Infusion of ex-vivo expanded human TCR-αß+ double-negative regulatory T cells delays onset of xenogeneic graft-versus-host disease.

Despite the demonstration of potent immunosuppressive function of T cell receptor (TCR)-αß+ double-negative regulatory T cells (DN Tregs ), scarce numbers and lack of effective expansion method limit their clinical applications. Here we describe an approach that allows for ∼3500-fold ex-vivo expansion of human DN Tregs within 3 weeks with > 97% purity. Ex-vivo-expanded DN Tregs suppress proliferation of polyclonally stimulated autologous T and B cells in vitro through direct cell-to-cell contact. In vivo, we demonstrate for the first time that infusion of human DN Tregs delayed an onset of xenogeneic graft-versus-host disease (GVHD) significantly in a humanized mouse model. Furthermore, preincubation of ex-vivo-expanded DN Tregs with a mechanistic target of rapamycin (mTOR) inhibitor rapamycin enhanced their immune regulatory function further. Taken together, this study demonstrates that human DN Tregs can be expanded ex vivo to therapeutic numbers. The expanded DN Tregs can suppress proliferation of T and B cells and attenuate GVHD, highlighting the potential clinical use of DN Tregs to mitigate GVHD.

Noradrenergic projections to the ventromedial hypothalamus regulate fat metabolism during endurance exercise.

The regulation of energy metabolism by the central nervous system during endurance exercise was examined. We conducted respiratory gas analysis by functionally paralyzing the ventromedial hypothalamus (VMH), the lateral hypothalamic area, and the paraventricular nucleus of the hypothalamus with local anaesthetic (lidocaine) during treadmill running at a velocity that allowed for efficient fatty acid oxidation. Our results showed that only the lidocaine treatment of the VMH attenuated fatty acid oxidation during endurance exercise. The monoaminergic neural activities at these nuclei during in vivo microdialysis in rats under the same conditions indicated a significant increase in the extracellular concentration of noradrenaline in all nuclei. Similarly, a significant increase in dopamine occurred at some points during exercise, but no change in serotonin concentration occurred regardless of exercise. Disruption of noradrenergic projections to the VMH by 6-hydroxydopamine attenuated the enhancement of fat oxidation during running. Blocker treatments clarified that noradrenergic inputs to the VMH are mediated by ß-adrenoceptors. These data indicate that information about peripheral tissues status is transmitted via noradrenergic projections originating in the medulla oblongata, which may be an important contribution by the VMH and its downstream mechanisms to enhanced fatty acid oxidation during exercise.

Synergistic upregulation of inducible nitric oxide synthase and cyclooxygenase-2 in gastric mucosa of Mongolian gerbils by a high-salt diet and Helicobacter pylori infection.

AIMS: The intake of salt and salty food is known as a risk factor for gastric cancer. We have previously demonstrated that a high-salt diet dose-dependently enhances Helicobacter pylori (H. pylori)-associated gastritis and stomach carcinogenesis in Mongolian gerbils. In this study, we focused on the influence of excessive salt intake on the expression of inflammatory mediators involved in progression of H. pylori-induced chronic gastritis. METHODS AND RESULTS: A total of 45 stomach samples from Mongolian gerbils were evaluated by immunohistochemistry. The animals were infected with H. pylori and fed basal (0.32%) or a high-salt (10%) diet, and sacrificed after 40 weeks. Proliferative activity and expression of cyclooxygenase-2 (COX-2) in gastric mucosa were significantly increased in H. pylori-infected gerbils. The additional high-salt diet significantly up-regulated the expression of inducible nitric oxide synthase (iNOS) and COX-2 in H. pylori-infected groups (P<0.01 and P<0.05, respectively), while no significant effects were noted in non-infected animals. There was significant synergistic interaction between H. pylori infection and 10% NaCl diet on the expression of iNOS (P<0.05) and also a tendency for enhanced COX-2 expression (P=0.0599). CONCLUSIONS: The present results suggest that a high-salt diet works synergistically with H. pylori infection to enhance iNOS and COX-2 expression in the gastric mucosa of Mongolian gerbils, and support the hypothesis that excessive salt intake may be associated with progression of H. pylori-induced gastritis.

