Mechanisms regulating IgA class-specific immunoglobulin production in murine gut-associated lymphoid tissues. II. Terminal differentiation of postswitch sIgA-bearing Peyer's patch B cells.

Our previous studies indicated that cloned T cells obtained from Peyer's patches (PP) (Lyt-1+, 2-, Ia+, and H-2K/D+) evoked immunoglobulin (Ig) class switching of PP B cells from sIgM to sIgA cells in vitro; however, these switch T cells could not in themselves provide optimal help for the differentiation of postswitch sIgA-bearing PP B cells to IgA-secreting cells. Thus, in the present report we described studies focused on mechanisms regulating terminal differentiation of the postswitch PP sIgA-bearing B cells. First, to explore the effect of T cell-derived B cell differentiation factor(s) (BCDF) and macrophage factor(s) (MF) on the terminal maturation of PP B cells, LPS-stimulated PP B cells were co-cultured for 7 d with cloned T cells in the presence or absence of the above factors. In the absence of PP cloned T cells the BCDF and MF had only a modest effect on IgA production, whereas in the presence of PP, but not spleen cloned T cells, IgA production was increased. Next, to investigate the effect of T cells derived from a gut-associated lymphoid tissue (GALT), mesenteric lymph nodes (MLN), as well as from spleen on terminal differentiation of postswitch sIgA PP B cells, LPS-driven PP B cells were precultured with the cloned T cells to induce a switch to sIgA, and subsequently cultured with MLN or spleen T cells or a Lyt-2+-depleted T cell subset in the presence of a T-dependent polyclonal mitogen, staphylococcal protein A. Alternatively, in the second culture period BCDF alone was added, instead of T cells and protein A. Here it was found that B cells pre-exposed to switch T cells from PP, but not spleen, were induced to produce greatly increased amounts of IgA in the presence of protein A and T cells or a Lyt-2+-depleted T cell subset as well as in the presence of BCDF alone. Furthermore, in the presence of BCDF alone many B cells expressed cytoplasmic IgA. These observations strongly support the view that the terminal differentiation of postswitch sIgA B cells is governed by helper T cells and macrophages, or factors derived from such cells. Such cells or factors do not affect preswitch B cells.

Mechanisms regulating IgA class-specific immunoglobulin production in murine gut-associated lymphoid tissues. I. T cells derived from Peyer's patches that switch sIgM B cells to sIgA B cells in vitro.

To explore mechanisms of T cell regulation governing mucosal IgA immune response, concanavalin A-induced cloned T cell lines from Peyer's patches (PP) as well as spleen were established. The cloned cell lines expressed Thy- 1.2(+), Lyt-l(+)2(-) and were radioresistant (1,500 rad). The capacity of the cloned T cells to regulate Ig synthesis was determined by measuring their effect on lipopolysaccharide (LPS)-induced polyclonal Ig synthesis by PP B cells. In initial studies Ig secreted by B cells was determined by double antibody radioimmunoassay. LPS in the absence of cloned T cells induced abundant amounts of IgM (average 8,860 ng/2 x 10(5) B cells) and IgG (average 1,190 ng/2 x 10(5) B cells), but little or no IgA. The addition of PP cloned T cells markedly suppressed production of IgM (88 percent at the highest T/B cell ratio, 4:1), but the addition of spleen cloned T cells suppressed only a little or not at all. IgG production was inhibited by both PP and spleen T clone cells (70 percent at the 4:1 T/B ratio), wheras IgA synthesis was enhanced by both clones, but only to a limited degree. In subsequent studies the expression of class-specific surface Ig (sIg) and cytoplasmic Ig (cIg) on/in unseparated PP B cells as well as Ig class- specific PP B cells and spleen B cells during culture with or without the cloned T cells was determined by immunofluorescence. The major findings were as follows: (a) Compared with unseparated B cell cultures and cultures of purified sIgM B cells derived from PP containing LPS alone, cultures containing LPS and PP cloned T cells showed a marked decrease in cIgM-, sIgG-, and cIgG-expressing cells that was accompanied by a striking increase in sIgA-bearing, but not cIgA-containing, cells. In contrast, unseparated B cell cultures and cultures of purified sIgM B cells derived from PP containing LPS and spleen cloned T cells did not show any increase in sIgA- bearing cells. (b) Compared with purified sIgG-bearing PP B cell cultures containing LPS alone, purified sIgG-bearing PP B cell cultures containing both LPS and PP cloned T cells showed no substantial change in sIgG- or cIgG- expressing cells, and no sIgA- or cIgA- expressing cells appeared. (c) Compared with sIgA-bearing PP B cell cultures containing LPS alone, purified sIgA-bearing PP B cell cultures containing both LPS and PP cloned T cells showed no increased proliferation, and cIgA cells did not occur. Cultures of purified sIgM B cells derived from spleen containing LPS and PP cloned T cells showed qualitatively similar changes. From these results we conclude that PP cloned T cells induced class-specific switching from sIgM- to sIgA- bearing B cells, whereas spleen cloned T cells lacked this property, although they may have induced an IgM {arrow} IgG or intersubclass IgG switch. These processes seem to be in part tissue dependent. Furthermore, the PP switch T cells appear to operate as true switch cells, which govern the pathway of DNA recombination events, rather than as classical helper cells, which act to expand already differentiated cells. Finally, these switch T cells probably account for the fact that PP are an important source of IgA B cells and also a major site of IgA heavy chain class switching during gut-associated mucosal B cell proliferation and differentation.

