In vitro cholesterol-lowering properties of Lactobacillus plantarum AN6 isolated from aji-narezushi.

Aji-narezushi is a traditional lactic acid-fermented fish. In this study, we screened for lactose-utilizing, acidophilic, bile-resistant and cholesterol-lowering lactic acid bacteria (LAB) from aji-narezushi for use as starter strains for fermented foods, as well as for use as probiotics. Of the 301 LAB isolates, 277 fermented lactose, and among these, 171 grew in de Man, Rogosa and Sharpe broth adjusted to pH 3·5. Thirty-four of the isolates were grown in a broth containing 3% (w/v) bile. All of the isolates were lactobacilli. Seven isolates that demonstrated cholesterol-lowering activity in ethanolic solution were selected. All of the isolates were identified as Lactobacillus plantarum. Lactobacillus plantarum AN6 showed the highest cholesterol-lowering activity. AN6 was more resistant to acid, salt and bile than the type strain NBRC15891(T). One-half of the cholesterol-lowering effect remained after boiling AN6 for 10 min. The Fourier transform infrared (FT-IR) analysis indicated that the content of cell wall polysaccharides in AN6 is higher than ones in the type strain. These results indicate that Lact. plantarum AN6 can be used as a profitable starter organism and probiotic.

Double-balloon endoscopy versus magnet-imaging enhanced colonoscopy for difficult colonoscopies, a randomized study.

BACKGROUND AND STUDY AIMS: Studies have estimated that failure of cecal intubation occurs with conventional colonoscopy in up to 10 % of cases. Double-balloon endoscopy (DBE) systems, magnetic endoscope imaging (MEI), and transparent cap have been shown to improve success rates for colonoscopy. This study evaluated the utility of DBE for complete examination of the colon compared with MEI plus cap (MEI-Cap) after incomplete or technically difficult colonoscopy in a randomized comparative manner. PATIENTS AND METHODS: A total of 94 patients with incomplete or technically difficult colonoscopy were randomly assigned to receive either DBE (n = 47) or colonoscopy with MEI-Cap (n = 47). The primary end point was cecal intubation rate within 30 minutes. Secondary end points included intubation time, pain score using a visual analog scale, abdominal pressure attempts, doses of sedative medication, and changes in patient position during colonoscopy. RESULTS: Patient characteristics were comparable in both groups. Cecal intubation rate within 30 minutes was significantly higher for DBE (45 /47, 95.7 %) than for MEI-Cap (34 /47, 72.3 %) (P = 0.0049). Mean time to reach the cecum was significantly lower in the DBE group (13.0 ±â€Š5.3 minutes) than in the MEI-Cap group (16.4 ±â€Š4.8 minutes; P = 0.0003). No complications were encountered in either group.   CONCLUSION: DBE is more useful for complete examination of the colon than MEI-Cap in patients with incomplete or technically difficult colonoscopy.

Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas.

We have created a double mutant of the herpes simplex virus (HSV) type 1 (termed G207) with favourable properties for treating human malignant brain tumours: replication-competence in glioblastoma cells (and other dividing cells), attenuated neurovirulence, temperature sensitivity, ganciclovir hypersensitivity, and the presence of an easily detectable histochemical marker. G207 has deletions at both gamma 34.5 (RL1) loci and a lacZ gene insertion inactivating the ICP6 gene (UL39). G207 kills human glioma cells in monolayer cultures. In nude mice harbouring subcutaneous or intracerebral U-87MG gliomas, intraneoplastic inoculation with G207 causes decreased tumour growth and/or prolonged survival. G207 is avirulent upon intracerebral inoculation of mice and HSV-sensitive non-human primates. These results suggest that G207 should be considered for clinical evaluation in the treatment of glioblastomas.

Treatment of human malignant meningiomas by G207, a replication-competent multimutated herpes simplex virus 1.

We have demonstrated that replication-competent attenuated mutants of herpes simplex virus type 1 (HSV-1) have therapeutic potential for malignant gliomas. Moreover, a recently described multiple mutant HSV (termed G207) has properties which may allow human clinical trials. G207 is able to replicate within and kill cells from three human malignant meningiomas in cell culture. In nude mice harboring s.c. human malignant meningioma (F5), G207 can inhibit growth in a dose-dependent fashion. In nude mice harboring intracranial subdural human malignant meningioma (F5), one injection of G207 caused significantly decreased tumor growth and one apparent cure with neither neurological dysfunction nor pathological changes in the surrounding brain. These results suggest that G207 should be considered for therapeutic trials in the treatment of malignant meningioma refractory to currently available therapies.

