Selective down-regulation of Th2 cell-mediated airway inflammation in mice by pharmacological intervention of CCR4.

BACKGROUND: The chemokine receptor CCR4 has been implicated in Th2 cell-mediated immune responses. However, other T cell subsets are also known to participate in allergic inflammation. OBJECTIVE: The role of CCR4 in Th1, Th2, and Th17 cell-mediated allergic airway inflammation was investigated. METHOD: We generated an allergic airway inflammation model by adoptive transfer of in vitro-polarized ovalbumin (OVA)-specific Th1, Th2, and Th17 cells. The effect of a low-molecular weight CCR4 antagonist, Compound 22, on this model was examined. RESULTS: Upon in vitro polarization of DO11.10 naïve T cells, Th1- and Th2-polarized cells dominantly expressed CXCR3 and CCR4, respectively, while Th17-polarized cells expressed CCR6 and CCR4. Intranasal OVA-challenge of mice transferred with each T cell subset induced accumulation of T cells in the lungs. Eosinophils were also massively accumulated in Th2-transferred mice, whereas neutrophils were preferentially recruited in Th1- and Th17-transferred mice. Compound 22, as well as anti-CCL17 or anti-CCL22 antibody selectively suppressed accumulation of Th2 cells and eosinophils in the lungs of Th2-transferred and OVA-challenged mice. Compound 22 also inhibited bronchial hyperresponsiveness but had little effect on goblet cell hyperplasia in Th2-transferred and OVA-challenged mice. CONCLUSIONS AND CLINICAL RELEVANCE: There were notable differences in allergic lung inflammation mediated by different T cell subsets. CCR4 blockage was selectively effective for suppression of Th2-mediated allergic inflammation by blocking infiltration of Th2 cells.

Investigation of new cytogenetic biomarkers specific to high-LET radiation using in vivo and in vitro exposed human lymphocytes.

PURPOSE: To find detectable cytogenetic biomarkers that can offer information about the radiation quality of in vivo exposure retrospectively. MATERIALS AND METHODS: Chromosome-type aberrations of peripheral lymphocytes of uterine cancer patients that received internal gamma- and external X-ray therapy or carbon beam therapy and of victims severely exposed to neutrons and gamma-rays in a criticality accident that occurred in Tokai-mura, Japan were analysed. Data obtained from in vitro irradiation experiments using 60Co gamma-rays and 10 MeV neutrons were compared with the in vivo exposure data. RESULTS: The ratio of acentric rings to dicentric chromosomes (termed RaD ratio) and that of excess fragments to dicentrics (termed EfD ratio) showed significant (p < 0.05) differences between the two groups of cancer patients, and these ratios for accidental victims were in between the values of the two groups of cancer patients. The in vitro studies using doses equivalent to 1 - 3 Gy of gamma-rays have confirmed that the EfD ratios were increased with the high LET (linear energy transfer) and RaD ratios decreased. CONCLUSION: The present data show that the RaD and EfD ratios can be used as cytogenetic biomarkers of exposure to high-LET radiation at least within a few years of exposure.

Aberration of genomic DNA in association with human hepatocellular carcinomas detected by 2-dimensional gel analysis.

Alterations of genomic DNAs in primary hepatocellular carcinomas (HCCs) were examined by restriction landmark genomic scanning (I. Hatada et al., Proc. Natl. Acad. Sci. USA, 88: 9523-9527, 1991) which is a 2-dimensional gel analysis that allows detection of deletion, amplification, or other rearrangements of genomic DNA. Sixteen HCC samples together with their normal counterparts were tested in this manner. Each HCC sample was micromanipulated to minimize possible carryover from non-malignant cells. DNAs from HCCs and their normal counterparts were cleaved with the restriction enzyme NotI, end labeled with 32P, and size fractionated by 2-dimensional electrophoresis using HinfI as the second cleavage enzyme. The resulting spots (about 2000) in HCC samples were compared with their normal counterparts. Five spots were more intense in 10-14 of the 16 HCCs (63-88%). The intensity of several spots was reduced to about half, suggesting the loss of one of two alleles. Some of these decreases were observed frequently in different HCC samples, whereas others were sporadic. Sixty of these spots reproducibly decreased in > 2 cases, with 27 showing a decrease in > 50% of the informative cases. The highest incidence was observed in 14 of 16 samples (88%). No significant correlations were observed between these changes in spots and hepatitis B virus or hepatitis B virus infection. The use of landmarks that show a reproducible increase or decrease in intensity is discussed in conjunction with future studies of genomic alterations inherent in HCC.

Identification of the IL-6-responsive element in an acute-phase-responsive human pancreatic secretory trypsin inhibitor-encoding gene.

