Carrier cell-based delivery of replication-competent HSV-1 mutants enhances antitumor effect for ovarian cancer.

Oncolytic viruses capable of tumor-selective replication and cytolysis have shown early promise as cancer therapeutics. We have developed replication-competent attenuated herpes simplex virus type 1 (HSV-1) mutants, named HF10 and Hh101, which have been evaluated for their oncolytic activities. However, the host immune system remains a significant obstacle to effective intraperitoneal administration of these viruses in the clinical setting. In this study, we investigated the use of these HSV-1 mutants as oncolytic agents against ovarian cancer and the use of human peritoneal mesothelial cells (MCs) as carrier cells for intraperitoneal therapy. MCs were efficiently infected with HSV-1 mutants, and MCs loaded with HSV-1 mutants caused cell killing adequately when cocultured with cancer cells in the presence or absence of HSV antibodies. In a mouse xenograft model of ovarian cancer, the injection of infected carrier cells led to a significant reduction of tumor volume and prolonged survival in comparison with the injection of virus alone. Our results indicate that replication-competent attenuated HSV-1 exerts a potent oncolytic effect on ovarian cancer, which may be further enhanced by the utilization of a carrier cell delivery system, based on amplification of viral load and possibly on avoidance of neutralizing antibodies.

AIDS, HIV and STD among Japanese and Japanese-Americans in San Francisco, California, USA.

In affluent countries, some people may go abroad specifically for HIV testing or care for greater anonymity, better treatment, and less discrimination. A secondary analysis of data from the San Francisco Department of Public Health was conducted to characterize trends in AIDS incidence, AIDS care, sexually transmitted disease (STD) incidence, and HIV counselling and testing among Japanese in San Francisco. A total of 96 AIDS cases were diagnosed among ethnic Japanese, of whom 32 were born in Japan. From 1985 to 2000, 144 gonorrhoea and chlamydia cases were identified among Japanese. Of 368 self-identified Japanese seeking HIV counselling from 1995 to 1997, three (0.9%) were HIV-positive. The HIV/AIDS and STD epidemics among Japanese in San Francisco temporally parallels, but lags behind in magnitude, the epidemics for San Francisco's population as a whole. Some persons from Japan actively seek HIV/STD services while abroad. Our study points to several areas needing further research and improvements.

Mechanism for maintenance of dominant T helper 1 immune responses in Lewis rats.

Lewis rats are prone to T helper (Th) 1 immune responses, whereas Brown Norway (BN) rats are susceptible to Th2 immune responses. Yet, the precise mechanism of induction of the different outcome between these two strains remained elusive. We investigated the expression levels of some cytokines, their receptors and accessory molecules responsible for the polarization of antigen-specific immune response into a predominant Th1 or Th2 profile in Lewis and BN rats. Lymph node (LN) cells collected from rats immunized with short ragweed (RW) were used directly or after stimulation in vitro with RW for 3 days. Expression of cytokines, their receptors and accessory molecules in these LN cells were tested by reverse transcriptase-PCR. Culture supernatant was used for ELISA to detect IL-12 protein. We observed clear differences between these strains in the expression of IL-12p40, which was high in LN cells of Lewis rats even before stimulation in vitro. In addition, a higher amount of IL-12 was present in the culture supernatant in Lewis rats. Upregulation of the expression of IL-12 receptor beta1, beta2, IFN-gamma receptor alpha and beta genes were more prominent in Lewis rats rather than BN rats. Furthermore, attenuated expression of CD40 and CD40 ligand by stimulation in vitro was noted only in BN rats. Changes in expression of these molecules by stimulation as well as higher basal level of IL-12p40 might have led to the activation of Th1 cells in Lewis rats.

Fifty-seven-kDa protein in royal jelly enhances proliferation of primary cultured rat hepatocytes and increases albumin production in the absence of serum.

We have previously shown that 57-kDa protein in royal jelly (RJ) was specifically degraded in proportion to both storage temperature and storage period, and we suggested that it could be useful as a marker of freshness of RJ (Kamakura, M., Fukuda, T., Fukushima, M. and Yonekura, M. (2001) Biosci. Biotechnol. Biochem. 65, 277-284.). Here, we investigated the physiological effects of 57-kDa protein on primary cultured rat hepatocytes in the absence of serum. The 57-kDa protein and RJ significantly stimulated hepatocyte DNA synthesis, whereas bovine serum albumin and RJ stored at 40 degrees C for 7 days, which lacks 57-kDa protein, did not. The mitogenic activity of 57-kDa protein was lost after treatment with trypsin. These results indicate that 57-kDa protein acts as a mitogen. The stimulatory effect of 57-kDa protein was dose-dependent and was more potent at lower than at higher cell densities. The 57-kDa protein also prolonged the cell proliferation of primary cultured rat hepatocytes, with an enhancement of albumin production compared to untreated cells. Therefore, 57-kDa protein is likely to promote liver regeneration and may have a cytoprotective action on hepatocytes.

