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Diabetic neuropathy is one severe complication of chronic diabetes. Though several theories exist, the pathophysiology of the disease is poorly understood. We have therefore developed an in vitro model to [...]

Most research on the effects of ageing on gut mucosal immunity has been performed using rodents. However, there are inherent difficulties in the extrapolation of rodent data to humans. This study was initiated to define age-related changes in the gastrointestinal (GI) mucosal immune response in non- human primates. Antibody responses were measured in young and old rhesus monkeys (Macaca mulatta) immunized intraduodenally with cholera toxin (Ctx)/cholera toxoid (Ctd). Antigen-specific immunoglobulin A (IgA) antibody levels were markedly lower while anti-Ctx IgG and IgM titres were higher in the intestinal lavage samples of old as compared to young animals. Total IgA concentrations in gut lavage were independent of age or immune status. Measurable titres of anti-Ctx IgA in the saliva of both age groups support the common mucosal immune hypothesis. Flow cytometric analysis was used to identify age-related shifts in the expression of cell surface antigens on peripheral blood lymphocytes. The relative number of both IgA + and Ctx + cells was dramatically reduced in the blood of old monkeys. Collectively, these data suggest that the GI mucosal immune response to Ctx is compromised in old rhesus macaques. The deficit in immune responsiveness, namely reduced anti-Ctx IgA antibody secretion into the intestinal lumen, may be a consequence of alterations in the process of maturation and homing of specific antibody-secreting B lymphocytes. [ABSTRACT FROM AUTHOR]

Although ageing is accompanied by systemic immunodeficiencies, the status of the mucosal immune system in the elderly remains unresolved. The gastrointestinal mucosal immune response was evaluated in young, mature and old male rats subjected to intra-intestinal immunization with cholera toxin (CTx). Five days following secondary immunization, the α-CTx-lgA titre in the bile of immunized rats was markedly reduced, i.e. the values measured in young rats were approximately five-fold higher than those of old animals. α-CTx-IgA levels in non-immunized rats were negligible and age-related shifts in other antibody titres (α-CFx IgG and IgM) were not significant. The antibody response to CTx was not reflected in the total IgA content of the samples. The number of α-CTx antibody-containing cells (ACCs) in the small intestinal lamina propria was significantly reduced in old immunized rats in comparison with the young or mature animals. These data suggest that ageing compromises both non-immune cell (antibody transport by hepatocytes) and immune cell (number of ACCs in the gut wall) functions in response to cholera toxin immunization in this animal model. [ABSTRACT FROM AUTHOR]

Non-transformed, rat intestinal epithelial cells (IEC-6), and human intestinal colonic carcinoma cells (CACO-2) have both been used to study processes of epithelial cell differentiation. However, only CACO-2 cells have been described as spontaneously expressing phenotypic changes of differentiation in culture. We report here that when IEC-6 cells are grown in post-confluent culture, they develop structural changes similar to those seen in cells induced to differentiate by culture on Englebreth-Holm-Swarm (EHS) extracellular matrix proteins. Correlated with this morphological change is loss of nuclear localization of c-myc protein and development of cell surface alkaline phosphatase (ALP) enzymatic activity. Messenger RNAs for liver and intestinal isoforms of ALP were expressed in both pre- and post-confluent cells. Inhibition of ALP activity in post-confluent cells by levamisole indicated the expressed ALP activity to be of the liver isoform. We suggest the expression of ALP activity, which occurs concomitantly with morphological alterations in post-confluent IEC-6 cells, represents increased expression and localization to the cell surface of the liver isoform of ALP. Cultured IEC-6 cells may provide a non-transformed, in vitro alternative to CACO-2 cells for study of epithelial cell differentiation., (Copyright 2003 Elsevier Science Ltd.)

Proliferation of the 7TD1 B cell hybridoma is dependent on the survival factor interleukin-6 (IL6). IL6 inhibits physiological cell death and allows expansion of populations of serum-stimulated cells. In this report, we demonstrate that cyclic AMP (cAMP)- and IL6-dependent signaling pathways can interact, controlling proliferation of 7TD1 cells through modulation of apoptosis. Cyclic AMP analogues inhibited proliferation, as well as other treatments that increased intracellular cAMP. The cAMP-induced inhibition could be reversed after 24 h by the removal of dibutyryl-cAMP from the culture medium and readdition of IL6. In the absence of IL6, cAMP induced a slow loss of viable cells. This decrease in viable cells in the presence of cAMP was accompanied by a marked increase in apoptosis. The increase in apoptotic cells after 48 h was preceded at 24 h by a parallel increase in DEVD-caspase activity after treatment with cell-permeable cAMP analogues. Increased DEVD-caspase activity and subsequent apoptosis could both be blocked by the addition of IL6. These coregulating actions may represent a cross-talk signaling mechanism modulating cytokine activation of cellular proliferation and survival., (Copyright 2001 Academic Press.)