Pancreatic secretory trypsin inhibitor (PSTI) has been suggested to be an acute-phase reactant in humans and to be induced by inflammatory cytokines such as the interleukins IL-1 and IL-6. We report that PSTI is synthesized in hepatoma cells and that the gene expression is augmented by IL-6. The start points (tsp) for basal and augmented transcription are exactly the same as the tsp in normal pancreas. Analysis of the PSTI gene revealed that a 40-bp DNA fragment located between kb -3.84 and -3.80 carries the element responsible for both transcriptional activity and IL-6-induced gene expression. This 40-bp fragment contains TTGNNGNAATG, the consensus sequence for the NF-IL6-binding site, which is also known as the IL-6-responsive element that is conserved among various acute-phase genes. The basal activity was augmented by another sequence that lies between kb -4.0 and -3.9.

A novel sperm-specific hypomethylation sequence is a demethylation hotspot in human hepatocellular carcinomas.

Certain human DNA regions are strikingly undermethylated at CpG sites in sperm compared to adult somatic tissues. These sperm-specific hypomethylation sequences are thought to function early in embryogenesis or gametogenesis. By using the restriction landmark genomic scanning (RLGS) cloning method, we have isolated a novel sperm-specific hypomethylation sequence, the status of which changes during spermatogenesis, embryonal growth and differentiation. This sequence is a part of a new 'NotI repeat' consisting of a 1.4 kb repetitive unit sequence named DE-1. The sequence is GC-rich and has high homology to a CpG DNA clone that was isolated by a methyl CpG protein binding column, indicating that it was normally highly methylated. We investigated the methylation status of this sequence. In the normal genome the sequence was methylated, but in the human hepatocellular carcinoma (HCC) genome, the target sequence was demethylated at the cytosine residue of the CpG dinucleotides with high frequency (75% in the previous study). These data suggest that this regional DNA hypomethylation may play a role in both cell differentiation and hepatocarcinogenesis.

Novel, potent, and selective phosphodiesterase-4 inhibitors as antiasthmatic agents: synthesis and biological activities of a series of 1-pyridylnaphthalene derivatives.

The structural requirements for potent and selective PDE4 inhibition were revealed in a 1-pyridylnaphthalene series, and the best compound (3kg, T-2585.HCl) was chosen for further biological evaluation (PDE4 inhibition IC50 = 0.13 nM, selectivity PDE3/4 ratio = 14 000). Compound 3kg showed potent antispasmogenic activities (ED50 = 0.063 mg/kg for reduction of antigen-induced bronchoconstriction, intravenously; ED50 = 0.033 mg/kg for reduction of histamine-induced bronchoconstriction, intraduodenally) in guinea pigs with little cardiovascular effects. Furthermore, 3kg induced significantly weaker emetic effects than RP73401 after oral administration in ferrets and intravenous administration in dogs (3kg, none of 4 ferrets vomited at a dose of 10 mg/kg, po and none of 8 dogs vomited at a dose of 0.3 mg/kg, iv; RP73401, 4 of 8 ferrets vomited at a dose of 3 mg/kg, po and 6 of 8 dogs vomited at a dose of 0.3 mg/kg, iv); that is compatible with the lower affinity for the high-affinity rolipram binding site (3kg, 2.6 nM; RP73401, 0. 85 nM). This may imply that 3kg has an improved therapeutic ratio because of a broad margin between the Ki value of binding affinity and the IC50 value of PDE4 inhibition (ratio = 0.050).

Recovery process of arthritis induced by 6-sulfanilamidoindazole (6SAI) in rats.

6-Sulfanilamidoindazole (6SAI) is known to induce not only an acute arthritis but also serositis and arteritis which resemble those induced by some vasodilators in rats. In this study, the recovery process of ankle lesions was examined histopathologically for up to 12 weeks of recovery period in rats bearing arthritis induced by administration of 6SAI (500 mg/kg) for 2 weeks. At 2 weeks of 6SAI-treatment, exudative synovitis and exudative/edematous periarthritis with marked formation of granulation tissues and periosteal reactive bone formation were noted in the ankles, but no remarkable neutrophil infiltration was detected in those lesions. The ankle swelling induced by 6SAI diminished by 4 weeks of recovery period, and the elevated plasma fibrinogen levels were normalized by 2 weeks of recovery period. Although fibrosis and newly-formed periosteal bone were still observed after 2 weeks of recovery period, no inflammatory lesion was detected at that point. At 4 or 12 weeks of recovery periods, the ankles showed an almost normal appearance. These results indicate that 6SAI-induced arthritis is reversible in nature and does not develop into chronic phase.

