Antihypertensive effect of a fixed-dose combination of losartan/hydrochlorothiazide in patients with uncontrolled hypertension: a multicenter study.

BACKGROUND: Achieving adequate blood pressure (BP) control often requires more than one antihypertensive agent. The purpose of this study was to determine whether a fixed-dose formulation of losartan (LOS) plus hydrochlorothiazide (HCTZ) (LOS/HCTZ) is effective in achieving a greater BP lowering in patients with uncontrolled hypertension. METHODS: The study was a prospective, multicenter, observational trial exploring the antihypertensive effect of a single tablet of LOS 50 mg/HCTZ 12.5 mg. A total of 228 patients whose BP had previously been treated with more than one antihypertensive agents without having achieved BP goal below 130/80 mmHg enrolled in the study. RESULTS: A significant decrease in systolic and diastolic BP was observed in both clinic and home measurement after switching from the previous treatment to LOS/HCTZ. There was a significant decrease in both B-type natriuretic peptide (BNP) and urinary albumin creatinine (Cr) excretion ratio (ACR), especially in patients with elevated values. In contrast, there was a significant increase in serum Cr concentration in conjunction with a decrease in estimated glomerular filtration rate (eGFR). Overall serum uric acid (UA) concentration increased, whereas in patients with hyperuricemia there was a significant reduction in this value. CONCLUSION: Switching to LOS/HCTZ provides a greater reduction in clinic and home BP in patients with uncontrolled hypertension. This combination therapy may lead to cardio-, reno protection and improve UA metabolism.

Expression of human and mouse genes encoding polkappa: testis-specific developmental regulation and AhR-dependent inducible transcription.

BACKGROUND: Human polkappa is a newly identified low-fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene-adduct in an error-prone manner, it bypasses benzo[a]pyrene-N2-dG lesions in a mostly error-free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand-activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin. RESULTS: We examined promoter structures of the human POLK and mouse Polk genes to study how their expressions are regulated. The mouse Polk gene is developmentally regulated in testis and utilizes two transcription start sites during spermatogenesis, while it utilizes only one site in tissues other than testis. Both of the mouse Polk and the human POLK genes have two AhR-binding sites in the promoter regions and the expression of the mouse Polk gene is indeed enhanced upon AhR-activation. CONCLUSIONS: The AhR activation increases expression of the mouse Polk gene and probably the human POLK gene, the product of which bypasses benzo[a]pyrene-N2-dG lesions in a mostly accurate manner. Thus, polkappa seems to function to reduce mutagenesis at benzo[a]pyrene-adducts, although it may also have a role related to spermatogenesis.

Use of secondarily revised VH genes in IgE antibodies produced in mice infected with the nematode Nippostrongylus brasiliensis.

Although a high level of IgE is produced after primary infection with Nippostrongylus brasiliensis (Nb), most of the IgE antibodies (Abs) are not specific to the worm. Analyses with Western blotting and enzyme-linked immunosorbent assay (ELISA) revealed that the IgE Abs from Nb-infected BALB/c mice did not show reactivity with Nb-derived excretory-secretory proteins (NES) and antigens present in the cell-free extracts of the worm. Monoclonal IgE Abs obtained from the Nb-infected mice were not reactive with these Nb antigen either. To characterize Nb-induced IgE response, we used (QM x C57BL/6)F1 (QBF1) mice that bear the knock-in 17.2.25 VHDJH segment (VHT) encoding a VH region specific to 4-hydroxy-3-nitrophenylacetyl hapten, and express VHT-encoded antigen receptors on 80-85% of their B cells. Consistent with the frequency of VHT-positive B cells, more than 80% of IgE Abs induced in QBF1 B cells that were cultured with LPS plus IL-4 were found to bear VHT-encoded H chains. In contrast, when QBF1 mice were infected with Nb, less than 10% of Nb-induced IgE Abs were found to use VHT. The QBF1-derived IgE did not react with Nb antigens either. Taken together, data suggest that Nb-induced IgE response in mice is not merely the result of polyclonal activation of B cells, but may involve a mechanism that revises Ig genes secondarily.

Restoration of immunocyte functions by thymosin alpha1 in cyclophosphamide-induced immunodeficient mice.

