Anticorpos/imunologia , Especificidade de Anticorpos , Técnicas de Cocultura , AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Eletrofisiologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Inflamação/genética , Inflamação/metabolismo , Interferon gama/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E/classificação , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E/imunologia , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4 , Linfócitos T/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/patologiaRESUMO
Exposure to oocysts of the protozoan Cryptosporidium parvum causes intestinal epithelial cell dysfunction in vivo and in vitro, but effective means by which mucosal injury might be prevented remain unclear. We examined the ability of transforming growth factor beta1 (TGF-beta1)-a cytokine synthesized and released by cells in the intestine-to preserve the barrier function of human colonic epithelia when challenged with C. parvum oocysts and then studied the mechanisms involved. Epithelial barrier function was monitored electrophysiologically, receptors for TGF-beta1 were localized by confocal microscopy, and TGF-beta1-induced protein kinase C activation was detected intracellularly by translocation of its alpha isozyme. TGF-beta1 alone enhanced intestinal epithelial barrier function, while exposure to C. parvum oocysts (> or =10(5)/monolayer) markedly reduced barrier function to < or =40% of that of the control. When epithelial monolayers were pretreated with TGF-beta1 at 5.0 ng/ml, the barrier-disrupting effect of C. parvum oocysts was almost completely abrogated for 96 h. Further investigation showed that (i) the RI and RII receptors for TGF-beta1 were present on 55 and 65% of human epithelial cell line cells, respectively, over a 1-log-unit range of receptor protein expression, as shown by flow cytometry and confirmed by confocal microscopy; (ii) only basolateral and not apical TGF-beta1 exposure of the polarized epithelial monolayer resulted in a protective effect; and (iii) TGF-beta1 had no direct effect on the organism in reducing its tissue-disruptive effects. In exploring mechanisms to account for the barrier-preserving effects of TGF-beta1 on epithelium, we found that the protein kinase C pathway was activated, as shown by translocation of its 80-kDa alpha isozyme within 30 s of epithelial exposure to TGF-beta1; the permeability of epithelial monolayers to passage of macromolecules was reduced by 42% with TGF-beta1, even in the face of active protozoal infection; and epithelial cell necrosis monitored by lactate dehydrogenase release was decreased by 50% 70 h after oocyst exposure. Changes in epithelial function, initiated through an established set of surface receptors, likely accounts for the remarkable barrier-sparing effect of nanogram-per-milliliter concentrations of TGF-beta1 when human colonic epithelium is exposed to an important human pathogen, C. parvum.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Colo/imunologia , Cryptosporidium parvum/patogenicidade , Mucosa Intestinal/imunologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Polaridade Celular , Colo/parasitologia , Cryptosporidium parvum/crescimento & desenvolvimento , Ativação Enzimática , Células Epiteliais/enzimologia , Células Epiteliais/parasitologia , Células Epiteliais/fisiologia , Humanos , Mucosa Intestinal/parasitologia , Mucosa Intestinal/fisiologia , Necrose , Proteína Quinase C/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Linfócitos T/imunologiaRESUMO
Although the tissue concentration of PGE(2) is heightened 3-fold or more during mucosal inflammation, the cellular targets of prostanoids in human mucosa and the resulting changes in cell physiology have not been fully explored. We used a panel of immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGE(2) to cells of histologically normal and inflamed human colonic mucosa, and then examined prostanoid-induced changes in mucosal lymphocyte function. Prostanoid receptors were selectively expressed on a limited number of human colonic mucosal cells; EP(4) alone was expressed on lamina propria mononuclear cells. Dual immunostaining in situ identified the CD3(+) T lymphocyte as a major EP(4) receptor-bearing cell in normal mucosa. Flow cytometry of isolated cells showed that 19.2% of lamina propria mononuclear cells were EP(4)(+), and almost 30% of these were CD3(+). In situ hybridization with digoxygenin-labeled RNA probes largely confirmed this localization. During inflammation, mucosal T lymphocytes showed a significant enhancement in EP(4) immunoreactive receptor protein. Computer-assisted densitometry of single cells demonstrated an increase in fluorescence intensity from 4.8 +/- 1.8 to 8.6 +/- 1.8 (p < 0.04). The effects of PGE(2) included a 35% reduction in T lymphocyte IL-2 secretion. COX 1(+) lamina propria cells nearly doubled in number during inflammation; expressed a T lymphocyte marker; but retained an unchanged quantity of immunoreactive COX 1 protein per cell. The number of newly appeared COX 2(+) lymphocytes remained <50% that of COX 1(+) cells. A major perturbation in the number and distribution of PGE(2) receptors and enzymes for prostanoid synthesis occurs in chronic inflammation of the colon, with consequences for mucosal T lymphocyte function.