Down regulation of gastric and intestinal phenotypic expression in Epstein-Barr virus-associated stomach cancers.

AIMS: We have previously demonstrated the importance of gastric and intestinal phenotypic expression for stomach carcinogenesis. In this study, we focused on Epstein-Barr virus (EBV)-associated stomach cancers, with special attention to Cdx2. METHODS AND RESULTS: We evaluated the expression of gastric and intestinal phenotypic markers by immunohistochemistry in 35 EBV-positive [EBV (+)] and 75 EBV-negative [EBV (-)] stomach cancers in Colombia. The lesions were divided phenotypically into gastric (G), gastric-and-intestinal mixed (GI), intestinal (I), and null (N) phenotypes. In the EBV (+) cases, the lesions were divided phenotypically into 9 G (25.7%), 1 GI (2.9%), 3 I (8.6%), and 22 N (62.9%) types. Similarly, the EBV (-) lesions were also classified phenotypically as 15 G (20.0%), 19 GI (25.3%), 24 I (32.0%), and 17 N (22.7%) types. The proportion of N type EBV (+) lesions was higher than for their EBV (-) counterparts (P<0.0001). The expression of Cdx2 and MUC2 was also found to be significantly lower in EBV (+) than in EBV (-) stomach cancers (P=0.0001; P<0.0001). Cdx2 expression in the intestinal metaplastic glands present in non-neoplastic mucosa surrounding EBV (+) lesions was also significantly lower than in EBV (-) tumors (P=0.016) despite no evidence of EBV infection. CONCLUSIONS: EBV (+) stomach cancers are characterized by low expression of intestinal phenotype markers, including Cdx2, and only occasional gastric phenotypic expression.

Loss of MUC2 expression correlates with progression along the adenoma-carcinoma sequence pathway as well as de novo carcinogenesis in the colon.

AIMS: We have previously demonstrated links between clinicopathological findings and phenotypes using several gastric and intestinal phenotypic markers in stomach and pancreatic cancers. However, the clinicopathological significance of the phenotype and Cdx2 expression has hitherto remained unclear in colorectal carcinogenesis. METHODS AND RESULTS: We examined the correlation between gastric and intestinal phenotypic expression in 91 primary early carcinomas of the colon. MUC2 expression demonstrated a significant decrease from tubular/tubulovillous adenomas with moderate atypia, through intramucosal carcinomas, to cancers with submucosal invasion (P<0.0001). Intramucosal de novo carcinomas (flat type carcinomas without adenomatous components) exhibited a greater decrease of MUC2 than intramucosal lesions with adenomatous components. Expression of MUC5AC also decreased significantly with progression according to the tubular/tubulovillous adenoma-carcinoma sequence, carcinomas with villous adenomatous components having a higher level compared with their tubular adenomatous counterparts, suggesting differences in the pathway of malignant transformation. Cdx2 nuclear expression was maintained in all of the adenomas and early carcinomas examined. CONCLUSIONS: Our data suggest that the reduction of MUC2 expression may be associated with the occurrence and progression of colorectal carcinomas in both adenoma-carcinoma sequence pathway and de novo carcinogenesis. Tumor-suppressive effects of Cdx2 may be preserved during early stages of colorectal carcinogenesis.

Gastric and intestinal phenotypic correlation between exocrine and endocrine components in human stomach tumors.

We have previously suggested that an origin of a stomach cancer is from a progenitor cell specializing toward exocrine cell (Exo-cell) lineages. To clarify whether our hypothesis is correct or not, we analyzed the expression of Exo-cell and endocrine cell (End-cell) markers in a series of lesions for comparison. We evaluated chromogranin A (CgA) expression in 37 early and 73 advanced stomach cancers, in 30 stomach adenomas, in 8 carcinoid tumors, and in 4 endocrine cell carcinomas (ECCs) with assessment of gastric and/or intestinal (G/I) phenotypes in both Exo-cell and End-cell by immunohistochemistry. CgA expression was observed in 10.8% of the early and 16.4% of the advanced stomach cancers, respectively. The End-cell G/I phenotypes were in line with the Exo-cell counterparts in the CgA-positive stomach cancerous areas, and there was strong association between Cdx2 expression and the intestinal End-cell markers. All of the adenoma cases had the intestinal Exo-cell phenotypic expression, with the positive link between Exo-cell and End-cell G/I phenotypes. All stomach carcinoids had CgA expression but no expression of Exo-cell markers. In conclusion, most stomach cancers might develop from a progenitor cell specializing towards Exo-cell lineages, but some cases possessed both Exo-cell and End-cell markers with maturely differentiated phenotypes. In such cases, Exo-cell and End-cell phenotypes were found to correlate strongly, suggesting the possibility of histogenesis from "cancer stem cells".