Thymosin alpha1 (Talpha1) is an oligopeptide hormone originally isolated from the thymus gland, and has been reported to have stimulating effects on the differentiation of T cells and NK cells. These immunostimulating properties have been considered to be useful for improving immune disorders associated with various diseases including cancer, AIDS and hepatitis. Here, we characterized immunostimulating properties of Talpha1 in experimental immunodeficiency of mice that was induced by the administration of cyclophosphamide (CY). Repeated injection of 30-300 microg/kg/day of Talpha1 after CY-treatment significantly accelerated the restoration of the reduced number of CD4+CD8+ T cells in the thymus. Talpha1 administration was effective in restoring the suppressed activities of helper T cells and cytotoxic T cells in CY-treated mice. Talpha1 also had stimulating effects on reduced activity of lymphokine-activated killer cells in CY-treated mice. These results indicate that Talpha1 is stimulatory for both humoral and cellular immune responses, thus providing the immunological basis for the clinical benefit of this compound.

[Comparative antibacterial activity of carbapenems against P. aeruginosa (1)].

Comparative antibacterial activity of imipenem (IPM), panipenem (PAPM), meropenem (MEPM) and biapenem (BIPM) was determined against 288 clinical isolates of P. aeruginosa collected from various hospitals in 2000. The order of activity by comparison of MIC50/MIC80/MIC90 was: MEPM (1/4/8 micrograms/ml) > BIPM (1/4/16 micrograms/ml) > IPM (2/4/16 micrograms/ml) > PAPM (8/16/32 micrograms/ml). Moreover, the order of activity against 75 strains of P. aeruginosa (MIC of CAZ, AZT was > or = 16 micrograms/ml and MIC of IPM, MEPM was < or = 8 micrograms/ml) by comparison of MIC50/MIC80/MIC90 was: BIPM (1/2/8 micrograms/ml) > or = MEPM (1/4/8 micrograms/ml) > or = IPM (2/2/8 micrograms/ml) > PAPM (8/16/16 micrograms/ml). Judging from both correlation between the MICs of carbapenems and relationship between class C beta-lactamase activity and drug susceptibility of carbapenems, it becomes apparent that carbapenems, especially BIPM and MEPM will be useful for treatment of antipseudomonal cephem resistant pseudomonas infection.

Prevention of collagen-induced arthritis in DBA/1 mice by oral administration of AZ-9, a bacterial polysaccharide from Klebsiella oxytoca.

Collagen-induced arthritis (CIA) is an excellent model of rheumatoid arthritis (RA) in humans that is induced in DBA/1 mice immunized with bovine type II collagen (CII). Here, we report that the induction of CIA was effectively suppressed by oral administration of AZ-9, a purified polysaccharide with the average molecular weight of approximately 200 kDa that was produced by a soil bacterium, Klebsiella oxytoca. When AZ-9 was administered at 125-250 mg/kg/day orally for 9 consecutive days after immunization with CII followed by its administration every 3 days, resulted in a marked reduction of the incidence and the severity of CIA. The serum level of anti-CII IgG2a and the production of IFN-gamma and IL-12 in the draining lymph node (LN) cells were significantly lower in AZ-9-administered mice than the untreated control. These findings suggest that orally administered AZ-9 suppressed CIA through attenuating a Th1-type response to CII. AZ-9 could be fragmented into smaller molecules (3-4 kDa) without losing its suppressive activity.

In vitro plasma protein binding and cellular uptake of ATX-S10(Na), a hydrophilic chlorin photosensitizer.

ATX-S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second-generation photosensitizer for photodynamic therapy (PDT) for cancer treatment. In this study, we examined plasma protein binding, cellular uptake and subcellular targets of ATX-S10(Na) in vitro. Protein binding ratios of 50 microg / ml ATX-S10(Na) in rat, dog and human plasma were 73.0%, 87.2% and 97.7%, respectively. Gel filtration chromatography revealed that 1 mg / ml ATX-S10(Na) bound mainly to high-density lipoprotein (HDL) and serum albumin at the protein concentration of 0.4%, with binding ratios of 46% and 36%, respectively. The free form of ATX-S10(Na) was mostly incorporated into T.Tn cells, and its cellular uptake was partially but significantly inhibited by endocytosis inhibitors such as phenylarsine oxide, chloroquine, monensin and phenylglyoxal, and by chilling the cells to 4 degrees C. However, ouabain, harmaline, sodium cyanide, probenecid and aspartic acid did not influence the uptake of ATX-S10(Na), suggesting that cellular uptake of ATX-S10(Na) was not related to sodium-potassium pump activity, sodium-dependent transporter activity, mitochondrial oxidative respiration, organic anion transporter activity or aspartic acid transporter activity. By fluorescence microscopy, lysosomal localization of ATX-S10(Na) was observed in T.Tn cells. However, electron microscopic observation revealed that many subcellular organelles such as mitochondria, endoplasmic reticulum, ribosomes, Golgi complex and plasma membrane were damaged by PDT using 25 microg / ml ATX-S10(Na) soon after laser irradiation at 50 J / cm(2), and tumor necrosis was rapidly induced. This result indicated that ATX-S10(Na) was widely distributed within the cell.