Effects of imidapril and captopril on streptozotocin-induced diabetic nephropathy in mice.

We investigated whether the prevention of the development of diabetic nephropathy by angiotensin-converting enzyme inhibitors is associated with decreases in renal angiotensin-converting enzyme activity and/or blood pressure in diabetic mice. C57Bl/6 mice were injected with streptozotocin (200 mg/kg, i.v.) and randomized to receive either imidapril (1 and 5 mg/kg) or captopril (10 and 50 mg/kg) or vehicle by gavage for 28 days. Each assay was performed on 8-10 mice from each treatment. At 28 days after the start of drug treatment, imidapril and captopril significantly reduced blood pressure of the diabetic mice, and this effect of captopril was stronger than that of imidapril. On the other hand, inhibition of renal angiotensin-converting enzyme activity by imidapril was stronger than that by captopril. Imidapril and captopril dose-dependently inhibited urinary albumin excretion to similar extents, but they failed to inhibit the renal hypertrophy and elevation of creatinine clearance. Total renal angiotensin-converting enzyme activity was significantly reduced in diabetic mice, but immunohistochemical localization of angiotensin-converting enzyme was intensive in the vasculature and glomeruli of the diabetic kidney. In conclusion, both effects on blood pressure and angiotensin-converting enzyme activity may be involved in the prevention of development of diabetic nephropathy by imidapril and captopril in streptozotocin-induced diabetic mice. The data suggest that the degrees of contribution of their effects on blood pressure and renal angiotensin-converting enzyme activity to the inhibition of urinary albumin excretion may be different between the two angiotensin-converting enzyme inhibitors.

Involvement of angiotensin II in development of spontaneous nephrosis in Dahl salt-sensitive rats.

We investigated the effect of angiotensin-converting enzyme inhibition on spontaneous nephrosis in Dahl salt-sensitive (Dahl/S) rats. Dahl/S rats fed on a normal sodium diet spontaneously developed nephrosis and mild hypertension from a young age. In young Dahl/S rats, an angiotensin-converting enzyme inhibitor, imidapril, attenuated the development of proteinuria accompanied by a decrease in blood pressure. Methylprednisolone, a potent therapeutic agent for proteinuria, did not affect the development of nephrosis. An angiotensin AT1 receptor antagonist, losartan, but not a Ca2+ channel blocker, verapamil, inhibited the development of nephrosis while both agents decreased blood pressure to a similar extent as imidapril. In mature Dahl/S rats, imidapril suppressed not only the development of proteinuria but also the glomerular lesions. It is concluded that the development of spontaneous nephrosis in Dahl/S rats is mediated by angiotensin II.

Enhanced attachment and elastase-releasing capacity of neutrophils after surgery.

Perioperative changes in neutrophil attachment level and neutrophil elastase-releasing capacity were examined in patients who underwent surgery for either esophageal or gastric cancer. The neutrophil attachment level was significantly increased in both groups to approximately three times that of the preoperative value. The elastase-releasing capacity of nonstimulated neutrophils was not significantly changed, but it was significantly increased in neutrophils stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). Moreover, both neutrophil attachment and elastase-releasing capacity of FMLP-stimulated neutrophils were more enhanced in patients with postoperative complications than in patients without complications. Peak levels of serum interleukin 6, serum alpha 1 proteinase inhibitor, and plasma neutrophil elastase were also significantly higher in patients with postoperative complications than in patients without. These results suggest that during the postoperative period, neutrophils may be primed and activated in response to inflammatory mediators such as cytokines, and that such an alteration of neutrophil functions may reflect the extent of inflammation and may, at times, be implicated in postoperative complications.

[Establishment of EGF, EGF-R and FN-R positive human adenocarcinoma cell line (GAC-1)].

A human adenocarcinoma cell line designated as GAC-1, was established from ascites of the 56-year old male patient with rapidly progressive gastric cancer. The doubling time was about 18.5 hours in vitro, and cell cycle analysis using flow cytometry showed marked increase of S phase (46.1%). Immunohistochemical demonstration of GAC-1 cells revealed positive staining of TGF-alpha, EGF, EGF-R, FN-R, laminin and negative staining of fibronectin. Histogram of them indicated aneuploidy with modal number 57 and they formed tumors in nude mice.

Hepatic changes of mice in the subacute phase of streptozotocin (SZ)-induced diabetes.