Neurotropism of swine haemagglutinating encephalomyelitis virus (coronavirus) in mice depending upon host age and route of infection.

Mice aged 1, 4 or 8 weeks were inoculated with haemagglutinating encephalomyelitis virus (HEV), strain 67N, by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) or oral route, with different doses. In 1-week-old mice, mortality and mean time to death were mostly the same regardless of the inoculation route, except for the oral route, which appeared to be the least effective. The virus killed 4-week-old mice readily by all routes of inoculation except the oral, and 8-week-old mice by i.c., i.n. or s.c. inoculation. In descending order of efficacy, the routes of HEV infection were: i.c., i.n., s.c., i.p., i.v. and oral. To follow the spread of HEV from peripheral nerves to the central nervous system (CNS), the virus was inoculated subcutaneously into the right hind leg of 4-week-old mice. The virus was first detected in the spinal cord on day 2, and in the brain on day 3. The brain titres became higher than those of the spinal cord, reaching a maximum of 10(7)PFU/0.2 g when the animals were showing CNS signs. Viral antigen was first detected immunohistochemically in the lumbar spinal cord and the dorsal root ganglion ipsilateral to the inoculated leg; it was detected later in the pyramidal cells of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum but not in the ependymal cells, choroid plexus cells or other glial cells. The infected neurons showed no cytopathological changes.

Tob is a negative regulator of activation that is expressed in anergic and quiescent T cells.

During a search for genes that maintain T cell quiescence, we determined that Tob, a member of an anti-proliferative gene family, was highly expressed in anergic T cell clones. Tob was also expressed in unstimulated peripheral blood T lymphocytes and down-regulated during activation. Forced expression of Tob inhibited T cell proliferation and transcription of cytokines and cyclins. In contrast, suppression of Tob with an antisense oligonucleotide augmented CD3-mediated responses and abrogated the requirement of costimulation for maximal proliferation and cytokine secretion. Tob associated with Smad2 and Smad4 and enhanced Smad DNA-binding. The inhibitory effect of Tob on interleukin 2 (IL-2) transcription was not mediated by blockade of NFAT, AP-1 or NF-kappaB transactivation but by enhancement of Smad binding on the -105 negative regulatory element of the IL-2 promoter. Thus, T cell quiescence is an actively maintained phenotype that must be suppressed for T cell activation to occur.

CD40 activation of carcinoma cells increases expression of adhesion and major histocompatibility molecules but fails to induce either CD80/CD86 expression or T cell alloreactivity.

A major obstacle for the development of cancer immunotherapy is the poor capacity of most tumor cells to present antigen. It has previously been shown that ligation of CD40 on the surface of malignant B cells results in the induction of efficient antigen presentation primarily because of upregulated expression of MHC, costimulatory, and adhesion molecules. Ongoing clinical trials are testing the impact of CD40 ligation as immunotherapy for B cell malignancies. Because CD40 is also widely expressed in carcinomas, we studied whether CD40 activation of these cells using soluble recombinant trimeric human CD40 ligand (srhCD40L) can also induce T cell responses. Here, we show that carcinoma cells upregulate expression of CD54 and MHC molecules following in vitro exposure to srhCD40L but do not upregulate CD80 or CD86. CD40-activated carcinoma cells failed to trigger mixed lymphocyte reactions, in sharp contrast to CD40-activated lymphoma cells for which CD40 activation, as expected, resulted in increased expression of MHC, adhesion, and costimulatory molecules, and generated brisk allogeneic lymphocyte reactions. Retroviral-mediated expression of CD80 in carcinoma cells, with or without CD40 activation, triggered mixed lymphocyte reactions, provided cells were treated with IFN-gamma. Thus, the cell surface phenotype induced on carcinoma cells following CD40 activation is not fully capable of inducing T cell proliferation; however, these results support ongoing efforts to exploit costimulation in clinical efforts aimed at increasing carcinoma immunogenicity.