Photodynamic therapy for experimental tumors using ATX-S10(Na), a hydrophilic chlorin photosensitizer, and diode laser.

ATX-S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second-generation photosensitizer for use in photodynamic therapy (PDT) for cancer treatment. The effectiveness of PDT using ATX-S10(Na) and a diode laser for experimental tumors was evaluated in vitro and in vivo. In-vitro PDT using ATX-S10(Na) and the diode laser showed drug concentration-, laser dose- and drug exposure time-dependent cytotoxicity to various human and mouse tumor cell lines. In Meth-A sarcoma-implanted mice, optimal PDT conditions were found where tumors were completely eliminated without any toxicity. Against human tumor xenografts in nude mice, the combined use of 5 mg / kg ATX-S10(Na) and 200 J / cm(2) laser irradiation 3 h after ATX-S10(Na) administration showed excellent anti-tumor activity, and its efficacy was almost the same as that of PDT using 20 mg / kg porfimer sodium and a 100 J / cm(2) excimer dye laser 48 h after porfimer sodium injection. Microscopic observation of tumor tissues revealed that PDT using ATX-S10(Na) and the diode laser induced congestion, thrombus and degeneration of endothelial cells in tumor vessels, indicating that a vascular shutdown effect plays an important role in the anti-tumor activity of PDT using ATX-S10(Na) and the diode laser.

Selective augmenting effects of nitric oxide on antigen-specific IgE response in mice.

Here, we report the enhancing effects of nitric oxide (NO) on an IgE antibody response in mice. Anti-trinitrophenyl (TNP) IgE production induced in vitro in TNP keyhole limpet hemocyanin (KLH)-primed spleen cells was inhibited by approximately 70% when an NO synthase (NOS) inhibitor, L-N(G)-monomethyl-L-arginine, was added at 10(-7)-10(-6) M to the lymphocyte culture. On the other hand, addition of NO-generating agents to the culture resulted in a marked enhancement of the IgE production. In contrast, anti-TNP IgM and IgG1 responses were affected only marginally when the IgE production was either suppressed or augmented by these agents. NO did not directly augment IgE class switching in normal B cells stimulated with lipopolysaccharide and interleukin (IL)-4. NO-mediated augmentation of the IgE response is considered to be of a physiological significance because administration of aminoguanidine (AG), an inhibitor of inducible NOS, to immunized mice resulted in a preferential suppression of anti-TNP IgE production in vivo. This may be explained by the observation that AG-administration increased interferon-gamma expression without changing that of IL-4 in the immunized mice. Taken together, these observations suggest a pathophysiological role of NO in the development of IgE-mediated allergic diseases.

In vitro antibacterial activity of LJC 11,036, an active metabolite of L-084, a new oral carbapenem antibiotic with potent antipneumococcal activity.

LJC 11,036 is the active metabolite of L-084, a novel oral carbapenem that exhibits potent broad-spectrum activity. Antibacterial activities of LJC 11,036 against clinical isolates from respiratory infections, such as Streptococcus pneumoniae (n = 52), Streptococcus pyogenes (n = 19), Haemophilus influenzae (n = 50), Klebsiella pneumoniae (n = 53), and Moraxella catarrhalis (n = 53), and from urinary-tract infections, such as Escherichia coli (n = 53) (MICs at which 90% of the isolates were inhibited [MIC(90)s], 0.1,

IL-4-dependent IgE class switching in an anti-trinitrophenyl B-cell hybridoma after engagement of antigen receptors.

A B-cell hybridoma, TP67.21 that expresses surface anti-trinitrophenyl (TNP) IgM but does not secrete the antibody spontaneously has been reported to differentiate into anti-TNP IgM-secreting cells in response to lipopolysaccharide or engagement of surface IgM. Here, we report isolation and characterization of a subclone, TP67.21E (TP.E) that undergoes isotype switching to IgE in an interleukin (IL)-4-dependent manner. TP.E cells secreted anti-TNP IgE depending on exogenous IL-4 when they were cultured with an anti-IgM antibody for 6-8 days. 8-Mercaptoguanosine, which has been shown to enhance IgE class switching in murine splenic B-cells further augmented the IgE response in TP.E cells. Immunofluorescence microscopy revealed that approximately 1.2% of the cultured cells became positive for intracellular IgE after the stimulation culture. The germline epsilon transcripts were expressed transiently on days 2-4 of the culture, while expression of the productive epsilon transcripts was induced 5 days after the start of the culture, thus suggesting that IgE class switching occurred in TP.E cells under these conditions.