[Characteristics of heterosexually acquired AIDS in Japan. An inter-country comparison using AIDS Surveillance data].

OBJECTIVES: To identify epidemiological characteristics of heterosexually acquired AIDS in Japan, with emphasis on potential influence on future trends. METHODS: National AIDS Surveillance data in Japan were compared with those in the UK and US, where detailed information is available from well-established surveillance procedures. Data on AIDS cases diagnosed until the end of 1996, particularly those acquired heterosexually, were analyzed by year of diagnosis, gender and age group. RESULTS: The number of heterosexually acquired AIDS cases in Japan has continued to increase, while those in the UK and US leveled out or decreased recently. The increase during a two-year period after reaching a certain number of cases per year was found to be 2.3-fold in Japanese, 2.4-fold in UK whites and 5-fold in US whites. The male to female ratio (M/F) for heterosexually acquired AIDS was 6.3 among Japanese, while the ratio was 1.1 and 0.5 in the UK and US, respectively. The age distribution at AIDS diagnosis demonstrated a peak from 35 to 54 years of age among Japanese males, as compared to 30 to 34 among males in the UK and the US. No significant difference was apparent in the age distribution among females in the three countries. CONCLUSIONS: The relatively small number of AIDS cases in Japan is attributable to the late introduction of HIV and the limited chance of heterosexual transmission from homosexual/bisexual men and injecting drug users. In addition, transmission has probably most often occurred between middle-aged Japanese males and non-Japanese females. As there is a growing risk of HIV infection among Japanese females and young Japanese males, new prevention strategies targeting these groups are urgently required.

Storage-dependent degradation of 57-kDa protein in royal jelly: a possible marker for freshness.

In order to find a marker for freshness of royal jelly (RJ), the composition change of RJ during storage was investigated. The contents of 10-hydroxy-2-decenoic acid, a bioactive component of RJ, and several vitamins did not change during storage at 40 degrees C for 7 days. However, a specific protein, designated royal jelly protein-1 (RJP-1), was gradually degraded during storage under various conditions (from 4 degrees C to 50 degrees C for up to 7 days). The specific degradation of RJP-1 was proportional to storage temperature and storage period. RJP-1 was purified to homogeneity and characterized as a monomeric glycoprotein with a molecular mass of 57 kDa. These results suggest that 57-kDa protein in RJ can be used as a marker for freshness of RJ, reflecting the conditions under which RJ has been stored.

Antifatigue effect of fresh royal jelly in mice.

We investigated the antifatigue effect of royal jelly (RJ), which had been stored at -20 degrees C from immediately after collection, in male Std ddY mice. The mice were accustomed to swimming in an adjustable-current swimming pool, then subjected to forced swimming five times during 2 wk, and the total swimming period until exhaustion was measured. They were separated into three groups with equal swimming capacity, which were administered RJ, RJ stored at 40 degrees C for 7 d (40-7d RJ), or the control solution including casein, cornstarch, and soybean oil before swimming. All mice were forced to swim for 15 min once; then the maximum swimming time to fatigue was measured after a rest period. The swimming endurance of the RJ group significantly increased compared with those of the other groups. The mice in the RJ group showed significantly decreased accumulation of serum lactate and serum ammonia and decreased depletion of muscle glycogen after swimming compared with the other groups, whereas there was no significant difference between the 40-7d RJ group and the control group in these parameters after swimming. A quantitative analysis of constituents in RJ showed that 5 7-kDa protein, which we previously identified as a possible freshness marker of RJ, was specifically degraded in RJ stored at 40 degrees C for 7 d, whereas the contents of various vitamins, 10-hydroxy-2-decenoic acid, and other fatty acids in RJ were unchanged. These findings suggest that RJ can ameliorate the physical fatigue after exercise, and this antifatigue effect of RJ in mice seems to be associated with the freshness of RJ, possibly with the content of 5 7-kDa protein.

Molecular characterization and nuclear localization of rat timeless-like gene product.

Among three period genes (per1, per2, per3) in mammals, only per2 gene was shown to be involved in the core clock mechanism. To elucidate the molecular function of rat PERIOD2 (rPER2), we searched for binding proteins to the PAS domain of rPER2. We isolated a binding protein to this domain and identified it as a TIMELESS-like protein (TLP) on the basis of mass analyses. Then, we isolated a rat TLP cDNA from the rat hypothalamus library. RNA blot analysis and in situ hybridization indicates that rTLP mRNA was expressed in all rat tissues from whole brain, the suprachiasmatic nucleus, eye, lung, heart, liver, kidney, placenta, and testis. When rTLP gene product was expressed in COS-1 cells, nuclear localization of rTLP was detected in 99.6% of transfected cells. These results suggest that the interaction of rPER2 with rTLP may influence the regulation of circadian clock components in nucleus after rPER2 is translocated into the nucleus.

