Administration route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens.

Dendritic cells (DCs) loaded with tumour antigens have been successfully used to induce protective tumour immunity in murine models and human trials. However, it is still unclear which DC administration route elicits a superior therapeutic effect. Herein, we investigated the vaccine efficiency of DC2.4 cells, a murine dendritic cell line, pulsed with ovalbumin (OVA) in the murine E.G7-OVA tumour model after immunization via various routes. After a single vaccination using 1 x 10(6)OVA-pulsed DC2.4 cells, tumour was completely rejected in the intradermally (i.d.; three of four mice), subcutaneously (s.c.; three of four mice), and intraperitoneally (i.p.; one of four mice) immunized groups. Double vaccinations enhanced the anti-tumour effect in all groups except the intravenous (i.v.) group, which failed to achieve complete rejection. The anti-tumour efficacy of each immunization route was correlated with the OVA-specific cytotoxic T lymphocyte (CTL) activity evaluated on day 7 post-vaccination. Furthermore, the accumulation of DC2.4 cells in the regional lymph nodes was detected only in the i.d.-and s.c.-injected groups. These results demonstrate that the administration route of antigen-loaded DCs affects the migration of DCs to lymphoid tissues and the magnitude of antigen-specific CTL response. Furthermore, the immunization route affects vaccine efficiency.

A case of acromegaly accompanied by adrenal preclinical Cushing's syndrome.

We encountered a 58-year-old woman with acromegaly accompanied by a cortisol-secreting adrenal tumor without clinical features of hypercortisolism. The simultaneous occurrence of these two endocrinopathies in one individual is extremely rare. She was diagnosed as having diabetes mellitus 8 years ago. Afterwards, in spite of insulin therapy, her hyperglycemia could not be well controlled. Her acromegaly and preclinical Cushing's syndrome were histopathologically proven to be due to a pituitary adenoma and an adrenocortical adenoma, respectively. Successful treatment for these endocrinopathies resulted in greatly improved blood sugar control because of a reduction in insulin resistance. In this case of preclinical Cushing's syndrome, replacement therapy with glucocorticoid was able to be discontinued at only 8 weeks after adrenalectomy, so that the period of necessary replacement was much shorter than that for overt Cushing's syndrome. This is the first report describing insulin resistance before and after treatment in a case of acromegaly accompanied by adrenal preclinical Cushing's syndrome.

[In vitro antibacterial activity of vancomycin in combination with panipenem against carbapenem-resistant MRSA].

The synergistic relationship between vancomycin (VCM) and carbapenem (CRB) has been reported in antibacterial activity against CRB-resistant strains of MRSA. The purpose of this study is to investigate the antibacterial activity against CRB-resistant MRSA using VCM, panipenem (PAPM), and a combination of both. 8 strains of CRB-resistant MRSA were used to examine the effects of these antibiotics by the broth microdiluton technique. The effect of pH (pH 6, 7, 8) on MIC of VCM alone was not observed in 7 out of 8 strains; MICs were between 1.0-2.0 micrograms/ml. PAPM alone, however, showed an enhancing tendency in alkaline condition in 6 out of 8 strains. There was no influence of pH on MICs in the combination use of VCM and PAPM, showing additive effect in 1 strain and synergistic in 6 strains. Killing-curves against PAPM-resistant MRSA were examined under the following drug combinations; 1/4 MIC of VCM (0.5 micrograms/ml) plus 1/4 MIC of PAPM (16 micrograms/ml), and 1/4 MIC of VCM plus 1/8 MIC of PAPM (8 micrograms/ml). The former drug combination showed synersistic effect; decrease from 1.05 x 10(5) to 6.45 x 10(4) CFU/ml after 6 hours' incubation and to less than 10 CFU/ml after 24 hours. The latter drug combination showed synergistic activity (2.68 x 10(2) CFU/ml) after 24 hours' incubation, but lost antibacterial activity after 48 hours. In conclusion, PAPM in combination with VCM showed synergistic effects on CRB-resistant MRSA. This combination therapy should be evaluated for the treatment of MRSA infection in patients with renal dysfunction.