Cell-adhesion proteins of the immunoglobulin superfamily in the nervous system.

The unique groups of cell-adhesion proteins, such as IgSF, play essential parts in the formation and maintenance of the nervous system. Recent crystallographic studies have revealed a possible common structure of cell-adhesion proteins. The IgSF proteins are sub-grouped into simple, complex and mixed types. Accumulating evidence reveals the importance of cell-adhesion proteins in neural morphogenesis, maintenance and regeneration. They play key roles in neuronal migration, neurite outgrowth promotion, neurite fasciculation, pathfinding, target recognition, synaptogenesis and myelination. Mutations of cell-adhesion proteins result in neurological disease; for example, mutations of PO in hereditary neuropathy and mutations of L1 in hereditary hydrocephalus, MASA syndrome and spastic paraplegia type 1. Perspectives of the studies of neural cell-adhesion proteins are discussed.

Intravesical and intravenous therapy of human bladder cancer by the herpes vector G207.

G207, a conditionally replicating herpes vector, efficiently kills human bladder cancer cells in vitro. To evaluate the therapeutic potential of G207, we have established three in vivo models similar to the clinical situation. In vivo, G207 was intraneoplastically, intravesically, or intravenously inoculated in nude mice. Intraneoplastic inoculation into subcutaneous tumor caused significant tumor growth inhibition. Intravesical inoculation of G207 also caused decreased tumor growth in an orthotopic human bladder cancer model. Furthermore, multiple intravenous inoculation markedly inhibited subcutaneous tumor growth. These results suggest that intravesical therapy with G207 is effective for localized bladder tumor, especially for carcinoma in situ (CIS), and intravenous therapy with G207 is promising for invasive or metastasized bladder tumor.

Glycopeptide of P0 protein inhibits homophilic cell adhesion. Competition assay with transformants and peptides.

Expression of major myelin glycoprotein P0 by P0 cDNA transfection into C6 glioma cells promoted homophilic cell adhesion of the cells. After the dissociated cells were incubated for various times, the number of particles at each time point was measured. The total number of particles decreased to 24% in 60 min for transformant (C6P0) cells, in contrast to only 68% for control (C6P0') cells. To confirm the homophilic mechanism of adhesion, mixed-cell aggregation experiments were performed. Among the four synthetic peptides corresponding to a part of the P0 sequence used, only peptide 3 (residues 90-96), which contained a carbohydrate attaching site, caused considerable inhibition of cell aggregation (approximately 50%). In addition, the glycopeptide (residues 91-95) obtained from bovine P0 markedly inhibited cell aggregation (by approximately 85%).

Expression of L1 and TAG-1 in the corticospinal, callosal, and hippocampal commissural neurons in the developing rat telencephalon as revealed by retrograde and in situ hybridization double labeling.

In the telencephalon, the corticospinal (CS), callosal, and hippocampal commissural neurons are the major types of neurons that have axons crossing the midline of the brain. To understand the mechanisms involved in crossing the midline structure and to examine whether the expression patterns of L1 and TAG-1 in the commissural neurons are similar to those in the spinal cord, we investigated L1 and TAG-1 expression in these neurons in rats by using a double-labeling technique involving retrograde labeling and in situ hybridization. Expression of L1 messenger RNA was detected in the retrogradely labeled CS projection neurons by 1,1;-dioctadecyl-3,3, 3;,3;-tetramethylindocarbocyanine perchlorate (DiI) injection into the pons at embryonic day (E) 19, but expression of TAG-1 messenger RNA was not detected in these neurons. Also, after their axons crossed the pyramidal decussation, continued expression of L1 but no expression of TAG-1 in the CS projection neurons was shown by an additional double-labeling experiment involving DiI injection into the spinal cord at postnatal day (P) 1. An immunohistochemical study showed that L1 was continuously present in each level of the CS tract at E21 and P3, but TAG-1 immunoreactivity was not found in any level at any stage. Finally, we examined the expression of L1 and TAG-1 messenger RNAs in the callosal and hippocampal commissure neurons after their axons had crossed the midline by using the double-labeling technique. In both cases, hybridization signals of the L1 and TAG-1 messenger RNAs were observed in the retrogradely labeled neurons at P3. These results suggest that the roles of L1 and TAG-1 in the formation of the commissures in the forebrain are different from their roles in the spinal cord.

Antitumoral effects of defective herpes simplex virus-mediated transfer of tissue inhibitor of metalloproteinases-2 gene in malignant glioma U87 in vitro: consequences for anti-cancer gene therapy.

