Low prevalence of maternal microchimerism in peripheral blood of Japanese children with type 1 diabetes.

AIM: To clarify the prevalence and degree of maternal microchimerism in Japanese children with type 1 diabetes, as well as its effect on phenotypic variation. METHODS: We studied 153 Japanese children with type 1 diabetes, including 124 children positive for ß-cell autoantibodies, and their 71 unaffected siblings. The number of circulating microchimeric cells per 105 host cells was estimated by the use of quantitative-polymerase chain reaction targeting non-transmitted maternal human leukocyte antigen alleles. The results were compared to previous data from white European people. Phenotypic comparison was performed between maternal microchimerism carriers and non-carriers with diabetes. RESULTS: Maternal microchimerism was detected in 15% of children with autoantibody-positive type 1 diabetes, 28% of children with autoantibody-negative type 1 diabetes, and 16% of unaffected siblings. There were no differences in the prevalence or levels of maternal microchimerism among the three groups or between the children with type 1 diabetes and their unaffected siblings. Furthermore, maternal microchimerism carriers and non-carriers exhibited similar phenotypes. CONCLUSIONS: Maternal microchimerism appears to be less common in Japanese children with type 1 diabetes than in white European people. Our data indicate that maternal microchimerism is unlikely to be a major trigger or a phenotypic determinant of type 1 diabetes in Japanese children and that the biological significance of maternal microchimerism in type 1 diabetes may differ among ethnic groups.

Urogenital symptoms in mitochondrial disease: overlooked and undertreated.

BACKGROUND AND PURPOSE: Bowel symptoms are well documented in mitochondrial disease. However, data concerning other pelvic organs is limited. A large case-control study has therefore been undertaken to determine the presence of lower urinary tract symptoms (LUTS) and sexual dysfunction in adults with genetically confirmed mitochondrial disease. METHODS: Adults with genetically confirmed mitochondrial disease and control subjects were recruited from a specialist mitochondrial clinic. The presence and severity of LUTS and their impact on quality of life, in addition to sexual dysfunction and bowel symptoms, were captured using four validated questionnaires. Subgroup analysis was undertaken in patients harbouring the m.3243A>G MT-TL1 mitochondrial DNA mutation. A subset of patients underwent urodynamic studies to further characterize their LUTS. RESULTS: Data from 58 patients and 19 controls (gender and age matched) were collected. Adults with mitochondrial disease had significantly more overactive bladder (81.5% vs. 56.3%, P = 0.039) and low stream (34.5% vs. 5.3%, P = 0.013) urinary symptoms than controls. Urodynamic studies in 10 patients confirmed that bladder storage symptoms predominate. Despite high rates of LUTS, none of the patient group was receiving treatment. Female patients and those harbouring the m.3243A>G MT-TL1 mutation experienced significantly more sexual dysfunction than controls (53.1% vs. 11.1%, P = 0.026, and 66.7% vs. 26.3%, P = 0.011, respectively). CONCLUSIONS: Lower urinary tract symptoms are common but undertreated in adult mitochondrial disease, and female patients and those harbouring the m.3243A>G MT-TL1 mutation experience sexual dysfunction. Given their impact on quality of life, screening for and treating LUTS and sexual dysfunction in adults with mitochondrial disease are strongly recommended.

Evaluating the efficacy of hydrogen peroxide vapour against foot-and-mouth disease virus within a BSL4 biosafety facility.