There is considerable evidence that the mucosal or secretory immune response in the gastrointestinal tract is compromised by aging. The generation of a mucosal immune response is an extremely complex process that involves antigenic stimulation of a specific subpopulation of immunologically competent cells in the Peyer's patches, differentiation and migration of these cells to the small intestinal lamina propria, initiation and regulation of local antibody production in the intestinal wall, and mucosal epithelial cell receptor-mediated transport of antibodies to the intestinal lumen. Available data suggest that gastrointestinal mucosal immunosenescence reflects deficits in: (1) the differentiation and/or migration (homing) of immunoglobulin A immunoblasts to the intestinal lamina propria, and (2) the initiation and/or regulation of local antibody production. The significant age-related increases in the incidence and severity of gastrointestinal infectious diseases, coupled with the potential for immunopharmacologic manipulation of the mucosal immune compartment, substantiate the merit of studies designed to resolve the etiology of mucosal immunodeficiency in the elderly.

A solid-phase radioimmunoassay for cAMP in tissues, body fluids, and cultured cells has been developed using 125I-2'-O-monosuccinyl adenosine-3':5'-cyclic monophosphate tyrosyl methyl ester and High Binding EIA microtiter strips coated overnight at 4 degrees C with a rabbit or sheep polyclonal anti-cAMP antibody. After washing and blocking of wells, samples or standards were added, followed by the addition of radiolabel. Bound 125I-cAMP was separated from free by washing with phosphate buffer containing Tween 20. Bound 125I-cAMP was inversely proportional to cAMP in samples or standards. Cyclic AMP content of unknowns was calculated from a standard curve run concurrently with each assay. Both antibodies showed sensitivity of approximately 1 fmol, an assay range between 15 and 1,000 fmol, a maximum displacement ratio of up to 11-12, and no cross-reactivity with other cyclic nucleotides. Recoveries were 86.5%-106.8%, intraassay coefficients of variation were 2.4%-6.0%, and interassay coefficients of variation were 7.4%-10.2% for both antibodies. The cAMP content of tissues (brain > heart > kidney, liver > muscle) from rat, rabbit, and guinea pig, cultured rat lymphocytes from three lymphoid tissues, and human serum and urine were tested. This solid-phase RIA is a reliable, sensitive, rapid, and relatively inexpensive method for determination of cAMP.

The Fischer rat shows an age-related loss of both hepatic blood to bile transport and secretory component-specific binding sites for polymeric immunoglobulin (Ig) A. This age-related loss of hepatic IgA receptor function is also shown by cultured hepatocytes. It is reported here that compared with young cells, binding and uptake of 125I-polymeric IgA by cultured hepatocytes was markedly reduced in cells from senescent animals. In addition, cells from old animals showed markedly diminished secretion of secretory component determined by enzyme-linked immunosorbent assay and expression of polymeric immunoglobulin receptor determined by incorporation of 35S-labeled amino acid and fluorography. It is suggested that the age-related decrease in IgA receptor-mediated transport from serum to bile results, at least in part, from decreased expression and secretion of total hepatic secretory component.

The hepatobiliary transport of asialoorosomucoid (ASOR) was examined in aging male Fischer 344 rats. The time course of transport of 125I-ASOR from blood to bile was identical in both senescent and young adult rats. Peak secretion occurred at approximately 35 minutes after injection via the femoral vein. Total secretion of radiolabeled ASOR (3.6% of injected dose), bile secretion and rate of secretion of radiolabeled ligand (approximately 2% of administered dose/hr/gm bile/liver) were not significantly different for the two age groups. Determination of the binding capacity for 125I-ASOR with liver plasma membrane-enriched preparations showed the membranes from old animals capable of binding approximately 50% less radiolabeled ligand as the young adult animals. Analysis of the distribution of 125I-ASOR autoradiographic grains along the liver lobule indicated extensive uptake of ligand in Zone 2 and 3 cells in senescent animals, whereas uptake in young rats was essentially limited to Zone 1 parenchymal cells. These results indicate that, contrary to the age-related loss of hepatic receptors for dimeric IgA and the concomitant reduction in hepatobiliary secretion of IgA, loss of ASOR binding capacity on liver plasma membranes from old animals is not reflected in diminished hepatobiliary secretion of ASOR. The loss of ASOR binding capacity is offset by the recruitment of Zone 2 and 3 hepatocytes along the liver lobule. This result suggests that hepatic metabolism and hepatobiliary secretion of macromolecules which exhibit a lobular gradient of uptake (e.g. ASOR) will be relatively less affected by loss of receptors compared to ligands which do not display such a gradient (e.g. IgA).

