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Biomedical subjects

T Kuwata

Publications and source records attributed to T Kuwata.

At least 19 recordsLinked to original sources
PubMed2005

A new three-dimensional automatic bodymarker system for transvaginal ultrasonography.

OBJECTIVE: To evaluate the accuracy and usefulness of a newly developed three-dimensional automatic bodymarker system for transvaginal ultrasonography. METHODS: A bodymarker system which utilizes an electromagnetic field to specify the probe position was developed. Software was written which enabled the positional information of the probe and the ultrasound image to be simultaneously displayed on a personal computer. The bodymarker was displayed as a three-dimensional graphic model. The differences between the estimated and the actual position--i.e. the error--for both position (x, y and z) and angles (azimuth, elevation and roll) of the probe were measured. The movement of the probe was then evaluated in healthy female volunteers and the comparative time required for ultrasound examination was determined. RESULTS: Both the position and the angles of the probe were accurately shown in the computer display. The errors were 1.0 mm and 0.7 degrees (median) for position and angle, respectively. The system was also shown to work well in healthy female volunteers. Calibration of the new system required only 5.0 seconds, compared with an average of 7.6 seconds for the conventional bodymarker. CONCLUSION: The newly developed three-dimensional automatic transvaginal bodymarker system provides an accurate indication of probe position and its tilting angle. It works well in healthy female volunteers and speeds up the examination time. It may be clinically useful for transvaginal ultrasound examination.

Calibration↗
PubMed2004

Protective effects of nef-deleted SHIV or that having IFN-gamma against disease induced with a pathogenic virus early after vaccination.

To clarify the involvement of primitive non-specific immune responses in the protective effects of a live, attenuated virus, each two rhesus macaques were intravenously immunized with an attenuated chimeric simian and human immunodeficiency virus (SHIV) in which the nef gene was deleted (SHIV-NI) or a SHIV having human IFN-gamma inserted into the deleted nef region (SHIV IFN-gamma). These immunized monkeys were intravenously challenged with a heterologous pathogenic SHIV (SHIV-C2/1) at four weeks post immunization (wpi). After vaccination, one of each SHIV-NI- or SHIV IFN-gamma-immunized monkeys showed a low level of SIV Gag-specific lymphocyte proliferative response but did not have neutralizing antibodies to both the parental and challenge viruses. After the challenge, the plasma viral RNA loads of the challenge virus were suppressed in all the immunized monkeys and the severe CD4+ T cell loss observed in the unimmunized monkeys was not found. Thus, both SHIV IFN-gamma and SHIV-NI infections could prevent from disease progression by a pathogenic virus early after immunization, suggesting that primitive non-specific immune response elicited by attenuated virus infection, in addition to highly acquired virus-specific immunity, contributes to the protective effect against a pathogenic virus.

AIDS Vaccines↗
PubMed2003

The quantity and diversity of infectious viruses in various tissues of SHIV-infected monkeys at the early and AIDS stages.

To detect the major sites of viral replication in immunodeficiency virus-infected individuals, we quantified proviral DNA and infectious viruses using quantitative PCR and a plaque assay, respectively, in various tissues of SHIV(KU-2)-infected monkeys in the early and AIDS stages of infection. Compared the quantity of infectious virus among PBMC and the lymphoid tissues, the mesenteric lymph node had the largest number of infectious viruses at the AIDS stage more than at the early stage of infection. These results suggested that the gastrointestinal tract was a major site of viral replication. In the brain, proviral DNA was detected at the early and AIDS stage of infection, but infectious viruses were detected at only the AIDS stage. Moreover, we analyzed the nucleotide sequences of the env V3 region in infectious virus clones isolated from each plaque. The viruses in the lymphoid tissues of the monkey that developed AIDS diverged from the inoculated virus and had the same three amino acid substitutions. However, the viruses in the brain were almost identical to the inoculated virus, suggesting that the virus entered the brain early after infection and persisted without replication and genetic diversion until the AIDS stage.

Amino Acid Sequence↗
PubMed2003

Augmentation of antigen-specific cytokine responses in the early phase of vaccination with a live-attenuated simian/human immunodeficiency chimeric virus expressing IFN-gamma.