Isolated Pulmonary Cryptococcosis Confused with Lung Tumor 5 Years After Kidney Transplantation: A Case Report.

BACKGROUND: In transplant recipients, due to the use of immunosuppressive therapy, it is occasionally difficult to distinguish between an infection and malignancy, especially in the case of a lung lesion. Here, we report a case of isolated pulmonary cryptococcosis after kidney transplantation that was difficult to distinguish from a lung tumor. CASE REPORT: A 52-year-old man underwent a kidney transplant from his mother when he was 44 years old. Immunosuppression was maintained with tacrolimus, methylprednisolone, and mycophenolate mofetil. His post-transplant course was uneventful and serum creatinine levels were maintained. Five years post-transplantation, a non-contrast computed tomography (CT) examination revealed a nodule measuring 3 mm in diameter in the middle lobe of the right lung. The nodule gradually increased to 12 mm in 2 years. Positron emission tomography/CT examination showed a maximum standardized uptake value of 0.5 for the nodule. Biochemical examination revealed no elevation in total leucocyte count and C-reactive protein levels. However, tumor markers were elevated: serum carcinoembryonic antigen, 5.9 ng/mL; pro-gastrin-releasing peptide, 84.6 pg/mL. Furthermore, the serum cryptococcus antigen was negative. Therefore, thoracoscopic partial lung resection was performed. Pathologically, a number of spherical fungi from the necrotic substance of the tumor were confirmed positive by periodic acid-Schiff and Grocott-Gomori staining. The patient was therefore diagnosed with pulmonary cryptococcosis. Two years later, the patient is alive and has shown no evidence of recurrence. CONCLUSIONS: In lung nodules after kidney transplantation, even if serum cryptococcus antigen is not identified, it is necessary to keep in mind the possibility of pulmonary cryptococcosis.

Complementary use of peritoneal and hemodialysis: therapeutic synergies in the treatment of end-stage renal failure patients.

The simultaneous use of peritoneal dialysis (PD) and hemodialysis therapy has been studied both in established PD patients who are experiencing problems with their dialysis treatment that might otherwise prompt a change in modality, and in patients new to dialysis. The application of combination therapy allows in incident patients a partial separation of solute clearance and ultrafiltration, optimizing each modality within that overall delivery. This article discusses the published experience of combination treatment, and considers the possible benefits of such an approach.

Dicoumarol potentiates cisplatin-induced apoptosis mediated by c-Jun N-terminal kinase in p53 wild-type urogenital cancer cell lines.

