Triamterene-β-cyclodextrin systems: Preparation,characterization and in vivo evaluation

The purpose of this research was to improve the solubility and therefore dissolution and bioavailability of triamterene, a poorly water soluble diuretic, by complexation with β-cyclodextrin. Triamterene has been reported to show low bioavailability after oral administration, with wide intersubject variation. This study presents the formulation of solid dispersions of triamterene with β-cyclodextrin—by cogrinding, kneading, and coevaporation, using low pH conditions—and their characterizations, evaluation of improvement in dissolution profiles, and in vivo advantage. Phase solubility studies indicated complex with possible stoichiometry of 1∶1 and a stability constant of 167.67M⁻¹. The solid dispersions were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, x-ray diffraction, and differential scanning calorimetry studies. The characterization studies confirmed inclusion of the phenyl ring of triamterene within the nonpolar cavity of β-cyclodextrin in the coevaporate. Remarkable improvement in in vitro drug release profiles in 0.1 N HCl and pH 6.8 phosphate buffer was observed with all dispersions, especially the coevaporate. The coevaporate, when administered orally in rats, also exhibited improved in vivo activity, as measured by net sodium ion excretion, as compared with triamterene powder. Thus, coevaporation of the drug and β-cyclodextrin from acidified alcohol provide the optimum condition for inclusion complexation to give a binary system with remarkable improvement in in vitro drug release profile and in vivo performance.     

A site in the fourth membrane-associated domain of the N-methyl-D-aspartate receptor regulates desensitization and ion channel gating

The N-methyl-d-aspartate (NMDA) receptor has four membrane-associated domains, three of which are membrane-spanning (M1, M3, and M4) and one of which is a re-entrant pore loop (M2). The M1-M3 domains have been demonstrated to influence the function of the ion channel, but a similar role for the M4 domain has not been reported. We have identified a methionine residue (Met(823)) in the M4 domain of the NR2A subunit that regulates desensitization and ion channel gating. A tryptophan substitution at this site did not alter the EC(50) for glycine or the peak NMDA EC(50) but decreased the steady-state NMDA EC(50) and markedly increased apparent desensitization, mean open time, and peak current density. Results of rapid solution exchange experiments revealed that changes in microscopic desensitization rates and closing rates could account for the changes in macroscopic desensitization, steady-state NMDA EC(50), and current density. Other amino acid substitutions at this site could increase or decrease the rate of desensitization and mean open time of the ion channel. Both mean open time and desensitization were dependent primarily upon the hydrophobic character of the amino acid at the position. These results demonstrate an important role for hydrophobic interactions at Met(823) in regulation of NMDA receptor function.     

Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells

CD4+ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-gamma or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-gamma producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-gamma alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-gamma- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-gamma and IL-4 producers.     

The role of polymerase eta in somatic hypermutation determined by analysis of mutations in a patient with xeroderma pigmentosum variant

To determine the possible role of polymerase eta (pol eta) in somatic hypermutation of B cells, a mutational analysis of 24 nonproductive rearrangements from a patient with xeroderma pigmentosum variant with a defect in pol eta was conducted. Although the mutational frequency of A and T bases decreased in WA (A/T, A) motifs, regardless of their RGYW (purine, G; pyrimidine, A/T) context, the overall mutational frequency of A or T bases was not affected. Moreover, the overall mutational frequency of the sequences examined was not decreased. There was an apparent increase in the number of insertions and deletions. The results are consistent with the conclusion that pol eta specifically targets WA motifs. However, its overall contribution to the somatic hypermutational process does not appear to be indispensable and in its absence other mechanisms maintain mutational activity.     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sj?gren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunoglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunoglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (V lambda 2E) and variable kappa chain genes (V kappa A27). Moreover, clonally related V(L) chain rearrangements were identified; namely, V kappa A27-J kappa 5 and V kappa A19-J kappa 2 in the parotid gland, and V lambda 1C-J lambda 3 in the parotid gland and the peripheral blood. V kappa and V lambda rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V(L) chain genes caused by selection and clonal expansion of B cells expressing particular V(L) genes. In addition, the data document an accumulation of B cells bearing mutated V(L) gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.     