Assuntos
Colite/imunologia , Colo/imunologia , Dinoprostona/metabolismo , Mucosa Intestinal/imunologia , Receptores de Prostaglandina E/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Colo/citologia , Colo/metabolismo , Colo/patologia , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Dinoprostona/farmacologia , Expressão Gênica , Humanos , Interleucina-2/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Secreções Intestinais , Isoenzimas/metabolismo , Proteínas de Membrana , Dados de Sequência Molecular , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/metabolismo , Prostaglandinas/farmacologia , RNA Mensageiro/análise , Coelhos , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E Subtipo EP4RESUMO
Freshly isolated human mucosal T lymphocytes in vitro can markedly diminish an important property of intestinal epithelium-its barrier function. On the other hand, cytokines and their cellular receptors, which maintain homeostasis of epithelia, limit epithelial permeability, and preserve barrier function, are not well characterized. Using a described human colonic epithelial cell monolayer system, we found that transforming growth factor-beta1 (TGF-beta1) preserved 75% or more of epithelial barrier function, quantitated electrophysiologically, even in the presence of cytokines generated by a high density of barrier-disruptive mucosa-derived mononuclear cells. In opposing the TGF-beta1 effect, cytokines able to reduce barrier function were spontaneously secreted by mucosal T cells and were increased in their barrier effect after T-lymphocyte activation. Further, neutralization of individual cytokines with specific monoclonal antibodies abrogated the lymphocyte-induced reduction in epithelial barrier function, and identified interferon gamma (IFN-gamma), interleukin (IL)-4, and IL-10, but not IL-6, as the primary cytokines whose barrier effects were curtailed by TGF-beta1. Receptors (RI and RII) for TGF-beta1 were found to be localized primarily to the apical and basal membranes of surface epithelium in colonic crypts. These findings provide the scientific basis for new strategies to pharmacologically enhance the barrier function of epithelia in mucosal organs regularly exposed to environmental antigens and to T-lymphocyte products.
Assuntos
Citocinas/antagonistas & inibidores , Mucosa Intestinal/efeitos dos fármacos , Receptores de Superfície Celular/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , AMP Cíclico/fisiologia , Homeostase/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Mucosa Intestinal/química , Contagem de Linfócitos/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/análise , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The ability for the delayed effects of phencyclidine to model schizophrenia-like symptomatology was investigated by assessing the effects of phencyclidine pretreatment on amphetamine-induced behavior. Corresponding changes in striatal, nucleus accumbens and anterior cingulate cortex c-Fos induction were also assessed in order to test the hypothesis that alterations in the neurochemistry of these regions accompany phencyclidine-induced changes in amphetamine-induced behaviors. Rats were treated with 15.0 mg/kg phencyclidine or vehicle 24 h prior to behavioral testing following vehicle, 0.5, 2.5 or 5.0 mg/kg amphetamine. Phencyclidine pretreatment significantly increased amphetamine-induced locomotion and rearing in response to 0.5 mg/kg amphetamine. Likewise, phencyclidine pretreatment produced an increase in the number of striatal cells expressing c-Fos following treatment with 0.5 mg/kg amphetamine. Phencyclidine pretreatment did not alter c-Fos induction in the nucleus accumbens, but did decrease the basal number of c-Fos-containing cells in the anterior cingulate cortex. While stereotypy rating revealed that phencyclidine pretreatment enhanced the behavioral response to 5.0 mg/kg amphetamine over time, no other alterations in behavior or c-Fos expression in response to the higher doses of amphetamine were induced by phencyclidine pretreatment. These data demonstrate that the delayed effects of a single dose of phencyclidine alter anterior cingulate cortex neurochemistry, and enhance the behavioral and striatal c-Fos response to a low dose of amphetamine. These findings suggest that the delayed effects of a single dose of phencyclidine may produce a reasonable animal model for schizophrenia.