Hepatic changes of mice in the subacute phase of streptozotocin (SZ)-induced diabetes were investigated biochemically and pathologically. Biochemically, the contents of serum glucose and of serum and liver lipids increased while the content of liver glycogen decreased in SZ-induced diabetic mice. Histopathologically, hypertrophy of hepatocytes due to an increase in number of intracytoplasmic acidophilic granules was common to SZ-induced diabetic mice. Electron microscopically, these hepatocytes were characterized by a prominent increase in number of mitochondria showing normal structure, a marked decrease of glycogen granules and poorly developed rough endoplasmic reticulum, which were common to so-called oncocytic cells. In some SZ-induced diabetic mice, bile duct hyperplasia with an appearance of cytomegalic hepatocytes was also observed.

[Intra-arterial infusion chemotherapy in locally recurrent breast cancer].

Intra-arterial infusion chemotherapy (IA) using implantable reservoir was performed in four locally recurrent breast cancers. The regimen consisted of several cycles of induction (1 cycle, 90-150mg: EPI) and the following maintenance infusion. The results were as follows. (1) D.F.I. of all cases were short and pre-treatment of 3 cases was not effective. (2) Clinical response was 2 CR and 1 MR. We confirmed that IA was an effective treatment for not only locally recurrence but also disseminated foci. (3) In 3 out of the four cases there was reduction of severe arm swelling. These findings suggested that IA using implantable reservoir was useful for the treatment of locally recurrent breast cancer in terms of anti-cancer effect and improved QOL.

Strain differences in vulnerability of hippocampal neurons to transient cerebral ischaemia in the rat.

Strain differences in the vulnerability of hippocampal neurons to an ischaemic insult were investigated in Sprague-Dawley, Wistar and Fischer 344 rats. Transient global brain ischaemia was produced for 5 minutes by a combination of bilateral carotid artery occlusion and oligaemic hypotension (40 mmHg) induced by exsanguination. The number of viable neurons in the CA1 subfield was counted under a light microscope 7 days after the ischaemic insult. The density of viable neurons in the CA1 subfield of normal rats was around 150 cells/mm of CA1 length in all the strains examined; after global brain ischaemia, this parameter in Sprague-Dawley, Wistar and Fischer 344 strain rats was approximately 110, 120, and 70, respectively. The results suggest that the hippocampal CA1 neurons of Fischer 344 strain rats are more vulnerable to ischaemic insult than those of the other strains. There were many F344 rats (8/21) which showed atypical vasculature patterns in the posterior region of the circle of Willis, suggesting less blood flow through anastomosis from basilar artery to posterior cerebral artery and/or ill-balanced blood flow between left and right hemispheres. These anatomical variations in the circle of Willis may in part contribute to the strain differences in the vulnerability to cerebral ischaemia.

Systemic histopathology of rats treated with 6-sulfanilamidoindazole, a novel arthritogenic sulfonamide.

6-Sulfanilamidoindazole (6SAI) is a sulfonamide that induces acute, self-limiting arthritis in rats, and 6SAI-induced arthritis is thought to be a model for testing anti-inflammatory agents. In this study, in order to clarify the location of arthritis and relationships between arthritis and other changes in this model, we have investigated the detailed pathologic changes in rats administered orally with 6SAI (125, 250, 500 mg/kg) daily for 4 wk in a time-course experiment. Moderate to severe arthritis was observed in rats of middle- and high-dose groups. Histologically, in the affected ankle, exudative synovitis and periarthritis were observed at 1 wk, granulation tissue formation with angiogenesis and periosteal new bone formation at 2 wk, and marked fibrosis of affected area at 4 wk, respectively. In addition to these changes, in periarticular and periosteal tissues of affected ankles, subendothelial insudation of small-sized arteries and medial fibrinoid degeneration of medium-sized arteries were observed at 1 and 2 wk and intimal thickening and medial hypertrophy at 4 wk, respectively. No arterial changes were observed in the unaffected ankles. Similar arterial changes were often observed in the liver, thyroid glands, and lungs and rarely in various organs and tissues. Acute inflammation of serous tissues such as mesentery, mediastinum, and capsule of spleen or thymus were also present in 6SAI-treated groups, and it was sometimes accompanied by arteritis. In addition, in 6SAI-treated rats, follicular hyperplasia of thyroid glands and pituitary changes, which are thought to be related to depression of thyroid hormone production by 6SAI, were observed. These results show that 6SAI induces not only arthritis but also arteritis, serositis, and thyroid change, and it is necessary to take the interaction between these changes into consideration when anti-inflammatory agents are tested in this model.

Phenytoin-induced cerebral thrombosis in rats: cerebral ultrastructure, water content and ischaemic volume in the acute phase.