A nef-deleted SHIV-NM-3rN (SHIV-NI) was previously shown to be nonpathogenic and to induce protective immunity. In the present study, a SHIV-NI expressing human interferon-gamma (SHIV-IFN-gamma) was constructed and the effect of co-expression of IFN-gamma on virus replication and immunopotentiation was investigated in macaques that were vaccinated with both viruses, by comparing cytokine responses during the first 4 weeks after vaccination. Peripheral blood mononuclear cells (PBMC) isolated from vaccinated macaques were stimulated with inactivated viral particles for 24 h, and the production of IL-2, IL-4, IL-6, IL-10, IL-12, TNF-alpha and IFN-gamma was determined by ELISA and flow cytometry. All of the vaccinated macaques showed increases in cytokine production. However, the production of IFN-gamma (Th1-type cytokine) was more rapidly induced by SHIV-IFN-gamma vaccination, and IFN-gamma-producing cells appeared to be still increasing at 4 weeks after vaccination, although the difference of virus replication during the time was not significant in contrast to in vitro replication in cultured PBMC. These results suggest that co-expression of IFN-gamma with SHIV can modulate the antiviral immune responses into the Th1 type response, which would probably provide more protective immunity.

Animals↗
PubMed2002

Infection of macaques with chimeric simian and human immunodeficiency viruses containing Env from subtype F.

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for investigating the pathogenicity of human immunodeficiency virus (HIV-1) and to develop an anti-HIV-1 vaccine. We attempted to construct SHIVs containing Env from various subtypes, because almost all SHIVs which have been reported so far have Env from HIV-1 that belongs to subtype B. Two infectious SHIVs containing Env from two strains of HIV-1, CMR304 and CMR306, which belong to subtype F and A, respectively, were newly obtained. These SHIVs essentially showed a coreceptor usage and a neutralization pattern that were similar to those of the parental HIV-1s. In macaque PBMC, SHIVcmr304 replicated with kinetics similar to that of prototypic SHIV-NM-3rN with HIV-1 NL432 Env, but SHIVcmr306 replicated poorly. Inoculation of four rhesus macaques with SHIVcmr304 resulted in an increase of plasma viral load in all the macaques, though viral RNA copies were 100-fold lower than that in the infection with NM-3rN. This SHIV containing Env from HIV-1 subtype F will be a valuable source for the analysis of HIV-1 subtype F and the evaluation of vaccine candidates as a genetically divergent challenge virus.

Animals↗
PubMed2002

Characterization of simian and human immunodeficiency chimeric viruses re-isolated from vaccinated macaque monkeys after challenge infection.

Monkeys that have been vaccinated with nef-deleted SHIVs were either fully or partially protected against challenge with acute pathogenic SHIV-89.6 P. Viruses isolated from these vaccinated monkeys were all found to be the 89.6 P challenge virus using PCR amplification and restriction enzyme analysis of the env region of the viruses. Analysis of the 3'-end of the env region and 5'-half of the nef region using a heteroduplex mobility assay revealed that the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys had quite an abundant and similar heterogeneous quasispecies population. In contrast, the viruses isolated from the vaccinated monkeys had different and fewer quasispecies indicating a selective immune pressure in the vaccinated monkeys. The in vitro replication of the viruses isolated from the vaccinated monkeys in human and macaque peripheral blood mononucular cells (PBMCs) as well as in established cell lines such as M8166 and HSC-F cells, were slow and delayed when compared to the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys. Further comparison revealed that in HSC-F cells the viruses from vaccinated monkeys again showed delayed and weak CD4(+) cell down-modulation as well as having little or no effect on cell growth or cell viability on HSC-F cells and monkey PBMC. Thus we noticed that these re-isolated 89.6 P viruses from the vaccinated monkeys had changed or had been selected for low pathogenic viruses in the monkeys. This suggests that though the vaccination did not completely prevent the replication of the challenge virus in the monkeys it did contain the challenge virus by suppressing the pathogenic variants. This further enhances the prospects of this nef-deleted SHIV as the bases for effective anti-HIV vaccine candidates.

AIDS Vaccines↗
PubMed2002

Characteristic transfer of colostrum-derived biologically active substances into cerebrospinal fluid via blood in natural suckling neonatal pigs.

The characteristic transfer of colostral components into cerebrospinal fluid (CSF) via serum after natural suckling has been studied by sodium dodecyl sulphate (SDS) electrophoresis, two-dimensional electrophoresis, immunoblot and enzyme linked immunosorbent assay methods in non-suckling pigs. Total protein concentrations in the serum increased immediately after first suckling, reached a peak value at 12 h, corresponding to a 2.3-fold compared with pre-suckling level. The protein concentration in CSF also increased and reached peak value at 6 h corresponding to 1.6-fold compared with presuckling level. IgG in serum not detected before suckling, increased steeply after suckling, IgG, IgM and IgA transported into the serum were observed in completely intact form by immunoblot method. The IgG transported into serum was quickly transferred into CSF after natural suckling in contrast to the case of bovine IgG. Serum concentration of transferrin was maintained at high level before suckling and was not changed by suckling. Transferrin also detected in CSF was not changed by suckling. Bovine lactoferrin (Lf) administered into the intestinal lumen was transported into serum (0.01%) and also detected in CSF after 6 h as undegraded form (3.1%). Thus, homologous IgG and bovine Lf are transported into CSF, suggesting that the transport of macromolecules into CSF is selective in neonatal pigs.