[Effect of vacuum cleaning of room floors and bed clothes of patients on house dust mites counts and clinical scores of atopic dermatitis. A double blind control trial].

By a randomized double blind control trial we studied the effect of vacuum cleaning of room floors, mattresses and quilts for twelve months on clinical symptoms and laboratory data of atopic dermatitis patients. All patients used the identical new vacuum cleaners. Thirty patients (3-12 years of age) with relatively stable skin conditions were randomly allocated to either of the following two groups. In the monitor group we visited patient's home every three weeks and mite specialists cleaned drastically the room floors, mattresses and quilts and the patient continued to clean in the same way in-between. In the control group we visited similarly but the cleaning was made insufficiently which was also followed by the patient. But, at 2 occasions (the first and the last visits), cleaning was made drastically also in the control group. Thus the mite numbers were counted precisely at the start and the end of the experiment both in the monitor and control groups. Each patient was seen every six weeks by the same doctor and estimated of his symptoms using our unique scoring system (Fig. 1). At the start and the end of the study, total IgE and specific IgE antibodies to house dust mites in the serum were evaluated. The monitor group showed a tendency to count smaller number of mites than the control group after a year, when there was a significant difference only in quilts. However, a statistically significant decrease in the mite counts was observed only in room floors and not in mattresses and quilts both in the monitor and control groups (Fig. 2). Clinical scores after a year significantly improved only in the monitor group and not in the control group (Fig. 3). Serum IgE value and specific antibody titer to house dust mites were not changed significantly after the trial in both groups. As a conclusion, vacuum cleaning of the patient's room improved the clinical symptoms of atopic dermatitis but this could not be related to the reduction of house dust mite number.

Dexamethasone-induced up-regulation of adrenomedullin and atrial natriuretic peptide genes in cultured rat ventricular myocytes.

To determine whether adrenomedullin (AM), a novel 51-residue vasodilator peptide originally isolated from human pheochromocytoma, is expressed by the heart, and whether the expression of cardiac AM gene is regulated by glucocorticoids, the effect of dexamethasone (DEX) on the expression of protein and mRNA of AM and atrial natriuretic peptide (ANP) was tested in cultured ventricular myocytes prepared from the neonatal rats. Northern blot analysis of rat ventricular myocytes with cDNAs for rat AM and ANP as probes revealed distinct bands corresponding to the sizes of rat AM mRNA (1. 6 kb) and rat ANP mRNA (1 kb), respectively. Dexamethasone increased steady-state levels of both AM and ANP mRNA as well as secretion of both AM and ANP immunoreactivity in a time- and dose-dependent manner. The stimulatory effects of dexamethasone were completely abolished by a glucocorticoid antagonist (RU38486) and a RNA synthesis inhibitor (actinomycin D). The approximate half-lives of basal and dexamethasone-induced AM mRNA were about 4 h, suggesting that dexamethasone-induced up-regulation of AM mRNA was unlikely due to its decreased degradation. These data suggest that glucocorticoids directly stimulate gene expression of AM as well as ANP in rat ventricular myocytes.

[In vitro combination effect of vancomycin and carbapenems against carbapenem-resistant MRSA].

We determined in vitro combined effects of vancomycin (VCM) plus carbapenems (CRBs) on 12 methicillin-resistant Staphylococcus aureus (MRSA) which are resistant to CRBs. Combinations of VCM plus imipenem (IPM) and VCM plus panipenem (PAPM) and VCM plus meropenem (MEPM) indicated synergistic effects, fractional inhibitory concentration (FIC) indices of < or = 0.05, against 67%, 75%, 67% of the strains, respectively. Against forty two percent of strains tested, 1 MIC of VCM was equal to 1 MBC, and similarly, IPM, PAPM and MEPM had 1 MIC = 1 MBC against 42%, 67% and 75% of the strains tested, respectively. Combinations of VCM plus IPM and VCM plus PAPM and VCM plus MEPM showed synergistic effects, hence a fractional bactericidal concentration (FBC) index of < or = 0.50, against 42%, 50%, 75% of the strains, respectively, and the combination of VCM plus MEPM was most synergistic. These results suggest that combination therapy of VCM with CRB is useful for the treatment of MRSA infection in patients with renal dysfunction.

