Lipopolysaccharide Induces CD25-Positive, IL-10-Producing Lymphocytes Without Secretion of Proinflammatory Cytokines in the Human Colon: Low MD-2 mRNA Expression in Colonic Macrophages

Despite the huge number of colonized Gram-negative bacteria in the colon, the normal colon maintains its homeostasis without any excessive immune response. To investigate the potential mechanisms involved, human colonic lamina propria mononuclear cells (LPMCs) obtained from uninflamed mucosa were cultured with lipopolysaccharide (LPS) prepared from Bacteroides vulgatus (BV-LPS) or Bacteroides fragilis (BF-LPS), as representatives of indigenous flora, or pathogenic Salmonella minnesota (SM-LPS). Colonic LPMCs failed to produce inflammatory cytokines in response to any type of LPS. Colonic macrophages barely expressed mRNA for MD-2, an essential association molecule for LPS signaling via Toll-like receptor 4. Further, BV-LPS induced CD25 and Foxp3 expression in lymphocytes and CD4(+)CD25(+) cells expressed IL-10 mRNA. Thus, the low expression of functioning LPS receptor molecules and induction of IL-10-producing CD4(+)CD25(+) lymphocytes by indigenous LPS may play a central role in the maintenance of colonic immunological homeostasis.     

Primary carcinosarcoma of the vagina

Primary carcinosarcoma of the vagina is a very rare tumor, with only eight cases diagnosed as carcinosarcoma in the literature that we are aware of. We recently encountered a case of primary carcinosarcoma of the vagina in a 75-year-old woman. The patient had a history of hysterectomy and bilateral ovariectomy for uterine corpus cancer at 55 years of age. Recurrence of the cancer was suspected 17 years after the operation and irradiation therapy was performed, but the patient died 3 years after the recurrence. Autopsy revealed a mass lesion in the pelvic cavity that originated in the vagina. Histological examination showed that the tumor contained anaplastic carcinoma, rhabdomyosarcoma, leiomyosarcoma and chondrosarcoma components, and it was diagnosed as carcinosarcoma. The histological diagnosis of the uterine corpus cancer was well-differentiated adenocarcinoma, and there was no sarcomatous component. The carcinosarcoma occurred 17 years after the hysterectomy, and it was concluded to be a primary carcinosarcoma of the vagina. This is the first case of primary vaginal carcinosarcoma in which the epithelial and sarcomatous components were clearly identified histologically and immunohistochemically.     

Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells

Since dendritic cells (DC) play pivotal roles in both innate and adaptive immunity, DC can be a good target for immuno-gene therapy. However, the optimal generation method for gene-modified DC has not yet been well exploited. CD34+ cells from cord blood (CB), bone marrow (BM), or peripheral blood (PB) were expanded in a medium containing stem cell factor (SCF), flt 3 ligand (Flt3L) and thrombopoietin (TPO) with or without HESS-5, a murine BM stromal cell line, for 2 weeks (the first expansion step), then differentiated to DC in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), flt 3 ligand (Flt3L), stem cell factor (SCF), tumor necrosis factor-alpha (TNF-alpha), IL-4, and lipopolysaccharide (LPS) for 9 days (the second differentiation step). DC progenitors were transduced with human immunodeficiency virus (HIV) vectors at different time points during the second step. Use of HESS-5 during the first step resulted in more DC generation than without it (cell expansion: CB, 10,461 vs. 354-fold; BM, 962 vs. 225-fold; peripheral blood mononuclear cell (PBMC), 8,506 vs. 240-fold; %DC: CB, 83.4% vs. 76.9%; BM, 83.6 vs. 69.8%; PBMC, 85.9 vs. 60.5%). Gene transduction to the in vitro expanded DC progenitors at day 3 during the second step, resulted in better final yield of the gene-modified DC than that to those at day 0 or day 6 (as much as 44% of DC expressed green fluorescence protein (GFP) as a transgene) and the transduction efficiency correlated with endocytic ability and percent of S phase. DC transduced with an HIV vector encoding a melanoma antigen, MART-1, were adequately recognized by specific anti-MART-1 CTL. The two-step culture method with HESS-5 is useful for rapid expansion of DC progenitors and subsequent lentiviral gene transduction to DC.     