Animals↗
PubMed2001

Induction of CCR7 expression in thymocytes requires both ERK signal and Ca(2+) signal.

CC chemokine receptor 7 (CCR7) expression is crucial for thymocyte trafficking across the corticomedullary junction in the thymus and for lymph node homing of naive T cells. However, the induction mechanism of CCR7 expression is vastly unknown. In isolated CD4+CD8+CCR7-thymocytes, a moderate 20-h pulse stimulation with a combination of the calcium ionophore ionomycin and the protein kinase C activator phorbol myristate acetate induced CCR7 expression and CD8 downregulation. Similar changes were induced in a CD4+CD8+CCR7- T cell line upon stimulation with the same combination of reagents, but not with either one alone. These changes were inhibited by U0126, an inhibitor of the extracellular signal-regulated kinase kinase (ERKK/MEK). The transfectants expressing a constitutively active form of the MEK kinase Raf-1 became CD4+CD8+CCR7+ upon stimulation with ionomycin alone. Thus, Raf-1-mediated signals and Ca(2+)-dependent signals are essential to induce CCR7 expression in CD4+CD8+ T cells and thymocytes as well as their differentiation.

Animals↗
PubMed2001

Apoptosis induced by in vitro infection with simian-human immunodeficiency chimeric virus in macaque and human peripheral blood mononuclear cells.

We investigated apoptosis induced by in vitro infection with the chimeric virus of simian immunodeficiency virus and human immunodeficiency virus (SHIV). Macaque and human peripheral blood mononuclear cells (PBMCs) were infected with pathogenic SHIV-89.6p (89.6p) or nonpathogenic SHIV-NM-3rN (NM-3rN). In macaque PBMCs, the extent of virus production and apoptosis induction in CD4(+) cells was much greater in 89.6p infection than in NM-3rN infection. The result was consistent with our previous study of in vivo SHIV infection. In human PBMCs, 89.6p replicated and induced apoptosis more extensively than did NM-3rN, when the cells were infected with the same infectious doses of the viruses. However, in cells infected with a high dose of NM-3rN, the levels of virus production and apoptosis induction were comparable to those in 89.6p infection. There was no significant difference in the extent of apoptosis induction between 89.6p and NM-3rN infection when growth curves of the two viruses matched. Thus, apoptosis induction by SHIV might depend quantitatively on the amount of virus production rather than on the strains of the virus. Moreover, the correlation between the extent of apoptosis induction and virus pathogenicity in macaque PBMCs has also been found in SHIV-infected macaques. This suggests that the profiles of SHIV infection in vitro reflect the in vivo phenomena. Therefore, the in vitro evaluation of apoptosis induction by SHIV could be useful as a safety test for the development of live-attenuated vaccines.

Animals↗
PubMed2001

Characterization of less pathogenic infectious molecular clones derived from acute-pathogenic SHIV-89.6p stock virus.

For a better understanding of the acute pathogenicity of SHIV-89.6P stock virus, which induces prominent CD4 cell loss within a month after inoculation in monkeys, we have constructed four infectious molecular clones (cl 18, cl 64, cl 69, and cl 71). Cl 64, cl 69, and cl 71, like the parental virus, showed a high in vitro replication ability and a pathogenic-like effect (CD4 downmodulation) in a monkey CD4(+) cell line, whereas cl 18 showed a lower replication ability and could not downmodulate CD4. Cl 64, which has characteristics similar to those of the parental virus in vitro, was inoculated into four rhesus monkeys. All monkeys showed a plasma viral load similar to that of the parental virus with a peak at 2 weeks after inoculation. However, the viral load gradually decreased and the virus failed to cause an AIDS-like disease in infected monkeys, but it induced a strong antiviral antibody response. These results demonstrate the polyclonal nature of the parental SHIV-89.6P virus stock and demonstrate that cl 64, aside from its high replicability, may differ qualitatively from the parental virus.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin.