Dendritic cells,chemokine receptors and autoimmune inflammatory diseases

Dendritic cells (DC) have been implicated in the induction of autoimmune diseases and have been identified in lesions associated with several autoimmune inflammatory diseases. Since DC are regarded as the professional antigen-presenting cell (APC) of the immune system and the only APC capable of activating na?ve T cells, they are likely to play a significant role in breaking tolerance of self-reactive lymphocytes and in supporting autoimmune responses in these diseases. A number of studies have revealed that small molecular weight chemotactic proteins known as chemokines are present within the autoimmune lesions and may contribute to the recruitment not only of DC populations, but also of immune cells such as T cells, B cells, neutrophils and monocytes into the site, and to the formation of organized lymphoid tissue structures within the target organ. The focus of this review will be a discussion of the role of chemokines in the recruitment of DC in human autoimmune inflammatory disorders, specifically the trafficking of DC into the inflammatory sites and the subsequent migration of differentiated DC from the inflammatory sites into the draining lymph nodes. Once DC are properly positioned within the lymph nodes, circulating antigen specific na?ve T cells can interact with DC and become activated, clonally expanded and stimulated to undergo differentiation into antigen-experienced memory T cells. Subsequent reactivation of memory T cells that enter the autoimmune lesions by DC present in the inflammatory lesion is thought to play a central role in tissue inflammation.     

Rheumatoid arthritis synovial stromal cells inhibit apoptosis and up-regulate Bcl-xL expression by B cells in a CD49/CD29-CD106-dependent mechanism

Inflammatory sites, such as rheumatoid arthritis (RA) synovial tissue, contain large numbers of activated B cells and plasma cells. However, the mechanisms maintaining B cell viability and promoting their differentiation are not known, but interactions with stromal cells may play a role. To examine this, purified human peripheral B cells were cultured with a stromal cell line (SCL) derived from RA synovial tissue, and the effects on apoptosis and expression of Bcl-2-related proteins were analyzed. As a control, B cells were also cultured with SCL from osteoarthritis synovium or skin fibroblasts. B cells cultured with medium alone underwent spontaneous apoptosis. However, B cells cultured with RA SCL cells exhibited less apoptosis and greater viability. Although SCL from osteoarthritis synovium and skin fibroblasts also rescued B cells from apoptosis, they were less effective than RA SCL. B cell expression of Bcl-xL was markedly increased by RA SCL in a contact-dependent manner, whereas B cell expression of Bcl-2 was unaffected. Protection of B cells from apoptosis and up-regulation of Bcl-xL by RA SCL were both blocked by mAbs to CD106 (VCAM-1), but not CD54 (ICAM-1). Furthermore, cross-linking of CD49d/CD29 (very late Ag-4) on the surface of B cells rescued them from apoptosis and up-regulated Bcl-xL expression. These results indicate that SCL derived from RA synovial tissue play a role in promoting B cell survival by inducing Bcl-xL expression and blocking B cell apoptosis in a CD49d/CD29-CD106-dependent manner.     

Cytokine, activation marker, and chemokine receptor expression by individual CD4(+) memory T cells in rheumatoid arthritis synovium

IL-10, IL-13, IFN-gamma, tumor necrosis factor (TNF)-alpha, LT-alpha, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4(+) memory T cells, whereas CD4(+) cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4(+) cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-alpha. A correlation between expression of IL-10 and CCR7, LT-alpha and CCR6, IFN-gamma and CCR5, and TRANCE and CXCR4 was also detected.     

Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus

CD4(+)CD25(+) T regulatory cells (Tregs) play an essential role in maintaining immunologic homeostasis and preventing autoimmunity. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear components. We hypothesized that altered function of CD4(+)CD25(high) Tregs might play a role in the breakdown of immunologic self-tolerance in patients with SLE. In this study, we report a significant decrease in the suppressive function of CD4(+)CD25(high) Tregs from peripheral blood of patients with active SLE as compared with normal donors and patients with inactive SLE. Notably, CD4(+)CD25(high) Tregs isolated from patients with active SLE expressed reduced levels of FoxP3 mRNA and protein and poorly suppressed the proliferation and cytokine secretion of CD4(+) effector T cells in vitro. In contrast, the expression of FoxP3 mRNA and protein and in vitro suppression of the proliferation of CD4(+) effector T cells by Tregs isolated from inactive SLE patients, was comparable to that of normal individuals. In vitro activation of CD4(+)CD25(high) Tregs from patients with active SLE increased FoxP3 mRNA and protein expression and restored their suppressive function. These data are the first to demonstrate a reversible defect in CD4(+)CD25(high) Treg function in patients with active SLE, and suggest that strategies to enhance the function of these cells might benefit patients with this autoimmune disease.