Assuntos
Anfetamina/farmacologia , Comportamento Animal/efeitos dos fármacos , Corpo Estriado/metabolismo , Fenciclidina/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Sinergismo Farmacológico , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoAssuntos
Colangite Esclerosante/complicações , Doença de Crohn/complicações , Glomerulonefrite Membranoproliferativa/complicações , Adulto , Colangiopancreatografia Retrógrada Endoscópica , Colangite Esclerosante/patologia , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Rim/patologia , MasculinoRESUMO
Regulation of immune cell activation in lymphocyte-bearing human tissues is a pivotal host function, and metabolites of arachidonic acid (prostaglandin E2 in particular) have been reported to serve this function at non-mucosal sites. However, it is unknown whether prostaglandin E2 is immunoregulatory for the large lymphocyte population in the lamina propria of intestine; whether low (nM) concentrations of prostaglandin E2 modulate immune responses occurring there; and whether adjacent inflammation per se abrogates prostaglandin E2's regulatory effects. To address these issues, intestine-derived lymphocytes and T hybridoma cells were assessed, T cell activation was monitored by release of independently quantitated lymphokines, and dose-response studies were performed over an 8-log prostaglandin E2 concentration range. IL-3 release by normal intestinal lamina propria mononuclear cells was reduced (up to 78%) in a dose-dependent manner by prostaglandin E2, when present in as low a concentration as 10(-10) M. PGE2 also inhibited (by > or = 60%) mucosal T lymphocytes' ability to destabilize the barrier function of human epithelial monolayers. Further, with an intestine-derived T lymphocyte hybridoma cell line, a prostaglandin E2 dose-dependent reduction in IL-3 and IL-2 (90 and 95%, respectively) was found; this was true for both mitogen- and antigen-driven T cell lymphokine release. Concomitant [3H] thymidine uptake studies suggested this was not due to a prostaglandin E2-induced reduction in T cell proliferation or viability. In contrast, cells from chronically inflamed intestinal mucosa were substantially less sensitive to prostaglandin E2, e.g., high concentrations (10(-6) M) of prostaglandin E2 inhibited IL-3 release by only 41%. We conclude that prostaglandin E2 in nM concentrations is an important modulator of cytokine release from T lymphocytes derived from the gastrointestinal tract, and it may play a central role in regulation of lamina propria immunocyte populations residing there.
Assuntos
Dinoprostona/fisiologia , Interleucina-2/metabolismo , Interleucina-3/metabolismo , Mucosa Intestinal/metabolismo , Linfócitos T/citologia , Animais , Linhagem Celular/citologia , Linhagem Celular/imunologia , Linhagem Celular/metabolismo , Colo/citologia , Colo/imunologia , Doença de Crohn/imunologia , Doença de Crohn/patologia , Células Epiteliais , Epitélio/embriologia , Epitélio/imunologia , Humanos , Hibridomas , Mucosa Intestinal/citologia , Camundongos , Ovalbumina/farmacologia , Linfócitos T/metabolismo , Timidina/metabolismo , Trítio/metabolismoRESUMO
Maintenance of the integrity of the single-cell-thick intestinal epithelium as an in vivo barrier between environmental Ags and mucosal immunocytes is pivotal for health. The T cell cytokine IFN-gamma consistently disrupts this epithelial barrier in vitro, but the substances in mucosa that may be responsible for sustaining or enhancing barrier function have not been clearly identified. Therefore, we characterized the effect on the epithelial barrier of TGF-beta 1 and three prominent neuropeptides (VIP, substance P, somatostatin) by using a model system in which barrier function of a mature polar human colonic epithelial (T84) cell monolayer is reflected in 1) the electrical potential difference across the apical to basolateral surface of each cell, 2) the transmonolayer permeability to macromolecules such as horseradish peroxidase, and 3) lactate dehydrogenase release into the medium indicating epithelial cell cytolysis. Whereas T84 monolayers exposed to TGF-beta 1 alone demonstrated a modest increase in electrical resistance and barrier integrity, TGF-beta 1 showed a striking ability to reduce the capacity of IFN-gamma to disrupt epithelial barrier function. Characterization studies demonstrated that this TGF-beta 1 effect was prolonged (e.g., days) after a single exposure, progressive over the dose range 0.1 to 2.5 ng/ml, reversible with increased concentrations of IFN-gamma, and more pronounced when TGF-beta 1 exposure was to basolateral rather than to apical epithelial membranes. Macromolecular (horseradish peroxidase) penetration of epithelium was not simultaneously altered by TGF-beta 1 and epithelial cellular injury was minimal as gauged by lactate dehydrogenase release. Additional studies using a human pathogen demonstrated that TGF-beta 1 delayed and decreased the barrier disruption caused by exposure to Cryptosporidium parvum. TGF-beta 1 may be the first of a new class of cytokines that maintains and/or enhances barrier function of human enterocytes, in part by countering the effect of a T cell cytokine.