This study was designed to clarify the mechanisms of hypocholesterolemic action of beta-lactoglobuline tryptic hydrolysate (LTH) and to identify the novel hypocholesterolemic peptide derived from LTH by screening using Caco-2 cells and animal studies. Serum and liver cholesterol levels were significantly lower in rats fed LTH than in those fed casein tryptic hydrolysate (CTH). The present study suggests that the inhibition of micellar solubility of cholesterol which causes the suppression of cholesterol absorption by a direct interaction between cholesterol mixed micelles, and LTH in the jejunal epithelia is part of the mechanism underlying the hypocholesterolemic action of LTH. Though no one could trace the hypocholesterolemic peptide to any protein origin, we identified, for the first time, a novel hypocholesterolemic peptide, Ile-Ile-Ala-Glu-Lys (IIAEK). Surprisingly, the present study provides the first direct evidence that a new hypocholesterolemic peptide derived from beta-lactoglobuline can powerfully influence serum cholesterol levels and exhibit a greater hypocholesterolemic activity in comparison with that of medicine, beta-sitosterol, in animal studies.

Administration, Oral↗
PubMed2001

Construction of SIV/HIV-1 chimeric viruses having the IL-5 gene and determination of their ability to replicate and produce IL-5.

During the progression of AIDS, there is a shift in abundance of immune cells from Th1-producing cells to Th2-producing cells. To determine whether this change might have an effect on HIV-1 replication in vivo, we constructed simian/human immunodeficiency chimeric viruses having the human IL-5 gene (a Th2-type cytokine) and examined the effect of the inserted gene on viral replication, IL-5 production and viral stability in vitro. The DNA of human IL-5 was inserted into vpr-deleted and nef-deleted infectious SHIVs. The obtained replication-competent viruses were used to infect human T-cell lines and monkey peripheral blood mononuclear cells. As a result, at the time of peak NI-IL5 virus production, IL-5 was produced with a significantly higher titer than 3sj-IL5. The functionality of the produced IL-5 was confirmed by IL-5-dependent cells. The replication of both SHIVs having IL-5 appeared to be faster than that of the parental viruses without the IL-5 gene. These results show that co-expression of IL-5 stimulates SHIV replication in vitro. Thus, it is expected that expression of IL-5 will also have an effect on viral replication and pathogenicity in vivo.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Transarterial intravenous coil embolization of dural arteriovenous fistula involving the superior sagittal sinus.

BACKGROUND: We report a rare case of traumatic dural arteriovenous fistula involving the superior sagittal sinus successfully treated by transarterial intravenous coil embolization. CASE PRESENTATION: A 38-year-old woman presented with tension headache. She had a past history of severe head injury at the age of three. Computed tomography scanning showed a heterogenous low-density area in the right frontal lobe, and magnetic resonance imaging demonstrated abnormal vascular structures in the same area. Angiography revealed a dural arteriovenous fistula involving the lateral wall of the fully patent superior sagittal sinus. The fistula was fed by scalp, meningeal, and cortical arteries, and drained into a cortical vein leading to the superior sagittal sinus. Femoral transarterial intravenous embolization with microcoils completely occluded the dural arteriovenous fistula. CONCLUSION: Severe head injury may lead to asymptomatic dural arteriovenous fistulas after a long time. Transarterial intravenous coil embolization can be effective in the treatment of dural arteriovenous fistulas involving the superior sagittal sinus.

Adult↗
PubMed2001

Using SHIVs to develop an anti-HIV-1 live-attenuated vaccine.

The use of chimeric simian and human immunodeficiency viruses (SHIVs) that encode HIV-1 Env and are infectious to macaques has made it possible to analyze the pathogenicity of HIV-1 in vivo, and to evaluate the efficacy of candidate vaccines in macaques. In addition, we believe that gene-deleted SHIVs could potentially be used as anti-HIV-1 live-attenuated vaccines. Gene-deleted SHIVs replicate transiently, are non-pathogenic and induce strong protection against challenge infection. The most important advantage of gene-deleted SHIVs is that their efficacy and safety can be evaluated in macaques before they are used in humans.

AIDS Vaccines↗
PubMed2001

Characterization of hepatitis B virus surface antigen-specific CD4+ T cells in hepatitis B vaccine non-responders.