3-3'-Methylene-bis [4-hydroxycoumarin] (dicoumarol), an inhibitor of NADPH:quinone oxidoreductase 1, has been reported to possess potential antineoplastic effects and the ability to abrogate p53 protein. In the present study, we investigated the cytotoxic effects of dicoumarol in combination with cisplatin (CDDP), using four bladder (RT112, 253J, J82 and UMUC3) and two prostate (LNCap and PC3) cancer cell lines. Single treatment with 100 microM dicoumarol suppressed cell proliferation but did not induce apoptosis at 24 h in all cell lines examined. On the other hand, pretreatment with dicoumarol enhanced cytotoxicity of CDDP in three cell lines with wild type of p53 (RT112, 253J and LNCap), but not in three other cell lines with mutant p53 or in RT112 stable transfectants with a dominant-negative mutant of p53. In RT112 and LNCap, CDDP induced p53 and p21 expression, while pretreatment of dicoumarol suppressed induction of p53/p21 and resulted in sequential activation of c-Jun N-terminal kinase (JNK) in a time-dependent manner. Furthermore, inhibition of JNK, using SP600125, completely suppressed activity of caspases and poly-(ADP-ribose) polymerase cleavage, leading to suppression of enhancement of CDDP-mediated apoptosis by dicoumarol. These results suggested that dicoumarol could enhance cytotoxicity of CDDP in urogenital cancer cells with wild-type p53 through the p53/p21/JNK pathways.

Effect of NOS inhibitor on cytokine and COX2 expression in rat pulpitis.

Various kinds of chemical mediators are synthesized in the course of pulpitis; thus, control of their production would assist in inducing a reduction in pulpal inflammation. We hypothesized that nitric oxide (NO) would be an important mediator of pulpal inflammation. Pulpal inflammation was induced by the application of LPS in rat incisor pulp, and inducible nitric oxide synthase (iNOS) expression was evaluated by reverse-transcription/polymerase chain-reaction and immunohistochemical staining. After LPS application, iNOS mRNA was first detected after 3 hrs, peaked at 6 hrs, and decreased thereafter. iNOS-positive cells were macrophages and neutrophils. An NOS inhibitor caused drastic decreases in the expression of pro-inflammatory cytokines and COX2 mRNA, which was highly induced in the LPS-induced pulpitis. These results indicate that NO synthesis is related to the initiation of mediator production, and that its down-regulation should contribute to the prevention of pro-inflammatory mediator synthesis.

Prevalence of Campylobacter pylori in esophagitis, gastritis, and duodenal disease.

The relationship between the presence of Campylobacter pylori and esophagitis was studied in patients undergoing paired biopsies of distal esophagus and gastric antrum during esophagogastroduodenoscopy. Biopsy specimens were examined for urease activity and for the presence of C pylori by culture and by histologic examination of hematoxylin-eosin- and Warthin-Starry-stained sections. Sixty-two patients were entered into the study. All esophageal biopsy specimens, regardless of histologic findings, were negative for the presence of C pylori by urease test, culture, and histologic examination. Of 35 patients with normal esophageal biopsy specimens, 11 (31%) had antral specimens that were positive for C pylori, while 11 (41%) of the 27 patients with esophagitis had antral specimens that were positive for the organism. Campylobacter pylori was detected in 14 (70%) of 20 patients with chronic gastritis, in 8 (67%) of 12 patients with endoscopically documented duodenal ulcers and erosions, but in only 3 (33%) of 9 patients with endoscopically defined duodenitis. We conclude that histologic esophagitis is not associated with increased prevalence of either gastric or esophageal C pylori. The well-described association of chronic gastritis and duodenal ulcers with C pylori was present in our study population.

The pathogenesis and therapeutic option of encapsulating peritoneal sclerosis.