[Peripheral lymphocyte subset of patients with pancreatic diabetes mellitus--about a decreased ratio of T-LGL and ability of host defense].

Patients with diabetes mellitus (DM) often suffer from various and severe infection. Patients with pancreatic DM have malnutrition due to chronic pancreatitis. Therefore it is believed that patients with pancreatic DM have a lower ability of host defence to infectious diseases than normal subjects. We examined the primpheral lymphocyte subsets (T cell, B cell, NK cell, CD3+ 56+ T) of eighteen patients (males, age = 52.8 +/- 12.7 years old, mean +/- SD) with pancreatic DM by two color flow-cytometry to evaluate the lymphocyte function. Ratios of T cell (T%, 66.9 +/- 9.3%, mean +/- SD). B cell (B%, 13.1 +/- 5.8%) and NK cell (Nk%, 19.3 +/- 8.7%) to total peripheral lymphocytes of patients with pancreatic DM were not significantly different from those (T% = 66.4 +/- 7.8%, B% = 13.5 +/- 6.7%, NK% = 19.9 +/- 9.1%) of thirteen normal subjects (males, age = 51.2 +/- 13.1). CD3+56+ T cell % (4.1 +/- 1.9%) of patients with pancreatic DM was lower than that (6.0 +/- 3.0%) of normal subjects (p < 0.05). CD3+56+ T cells have cytotoxic activity and it is likely that this activity is similar to that of NK cells. These results suggested that a decrease in peripheral CD3+56+ T cell % is a factor showing a weak host defense mechanism to infectious diseases in patients with pancreatic DM.

Effect of omeprazole on changes in gastric and upper small intestine pH levels in patients with chronic pancreatitis.

Gastric and upper small intestine pH levels were measured continuously over 24 hours in patients with chronic pancreatitis, and values obtained before and after the administration of omeprazole were compared. Additionally, omeprazole was administered for 2 weeks and the fecal excretion of fat was compared before and after drug therapy. Postprandial gastric pH levels, initially 2.9 to 3.2, increased by 1.6 to 2.1 after treatment. Postprandial upper small intestine pH levels, initially 5.1 to 5.5, increased by 0.7 to 1.0. The lowest pH value of the upper small intestine was 2.2 to 2.4 postprandially; this was increased by > 1.0 after omeprazole, and the amplitude of pH variation was reduced. The cumulative proportions of intraintestinal pH strata of < or = 3, < or = 4, or < or = 5, and higher, initially being 16.4% to 17.1%, 27.4% to 31.7%, and 52.6% to 57.8%, respectively, were remarkably improved after drug treatment. Gastric pH and upper small intestine pH levels showed a positive correlation; an increase in gastric pH levels by 2 corresponded to an increase in small intestine pH levels by 1. After omeprazole administration, mean fecal excretion of fat was decreased to 4.1 +/- 2.6 g/d (range, 1.1 to 9.8 g/d) from 6.5 +/- 3.9 g/d (range, 1.6 to 13.5 g/d). Decreases in excretion of fat averaged 3.4 g/d (range, 2.2 to 4.5 g/d) in patients with steatorrhea. It was concluded that steatorrhea due to chronic pancreatitis can be improved to some extent by improving upper small intestine pH levels following the elevation of gastric pH levels after administration of omeprazole.

Nonselective ETA/ETB receptor antagonist blocks proliferation of rat vascular smooth muscle cells after balloon angioplasty.