TMC-69, a new antitumor antibiotic with Cdc25A inhibitory activity, produced by Chrysosporium sp. TC1068. Taxonomy, fermentation and biological activities.

A new antibiotic designated TMC-69 has been isolated from the fermentation broth of a fungal strain Chrysosporium sp. TC 1068. TMC-69 exhibited moderate in vitro cytotoxic activity. TMC-69-6H, a derivative of TMC-69 prepared by hydrogenation, possessed more potent in vitro cytotoxicity than TMC-69, and exhibited in vivo antitumor activity against murine P388 leukemia and B16 melanoma. TMC-69-6H was found to specifically inhibit Cdc25A and B phosphatases.

Serial changes of herniated intervertebral discs after posterior lumbar discectomy: the relation between magnetic resonance imaging of the postoperative intervertebral discs and clinical outcome.

To evaluate the relation between the morphologic changes of postoperative intervertebral discs and the clinical outcome after posterior lumbar discectomy, the size of the bulging disc was analyzed prospectively on serial follow-up magnetic resonance images in 26 randomly selected patients. The bulging of postoperative intervertebral discs involved three patterns of reduction: early reduction (n = 15), gradual reduction (n = 6), and late reduction (n = 5). There was a significant difference in the serial changes of subjective symptoms and neurologic function among the three patterns. A late reduction of postoperative disc bulging could cause late recovery of subjective symptoms and neurologic disturbance.

Susceptibility of rats of different ages to inoculation with swine haemagglutinating encephalomyelitis virus (a coronavirus) by various routes.

Haemagglutinating encephalomyelitis virus, strain 67N, was used to inoculate 1-, 2-, 4- and 8-week-old rats by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) and oral routes with graded doses. The routes of infection, in descending order of efficacy, were: i.ci.ns.ci.pi.v. and oral. Rats aged 1 and 2 weeks were generally similar in terms of mortality and mean time to death, regardless of inoculation route, except for the oral route, which had little effect. In comparison with the 1- and 2-week-old rats, the 4-week-old rats were less susceptible to the virus by all routes. Eight-week-old rats inoculated by the i.ci.n. or s.c. routes died, but all those inoculated by other routes survived. To follow the spread of virus in the central nervous system, 4-week-old rats inoculated by the i.c. route were examined. The virus was first detected in the brain on day 1 and in the spinal cord on day 2. The viral titres in both tissues reached a plateau of 10(7) plaque-forming units (PFU)/0.2 g by day 4, at which time clinical signs had developed. By immunohistochemical analysis, virus-specific antigen was found first in the pyramidal cells of the hippocampus and cerebral cortex, and later in the large-sized neurons of the pons and spinal cord. Still later (day 4) immunolabelling was found in Purkinje cells of the cerebellum, but not in the ependymal cells, choroid plexus or other glial cells.

Immunohistochemical study of age-dependent brain lesions in mice infected intracerebrally with Kasba (Chuzan) virus.

When Kasba (Chuzan) virus (an orbivirus) was injected intracerebrally into 1-, 2- or 4-week-old mice, non-purulent necrotizing encephalitis developed and the mice showed nervous symptoms and became moribund. The necrotic lesions were more severe in younger animals. In 1-week-old mice, viral titres rose until 7 days post-infection, while in 2- and 4-week-old animals the titres reached a peak on day 3 and then declined gradually. Glial fibrillary acidic protein-positive astrocytes increased in the white matter, hippocampus and subpial area of the cerebral cortex of infected animals, and lectin-RCA-1-positive cells, thought to be microglial cells, increased in the necrotic lesions. The number of these glial cells increased even after viral titres had declined. In this study there were no survivors in any age group, but survival time increased with age.

Severe fenitrothion poisoning complicated by rhabdomyolysis in psychiatric patient.