Since the first peritoneal dialysis (PD) patients with encapsulating peritoneal sclerosis (EPS) were reported in 1980, EPS has been considered primarily a fatal complication. The incidence of EPS in PD patients has been reported to be from 0.7% to 7.3%, and the rate appears to be higher in patients receiving long-term treatment. Most data from Japan has shown an overall incidence of 2.5% with an evident negative effect of increasing duration of PD, which also augments mortality. Since EPS occurred after withdrawal from PD in more than half of the patients, strict monitoring is necessary when a long-term PD patient is withdrawn from PD. Maintaining patients on standard PD for more than 8 years using conventional solutions is associated with a substantial risk for development of EPS. Appropriate treatment according to the disease stage is most important in EPS treatment. Therefore, when examining a PD patient complaining of gastrointestinal symptoms, the possibility of EPS has to be kept in mind. Basic therapeutic tactics for EPS include an appropriate use of steroids. If the state of bowel obstruction persists, laparotomy and enterolysis should be performed to obtain complete cure. It is now recognized that EPS is not a fatal complication of PD.

T cell regulation of IgA immunoglobulin production in gut-associated lymphoid tissues.

To explore mechanisms of gut-mucosal IgA immune response, we have established Con A-induced cloned T cell lines originating from PP and spleen. These cloned cells expressed Thy-1.2+, Lyt-1+2-, Ia (I-A and I-E) and H-2 (K/D) surface antigens. Cloned T cells derived from PP were found to suppress LPS-induced IgM and IgG synthesis and secretion of co-cultured PP B cells; in addition, whereas the PP cloned T cells did not bring about IgA production, they did cause the appearance of large numbers of cells expressing sIgA. In contrast, cloned T cells derived from spleen had little or no effect on LPS-induced IgM synthesis and secretion by PP B cells; in addition, whereas they did suppress IgG production, they neither brought about IgA production nor the appearance of cells expressing sIgA. These studies provide evidence for the existence of a new type of T cell in PP, a switch T cell, which is able to induce B cells to undergo class-specific switches from IgM to IgA; the PP switch T cells appear to govern the pathway of DNA recombination (or RNA splicing) rather than cellular events resulting in terminal differentiation. Thus, these switch T cells are probably responsible for the fact that PP are a major source of mucosal IgA B cells. In additional studies, we show that post-switch IgA B cells, i.e. cells precultured with PP cloned T cells, have the capacity to undergo terminal differentiation into IgA producing plasma cells. provided they are exposed to helper T cells (uncloned) and an appropriate mitogenic stimulus (staphylococcal protein A). We can conclude, therefore, that the development of PP B cells into IgA-producing plasma cells in gut-associated lymphoid tissues (GALT) appears to require at least two steps: one which involves heavy chain switching to IgA and which is governed by IgA class-specific switch T cells in PP, and one which involves differentiation of post-switch B cells and which is governed by helper T cells in lymphoid tissues outside of PP (such as MLN and spleen).

Effects of phorbol myristate and ionomycin on in vitro growth of aged Peyer's patch T and B cells.

The proliferative responses of Peyer's patch (PP) T cells from aged BALB/c mice to concanavalin A (Con A) are considerably reduced, as compared to those of the young (P < 0.001). This reduced reactivity of aged T cells could be partly, but not entirely, corrected by interleukin 2 (IL-2) (P < 0.001). PP T cells from aged mice responded synergistically to a protein kinase C (PKC) activator, phorbol myristate acetate (PHA), plus a calcium ionophore, ionomycin, at much lower concentrations than to Con A (P < 0.001); however, the maximal proliferative response still remained nearly at 8/10th of the young (P < 0.01) and higher levels of PMA (but not of ionomycin) were required (P < 0.001). Addition of IL-2 restored the diminished response to the levels of the young T cells (P < 0.05), but that of Con A did not (P > 0.05). The proliferative responses of PP B cells to lipopolysaccharide (LPS) do not differ from those of the young (P > 0.05), but the spontaneous proliferation of aged (unstimulated) B cells is enhanced nearly twofold versus that of the young (P < 0.001). Like the PP T cells, PP B cells from aged mice also responded synergistically to PMA plus ionomycin but to a lesser degree than those of the young under the same stimulation (P < 0.01). Their maximal proliferation required higher levels of PMA, but not of ionomycin and was also diminished (P < 0.01), compared to that of the young. B cell stimulatory co-factors, IL-4 and IL-6, failed to affect the response of aged and young B cells to PMA plus ionomycin (P > 0.05), whereas LPS remediates the reduced response of aged B cells to PMA plus ionomycin. Thus, T and B cells from senescent PP demonstrate an impaired proliferative responsiveness via the Ca-dependent PKC pathway. A T cell mitogen and B cell stimulatory cytokines did not alter this activation pathway, once optimally stimulated. Whereas, T cell stimulatory cytokine IL-2 and B cell mitogen LPS could restore the age-associated decline of the corresponding lymphocyte subsets, T and B cells, in activation of the Ca-dependent pathway. The altered transmembrane signal transduction appears to be intrinsically defective in these aged PP T and B cells.