Delays and continuation of hospital visits among HIV-infected persons and AIDS cases in Japan.

This study attempts to clarify the distribution patterns of delay between HIV transmission and the first hospital visit among HIV-infected persons and AIDS cases in Japan except those infected through blood products. Such hospital visit patterns were analyzed, and the rates of reporting for HIV/AIDS surveillance among diagnosed HIV-infected persons and AIDS cases in hospitals were shown. From 1991 to 1997, a survey and subsequent follow-up were conducted among HIV-infected persons and AIDS cases diagnosed at 74 hospitals in Tokyo. The numbers of HIV-infected persons and AIDS cases were 590 and 208, respectively. The percentage of patients whose estimated date of HIV transmission was obtained ranged 23-41% among Japanese and non-Japanese HIV-infected persons and AIDS cases. Among these patients, 28% to 86% showed a 3-year delay between HIV transmission and their first hospital visit. The rate of HIV-infected persons who continued to visit hospitals within 1 year after their first visit was 77% for Japanese and 45% for non-Japanese; among those after 1 year or more following their first hospital visit the rate was more than 80% among Japanese and over 70% among non-Japanese. The rate of reporting to HIV/AIDS surveillance among diagnosed HIV-infected persons and AIDS cases was 90% or more after 1994 in Japan. The delay between HIV transmission and the first hospital visit was suggested to be very long. Not a few patients stopped visiting hospitals after only a short time. Most diagnosed HIV-infected persons and AIDS cases were reported to the surveillance system of Japan.

Cholecystokinin A and B receptor mRNA expression in human pancreas.

Little information is available on the expression of cholecystokinin (CCK) receptors in the human pancreas, especially in the developing pancreas. We evaluated expression patterns for the CCK receptors in human pancreas at three different ages: fetus, infant, and adult. Expressions of CCK-A and CCK-B receptor messenger RNA (mRNA) were studied in human midtrimester fetus (14-15 weeks' gestation), infant (50 days old), and adult pancreas by reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis. Expression levels of mRNA for both receptors also were evaluated by Northern blot analysis of adult pancreas. Northern blot analysis showed a strong signal for CCK-B receptor mRNA in adult pancreas, but no detectable signal for CCK-A receptor mRNA. However, RT-PCR/Southern blotting showed the presence of CCK-A receptor mRNA in adult pancreas. This was confirmed by sequencing of the complementary DNA (cDNA). RT-PCR/Southern blot analysis also showed CCK-A and CCK-B receptor mRNA expression in fetal and infant pancreas. These results show that the both CCK receptor types are expressed in human pancreas at stages of early gestation, but there is predominant expression of CCK-B receptor in adult pancreas.

Mutations in exons 2 and 3 of the cationic trypsinogen gene in Japanese families with hereditary pancreatitis.

BACKGROUND/AIMS: Single-point mutations in the cationic trypsinogen gene have been reported in hereditary pancreatitis kindreds in the white population. The aim of the present study was to investigate whether similar gene mutations are present in Japanese hereditary pancreatitis kindreds. METHODS: All five exons of the cationic trypsinogen gene were amplified by polymerase chain reaction and sequenced in six Japanese families with hereditary pancreatitis. RESULTS: Two types of single-point mutation in the cationic trypsinogen gene, which were identical with those reported in white families with hereditary pancreatitis, were observed in separate Japanese families with hereditary pancreatitis: 21Asn (AAC) to Ile (ATC) (N21I) in exon 2 and 117Arg (CGC) to His (CAC) (R117H) in exon 3. Pancreatitis occurred at more advanced ages in patients with the N21I mutation than in those with the R117H mutation. Besides normal polymorphisms in exons 4 and 5, no mutation was found in patients in the remaining four families with hereditary pancreatitis, 21 patients with sporadic chronic pancreatitis, or five normal subjects. CONCLUSIONS: These results show heterogeneity, but no racial specificity, in the cationic trypsinogen gene mutations in hereditary pancreatitis kindreds. A distinctive clinical feature for each of the mutation types is suggested: adult onset for the N21I mutation and childhood onset for the R117H mutation.

Trends in HIV and AIDS based on HIV/AIDS surveillance data in Japan.