An evaluation was made of the efficacy of 35% hydrogen peroxide vapour (HPV) against foot-and-mouth disease virus (FMDV) in a biosafety facility. Biological indicators (BIs) were produced using three serotypes of FMDV, all with a titre of ≥106 TCID50 per ml. Fifteen BIs of each serotype were distributed across five locations, throughout a 30-m3 airlock chamber, producing a total of 45 BIs. Thirty-five percent HPV was generated and applied using a Bioquell vaporization module located in the centre of the chamber. After a dwell period of 40 min, the HPV was removed via the enclosures air handling system and the BIs were collected. The surfaces of the BIs were recovered into Glasgow's modified Eagle's medium (GMEM), cultivated in BHK21 Cl13 cell culture and analysed for evidence of cytopathic effect (CPE). No CPE was detected in any BI sample. Positive controls showed CPE. The experimentation shows that FMDV is susceptible to HPV decontamination and presents a potential alternative to formaldehyde. SIGNIFICANCE AND IMPACT OF THE STUDY: Foot-and-mouth disease virus (FMDV) is an important pathogen in terms of biosafety due to its infectious nature and wide range of host animals, such as cattle, sheep, goats and pigs. Outbreaks of FMDV can have a severe impact on livestock production, causing morbidity, mortality, reduced yields and trade embargoes. Laboratories studying FMDV must possess BSL4 robust bio-decontamination methods to prevent inadvertent release. Formaldehyde has been the primary agent for environmental decontamination, but its designation as a human carcinogen has led to a search for alternatives. This study shows 35% hydrogen peroxide vapour has the potential to be a rapid, effective, residue-free alternative.

The long terminal repeat negative control region is a critical element for insertional oncogenesis after gene transfer into hematopoietic progenitors with Moloney murine leukemia viral vectors.

Integrating vectors based on γ-retroviruses and containing full-length long terminal repeats (LTRs) have been associated with activation of oncogene expression and leukemogenesis in human gene therapy trials. Identification of the specific molecular elements of the LTRs that have a role in insertional oncogenesis events is important as it can lead to the development of safer gene transfer vectors. The negative control region (NCR) of the LTR is a particularly well-conserved sequence among mammalian γ-retroviruses with demonstrated regulatory activity of gene transcription in hematopoietic cells, which led us to hypothesize that this region may have a role in insertional oncogenesis after γ-retroviral vector (GV)-mediated gene transfer into hematopoietic progenitors. We used an in vitro assay of murine bone marrow cell immortalization to compare the immortalization capabilities of a series of GVs carrying murine leukemia virus (MLV) LTR deletion mutants. Compared with GV carrying the full-length MLV LTR, deletion of the complete LTR enhancer sequence showed significant reduction of immortalization rates. However, the use of a mutant LTR deleted of the enhancer sequence, with exception of the NCR, did not affect immortalization. Importantly, the inclusion of an LTR mutant devoid only of the NCR did show significant reduction of immortalization rates compared with the full LTR sequence. Therefore, our data point to the NCR as a key element for immortalization and justify additional studies to evaluate its specific role in MLV-mediated insertional oncogenesis.

Pelvic organ dysfunction is more prevalent and severe in MSA-P compared to Parkinson's disease.

AIMS: It is usually difficult to distinguish between idiopathic Parkinson's disease (PD) and parkinsonian-type multiple system atrophy (MSA-P) in the early stage. However, it is important to make a careful early-stage diagnosis. Therefore, we determined whether an examination of pelvic organ dysfunction would be helpful to distinguish between PD and MSA-P. METHODS: We recruited 61 patients with PD and 54 patients with MSA-P who were examined at our neurology clinic. The mean ages of the patients with PD and MSA-P were 67 and 64 years, respectively. The mean disease duration of both groups was 3.2 years. We administered a questionnaire on pelvic organ dysfunction to the PD and MSA-P groups. The questionnaire had sections focusing on bladder, bowel, and sexual function. Dysfunction, as described in the responses, was evaluated as normal, mild (>once a month), moderate (>once a week), or severe (>once a day). The Mann-Whitney U-test was used for statistical analysis. RESULTS: Compared with the PD group, the prevalence and severity of pelvic dysfunction in the MSA-P group was significantly higher for urinary urgency (MSA-P 76%, PD 58%, P<0.05), retardation in initiating urination (79%, 48%, P<0.05), prolongation in urination (79%, 72%, P<0.05), and constipation (58%, 31%, P<0.05). The quality-of-life index among pelvic organ dysfunctions indicated that urinary and bowel function was significantly more impaired in the MSA-P group than in the PD group. CONCLUSIONS: Urinary urgency, retardation in initiating urination, prolongation in urination, and constipation are more prevalent and severe in MSA-P compared to PD.