To elucidate the role of endothelin receptor subtypes in the abnormal proliferation of vascular smooth muscle cells (VSMC) associated with vascular injury, we have investigated the effects of a novel and potent nonselective ETA/ETB receptor antagonist (TAK-044) on the proliferation of rat VSMC in vitro and in vivo. TAK-044 dose-dependently inhibited DNA synthesis stimulated by 10(-7) M ET-1 in cultured rat VSMC from the late passage with the approximate IC50 of 6 x 10(-8) M. After balloon angioplasty, the neointimal lesion in the injured carotid arteries in the TAK-044-treated group (0.052 +/- 0.014 mm2) was significantly (p < 0.05) decreased compared to that in control group (0.26 +/- 0.045 mm2), while the medial surface area was not affected. The intima/media ratio in the TAK-044 group (31 +/- 6%) also significantly (p < 0.05) decreased from that of the control group (148 +/- 25%). Our data suggest that nonselective ETA/ETB receptor antagonists may be therapeutic potential for prevention against the intimal thickening associated with vascular injury.

Distribution of kallikrein-binding protein mRNA in kidneys and difference between SHR and WKY rats.

We investigated kallikrein-binding protein (KBP) mRNA distribution in the kidney of Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHR), and Wistar-Kyoto strain (WKY) rats. Northern blot analysis revealed that KBP mRNA was located mainly in the medulla and with lower amounts in SHR than in WKY rats. KBP mRNA in microdissected nephron segments was detected by reverse transcription and polymerase chain reaction (RT-PCR) followed by Southern blot analysis. In SD rats, the most abundant signals were consistently found in inner medullary collecting duct (IMCD), with small amounts in outer medullary collecting duct, proximal convoluted tubule, and glomerulus. No signals were found in connecting tubule and cortical collecting duct. The nephron distribution of KBP mRNA was similar in WKY and SD rats. Only a small amount of signal was found, however, in IMCD of SHR. In conclusion, 1) KBP mRNA was predominantly distributed in the medullary segments of the distal nephron, downstream from the known kallikrein activity site in the collecting duct, and 2) KBP mRNA expression was significantly decreased in the kidney of SHR.

Induction of nitric oxide synthase gene by interleukin-1 beta in cultured rat cardiocytes.

BACKGROUND: Impaired myocardial contractility in septic shock is protracting, which may be caused by cytokine-induced nitric oxide (NO) synthesis in the heart. However, the cellular mechanism by which cytokines induce nitric oxide synthase (NOS) in cardiocytes remains obscure. METHODS AND RESULTS: We studied the effect of human recombinant interleukin-1 beta (IL-1 beta) on synthesis of NO2-/NO3- (NOx) and the expression of NOS mRNA and protein in cultured neonatal rat cardiocytes. IL-1 beta dose-dependently (0.1 to 10 ng/mL) stimulated NOx production as a function of time (6 to 48 hours). Northern blot analysis using complementary DNAs for rat brain-type constitutive (c) NOS and mouse macrophage-type inducible (i) NOS as probes showed that IL-1 beta induced expression of mRNA for iNOS but not for cNOS, starting after 6 hours and reaching a maximum after 48 hours in cardiocytes. IL-1 beta similarly induced iNOS mRNA expression in cultured adult rat cardiocytes in a time-dependent manner. Western blot analysis using specific antibody against the N-terminal fragment of mouse iNOS revealed the expression of 130-kD iNOS-like protein in IL-1 beta-treated cardiocytes. Northern blotting and immunocytochemical study revealed that IL-1 beta-induced iNOS mRNA and iNOS-like immunoreactivity were exclusively localized to cardiac myocytes but also to nonmyocytes, to a lesser extent. NG-mono-methyl-L-arginine, an NOS inhibitor, completely blocked the IL-1 beta-induced NOx production, whose effect was reversed by L-arginine but not by D-arginine. Dexamethasone inhibited the IL-1 beta-induced NOx production as well as iNOS mRNA expression. Cycloheximide and actinomycin D completely inhibited the IL-1 beta-induced NOx production and iNOS mRNA expression. Neither a calmodulin inhibitor (W-7), a protein kinase C inhibitor (calphostin C), nor a Ca2+ channel antagonist (nicardipine) showed any effect on the IL-1 beta-induced NOx production. CONCLUSIONS: These data demonstrate that IL-1 beta induces macrophage-type iNOS mRNA expression mainly by cardiac myocytes but also by nonmyocytes to a lesser extent, and subsequent de novo protein synthesis of iNOS leads to excessive local production of NO by cardiocytes.