A new rat model for multifocal cerebral thrombosis has recently been reported (Tani et al., 1994; 1995). Ultrastructural changes in the cerebral neocortex in the acute phase were investigated in order to characterize the early pathological events in this model. A bolus injection of alkaline phenytoin solution (pH 10.8) into one internal carotid artery in the rat caused severe endothelial injury accompanied by thrombosis in the cerebral vasculature within 5 minutes, and severe oedema of the ipsilateral hemisphere within an hour. Cerebral water content was measured by the simple dry-wet method, and cerebral surface area and the surface area and volume of the ischaemic zone were measured using computer-aided image analysis. Good correlations were demonstrated between cerebral water content and cerebral surface area, and between the surface area and volume of the ischaemic zone. We report here that quantitative evaluation of acute cerebral damage induced by phenytoin solution is possible with high reliability using simple image analysis.

Prolonging action of imidapril on the lifespan expectancy of cardiomyopathic hamsters.

We studied the effect of imidapril, a novel angiotensin-converting enzyme (ACE) inhibitor, on lifespan expectancy of cardiomyopathic (CM) hamsters of BIO 14.6 strain, one of the representative models of congestive heart failure (CHF). Imidapril was consecutively administered to hamsters by mixing it in their diet at a concentration of 480 ppm (approximately 30 mg/kg/day) or 1,600 ppm (approximately 120 mg/kg/day) from age 26 weeks. Only several control hamsters died before age 54 weeks, but their survival rate decreased to 23.7% at age 73 weeks. The survival rates of 480-ppm and 1,600-ppm imidapril groups at age 73 weeks were as high as 75.7 and 68.4%, respectively (p < 0.01 vs. control hamsters). Macroscopic and microscopic pathology in imidapril-treated groups was milder than that in control animals in general, but differences were not statistically significant when animals were divided into survivors and fatalities except for the presence of mural thrombus in the heart. We further studied the effects of imidapril on blood pressure (BP), in vivo cardiac function, cardiac beta-adrenoceptor distribution, and plasma catecholamine levels after dietary treatment with 480 ppm imidapril for 8-10 weeks from age 37 weeks. Imidapril-treated animals showed improved cardiac function under urethane anesthesia. These results indicate that imidapril prolongs lifespan expectancy of CM hamsters and suggest that a hemodynamic effect of imidapril is involved in its beneficial effect.

Overexpression of pancreatic secretory trypsin inhibitor in pancreatic cancer. Evaluation of its biological function as a growth factor.

Four clones of pancreatic secretory trypsin inhibitor (PSTI)-overexpressing cells (TF-PANC clones 1, 6, 8, and 36) were established to evaluate the physiological function of PSTI secreted by cancer cells, by means of introducing a PSTI-expression vector (pRSV-PSTI) into the human pancreatic adenocarcinoma cell line (PANC-1). No obvious changes were observed in the histological features of these transplanted tumors in nude mice, in the growth of TF-PANC and PANC-1, or that of 3T3 fibroblasts when cocultured with them. Addition of recombinant human PSTI to the cultured media resulted in no increase in proliferation of fibroblasts (3T3 and WI-38) or of four pancreatic cancer cell lines (PANC-1, CAPAN-I, MIAPaCa-2, and Hs766T). These results suggest that the estimation of tumor-bearing PSTI as a paracrine or autocrine growth factor in recent studies should be given careful consideration.

Expression of the pancreatic secretory trypsin inhibitor gene in the liver infected with hepatitis B virus.

Pancreatic secretory trypsin inhibitor, an acute phase reactant protein, is expressed in the liver in response to inflammatory cytokines, especially in hepatocellular carcinoma. Northern blots of 25 dissected liver tissues from non-hepatitis, chronic hepatitis and cirrhosis patients revealed that 10 (40%) expressed pancreatic secretory trypsin inhibitor. The expression seemed to be closely associated with hepatitis B viral infection, since among the 11 hepatitis B virus-infected samples, nine (81%) were pancreatic secretory trypsin inhibitor-positive. In contrast, this augmented expression was absent in non-infected livers (0/5; 0%), and rare in those infected with hepatitis C virus (1/9; 11%). There was no significant correlation between the pancreatic secretory trypsin inhibitor expression in the liver and the serum level of glutamic pyruvic transaminase, the hepaplastin test, the 15-min retention rate of indocyanine green, or the histological findings of the liver tissues such as lymphocyte infiltration and pseudolobular formation. Furthermore, we identified an almost three-fold increase in the transactivation of pancreatic secretory trypsin inhibitor gene expression in HepG2 cells after transient transfection with HBV-DNA or the X gene in an expression vector. These results suggest that the induction of pancreatic secretory trypsin inhibitor gene expression in livers with chronic hepatitis and cirrhosis is directly affected by hepatitis B virus.