The structure of the avian fast skeletal muscle troponin T gene: seven novel tandem-arranged exons in the exon x region

To elucidate the mechanism that produces enormous molecular diversity in troponin T (TnT) of fast skeletal muscle, we determined the 5-half genomic sequence of the chicken fast muscle TnT gene. The sequence of ca. 16 kb included seven exons (exons 1, 2, 3, 4, w, 5, and 6), which have been reported previously and presumed by sequencing TnT cDNAs. Additionally we found six 15 nt and one 18 nt sequences in the region between exons 5 and 6 (i.e. the exon x region). They were encompassed by consensus splice donor and acceptor sites and preceded by putative branch sites, and designated herein as exons xa to xg. Our result shows that the sequence derived from exons x1, x2, and x3, the exons presumed previously by cDNA sequencing, is actually encoded by the seven exons xa to xg, establishing the precise gene structure in the exon x region. Based on our data, together with that on the 3-half genomic sequence of the quail fast muscle TnT gene, we conclude that the avian fast skeletal muscle TnT gene includes 27 exons, 16 of which are alternatively spliced.     

Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes

The genus Thermococcus, comprised of sulfur-reducing hyperthermophilic archaea, belongs to the order Thermococcales in Euryarchaeota along with the closely related genus Pyrococcus. The members of Thermococcus are ubiquitously present in natural high-temperature environments, and are therefore considered to play a major role in the ecology and metabolic activity of microbial consortia within hot-water ecosystems. To obtain insight into this important genus, we have determined and annotated the complete 2,088,737-base genome of Thermococcus kodakaraensis strain KOD1, followed by a comparison with the three complete genomes of Pyrococcus spp. A total of 2306 coding DNA sequences (CDSs) have been identified, among which half (1165 CDSs) are annotatable, whereas the functions of 41% (936 CDSs) cannot be predicted from the primary structures. The genome contains seven genes for probable transposases and four virus-related regions. Several proteins within these genetic elements show high similarities to those in Pyrococcus spp., implying the natural occurrence of horizontal gene transfer of such mobile elements among the order Thermococcales. Comparative genomics clarified that 1204 proteins, including those for information processing and basic metabolisms, are shared among T. kodakaraensis and the three Pyrococcus spp. On the other hand, among the set of 689 proteins unique to T. kodakaraensis, there are several intriguing proteins that might be responsible for the specific trait of the genus Thermococcus, such as proteins involved in additional pyruvate oxidation, nucleotide metabolisms, unique or additional metal ion transporters, improved stress response system, and a distinct restriction system.     

Neurons in the rostral cingulate motor area monitor multiple phases of visuomotor behavior with modest parametric selectivity

We examined the cellular activity in the rostral cingulate motor area (CMAr) with respect to multiple behavioral factors that ranged from the retrieval and processing of associative visual signals to the planning and execution of instructed actions. We analyzed the neuronal activity in monkeys while they performed a behavioral task in which 2 visual instruction cues were given successively with an intervening delay. One cue instructed the location of the target to be reached; the other cue instructed which arm was to be used. After a second delay, the monkey received a motor-set cue to be prepared to initiate the motor task in accordance with instructions. Finally, after a GO signal, the monkey reached for the instructed target with the instructed arm. We found that the activity of neurons in the CMAr changed profoundly throughout the behavioral task, which suggested that the CMAr participated in each of the behavioral processing steps. However, the neuronal activity was only modestly selective for the spatial location of the visual signal. We also found that selectivity for the instructional information delivered with the signals (target location and arm use) was modest. Furthermore, during the motor-set and movement periods, few CMAr neurons exhibited selectivity for such motor parameters as the location of the target or the arm to be used. The abundance and robustness of the neuronal activity within the CMAr that reflected each step of the behavioral task and the modest selectivity of the same cells for sensorimotor parameters are strikingly different from the preponderance of selectivity that we have observed in other frontal areas. Based on these results, we propose that the CMAr participates in monitoring individual behavioral events to keep track of the progress of required behavioral tasks. On the other hand, CMAr activity during motor planning may reflect the emergence of a general intention for action.     