Assuntos
Criptosporidiose/fisiopatologia , Cryptosporidium parvum/patogenicidade , Interferon gama/antagonistas & inibidores , Mucosa Intestinal/imunologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Linhagem Celular , Polaridade Celular/imunologia , Criptosporidiose/imunologia , Mucosa Intestinal/anatomia & histologia , Potenciais da Membrana/imunologia , Neuropeptídeos/fisiologiaRESUMO
A cluster-sampling, cross-sectional study was conducted for assessing the prevalence of Cryptosporidium infection in children less than 16 years of age from three villages, Dondian, Linshan, and Fuziyin, in rural Anhui in eastern China. Among 320 apparently healthy children less than 10 years of age from Dondian who had stool specimens collected, cryptosporidial oocysts were found in stools of three children from Dondian, and no positive specimens were found in 239 children studied from Linshan. In addition, a total of 610 serum samples from children in these three villages were tested for specific IgG antibody to Cryptosporidium with an enzyme-linked immunosorbent assay (ELISA) and the prevalence rates were 42.3%, 51.7%, and 57.5%, respectively, in Dondian, Linshan, and Fuziyin. Seroprevalence increased progressively with age. No detectable antibody was found in infants between two and six months of age, and seropositivity steadily increased after one year of age. Among 36 sera from adults 15-60 years of age without diarrheal illness in Huanglu villages of rural Chaohu, 50% (18 of 36) were positive. As expected, a good correlation was found in the specific IgG antibody between the paired serum specimens from 30 matched mother-neonates who showed transplacental transfer of IgG. However, little or no IgM antibody was seen in the neonates even though several mothers had a positive anticryptosporidial IgM enzyme-linked immunoassay result. Forty randomly selected serum samples from children less than four years of age in a similarly impoverished semiurban community in Fortaleza, Brazil, where the majority of households also have pit toilets and shared community water supplies and 172 serum samples from patients one month to 29 years of age admitted to the University of Virginia Hospital without diarrhea were also examined. In Fortaleza, almost all children acquired antibody by their second year of life, demonstrating the high prevalence of this infection. In rural Anhui, only about half the children were infected by 5-7 years of age. The overall prevalence rate (16.9%) of seropositivity among children and young adults in Virginia was much lower than in China and Brazil. These results indicate that cryptosporidial infection is ubiquitous, and is highly endemic in these impoverished communities. The difference between China and Brazil may reflect earlier weaning, hygiene practices, poorer water or sanitation, multiple siblings in family and geographic environment in Brazil.
Assuntos
Anticorpos Antiprotozoários/sangue , Criptosporidiose/epidemiologia , Cryptosporidium/imunologia , Adolescente , Adulto , Fatores Etários , Animais , Brasil/epidemiologia , Criança , Pré-Escolar , China/epidemiologia , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lactente , Masculino , Prevalência , Reprodutibilidade dos Testes , População RuralRESUMO
A monolayer of mature polarized colonic epithelial cells (T84) able to generate and maintain a barrier to macromolecular flow was used to study pathophysiologic events that occur on microvillus cell exposure to Cryptosporidium parvum. By 24-48 h, several life cycle forms were seen in parasitophorous vacuoles near the apical cell surface, along with a time- and oocyst dose-dependent reduction in epithelial barrier function. As few as 10(5) organisms constituted a successful infecting dose, and heat inactivation of organisms markedly reduced the monolayer barrier alteration. Horseradish peroxidase flux studies demonstrated a substantial increase in macromolecular permeability of the monolayer, and lactate dehydrogenase determinations indicated modest injury of the T84 epithelial cells on exposure to oocysts. Thus, disruption of the epithelial cell barrier, not just opening of transcellular channels for ion flow as reported previously, is responsible for the effects of C. parvum oocysts on intestinal epithelium.
