Anin vitro invasion model for human renal cell carcinoma cell lines mimicking their metastatic abilities

We developed a modifiedin vitro invasion assay system using monolayers of vascular endothelial cells. A type I collagen gel was formed in plastic dishes, and overlaid with type IV collagen. Calf pulmonary arterial endothelial (CPAE) cells were seeded onto these plates, and incubated until they reached confluence. Five human renal cell carcinoma cell lines with various metastatic potentialsin vivo were then seeded on the monolayer CPAE cells, and their colony formation and invasion activities were examined for 9 days. At day 4, the highly metastatic cell lines increased the number of colony foci on monolayer CPAE cells several fold higher than their poorly metastatic counterpart. The horizontal spreading patterns were also different between poorly and highly metastatic cell lines. On day 9, the number of carcinoma foci that penetrated the monolayer of CPAE cells and type IV collagen sheets into type I collagen gels in highly metastatic cell lines greatly increased as compared with that of poorly metastatic cell lines. Ourin vitro invasion assay using monolayer CPAE cells would be useful to evaluate protease activities and colony formation during invasion.     

Genotype,serotype, and phylogenetic characterization of the complete genome sequence of hepatitis B virus isolates from Malawian chronic carriers of the virus

Hepatitis B virus (HBV) genotypes have distinct geographical distribution. HBV sequences among hepatitis B carriers in Malawi have not been evaluated thus far. HBsAg serotype and genotype of HBV was determined in 20 serum samples from Malawian chronic HBV carriers, and two complete genomes and 13 entire pre-S2/S genes were sequenced directly. Genotype A HBV isolates were found in all of the samples, and serotype with adw2 and ayw2 were detected in three and 17 samples, respectively. In phylogenetic analyses, two complete genomes were classified into a subgroup A' that was described previously in South African isolates of the virus, and were separated from HBV isolates in Western countries with nucleotide differences ranging from 4.1-6.2%. The separation of subgroup A' was also evident in the tree topology of the entire pre-S1/S2, X and precore/core region, but not evident in the small-S region. The nucleotide divergences in subgroup A' were higher than those among genotype A without subgroup A' in the complete genomes as well as each of four open reading frames. All of the 13 pre-S2/S sequences were classified into the subgroup A', and clustered with known HBV isolates with ayw2 in carriers from South Africa and Zimbabwe. Three amino acids in the pre-S2/S gene were characteristic of subgroup A' with ayw2. In conclusion, unique HBV isolates of subgroup A' with ayw2 are prevalent in Malawi, and subgroup A' with a relatively higher nucleotide diversity may be a HBV isolate characteristic of the indigenous population of some African countries.     

The influence of social environmental condition on the production of stress-induced 22 kHz calls in adult male Wistar rats

Adult rats emit 22 kHz ultrasonic vocalizations (USVs) in response to aversive stimuli, and these sounds are suggested to have communicative information among conspecifics. It is conceivable that social environment during development of rats has relevance to the emission of 22 kHz USVs. To examine the effects of social environment after weaning on production of stress-induced USVs, we compared the amount of emission of USVs among three groups of rats reared under different conditions after weaning. One group of rats was housed individually, and the other two groups were housed in pairs, in which social hierarchy of the pair was determined by social dominance-subordination relationships. The USVs were induced by acute mild somatic stimuli on the back and neck. Individually reared rats emitted much fewer USVs than pair-reared rats. In addition, socially subordinate rats emitted more USVs compared with socially dominant ones. These results suggest that not only social interaction but also the status in social hierarchy may play an important role in the process of the development of USVs induced by somatic stimuli.     

A digest of the Evidence-Based Clinical Practice Guideline for Nephrotic Syndrome 2020

Clinical and Experimental Nephrology -     

Prevention of postischemic reperfusion injury: The improvement of myocardial tissue blood flow after ischemia by terminal Nicorandil-Mg cardioplegia

We evaluated the preventive effect of postischemic reperfusion injury by Nicorandil-Mg cardioplegia given just prior to reperfusion as terminal cardioplegia. Twenty seven dogs were placed on cardiopulmonary bypass and the aorta was cross-clamped for 90 min under hypothermic (17–19°C) cardioplegic arrest. The canine hearts were divided into three groups: in group A (n=10) the hearts were reperfused without any treatment; in group B (n=9) the hearts received coronary perfusion with Nicorandil-Mg solution (Nic, 8 mg/l; Mg, 20 mEq/l; glucose, 50 g/l) for 2 min just prior to reperfusion; and in group C (n=8) the hearts received coronary perfusion with Nicorandil-Mg free solution (glucose, 50g/l). During and after ischemia, the myocardial tissue PCO₂ (t-PCO₂) was continuously monitored by an ion-sensitive field effective transistor (ISFET) sensor. In addition, the myocardial tissue blood flow (TBF), oxygen consumption, and lactate flux were then calculated at 5, 10, 20, and 40 min of reperfusion. In the initial reperfusion period, Group B showed an improved TBF compared to group A and C (at 5 min, group B was 42.7±11.9; group A was 29.4±11.2, P<0.025; and group C was 33.9±9.2% of the preischemic control level, P<0.05). T-PCO₂ in group B was significantly decreased at 5 min of reperfusion (group B, 127.5±22.5 42.5±9.7; group A, 117.5±23.0 85.2±17.4, P<0.001; group C, 122.3 mmHg 68.2±18.7 mmHg, P<0.01), and group B had a better metabolic recovery. These results suggest that terminal Nicorandil-Mg cardioplegia might reduce the rate of postischemic reperfusion injury.     