Up-regulation of ETB receptor subtype mRNA by angiotensin II in rat cardiomyocytes.

The effect of angiotensin (ANG) II on the expression of endothelin (ET) receptor subtype (ETA, ETB) mRNA in cultured neonatal rat cardiomyocytes was studied. ANG II (10(-7) M) increased steady-state mRNA levels of ETB receptor, but not ETA receptor, during 6-12 hr incubation, whose effect was dose-dependent (10(-9)-10(-7) M). ANG II receptor type-1 (AT1) antagonist (CV-11974) completely inhibited the ANG II-induced up-regulation of ETB receptor, whereas the inhibitory effect by ANG II receptor type-2 (AT2) antagonist (PD 123319) was incomplete. These results suggest that ANG II up-regulates cardiac ETB receptor, predominantly via AT1 receptor.

Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes.

To elucidate the cellular mechanism by which angiotensin II (ANG II) induces cardiac hypertrophy, we investigated the possible autocrine/paracrine role of endogenous endothelin-1 (ET-1) in ANG II-induced hypertrophy of neonatal rat cardiomyocytes by use of synthetic ET-1 receptor antagonist and antisense oligonucleotides to preproET-1 (ppET-1) mRNA. Northern blot analysis and in situ hybridization revealed that ppET-1 mRNA was expressed in cardiomyocytes, but, to a lesser extent, in nonmyocytes as well. ANG II upregulated ppET-1 mRNA level by threefold over control level as early as 30 min, and it stimulated release of immunoreactive ET-1 from cardiomyocytes in a dose- and time-dependent manner. ET-1 stimulated ppET-1 mRNA levels after 30 min in a similar fashion as ANG II. Tetradecanoylphorbol-acetate (10(-7) M) mimicked the effects of ANG II and ET-1 on induction of ppET-1 mRNA. ANG II-induced ppET-1 gene expression was completely blocked by protein kinase C inhibitor H-7 or by down-regulation of endogenous protein kinase C by pretreatment with phorbol ester. ET-1 and ANG II stimulated twofold increase [3H]leucine incorporation into cardiomyocytes, whose effects were similarly and dose dependently inhibited by endothelin A receptor antagonist (BQ123). Introduction of antisense sequence against coding region of ppET-1 mRNA into cardiomyocytes resulted in complete blockade with ppET-1 mRNA levels and [3H]leucine incorporation stimulated by ANG II. These results suggest that endogenous ET-1 locally generated and secreted by cardiomyocytes may contribute to ANG II-induced cardiac hypertrophy via an autocrine/paracrine fashion.

Insulin-like growth factor-I induces hypertrophy with enhanced expression of muscle specific genes in cultured rat cardiomyocytes.

BACKGROUND: Cardiac hypertrophy is commonly observed in acromegalic patients, in whom serum insulin-like growth factor-I (IGF-I) levels are elevated. In the present study, we examined whether IGF-I induces hypertrophy in cultured neonatal rat cardiomyocytes through its specific receptor and whether IGF binding protein-3 (IGFBP-3), which is a major circulating carrier protein for IGF-I, inhibits IGF-I-induced cardiac hypertrophy in vitro. METHODS AND RESULTS: Because the response of cardiac hypertrophy is characterized by the induction of expression for muscle-specific genes, the effect of IGF-I on steady-state levels of mRNA for myosin light chain-2 (MLC-2) and troponin I and for skeletal and cardiac alpha-actin isoforms was evaluated by Northern blot analysis. IGF-I (10(-7) M) increased mRNA levels for MLC-2 and troponin I as early as 60 minutes with maximum levels by 6 hours, which were maintained for as long as 24 hours. IGF-I (10(-7) M) also increased transcripts for skeletal alpha-actin but not for cardiac alpha-actin. The cell size as evaluated morphometrically was almost doubled after 48-hour treatment with IGF-I. IGF-I induction of protein synthesis was dose dependent (10(-10) to 10(-7) M) with a maximal 2.2-fold increase seen at 10(-8) M. In contrast to the hypertrophic effect of IGF-I, growth hormone affected neither protein synthesis nor expression for muscle-specific genes. Binding study using 125I-IGF-I revealed the presence of specific binding sites for IGF-I in rat cardiomyocytes. IGFBP-3 induced a dose-dependent inhibition of protein synthesis stimulated by IGF-I; IGFBP-3 (10(-7) M) completely inhibited the [3H]leucine uptake stimulated by IGF-I (10(-8) M). IGFBP-3 similarly inhibited the IGF-I-stimulated gene expressions for MLC-2 and troponin I. CONCLUSIONS: These results suggest that IGF-I directly causes cardiac hypertrophy and that its effect can be blocked by IGFBP-3.