We set up experiments to evaluate the effects of defective herpes simplex virus (HSV)-mediated in vitro gene transfer of tissue inhibitor of metalloproteinases-2 (TIMP-2) in malignant glioma cells. Intrinsic TIMPs are known to be inhibitors of the strong invasive activities of matrix metalloproteinases in malignant gliomas. The defective HSV vectors dvSRaTIMP2 was engineered to express human TIMP-2 (hTIMP-2) with a combination of replication-competent HSV mutant, temperature-sensitive HSV-tsK, and amplicon plasmid-containing hTIMP-2. The hTIMP-2 gene was driven by the simian virus 40 promoter. The helper virus (HSV-tsK) was thermosensitive; consequently, this vector could proliferate only at 31.5 degrees C. After infection of U87 human glioblastoma cells with the vector in vitro, expression of TIMP-2 was confirmed by reverse zymography. The U87 cells infected in vitro either with dvSRaTIMP2 or HSV-tsK were efficiently destroyed under replication-permissive conditions (at 31.5 degrees C) and significantly lowered under replication-nonpermissive conditions (at 37 degrees C). The invasive activity of U87 was clearly inhibited by dvSRaTIMP2 infection at both 31.5 degrees C and 37 degrees C. Our studies suggest that TIMP-2 expressing the defective HSV vector is possibly useful for the treatment of malignant brain tumors.

A prospective study of human herpesvirus-6 infection in renal transplantation.

Sixty-five kidney transplant recipients and their (22 living related and 43 cadaveric) donors were studied prospectively to determine the relationship between kidney transplantation and human herpesvirus-6 (HHV-6) infection. The virus isolation from peripheral blood and other tissues and sequential determination of neutralizing antibodies to HHV-6 were performed during 3 months following the transplantation. All of the donors and their recipients examined had neutralizing antibodies to HHV-6 at the time of renal transplantation and the virus was not isolated from them. HHV-6 was isolated from 3 renal tissues (2 living related and 1 cadaveric) obtained during transplant surgery, but not from their blood at that time. HHV-6 viremia occurred in 9 (14%) of the 65 recipients around 2 to 4 weeks after the transplantation. An additional 27 recipients showed a significant rise in the antibody titer. Thus, the infection with HHV-6 was confirmed in 36 (55%) of the 65. These results indicate that the virus is activated in many cases in the early posttransplant period and that HHV-6 establishes in vivo latency in the kidney tissue. There was no correlation between HHV-6 infection and acute rejection or the antirejection prophylaxis.

Clinical features and viral excretion in an infant with primary human herpesvirus 7 infection.

OBJECTIVE: To find clinical features of a virologically-confirmed patient with primary human herpesvirus 7 (HHV-7) infection and a relationship of the excretion of viruses between HHV-7 and human herpes-virus 6 (HHV-6). PATIENT AND METHODS: A 13-month-old boy who had a known prior history of exanthem subitum at 6 months of age developed fever for 3 days and a skin rash appeared as the fever was resolving. The course was accompanying with nonspecific signs and symptoms such as anorexia, irritability, mild diarrhea, palpebral edema, mild inflammation of pharynx, and mild occipital and cervical lymphadenopathy. Heparinized blood samples were used for isolation of HHV-6 and HHV-7 and detection of both virus DNA sequences by a nested polymerase chain reaction (PCR) amplification. Samples from other body sites were also tested for their DNA sequences using the PCR. Both virus antibody activity was measured by an indirect immunofluorescent assay or a neutralization test. RESULTS: Cultured mononuclear cells from the patient at the acute stage of the disease produced morphologic changes, which reacted only with the monoclonal antibody to HHV-7 but not with the antibody to HHV-6. Both viruses were not isolated from blood obtained at the convalescent stage. An antibody response of the patient indicated a seroconversion to HHV-7 but not to other microbial agents including HHV-6 and Mycoplasma pneumoniae. Both virus DNA sequences were detected in peripheral blood mononuclear cells at acute and convalescent stages. HHV-7 DNA was excreted into saliva and transiently into stool at an early convalescent stage followed by HHV-6 excretion into saliva. No HHV-7 and HHV-6 was excreted into urine. CONCLUSIONS: Clinical features of a virologically confirmed patient with primary HHV-7 infection were comparable with those of primary HHV-6 infection and HHV-7 infection may reactivate HHV-6.

Human herpesvirus-6 infection (exanthem subitum) without rash.