AIM: To study the mechanisms of hepatitis B vaccine non-response, we examined hepatitis B virus surface antigen (HBsAg)-induced proliferation of peripheral blood mononuclear cells (PBMC) obtained from hepatitis B (HB) vaccinees. METHODS: Subsequently, we have examined the features of HBsAg-reactive CD4+ T cells in HB vaccine non-responders (NR). Based on serum antibodies to HBsAg (anti-HBs) titers, we divided these vaccinees into three groups: high responder (HR), middle responder (MR) and non-responder (NR), and examined HBsAg-induced proliferation of their PBMC. RESULTS: We found that the in vitro response of PBMC to stimulation with HBsAg was correlated with their serum anti-HBs titer (mean stimulation index was 10.71 in HR, 4.37 in MR and 1.96 in NR). However, by the deletion of CD8+ T cells, the increased response was observed in two of four NRs. CONCLUSIONS: The present results have also shown that at least four distinct HBsAg-reactive CD4+ clones existed (variable gene of T cell receptor beta (V beta) 17 + clone restricted with HLA-DR locus (DR4), V beta 8 + clone restricted with HLA-DQ locust (DQ7), and both V beta 5.1 + clone and V beta 20 + clone restricted with either DR9 or DQ3) in NRs. The results demonstrated that heterogeneous HBsAg-reactive CD4 clones existed in some HB vaccine NRs.

Adult↗
PubMed2001

The effect of casein phosphopeptides on calcium absorption from calcium-fortified milk in growing rats.

The effect of casein phosphopeptides (CPP) prepared from bovine casein by enzymatic hydrolysis (extrinsic CPP) on Ca absorption from Ca-fortified milk was studied in young male rats, in comparison with that produced from casein in the small intestine (intrinsic CPP). The gastrointestinal Ca disappearance (Ca ingested - (gastric Ca + intestinal Ca )) was calculated as an indirect measurement of Ca absorption. After being fasted overnight, the animals were given 2.0 ml Ca-fortified milk (30 g fat, 35 g protein, 2.7 g Ca/kg) without or with 1.0 mg extrinsic CPP/ml, by gastric intubation. The intestinal soluble Ca level after 15 min and the gastrointestinal Ca disappearance after 15 and 30 min in the rats given Ca-fortified milk with 1.0 mg extrinsic CPP/ml were significantly higher than these figures in the rats given Ca-fortified milk without CPP (P < 0.05). When the rats were given unfortified milk (1.35 g Ca/kg) in another reference experiment, no significant effect on intestinal soluble Ca and gastrointestinal Ca disappearance was apparent from the addition of CPP to milk. Ca availability was estimated by measuring 45Ca-deposits in the bones of rats 48 h after being given 2.0 ml Ca-fortified milk labelled with 45Ca (180 kBq/2 ml) with or without 0.25 mg CPP/ml. The levels of 45Ca radioactivity of the femur and tibia from the rats given Ca-fortified milk with extrinsic CPP were significantly higher than those from the control group (P < 0.05). These results suggest that the addition of CPP to Ca-fortified milk could increase Ca absorption by growing rats mainly from CaCO3 added to the milk. The mechanism of CPP related to the interaction of CPP and Ca in the gastrointestinal tract is discussed.

Animals↗
PubMed2001

Bacterial translocation in neonatal rats: the relation between intestinal flora, translocated bacteria, and influence of milk.

BACKGROUND: A high incidence of bacterial translocation in neonates results not only from immaturity of host-defense functions, but also from the dominant colonization of aerobic bacteria in the intestine. Bacterial colonization develops differently among breast-fed, formula-fed, premature, and full-term infants. The purpose of this study was to examine the incidence of bacterial translocation and to identify the translocated bacterial species, relating these findings to the intestinal microflora and to the type of feeding in neonatal rats. METHODS: Animals were divided into three groups: breast-fed normal pups (MR group), formula-fed pups fed via an intragastric cannula implanted esophageally (AR group), and breast-fed pups after the removal of the cannula (Sham group). Artificial rearing was achieved using a machine feeding system. Culture and identification of the bacteria in the intestine, mesenteric lymph nodes, liver, portal blood, and lungs were made using a simplified version of Mitsuoka's method. RESULTS: At 14 days of age, the dominant bacteria in the feces of the MR and Sham Groups were Enterobacteriaceae, Lactobacillus, and Enterococcus, but Enterobacteriaceae and Clostridium were significantly more common in the AR group than in the MR group. The dominant bacteria in the mesenteric lymph nodes were Enterobacteriaceae, Lactobacillus, and Staphylococcus. The extent of systemic bacterial translocation decreased earlier in the Sham group than in the AR group. CONCLUSIONS: The frequency with which species of bacteria were cultured from mesenteric lymph nodes and other peripheral sites did not mirror the composition of the intestinal flora. Among the translocated bacteria, Staphylococcus may be especially hard to recognize and difficult for the host-defense systems to destroy. Breast-feeding inhibited systemic bacterial translocation in the suckling period of the rat.

Animal Feed↗