Impaired humoral immune responses to mycobacterial antigen in aged murine gut-associated lymphoid tissues.

Senescence-related alterations of local gut mucosal immune responses to enteric mycobacterial antigen (Ag) were examined. Both aged (greater than 24 months old) and young adult (4-5 months old) BALB/c mice were enterically immunized with crude Mycobacterium paratuberculosis (M. paratbc) protoplasmic Ag, and in vitro Ag- and class-specific immunoglobulin (g) production by lymphocytes from gut-associated lymphoid tissues (GALT) (Peyer's patches, PP; mesenteric lymph nodes, MLN) and non-GALT (spleen, SPN) were determined against semipurified M. paratbc Ag. Ag-specific spontaneous immunoglobulin production by aged B cells from both GALT and non-GALT was enhanced only to a minor extent. Similarly, the functional activity of the Ag-specific T (Th) (CD3+, CD4+) cell in both GALT and non-GALT was not profoundly affected by senescence (qualitative preservation). However, that of the suppressor T (Ts) (CD3+, CD8+) cell was considerably diminished (qualtative defect). Thus, oral tolerance (systemic immunologic hyporesponsiveness) to M. paratbc Ag in aged mice is impaired. These age-related changes, manifested as hyperreactive humoral responses to the enteric microbial Ag, are due, at least in part, to hyporeactivity of the Ts cell, resulting in relative hyperfunction of the Ag-specific Th cell, despite the quantitative defect of the latter cell.

In vivo immunologic intervention in age-related T cell defects in murine gut-associated lymphoid tissues by IL2.

Our recent studies indicate that the aging process impairs gut mucosal humoral immune responses to mycobacterial antigen (Ag), largely owing to defects in T cell function--in particular, that of suppressor T cells. To correct the age-associated Ag-specific T cell-mediated immune alteration recombinant IL2 (50,000 units/s.c./mouse/day) was administered for 3 weeks to the aged (greater than 24 months) mice (BALB/c), which were divided into 4 groups (Gr) [Gr. 1, fed intragastrically (i.g.) with saline; Gr. 2, immunized i.g. with Mycobacterium paratuberculosis (M. paratbc) protoplasmic Ag; Gr. 3, administered IL2 alone; Gr. 4, immunized i.g. with the Ag and given IL2]. In addition, young adult mice were also grouped and treated as the aged. First, we examined the effect of exogenous IL2 on Ag-specific immunoglobulin (Ig) production by gut-associated lymphoid tissues (GALT) (Peyer's patches, PP; mesenteric lymph nodes, MLN) and non-GALT (spleen, SPN) cells. Aged Gr. 4 (treated with both Ag and IL2) GALT and SPN unfractionated cells showed significantly reduced production of Ag-specific IgM, IgG, and IgA, as compared to aged Gr. 2 (treated with Ag alone) cells. Second, in co-culture experiments with aged T and B cells, aged GALT-derived CD8+ suppressor T (Ts)-depleted T cell subsets of Gr. 4 helped Ag-specific IgM and IgA production by GALT B cells, but to a slightly lesser extent, than those of the Gr. 2. GALT CD4+ T cells of aged Gr. 4 augmented IgM and IgA production by GALT B cells nearly to the levels of the corresponding cocultures of the Gr. 2. In contrast to aged Gr. 2 cocultures, GALT CD4+ plus CD8+ cells of aged Gr. 4 decreased IgM and IgA production to a considerable extent, and in those of SPN, IgG production was also diminished. The humoral immune responses of aged unprimed Gr. 1 (treated with saline) and Gr. 3 (treated with IL2 alone) GALT and SPN cells remained almost unchanged. Similarly, in all Gr. from young adult mice, oral tolerance was maintained regardless of IL2 administration. Third, together with the deletion experiments of the Ts cells, the results of the cross experiments, in which the young adult B and CD4+ Th cells and aged CD8+ Ts cells were cocultured, clearly support the view that the corrective mechanism of the humoral immune responses in aged GALT by exogenous IL2 is attributed to the partial recovery of the Ts cell functions.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging-associated intrinsic defects in IgA production by murine Peyer's patch B cells stimulated by autoreactive Peyer's patch T cell hybridoma-derived B cell stimulatory factors (BSF).