BACKGROUND: In recent years a decline in the number of new AIDS cases has been observed in several industrialized countries. It is important to know whether these recent trends observed in North America and Europe are also occurring in Japan. METHODS: The number of people reported with HIV and AIDS by nationality, route of infection, and sex was calculated based on the HIV/AIDS surveillance data available in Japan through December 1997. The effect of reporting delay, which was defined as those HIV and AIDS cases reported in the calendar year following diagnosis, on the trends was examined. The coverage rate in reporting HIV cases was estimated as the ratio of the reported AIDS cases with prior report as an HIV-positive to the total number of reported AIDS cases. RESULTS: The cumulative number of reported cases of HIV among Japanese and non-Japanese residents of Japan up to the end of 1997 were 1,300 and 1,190, respectively. The cumulative number of reported cases of AIDS among Japanese and non-Japanese up to the end of 1997 were 758 and 298, respectively. The number of reported cases of HIV among Japanese was found to be still increasing, with the major contribution from male cases. The increasing trend in the number of reported AIDS cases among Japanese began to slow in 1996 and 1997. The number of reported cases of HIV among non-Japanese residents of Japan peaked in 1992, and has decreased since then, and remained constant after 1994. In contrast, the number of reported AIDS cases among these non-Japanese tended to increase gradually. There was a slight reporting delay for people with HIV and AIDS. The estimated coverage rate in reporting HIV cases tended to decrease in 1996 and 1997 (1/7.2, 1/10.2, respectively). We point out several reasons for this recent decline and suggest the possibility of an ostensible decline in the estimates. CONCLUSIONS: We suggest that the number of people with HIV among Japanese has continued to increase, and that the increase in the number of AIDS cases among Japanese is now slowing.

Clinical factors influencing posttransfusion platelet increment in patients undergoing hematopoietic progenitor cell transplantation--a prospective analysis.

BACKGROUND: Platelet transfusion refractoriness remains problematic in the management of patients who have undergone hematopoietic progenitor cell transplantation. Bone marrow transplantation itself is reported to be a relevant factor hampering efficient platelet transfusions. However, a prospective analysis assessing factors affecting platelet transfusion efficacy in the setting of hematopoietic progenitor cell transplantation has yet to be conducted. STUDY DESIGN AND METHODS: To identify factors independently influencing platelet transfusion efficacy after hematopoietic progenitor cell transplantation, a prospective study was performed to determine the effectiveness of platelet transfusions by estimating posttransfusion (16-hour) corrected count increments (CCI) in 42 consecutive patients (26 who received allogeneic transplants and 16 who received autologous transplants) with 439 available platelet transfusions. RESULTS: The mean CCI and percentage of CCI <4500 for all transfusions were 6161.1 +/- 7775.2 per microL and 42.1 percent, respectively. Multiple linear regression analyses revealed high total bilirubin, total body irradiation, high serum tacrolimus, and high serum cyclosporin A to be major factors independently predicting a lower CCI. HLA antibodies with restricted specificity and platelet antibodies were detected transiently in 17 and 14 percent of the patients, respectively. The presence of these antibodies was not, however, associated with a poor response to platelet transfusions. CONCLUSION: Platelet transfusion efficacy in hematopoietic progenitor cell transplant recipients is markedly influenced by clinical factors specific to the procedure as well as those already recognized in other settings. Alloimmunization is not, however, a major factor associated with a poor response to platelet transfusions after this procedure.

The phospholipid flippase activity of gastric vesicles.

We found that isolated gastric vesicles contain a novel Mg2+-ATP-dependent phospholipid translocation (flippase) activity. Fluorescence analogue of phosphatidylcholine, 2-(12-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3- phosphocholine, was ATP-dependently translocated from the outer (cytosolic) to inner (luminal) leaflet of the lipid membrane bilayer of hog gastric vesicles. The translocation was saturable and depended on time and the ATP concentration (Km = 3.1 microM). The basal Mg2+-ATPase activity of gastric vesicles in the absence of K+ showed high (Km = 1.6 microM) and low (Km = 80 microM) affinities for ATP, indicating that the present flippase activity is driven mostly by the high affinity Mg2+-ATPase activity. It required Mg2+ but not K+. Verapamil, which is an inhibitor of mouse mdr2 phosphatidylcholine flippase, did not inhibit the present flippase activity. Isolated sarcoplasmic reticulum vesicles that contain Ca2+-ATPase did not show any flippase activity. Fluorescence analogues of phosphatidylserine and phosphatidylethanolamine were similarly translocated by the gastric flippase. These phospholipid flippase activities were inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) (IC50 = 0.14-0.25 microM), a specific K+-ATPase inhibitor of gastric H+,K+-ATPase rich in gastric vesicles. IC50 value for the SCH 28080-inhibitable Mg2+-ATPase activity was about 0.13 microM, indicating that the phospholipid translocation was driven mostly by the SCH 28080-sensitive Mg2+-ATPase activity. Possible physiological roles of flippases were discussed in relation with the gastric acid secretory and cytoprotective mechanisms.