The source and significance of immunoglobulin A in bile remains controversial. In the rat, and several other species, immunoglobulin A is transported through hepatocytes by a specific receptor, secretory component. In humans, immunohistochemical methods have indicated a distinct lack of receptors for immunoglobulin A on hepatocytes. Binding assays with 125I-immunoglobulin A and membranes from hepatocytes and intestinal cells of the rat display secretory component-dependent binding. Primate intestinal cells also show secretory component-specific binding of immunoglobulin A. Primate liver, on the other hand, does not show immunoglobulin A binding mediated by the polymeric immunoglobulin receptor.

Although ageing is accompanied by systemic immunodeficiencies, the status of the mucosal immune system in the elderly remains unresolved. The gastrointestinal mucosal immune response was evaluated in young, mature and old male rats subjected to intra-intestinal immunization with cholera toxin (CTx). Five days following secondary immunization, the alpha-CTx-IgA titre in the bile of immunized rats was markedly reduced, i.e. the values measured in young rats were approximately five-fold higher than those of old animals. alpha-CTx-IgA levels in non-immunized rats were negligible and age-related shifts in other antibody titres (alpha-CTx IgG and IgM) were not significant. The antibody response to CTx was not reflected in the total IgA content of the samples. The number of alpha-CTx antibody-containing cells (ACCs) in the small intestinal lamina propria was significantly reduced in old immunized rats in comparison with the young or mature animals. These data suggest that ageing compromises both non-immune cell (antibody transport by hepatocytes) and immune cell (number of ACCs in the gut wall) functions in response to cholera toxin immunization in this animal model.

In the rat, asialoorosomucoid and rat dimeric immunoglobulin A are both taken up by hepatocytes via receptor-mediated endocytosis. The fate of these two proteins, however, differs significantly. Rat dimeric IgA is taken up into smooth vesicles, transported to the bile canaliculus and secreted intact into the bile, whereas asialoglycoproteins are internalized via coated vesicles and transported to lysosomes for degradation. Recently, several studies both in the rat and in cultured human hepatoma cells have suggested that the receptor for asialoglycoproteins may play a role in the hepatic uptake and processing of human polymeric IgA. Using receptor-binding techniques, we have provided quantitative data for the competition of human monomeric, polymeric and secretory IgA with asialoorosomucoid for its receptor on liver plasma membrane preparations from rat, monkey and man. Some IgA molecules required desialylation with neuraminidase to enhance markedly their efficacy for asialoorosomucoid inhibition. Quantitatively, human IgA molecules showed an affinity for the ASOR receptor similar to that for asialoceruloplasmin. Rat dimeric IgA does not compete for this binding site. We conclude that human IgA can compete with ligands for the asialoglycoprotein receptor of rat, monkey and human liver. This receptor may provide an alternative pathway for the hepatic processing of IgA and IgA immune complexes when secretory component-mediated uptake is not available as in the monkey and man, particularly under pathological conditions where serum IgA concentrations accumulate to abnormally high levels.

The receptor for polymeric immunoglobulins is responsible for the transport of immunoglobulin A (IgA) through epithelial cells and its subsequent delivery to mucosal surfaces. We have extended our previous studies of the IgA receptor in the liver of the aging Fischer rat to include the small intestine. Basolateral membrane-enriched fractions prepared from rat small intestinal enterocytes exhibit a single binding site for dimeric IgA. This receptor is specific for molecules that interact with rat secretory component, e.g., rat dimeric IgA and IgM and human polymeric IgA but not human monomeric IgA or rat secretory IgA. Inhibition of binding by rabbit-antirat secretory component also indicated that binding is specific for secretory component. Both liver and intestinal membranes showed virtually identical binding specificity. Membranes from crypt cells show increased IgA binding (320 fmol bound per milligram protein) compared with villous cells (105 fmol bound per milligram protein); however, other than increased binding, crypt cells show the same binding characteristics as villous cells. In contrast to our previous findings, in which liver plasma membranes from old rats showed a four-fold decrease in IgA binding compared with young adult rats, membrane fractions from rat enterocytes showed no alterations in dimeric IgA binding with increased age.