Non-traumatic rhabdomyolysis associated with organophosphate intoxication has not been generally reported. We report here in a severe case of fenitrothion poisoning complicated by rhabdomyolysis. A 43-year-old woman ingested approximately 100 ml of fenitrothion emulsion (50%) in an attempt to commit suicide. On day 3 after admission, her creatine phosphokinase (CPK) peaked at 47,762 IU/L. She received supportive treatment included sodium bicarbonate and fluid resuscitation. However, muscarinic symptoms including excessive miosis and salivation developed on day 5 when her CPK levels decreased. The delay in cholinergic symptoms might have been due to the trihexyphenidyl she took with the antipsychotic drugs. Fortunately, the present patient recovered from the acute cholinergic crisis, and acute renal failure was prevented by early diagnosis. This is a case of organophosphate poisoning complicated by rhabdomyolysis in a psychiatric patient. The masking of acute cholinergic symptoms should be taken into consideration in such patients.

Identification of the transcriptional regulatory sequences of human calponin promoter and their use in targeting a conditionally replicating herpes vector to malignant human soft tissue and bone tumors.

The calponin (basic or h1) gene, normally expressed in maturated smooth muscle cells, is aberrantly expressed in a variety of human soft tissue and bone tumors. In this study, we show that expression of the calponin gene in human soft tissue and bone tumor cells is regulated at the transcriptional level by the sequence between positions -260 and -219 upstream of the translation initiation site. A novel conditionally replicating herpes simplex virus-1 vector (d12.CALP) in which the calponin promoter drives expression of ICP4, a major trans-activating factor for viral genes was constructed and tested as an experimental treatment for malignant human soft tissue and bone tumors. In cell culture, d12.CALP at low multiplicity of infection (0.001 plaque-forming unit/cell) selectively killed calponin-positive human synovial sarcoma, leiomyosarcoma, and osteosarcoma cells. For in vivo studies, 10 animals harboring SK-LMS-1 human leiomyosarcoma cells were randomly divided and treated twice on days 0 and 9 intraneoplastically with either 1 x 10(7) plaque-forming units of d12.CALP/100 mm(3) of tumor volume or with medium alone. The viral treatment group showed stable and significant inhibition of tumorigenicity with apparent cure in four of five mice by day 35. Replication of viral DNA demonstrated by PCR amplification and expression of the inserted LacZ gene visualized by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemistry was associated with oncolysis of d12.CALP-treated tumors, while sparing normal vascular smooth muscle cells. In mice harboring two SK-LMS-1 tumors, replication of d12.CALP was detected in a nontreated tumor distant from the site of virus inoculation. These results indicate that replication-competent virus vectors controlled by the calponin transcriptional regulatory sequence may be a new therapeutic strategy for treatment of malignant human soft tissue and bone tumors.

Mutation spectrum of o-aminoazotoluene in the cII gene of lambda/lacZ transgenic mice (MutaMouse).

The o-aminoazotoluene (AAT) has been evaluated as a possible human carcinogen by the International Agency for Research on Cancer. In rodents, it is carcinogenic mainly in the liver, and also in lung following long term administration. We previously examined in lambda/lacZ transgenic mice for the induction of lacZ mutations in liver, lung, urinary bladder, colon, kidney, bone marrow, and testis. AAT induced gene mutations strongly in the liver and colon. In the present report, we reveal the molecular nature of mutations induced by AAT in the lambda cII gene (the cII gene, a phenotypically selectable marker in the lambda transgene, has 294bp, which makes it easier to sequence than the original target, the 3kb lacZ gene). The cII mutant frequency in liver and colon was five and nine times higher, respectively, in AAT-treated mice than in control mice. Sequence analysis revealed that AAT induced G:C to T:A transversions, whereas spontaneous mutations consisted primarily of G:C to A:T transitions at CpG sites.

Effects of subtotal resection of the fundus on development of intestinal metaplasia induced by X-ray irradiation in Donryu rats.

Eight-week-old male Crj:Donryu rats underwent subtotal resection of the fundus and X-ray irradiation. Six months later the animals were autopsied and examined for intestinal metaplasia. The numbers of alkaline phosphatase-positive foci with the two treatments in combination were significantly increased, compared to the operation alone and non-treatment groups. Histologically assessed intestinal metaplasia was also increased in the combined treatment group. In conclusion, subtotal resection of the fundus combined with X-ray irradiation is an effective induction protocol for intestinal metaplasia.

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay.

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.