Immune functions deteriorate with age, primarily as a result of alterations in the number and subpopulations of T cells of the immune system. In contrast, the B cell component of the immune system is generally affected by senescence only to a minor extent. In the present report, we stimulated murine Peyer's patch (PP) B cells by nonspecific multifunctional B cell stimulatory factors (BSF) secreted by one of several autoreactive (self-major histocompatibility complex (MHC)-class II antigen-responsive) T cell hybridoma clones derived from PP of syngeneic mature adult mice, and then determined in vitro whether aging-associated intrinsic defects could be demonstrated in the proliferation of, and the synthesis and secretion of mucosal IgA by, the BSF-activated B cells. This approach could be a useful new in vitro method for assessing the effect of senescence on B cell Ig production, especially that of IgA, in the gut-associated lymphoid tissue (GALT). Aged PP B cells stimulated by the autoreactive PP T cell-derived BSF proliferated more (P less than 0.05), contained larger amounts of IgA (nearly 10 times) and also secreted considerably more IgA (nearly 4.5 times) than did mature adult PP B cells. However, the ratio of intracellular dimeric (d) IgA to total IgA in the aged B cell lysates was significantly reduced (by approx. 44%) as was also the secreted dIgA (by approximately 50%). The augumentation of not only the proliferation, but also the synthesis and secretion of IgA in vitro along with reduced dIgA/total IgA ratios of BSF-stimulated aged PP B cells appears to be due to aging-related intrinsic defects. Alterations in intracellular regulatory mechanisms of B cells, mediated by B cell receptors for autoreactive T cell-derived BSF, could be largely responsible for the observed polyclonal B cell hyperreactivity, associated with senescence.

Morphologic, biochemical and physiologic alterations in a case of idiopathic hypoalbuminemia (analbuminemia).

A patient with idiopathic hypoalbuminemia is described. A study of albumin kinetics demonstrated slowed albumin degradation suggesting low albumin synthesis. Morphologic observation of hepatocellular alterations suggested decreased protein synthesis. The intra- and extravascular space was low with an abnormally large postural shift of intravascular fluid into the extravascular compartment. Disturbances in the concentration of plasma lipids and of several plasma proteins were detected. The effect of albumin infusion on these physiologic and biochemical abnormalities suggests that most occurred as a secondary response to the hypoalbuminemic state. Evaluation of the patient's kindred revealed no members with hypoalbuminemia.

Potent and selective ET-A antagonists. 1. Syntheses and structure-activity relationships of N-(6-(2-(aryloxy)ethoxy)-4-pyrimidinyl)sulfonamide derivatives.

Modifications to the ET(A/B) mixed type compounds 1 (Ro. 46-2005) and 2 (bosentan) were performed. Introduction of a pyrimidine group into 1 resulted in a dramatic increase in affinity for the ET(A) receptor, and the subsequent optimization of substituents on the pyrimidine ring led us to the discovery of N-(6-(2-((5-bromo-2-pyrimidinyl)oxy)ethoxy)-5-(4-methylphenyl)-4-pyrimidinyl)-4-tert-butylbenzenesulfonamide (7k), which showed an extremely high affinity for the human cloned ET(A) receptor (K(i) = 0.0042 +/- 0.0038 nM) and an ET(A/B) receptor selectivity up to 29 000 (K(i) = 130 +/- 50 nM for the human cloned ET(B) receptor). The compound was designed on the hypothesis that the hydrogen atom of the hydroxyl group in 1 and 2 played a role not as a proton donor but as an acceptor in the possible hydrogen bonding with Tyr129. Since the incorporation of a pyrimidinyl group into the hydroxyethoxy side chain of the nonselective antagonist (1) dramatically enhanced both the ET(A) receptor affinity and selectivity, and since similar results were obtained from the benzene analogues, we put forward the hypothesis that a "pyrimidine binding pocket" might exist in the ET(A) receptor.