Two patients with a three-day febrile episode in whom exanthem subitum was expected showed no appearance of skin eruption after subsidence of the fever. Virus was isolated from peripheral blood mononuclear cells in the acute stage, which was identified by the cytopathic effects of infected cells and the specific immunofluorescent staining of the cells with convalescent sera from exanthem subitum, but the virus could not be isolated from those in the convalescent stage. The morphology of the virus was similar to herpes group virus. There were seroconversions against representative strain of the causative agent of exanthem subitum in the two patients. The results indicated the presence of atypical clinical course of exanthem subitum.

Protection against varicella in family contacts by immediate inoculation with live varicella vaccine.

A live varicella vaccine (Oka strain) was given to susceptible household contacts of varicella to test the protective efficacy of the vaccination. Twenty-six contacts of 21 families were vaccinated, usually within three days after onset of the index cases. None of the vaccinated children developed clinical symptoms of varicella. Eighteen of 24 sera obtained before vaccination were found to be seronegative by complement fixation and neutralization tests. Seroconversion was observed in all of the 18. On the other hand, all of 19 unvaccinated contacts in the 15 families, who served as controls, showed typical manifestations of varicella from 10 to 33 days after onset of the index varicella cases. In three families, where only one sibling contact received vaccine and the other was an unvaccinated control, none of the vaccinated children showed any clinical symptoms while unvaccinated controls exhibited typical varicella symptoms 10 to 14 days after the onset of the index cases. These results indicate that varicella is prevented in household contacts by vaccination within three days following exposure.

Protective efficacy of vaccination in children in four episodes of natural varicella and zoster in the ward.

It was previously reported that a live varicella vaccine (Oka strain) has been developed and that the immediate vaccination of hospitalized children was effective for prevention of spread of varicella in a ward. Six to nine months later, there were four separate episodes of varicella and zoster in the same ward. Eighteen children (11 with nephrotic syndrome, 6 with nephritis, and 1 with hepatitis) with no history of varicella were inoculated with a live vaccine before or immediately after admittance or occurence of the varicella and zoster cases. Twelve of them had been receiving steroid therapy and 15 of the 18 were found to be seronegative by complement fixation and neutralization tests before the vaccination. All of them became seropositive after vaccination without any clinical symptoms. The longest period between vaccination and exposure was nine months. None of the vaccinees exhibited varicella symptoms after exposure. Serological follow-up of ten vaccinated children was done, and booster responses were observed in some of them after exposure. These results suggest that the live vaccine affords immunity to the recipients. If hospitalized children are vaccinated before or immediately after exposure, isolation of the patient is unnecessary.

Immunoglobulin subclass antibodies to varicella-zoster virus.

Commercially available mouse monoclonal antibodies to human IgG subclass (IgG1 to IgG4) were applied to an enzyme-linked immunosorbent assay to measure IgG subclass-specific antibodies to varicella-zoster virus in children naturally infected with varicella-zoster virus and in varicella vaccine recipients. In children naturally infected with varicella-zoster virus, IgG 1 antibody was detected 2 weeks after onset of the disease in all cases, its activity increased at 1 month after onset, and almost equal antibody value was maintained 10 years after infection. This pattern of antibody response was similar to that of total IgG antibody to varicella-zoster virus after natural infection. On the other hand, low antibody activity was found in IgG2 only at 1 month of the disease. The highest antibody level of IgG3 was shown 2 weeks after onset of the disease; then, it gradually decreased, and no antibody activity was detected 10 years later. IgG4 antibody was first detected 1 month after onset and an almost equal level of antibody was shown 10 years after the disease. After inoculation of children with a live varicella vaccine, in contrast, IgG subclass antibody responses to vaccine recipients were almost equal to those after natural infection.

Distribution of antibodies to a causative agent of exanthem subitum (human herpesvirus-6) in healthy individuals.

The transfer of IgG antibodies to a causative agent of exanthem subitum (human herpesvirus-6) from mother to infant was examined with an indirect immunofluorescence assay. Of 20 mothers, 85% had the antibody more than 1:10, and the significantly higher level of the antibody was found in the cord blood with a positive rate of 95%. A mean ratio of cord blood to maternal antibody titer was 1.63. A total of 301 sera from healthy individuals was examined for the age-specific prevalence of antibody to the virus. In the first 2 months of life, 87% of infants had the antibody; the positive rate and the level of antibody decreased during the first 6 months of life with the lowest positive rate of 6% at 4 to 5 months of age. After 6 months of age, they increased gradually and reached the highest level at 1 year of age with the positive rate of 86%. From 2 years of age, the prevalence of the antibody was almost stable (69% to 76%) and similar to those in adolescents and adults.