PCR analysis of the Y chromosome long arm in azoospermic patients: evidence for a second locus required for spermatogenesis

We analyzed DNA from 63 Japanese men with either azoospermiaor severe oligospermia whose Y chromosomes were cytogeneticallynormal. A total of 16 loci were examined: 15 loci on the longarm between DYS7E and DYZ1, and the YRRM1 locus, a candidategene for the azoospermic factor, AZF. One patient with a perlcentricinversion of the Y chromosome was also included. We detectedmicro-deletions in ten individuals. The YRRM1 gene was Involvedin only three of them. The remaining seven patients showed deletionbetween DYS7C and DYS239 in common, indicating the presenceof at least one additional gene, deletion of which causes azoospermia.     

Apparent superiority of H2-receptor stimulation and simultaneousβ-blockade over conventional treatment withβ-sympathomimetic drugs in post-acute myocardial infarction: Cardiac effects of impromidine — a new specific H2-receptor agonist — in the surviving catecholamine-insensitive myocardium

Left ventricular infarction (AMI) was produced in experimental animals and the contractile response to -adrenergic and H₂-histaminergic stimulation by isoproterenol and impromidine tested in the isolated perfused heart preparation. Adenylate cyclase activity as well as binding characteristics of [³H]-dihydroalprenolol ([³H]-DHA), [³H]-methyl-tiotidine ([³H]-TIOT) and [³H]-quinuclidinyl benzilate ([³H]-QNB) to cardiac ₁-, H₂- and cholinergic muscarinic receptors were determined in sarcolemmal membrane preparations of the right ventricle of the same hearts. In addition, an attempt was made to elucidate the therapeutic value of post-AMI treatment with impromidine in the presence and absence of-sympathomimetic, in contrast to administration of prenalterol and the conventional therapy with -sympathomimetic drugs, e.g. dobutamine. Three days post-AMI the dose-response curve for isoproterenol of right ventriculardP/dt _max was significantly depressed, while the inotropic effect of impromidine was not impaired. Stimulation of adenylate cyclase activity by isoproterenol was reduced by 80% whereas impromidine and NaF stimulation rates were unaltered. Receptor-binding studies indicated a 90% loss and 10-times lowered affinity (K _D) of the remaining -receptors while specific [³H]-TIOT- and [³H]-QNB-binding was unchanged.Administration of dobutamine increased mortality rates and extension of infarct size, led to a further decrease in contractile response to isoproterenol, induced complete insensitivity of adenylate cyclase to isoproterenol stimulation and caused pronounced additional reduction of number and affinity of [³H]-DHA-binding sites. In contrast, all above alterations were prevented by treatment with either prenalterol or combined administration of impromidine plus metoprolol. It is concluded, that these alterations in the non-ischemic, uninvolved myocardium post-AMI are the result of catecholamine-induced specific damage of sarcolemmal -receptors. Furthermore, treatment with H₂-agonists in combination with -blocking agents may have beneficial effects, whereas conventional therapy with -sympathomimetic drugs tends to worsen the already depressed function of the -adrenergic stimulation mechanism.Supported by grants Ba 666/1 and Ba 666/2-2 from the Deutsche Forschungsgemeinschaft (DFG).Data presented in this paper are part of a doctoral thesis by Dr S.B. Felix.     

Immunomodulation by vitamin B12: augmentation of CD8+ T lymphocytes and natural killer (NK) cell activity in vitamin B12-deficient patients by methyl-B12 treatment

It has been suggested that vitamin B12 (vit.B12) plays an important role in immune system regulation, but the details are still obscure. In order to examine the action of vit.B12 on cells of the human immune system, lymphocyte subpopulations and NK cell activity were evaluated in 11 patients with vit.B12 deficiency anaemia and in 13 control subjects. Decreases in the number of lymphocytes and CD8+ cells and in the proportion of CD4+ cells, an abnormally high CD4/CD8 ratio, and suppressed NK cell activity were noted in patients compared with control subjects. In all 11 patients and eight control subjects, these immune parameters were evaluated before and after methyl-B12 injection. The lymphocyte counts and number of CD8+ cells increased both in patients and in control subjects. The high CD4/CD8 ratio and suppressed NK cell activity were improved by methyl-B12 treatment. Augmentation of CD3-CD16+ cells occurred in patients after methyl-B12 treatment. In contrast, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, lectin-stimulated lymphocyte blast formation, and serum levels of immunoglobulins were not changed by methyl-B12 treatment. These results indicate that vit.B12 might play an important role in cellular immunity, especially relativing to CD8+ cells and the NK cell system, which suggests effects on cytotoxic cells. We conclude that vit.B12 acts as an immunomodulator for cellular immunity.