Expression of recombination activating genes in germinal center B cells: involvement of interleukin 7 (IL-7) and the IL-7 receptor.

Mouse germinal center (GC) B cells have been shown to undergo secondary V(D)J (V, variable; D, diversity; J, joining) recombination (receptor editing) mediated by the reexpressed products of recombination activating gene (RAG)-1 and RAG-2. We show here that interleukin (IL)-7 as well as IL-4 was effective in inducing functional RAG products in mouse IgD+ B cells activated via CD40 in vitro. Blocking of the IL-7 receptor (IL-7R) by injecting an anti- IL-7R monoclonal antibody resulted in a marked suppression of the reexpression of RAG-2 and subsequent V(D)J recombination in the draining lymph node of immunized mice, whereas RAG-2 expression was not impaired in immunized IL-4-deficient mice. Further, these peripheral B cells activated in vitro or in vivo were found to express IL-7R. These findings indicate a novel role for IL-7 and IL-7R in inducing receptor editing in GC B cells.

Expression and function of recombination activating genes in mature B cells.

Recombination activating genes, RAG-1 and RAG-2, encode proteins that catalyze the rearrangement of immunoglobulin genes in B cells and T cell receptor genes in T cells to generate the diversity of these important recognition molecules in immune system. It has been believed that these gene rearrangements occur exclusively in premature stages of B and T lymphocytes, consistent with the observation that RAG expression is downregulated in mature lymphocytes. However, recent studies have revealed that even mature B cells in peripheral lymphoid tissues can reexpress RAG-1 and RAG-2 proteins following immunization. Strikingly, RAG-expressing B cells are localized in the germinal centers (GCs) of secondary lymphoid tissues in which somatic hypermutations, isotype switching, and affinity maturation of antibodies take place. Recently, it has been shown that RAG proteins thus induced are functional and can mediate the secondary rearrangement of Ig genes (receptor editing) at mature stages of B cells. Evidence is accumulating suggesting that GCs are regarded as a primary lymphoid tissue. In the present review, we briefly summarize recent advances in the expression and the characterization of RAG proteins and discuss their possible role in mature B cells in relation to the diversification and the selection of B cell repertoire in GCs.

Characterization of ASK mice, a strain highly sensitive to anaphylactic shock.

A mouse strain named ASK that was originally isolated from El (epilepsy) mice has been shown to be highly sensitive to anaphylactic shock. Here, we characterized the bases of the sensitivity of ASK mice in comparison with the parental strain, El. More than 90% of ASK mice, but not El mice that had been sensitized either actively or passively, died within 1 h following an antigen challenge. The anaphylactic death was effectively blocked by diphenhydramine. Plasma histamine levels increased by 30-50 fold in ASK after the antigen challenge, but only a 2-3-fold increase was observed in El mice. All (El x ASK) F1 mice, either male or female, showed an ASK-like phenotype, suggesting that the impaired plasma histamine response in El mice is due to some recessive mutation(s). Consistent with the plasma histamine responses, cultured mast cells derived from El bone marrow showed impaired potency to degranulate in response to surface IgE engagement, in contrast to ASK mast cells which undergo normal degranulation. Another characteristic feature of ASK mice is their sensitivity to histamine, since 75% of the mice were killed by the subcutaneous administration of 100-200 mg/kg histamine, while C3H and BALB/c mice were resistant to even 600 mg/kg histamine. Taken together, the major bases of the susceptibility to anaphylactic shock in ASK mice are thought to be the enhanced sensitivity to histamine and the recovered degranulation machinery in mast cells that is impaired in El mice.

Rearrangement of lambda light chain genes in mature B cells in vitro and in vivo. Function of reexpressed recombination-activating gene (RAG) products.

V(D)J (V, variable; D, diversity; J, joining) combination of immunoglobulin (Ig) genes established in premature B cells has been thought to be conserved throughout differentiation at mature stages. However, germinal center (GC) B cells have been shown to reexpress recombination-activating gene (RAG)-1 and RAG-2 proteins in immunized mice. Here, we present several lines of evidence indicating that RAG proteins thus induced are functional as the V(D)J recombinase. DNA excision product reflecting Vlambda1 to Jlambda1 rearrangement was generated in parallel with the expression of RAG genes in mature mouse B cells that were activated in vitro with LPS and IL-4. Similar lambda chain gene rearrangement was observed in the draining lymph node of immunized mice. Further, B cells that underwent lambda gene rearrangement were shown by in situ PCR to be localized within GCs. Thus, secondary rearrangement of Ig genes (receptor editing) can occur in mature B cells.