Potent and selective ET-A antagonists. 2. Discovery and evaluation of potent and water soluble N-(6-(2-(aryloxy)ethoxy)-4-pyrimidinyl)sulfonamide derivatives.

In the preceding article,(1) we outlined the discovery and structure-activity relationship of a potent and selective ET(A) receptor antagonist 1 and its related compounds. Metabolites of 1 having potent selective ET(A) receptor antagonist activity were identified. This study suggested the metabolic pathways of 1 were considerably affected by species. Consequently, structural modification of 1 intended to improve the complexity of the metabolic pathway, and water solubility was performed. The subsequent introduction of a hydroxyl group into the tert-butyl moiety of 1 led to the discovery of our new clinical candidate, 6b, which showed a higher water solubility, a uniform metabolic pathway among species, and very high affinity and selectivity for the human ET(A) receptor (K(i) for ET(A) receptor: 0.015 +/- 0.004 nM; for ET(B) receptor: 41 +/- 21 nM).

Na(+)-glucose cotransporter (SGLT) inhibitors as antidiabetic agents. 4. Synthesis and pharmacological properties of 4'-dehydroxyphlorizin derivatives substituted on the B ring.

In our studies of Na(+)-glucose cotransporter (SGLT) inhibitors as antidiabetic agents, a series of novel 4'-dehydroxyphlorizin derivatives substituted on the B ring was prepared and their effects on urinary glucose excretion were evaluated in rats. Introduction of only a small alkyl group at the 4'-position increased the activity, and 3-(benzo¿bfuran-5-yl)-2',6'-dihydroxy-4'-methylpropiophenone 2'-O-beta-D-glucopyranoside (4) showed the most potent effect. To overcome hydrolysis of compound 4 by beta-glucosidase in the digestive tract, the OH groups on the glucose moiety of compound 4 were modified. Three prodrugs (5, 42, and 55) were more potent than the parent compound 4 by oral administration, and finally 3-(benzo¿bfuran-5-yl)-2',6'-dihydroxy-4'-methylpropiophenone 2'-O-(6-O-methoxycarbonyl-beta-D-glucopyranoside) (5) was selected as a new promising candidate. Compound 5 was metabolized mainly by liver esterase to the active form (4), which was about 10 times more potent than 5 in inhibiting SGLT. In oral glucose tolerance test in db/db mice, compound 5 dose-dependently suppressed the elevation of glucose levels. Single administration of 5 reduced hyperglycemia concurrently with increase of glucose excretion into urine in diabetic KK-A(y) mice. Furthermore, compound 5 suppressed the elevation of blood glucose levels but did not lower it below the normal level even in fasted conditions in KK-A(y) mice. Additionally, long-term treatment with 5 dose-dependently reduced hyperglycemia and HbA1c in KK-A(y) mice. These pharmacological data strongly suggest that compound 5 has a therapeutic potential in the treatment of NIDDM.

Activation of calcium (Ca)-dependent protein kinase C in aged mesenteric lymph node T and B cells.