Assuntos
Cryptosporidium parvum/crescimento & desenvolvimento , Intestinos/parasitologia , Animais , Bovinos , Linhagem Celular , Permeabilidade da Membrana Celular , Cryptosporidium parvum/ultraestrutura , Impedância Elétrica , Eletrofisiologia , Epitélio/parasitologia , Epitélio/patologia , Epitélio/fisiopatologia , Peroxidase do Rábano Silvestre/metabolismo , Intestinos/patologia , Intestinos/fisiopatologia , L-Lactato Desidrogenase/metabolismo , Microscopia Eletrônica , Fatores de TempoRESUMO
Mucosal and submucosal mast cell hyperplasia is a feature of the chronic inflammatory bowel diseases--ulcerative colitis and Crohn's disease. The mast cells are often seen to be degranulated in areas of active disease, suggesting that the inflammatory mediators released from these cells contribute to the pathophysiology of these disorders. We examined the hypothesis that epithelial cell-derived proteins, intestinal epithelial cell-associated components (ECAC), interact with the mast cells of patients with chronic inflammatory bowel disease to trigger the local release of mast cell mediators. Aliquots of human intestinal mucosal mast cell suspensions obtained from surgically resected specimens of colon or small intestine (ulcerative colitis, 12; Crohn's disease, 3; histologically normal controls, 8) were incubated with 1-100 micrograms/ml of colon or small bowel-derived murine ECAC or control kidney protein, or 1 microgram/ml goat anti-human IgE positive control for 30 min at 37 degrees C. Supernatants were analyzed in duplicate for histamine content by fluorometric assay. The median percent total histamine released by chronic inflammatory bowel disease mast cell suspensions to colonic epithelium-derived protein (ECAC-C) was 4% histamine (range 0-20%), such that the distribution of histamine release values in inflammatory bowel disease specimens was significantly different from the distribution of values in mast cells taken from normal mucosa (median 0%, P < 0.05). The median histamine release by all chronic inflammatory bowel disease specimens was also increased in response to the ECAC preparations derived from small bowel epithelium in that a third of the inflammatory bowel disease specimens showed greater than 10% histamine release to ECAC.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Colo/metabolismo , Liberação de Histamina , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Mastócitos/metabolismo , Proteínas/farmacologia , Animais , Separação Celular/métodos , Colo/citologia , Colo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida/métodos , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Liberação de Histamina/efeitos dos fármacos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Rim , Mastócitos/efeitos dos fármacos , Camundongos , Proteínas/análise , Proteínas/isolamento & purificação , Proteínas/metabolismoRESUMO
In mucosa-bearing organs with inherent lymphoid populations, classical modes for control of the immune response may be augmented by products of extrinsic sensory afferent nerve endings which arborize through the lamina propria compartment containing large numbers of T and B lymphocytes. Therefore, we sought to determine the role of neuropeptides (substance P, vasoactive intestinal peptide, and somatostatin) in immune response regulation by using a homogeneous line of T lymphocytes (AO40.1 hybrid), whose activation is driven by a specific Ag (OVA) and where the end point (IL-2 release) could not be contributed to by accessory or other cells. IL-2 was quantitated by the rate of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolism with the use of a murine CD4+ IL-2-dependent T lymphocyte line, and dose-response effects of each neuropeptide were examined over a broad concentration range (10(-14)-10(-6) M) encompassing that regarded as physiologic. Vasoactive intestinal peptide stimulated IL-2 release at low concentrations with a marked effect at 10(-14) M that gradually returned to control levels by 10(-7) M. Somatostatin was associated with a substantial augmentation of AO40.1 T lymphocyte IL-2 release at 10(-10) to 10(-8) M concentrations, whereas substance P demonstrated a stimulatory effect only at high concentrations (10(-9) to 10(-6) M). Concomitant [3H]thymidine uptake studies suggested that changes in cell proliferation or viability did not account for neuropeptide-induced effects in our system. With several exceptions, similar results were found with mitogen (Con A)-stimulated AO40.1 cells and human colonic lamina propria mononuclear cells. It was concluded that the three study neuropeptides, over a broad range of concentrations, have profound stimulatory (and occasionally inhibitory) effects upon the function of a cloned T lymphocyte hybrid cell responding to specific Ag and that these events may reflect those of Ag-driven mucosal T lymphocytes exposed to neuropeptides in vivo.