Long-term protective immunity of recipients of the OKA strain of live varicella vaccine.

In spite of close contacts with patients who had varicella, 101 of 106 (95%) healthy and sick children (142 of 147 (97%) exposures of these children) who had received the OKA strain of live varicella vaccine 7 to 10 years earlier were protected against the disease completely. Among them, 37 of 38 (97%) vaccine recipients who received immunologic testing had varicella-zoster virus (VZV) antibodies tested by fluorescent antibody to membrane antigen method with a geometric mean titer of 1:9.3, and 37 of the 38 (97%) showed positive skin reaction to varicella-zoster virus antigen with erythema (mean diameter 13.4 mm). These findings were compared with those for 29 children who had contracted typical varicella 7 to 10 years earlier, whose seropositive rate was 100% with a geometric mean titer of 1:10.5, and 97% of whom (28/29) had positive skin reaction with mean diameter of 12.9 mm. These results indicate that the vaccine-induced protective immunity persists for approximately one decade and is almost equal to the long-term immunity following natural infection.

Clinical features of infants with primary human herpesvirus 6 infection (exanthem subitum, roseola infantum).

OBJECTIVE: To clarify clinical features of patients with primary human herpesvirus 6 (HHV-6) infection (roseola infantum, exanthem subitum) in a large-scale study. SUBJECTS AND METHODS: Clinical signs and symptoms were analyzed in 176 infants in whom exanthem subitum was initially suspected and primary HHV-6 infection was later confirmed. The infection was proved by isolation of the virus from blood, a significant increase in the neutralizing antibody titers to the virus, or both. RESULTS: The primary HHV-6 infection, which occurred throughout the year, was observed in 94 boys and 82 girls (mean age, 7.3 months). Fever developed in 98% (mean maximum fever, 39.4 degrees C) and lasted for 4.1 days. Macular or papular rashes appeared in 98%, on face, trunk, or both, mostly at the time of subsidence of the fever, and lasted for 3.8 days. Other clinical manifestations occurred as follows: mild diarrhea in 68%, edematous eyelids in 30%, erythematous papules in the pharynx in 65%, cough in 50%, and mild cervical lymph node swelling in 31%. Twenty-six percent had bulging of the anterior fontanelle and 8% had convulsions. CONCLUSIONS: Clinical features of patients with virologically confirmed exanthem subitum were comparable with those described before discovery of HHV-6.

Postexposure prophylaxis of varicella in family contact by oral acyclovir.

OBJECTIVE: To determine whether varicella can be prevented by administration of oral acyclovir (ACV) during the incubation period of the disease. SUBJECTS AND METHODS: ACV (40 or 80 mg/kg daily in four divided doses) was given orally to 25 exposed infants and children for 7 days, starting 7 to 9 days after exposure from the index case in their families. Their clinical features were compared with those of 25 age-matched control subjects who had been exposed in their families but did not receive ACV. A fluorescent antibody to membrane antigen assay was used for determination of the antibody to varicella-zoster virus, and a nested polymerase chain reaction method was used for detection of viremia. RESULTS: Among the 25 who received ACV, 4 (16%) developed the disease and 1 (4%) had a fever. On the other hand, all of 25 control subjects developed the disease and 17 (68%) had a fever. The incidence of fever and the severity of skin rashes were significantly lower (P < .01) in the subjects who received oral ACV than in the control group. Seroconversion was observed in 84% of subjects who received ACV. In some cases, varicella-zoster virus DNA was detected by polymerase chain reaction amplification in peripheral blood mononuclear cells from blood drawn approximately 14 days after exposure. CONCLUSIONS: Varicella can be prevented or modified by administration of oral ACV late in the incubation period.

Human herpesvirus-6 DNA in cerebrospinal fluid of a child with exanthem subitum and meningoencephalitis.

The involvement of human herpesvirus-6 (HHV-6) and herpes simplex virus infections was evaluated virologically and serologically in a 13-month-old girl with meningoencephalitic illness occurring in the pre-eruptive stage of exanthem subitum. An isolation of HHV-6 from blood and seroconversion to the virus confirmed the primary infection with the virus. HHV-6 gene sequences were detected in cerebrospinal fluid of acute stage of the disease by polymerase chain reaction. There was no evidence of herpes simplex virus infection in blood and cerebrospinal fluid. The patient recovered from the disease without any sequelae, although abnormal electroencephalography and cerebral computed tomography findings were observed temporally in the acute stage of the disease. These findings strongly suggest that HHV-6 invades the central nervous system and causes meningoencephalitis.