We studied the effects of aging on the activities and translocation of Ca-dependent protein kinase C (PKC) in resting mesenteric lymph node (MLN) T and B cells during the activation process induced by T and B cell mitogens and B cell-stimulatory interleukins, including IL-4, IL-5 and IL-6. The activation process in senescent, resting (high density (HD)) MLN T cells is impaired, when these cells are stimulated with T cell mitogen, Con A. The defect in activation is associated with a reduction in both the new production of inositol-1,4,5-triphosphate (IP3) (an indicator for the production of intracellular free Ca) and the induction of Ca-dependent PKC. In contrast, the activation of the aged B cells with LPS plus/minus interleukins (IL-4, IL-5 and IL-6) is not impaired, being at least associated with a Ca-independent pathway of PKC activation. The elevated IP3 content and total (cytosol plus membrane) PKC activity in both resting T and B cells from aged MLN along with the greater difference in T cells than in B cells suggest that the in vivo Go cell cycle status of these cells may differ from that of the young, involving more in T cells. Finally, the MLN and splenic T cell Ca-dependent and B cell Ca-independent PKC activation do not differ between both age groups.

Heat shock cellular stress on aged gut-associated lymphocytes; mRNA expression of inducible heat shock protein gene and protooncogenes.

To investigate the effects of senescence and cellular stress on gut mucosal lymphocytes, we used heat shock (HS) to determine the mRNA expression of an inducible HSP70-family gene (HSP68) and two nuclear protooncogenes (c-fos and c-myc) from gut-associated (GA) lymphocytes of aged (> or = 24 month) and young (3-4 month) mice. First, with temperatures of 37-44 degrees C, the maximal HS effect on expression of inducible HSP68 mRNA was at 41 degrees C, and that of c-fos and c-myc at 42 degrees C; with HS durations of 0-6 h at 42 degrees C, the maximal effect was at 1 or 2 h in the young GA lymphocytes. Second, without HS (37 degrees C), inducible HSP68 mRNA was not expressed in the GA lymphocytes of both age groups. The expression of c-fos and c-myc mRNA was greater in the aged GA lymphocytes than in the young, and c-myc mRNA was more highly expressed than c-fos mRNA in both age groups. Third, with HS (42 degrees C) for 30 min to 6 h, followed by recovery (37 degrees C) for up to 6 h, the peak expression of inducible HSP68 mRNA in the aged GA lymphocytes was reduced, but occurred at the same time as seen in the young (2 h) and declined faster during the recovery period. The peak expression of c-fos mRNA in the aged GA lymphocytes was earlier than that of the HSP68 mRNA in both age groups (1 h); the intensity of expression was decreased at all time points in the aged and declined more rapidly during the recovery period, whereas the peak expression of c-myc mRNA in the aged GA lymphocytes after HS was retarded in time (2 h) but also reduced in amount, when compared to that of the young, and during recovery the expression declined more rapidly in the aged than in the young. However, the recovery kinetics of the above three mRNAs remained unaltered in the lymphocytes from two different ages. To summarize our conclusions: First, prior to HS cellular stress, aged GA lymphocytes did not show mRNA expression of HSP68 but augmented mRNA expression of the nucleoprotooncogenes c-fos and c-myc. These findings suggest that senescent lymphocytes are more active metabolically in vivo than are young ones without significant activation of an inducible HSP70 family gene.(ABSTRACT TRUNCATED AT 400 WORDS)

EM changes and other toxic effects of firemaster BP-6 (polybrominated biphenyls) in the mouse.

Groups of Swiss ICR mice were fed 1000 ppm polybrominated biphenyls (FireMaster BP-6) in rodent chow for 4, 8, 11, and 14 days. Control groups were fed standard rodent chow without FireMaster BP-6. Animals were killed at the end of each feeding period and the livers examined by electron microscopy. EM changes noted were progressive increase in size of hepatocytes, a decrease in rough endoplasmic reticulum, a marked increase in smooth endoplasmic reticulum, mitochondrial degeneration, increased lysosomes, and a decrease in glycogen. In addition, there was increasing proliferation of microvilli in bile canaliculi with increasing feeding times. A group of mice fed 1000 ppm FireMaster BP-6 in rodent chow for 11 days had livers with a mean of 13.93% of total body weight as compared with 6.49% for the control group (P=0.02). Tissue distribution following ingestion of 100 ppm FireMaster BP-6 for 14 days was studied. Twelve weeks post-feeding, the tissue concentrations of hexabromobiphenyl in order of highest concentration to lowest were as follows: perithymic fat, perirenal fat, adrenal glands, thymus gland, liver and stomach.