Assuntos
Adjuvantes Imunológicos/farmacologia , Neuropeptídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Concanavalina A/farmacologia , Relação Dose-Resposta a Droga , Humanos , Hibridomas/imunologia , Interleucina-2/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Linfócitos T/imunologia , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
The single cell-thick intestinal epithelium forms a crucial barrier between the host and environment, and is modeled in vitro by a monolayer of polarized, highly differentiated T84 epithelial cells impermeable to most macromolecules because of functional intercellular tight junctions. Absence of a permeability defect across the monolayer, either transcellular or paracellular, is indicated by development of a transepithelial electrical resistance of > or = 1000 ohm-cm2, reported to be markedly diminished by exposure to a T lymphocyte cytokine, IFN-gamma. We sought to define this phenomenon in four ways by determining its duration and reversibility; the uniqueness of type II (gamma) IFN as opposed to type I (alpha) IFN; the surface of the polarized columnar epithelium likely involved in responding to IFN-gamma; and whether a specific surface membrane receptor on the epithelial cell participates in the response. Using a special apparatus that allows differential cytokine exposure of monolayer surfaces, our data demonstrate 1) only the monolayer's basolateral surface is IFN-gamma responsive, whereas the apical (microvillous) surface is no; 2) the alteration in electrical resistance of epithelium is prolonged (5 days), even after a single (24 h) exposure to IFN-gamma, but nevertheless is reversible; 3) the effect is likely receptor-ligand mediated, because it can be partially blocked by IFN-gamma receptor-specific monoclonal Ig; 4) an alteration in tight junction function (a paracellular pathway) rather than cell necrosis or a transcellular pathway is responsible for IFN-gamma-induced monolayer dysfunction because permeability to a 44,000-Da macromolecule (horseradish peroxidase) did not increase, and intracytoplasmic T84 cell enzymes were not released into the media; and 5) the biologic phenomenon could not be induced by a species (alpha) of class I IFN, making IFN-gamma reasonably unique in this regard. Given the proximity; activation status, and capacity of T lymphocytes for cytokine production in mucosa, we suggest that IFN-gamma-induced changes in epithelial permeability may be a major cause of altered intestinal barrier function in vivo.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Interferon gama/farmacocinética , Mucosa Intestinal/metabolismo , Receptores de Interferon/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Ligação Competitiva , Células Cultivadas , Células Epiteliais , Epitélio/metabolismo , Humanos , Interferon gama/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Necrose , Ligação Proteica , Receptores de Interferon/fisiologia , Fatores de TempoRESUMO
Near-total pancreatectomy in a neonate presenting with persistent hypoglycemia offered an unusual opportunity to grow preparations enriched in beta-cells. Morphology, chromosomal analysis, and immunohistochemistry were used to characterize a subculture (Nesi B) that remained stable through passage 11. Insulin secretion of Nesi B was constitutive at 38-74 nU/microliters of medium/24 h, increasing modestly in the presence of isobutylmethylxanthine, an inhibitor of cyclic AMP phosphodiesterase. Endocrine cells, exclusive of those in islet regions, were widely distributed throughout the original tissue section, and immunostaining of the Nesi B subculture demonstrated well-differentiated heavily granulated insulin-positive cells, each with a normal modal number of chromosomes (46). Nesi B cell-associated macromolecules were isolated in 3 x 10(-3) ethylenedinitrilotetraacetate/phosphate-buffered saline and were found to be reactive with a heterologous immune serum elicited to a cloned rat pancreatic beta-cell line (RIN-5F). Western (immuno) blotting showed this immunoreactivity to reside primarily in a 95-kDa fraction of Nesi B-derived components. These results indicate that human nesidioblasts can be cultured for a sufficient number of passages to allow isolation and immunochemical characterization of pancreatic beta-cell macromolecules shared between rat and human and that may serve as organ-specific antigens for inflammatory disorders of the pancreas.