CD98 regulates the phosphorylation of HER2 and a bispecific anti-HER2/CD98 antibody inhibits the growth signal of human breast cancer cells

Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.     

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Interleukin-1β-induced, nitric oxide-dependent and -independent inhibition of vascular smooth muscle contraction

Stimulation of vascular smooth muscle by bacterial lipopolysaccharide has been shown to produce interleukin-1β and to induce vasodilation in septic shock. To understand the mechanisms of interleukin-1β-induced relaxation, we examined the effects of interleukin-1β on contractility and cyclic GMP contents of vascular smooth muscle. After treatment of the rat aorta with interleukin-1β (20 ng/ml) for 6 h, the cyclic GMP content increased and the contraction induced by phenylephrine (1 μM) was partially inhibited. An inhibitor of nitric oxide (NO) synthase, N^G-monomethyl-

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-arginine (

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-NMMA, 100 μM), prevented the inhibitory effect of interleukin-1β. After treatment with interleukin-1β for 24 h, the phenylephrine-induced contraction was inhibited more strongly. Neither

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-NMMA (100 μM) nor aminoguanidine (100 μM) reversed the inhibition, whereas methylene blue (10 μM) partially reversed the inhibition. After treatment with interleukin-1β for 12 or 24 h, the cyclic GMP content increased but to a level lower than that obtained with a 6-h treatment. The effects of sodium nitroprusside (1 μM) to inhibit the phenylephrine-induced contraction and to increase the cyclic GMP content were markedly suppressed by the 24-h interleukin-1β treatment. In contrast, the 24-h interleukin-1β treatment did not change the ability of 8-bromo-cGMP to relax the phenylephrine-stimulated aorta. Addition of

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-NMMA (1 mM) during the 24 h treatment prevented NO production and preserved the sodium nitroprusside-induced cGMP generation by interleukin-1β. The 24 h interleukin-1β treatment increased the threshold concentration of KCl needed to induce contraction without changing the maximum contraction. In the presence of 25.4 mM KCl or the non-selective K⁺ channel inhibitor, tetraethylammonium, the inhibitory effect of the 24-h interleukin-1β treatment on phenylephrine-induced contraction was restored. These results suggest that interleukin-1β inhibits vascular smooth muscle contraction by a time-dependent, dual mechanism. After a 6-h treatment with interleukin-1β, the NO/cyclic GMP system is activated. After a 24-h interleukin-1β treatment, in contrast, the NO/cyclic GMP system may be desensitized and the contraction of vascular smooth muscle is inhibited by another mechanism, possibly membrane hyperpolarization.     

Accumulation of L-[2-(F-18)]fluorophenylalanine in peri-infarct area in a patient with acute cerebral infarction

We studied the brain uptake of amino acid in a patient with acute cerebral infarction with L-[2-(F-18)] fluorophenylalanine and positron emission tomography. The increased accumulation of the ligand was specifically found in the peri-infarct area where oxygen metabolism was still maintained but decreased later in the 72-day follow-up period. The kinetic analysis revealed that increased accumulation was not due to increased transport from the blood to the brain but to delayed washout from the brain to the blood. Although the mechanism is still unknown, abnormally high accumulation of L-[F-18]fluorophenylalanine may predict delayed neuronal changes after ischemic insults of the brain.     

Prevalence of second generation antibody to hepatitis C virus among voluntary blood donors in Osaka,Japan

To clarify the demographic characteristics of the prevalence of hepatitis C virus (HCV) infection in Osaka, Japan, where hepatocellular carcinoma is common, we investigated the screening data of antibody to HCV (anti-HCV, DAINABOTHCVPHA, second generation assay) in 197,600 voluntary blood donors residing in Osaka. The study found that age-standardized prevalence of anti-HCV was significantly higher than that of HBsAg (2.25cf 0.86 percent among males,P<0.001; 2.17cf 0.55 percent among females,P<0.001. It was much higher in the blood donors aged 55–64 years than in those aged 16–54 years (8.49cf 1.32 percent among males,P<10^–5; 7.26cf 1.42 percent among females,P<10^–5). The prevalence of anti-HCV among males was significantly higher than that of females in the younger (25–34 years) generations (1.02 to 1.49 percentcf 0.71 to 1.13 percent,P<0.05). A similar tendency was observed in the prevalence of high-titer (2¹²) anti-HCV. The number of coinfection (both HBsAg and anti-HCV seropositive) was very small, and it was not statistically different from the expected number.