Endothelin-1 enhances calcium entry through T-type calcium channels in cultured neonatal rat ventricular myocytes.

Endothelin-1 (ET-1), a 21-amino acid vasoconstrictive peptide, increases intracellular Ca2+ level and has hypertrophic action on ventricular myocytes. To elucidate a possible role of Ca2+ entry through sarcolemmal Ca2+ channels on this ET-1 action, we examined effects of ET-1 on L-type (ICa,L) and T-type (ICa,T) Ca2+ currents in cultured neonatal rat ventricular myocytes using the patch-clamp technique. ET-1 at a concentration of 10 nM increased the maximum current density of ICa,T from -3.0 +/- 1.4 microA/cm2 in the control condition to -4.4 +/- 1.6 microA/cm2 (p < 0.01). Although the peak amplitude of ICa,L was decreased during ET-1 application (from -9.7 +/- 1.9 microA/cm2 in the control condition to -5.0 +/- 1.4 microA/cm2 [p < 0.01]), this magnitude of decrease in ICa,T (52 +/- 19%) was comparable to that of spontaneous "run-down" of ICa,L (47 +/- 26%). The enhancement of ICa,T by ET-1 was dose dependent; it was initiated as low as 0.32 nM, and the maximal response was attained at approximately 10 nM, with a half-maximal dose of 1.26 nM. The enhancement of ICa,T by ET-1 was antagonized by protein kinase C inhibitors staurosporine (0.2 microM) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 20 microM) applied to the pipette solution. Extracellular application of tumor-promoting phorbol esters, phorbol 12,13-dibutyrate (PDBu) and 4 beta-phorbol 12-myristate 13-acetate, augmented ICa,T. PDBu (0.2 microM) increased the maximal current density of ICa,T from -4.2 +/- 0.5 microA/cm2 in the control condition to -5.5 +/- 1.0 microA/cm2 (p < 0.01). In the presence of H-7 (20 microM) in the pipette solution, PDBu failed to enhance ICa,T, and an inactive isomer of PDBu (4 alpha-phorbol 12,13-dibutyrate, 0.2 microM) did not augment ICa,T. Thus, ET-1 enhances Ca2+ entry through the sarcolemmal T-type Ca2+ channel, possibly through a pathway involving activation of protein kinase C. This ET-1 action may be involved in the rise of the intracellular Ca2+ level and may contribute to the induction of cardiac hypertrophy by ET-1.

A partial defect in technetium-99m pyrophosphate image suggesting cardiac rupture following acute myocardial infarction.

We present the case of a 70-year-old woman with acute myocardial infarction who died of cardiac rupture on the 2nd hospital day. Dual isotope single photon emission computed tomography (SPECT) using thallium-201 chloride and technetium-99m pyrophosphate (PYP) performed on the 2nd hospital day showed a large perfusion defect in the anteroseptal wall on 201Tl image and a increased accumulation on 99mTc-PYP image in the anterior area consistent with a partial defect. Autopsy performed 1 h after death revealed a tear in the left ventricular anterior wall consistent with the defect on the 99mTc-PYP image. We propose that the finding of a partial defect in 99mTc-PYP is an interesting finding which may be associated with cardiac rupture following acute myocardial infarction.