Molecular cloning of a novel murine cell-surface glycoprotein homologous to killer cell inhibitory receptors.

We have isolated a cDNA clone encoding a novel murine cell-surface glycoprotein. This polypeptide is predicted to be composed of a signal peptide of 23 amino acids, an extracellular region of 620 amino acids that contains six immunoglobulin-like domains with five potential N-glycosylation sites, a transmembrane sequence of 20 amino acids, and a cytoplasmic tail of 178 amino acids with four sets of sequences similar to the immunoreceptor tyrosine-based inhibition motif. The relative molecular mass of the mature polypeptide is calculated to be 90,520 Da. The polypeptide, designated as p91, shows striking homologies to human killer cell inhibitory receptors, a murine gp49B1 protein, a bovine Fcgamma2 receptor, and a human Fcalpha receptor. The mRNA of p91 was especially abundant in murine macrophages. Western blot analysis using p91-specific anti-peptide sera detected a 130-kDa polypeptide in macrophages. Surface biotinylation and immunoprecipitation analysis verified the surface expression of the translation products on COS-1 cells transfected with the p91 cDNA, but the cells failed to show any Fc binding activity.

Characterization of B cells expressing recombination activating genes in germinal centers of immunized mouse lymph nodes.

Products of recombination activating genes (RAG-1 and RAG-2) involved in the rearrangement of Ig genes have been shown to be expressed only in immature stages of B cells. However, we have recently found that RAG genes were re-expressed in mature mouse B cells activated in vitro and in germinal centers (GCs) of immunized mouse lymph nodes (LNs). Here, we report that RAG transcripts and their proteins were expressed in parallel with the formation of GCs in popliteal LNs from mice immunized in the hind footpads. Immunocytochemical analysis revealed that RAG+ B cells were localized within GCs and were present as apoptotic tingible body cells. RAG expression is not considered a nonspecific result of apoptosis, since apoptotic B cells generated by surface Ig-engagement did not express RAG genes. These results suggest a novel role of RAG products in the differentiation of B cells in GCs.

Reexpression of RAG-1 and RAG-2 genes in activated mature mouse B cells.

Recombination activating genes (RAG-1 and RAG-2), involved in V(D)J rearrangement of immunoglobulin genes, have been thought to be expressed only in immature stages of B-cell development. However, RAG-1 and RAG-2 transcripts were found to be reexpressed in mature mouse B cells after culture with interleukin-4 in association with several different co-stimuli. Reexpression was also detected in draining lymph nodes from immunized mice. RAG-1 and RAG-2 proteins could be detected by immunofluorescence microscopy in the nuclei of B cells cultured in vitro and in the germinal centers of draining lymph nodes. These findings suggest that RAG gene products play a heretofore unsuspected role in mature B cells.

Suppression of in vitro cellular immune response by nitrogen-containing terpene alcohol derivatives.

A series of nitrogen-containing terpene alcohol derivatives were tested for their immunosuppressive effects in vitro, and KYKC-407 (erythro-1-(1-imidazolyl)-3,7,11-trimethyl-dodecane-2(S),3(R)-diol) was found to be the most effective in suppressing the induction of cytotoxic T cell (CTL) activity. CTL induction in the coculture of C3H spleen cells with mitomycin C-treated BALB/c spleen cells was suppressed by more than 90% when 4 microM KYKC-407 was added to the culture. Under these conditions, lymphocyte proliferation was also suppressed to a similar extent by this compound. Flow cytometric analysis revealed that KYKC-407 strongly inhibited the proliferation of CD8+ T cell subset, but showed less suppressive effects on CD4+ T cells. In contrast to cyclosporin A, KYKC-407 at 1-4 microM did not inhibit the proliferative response to concanavalin A. Further, KYKC-407 suppressed CTL induction even in the presence of exogenous IL-2, thus suggesting that the compound does not exert its effects through inhibiting IL-2 production.

Subconjunctival immunization of mice for inducing IgE antibody response in parotic lymph node.

Subconjunctival immunization of mice with dinitrophenyl (DNP)-Ascaris plus alum led to the induction of a local anti-DNP IgE response in 8 days. Anti-DNP IgE was found to be secreted from isolated lymphocytes in the parotic lymph node neighboring the immunization site but not from those in the spleen and the mesenteric lymph node. The IgE response was also confirmed by the detection of C epsilon transcript in the parotic lymph node cells. Ocular topical application of betamethazone resulted in considerable suppression of the IgE response in the parotic lymph node, thus suggesting that this immunization protocol is useful for evaluating ocular topical anti-allergic drugs that are expected to suppress local IgE responses.