Expression of Tac antigen on activated normal human B cells.

Two-color fluorescence analysis revealed that Tac antigen, which was previously reported to be restricted to T cells, was expressed on a proportion of normal B cells activated by Staphylococcus aureus Cowan I (SAC). Immunoaffinity-purified interleukin 2 (IL-2) induced the proliferation of SAC-activated B cells, and the proliferation was completely inhibited by anti-Tac antibody, which blocked the membrane binding and action of IL-2. These results suggest that an IL-2 receptor system is directly involved in the B cell immune response.

Transmembrane signaling of interleukin 2 receptor. Conformation and function of human interleukin 2 receptor (p55)/insulin receptor chimeric molecules.

Chimeric genes were constructed which gave rise to the expression of novel receptor molecules consisting of the extracellular domain of the human interleukin 2 receptor (IL-2-R; p55 or Tac antigen) joined to the transmembrane domain and either full-length or truncated cytoplasmic domain of the human insulin receptor (Ins-R). Expression studies using mouse T cell line EL-4 revealed that the chimeric receptors are able to manifest properties indistinguishable from the authentic IL-2-R. On the other hand, stimulation of the tyrosine kinase activity by IL-2 was not observed in the chimeric receptor with the entire cytoplasmic domain of the Ins-R. These findings thus shed light on the structural conformation and functioning of the IL-2-R complex.

Pseudo-high affinity interleukin 2 (IL-2) receptor lacks the third component that is essential for functional IL-2 binding and signaling.

Functional studies of the interleukin 2 receptor (IL-2R) of two (ED515-D and Kit225) IL-2-dependent and three (ED515-I, 3T3-alpha beta 11, and Hut102) IL-2-independent cell lines were done. All of these cell lines appeared to express high as well as low affinity IL-2R. However, ED515-I and 3T3-alpha beta 11, which expressed the IL-2R beta chain, did not bind IL-2 at all when IL-2 binding to their IL-2R alpha chain was blocked with anti-Tac monoclonal antibody, whereas the intermediate affinity binding in ED515-D, Kit225, and Hut102 cells remained. We tentatively called the high affinity IL-2R of the former cells pseudo-high affinity IL-2R. The dissociation constant of pseudo-high affinity IL-2R was higher than that of ordinary high affinity IL-2R. Internalization of cell-bound 125I-IL-2 into ED515-I and 3T3-alpha beta 11 cells was less efficient than that into ED515-D cells. The addition of IL-2 neither promoted cell growth nor upregulated IL-2R alpha chain expression in ED515-I and 3T3-alpha beta 11 cells. Furthermore, tyrosine phosphorylation of the cellular proteins (p120, p98, p96, p54, and p38) was induced or enhanced in response to the addition of IL-2 in ED515-D and Kit225 cells, but not in the cell lines expressing pseudo-high affinity IL-2R. Finally, 125I-IL-2 crosslinking followed by SDS-PAGE analysis showed an 80-kD band corresponding to p65 + IL-2, in addition to bands corresponding to IL-2R alpha and beta chain + IL-2 in cells bearing ordinary high affinity IL-2R but not in cells with pseudo-high affinity IL-2R. Taken together, we consider that another protein whose molecular mass is approximately 65 kD is functionally important in IL-2 binding and subsequent signal transduction and may be the third component of IL-2R.

Characterization of a soluble suppressor of human B cell immunoglobulin biosynthesis produced by a continuous human suppressor T cell line.

A human suppressor T cell maintained in long-term culture with conditioned medium containing interleukin 2 elaborates a suppressor factor(s) that specifically inhibits human polyclonal B cell immunoglobulin biosynthesis. This soluble immune suppressor supernate of immunoglobulin production (CTC-SISS-B) shares a number of features with the previously described suppressive mediator elaborated by concanavalin A-activated human peripheral T cells (SISS-B) including: (a) the inhibition by a noncytotoxic mechanism, (b) the suppression of immunoglobulin biosynthesis either through direct action on the B cell or indirect action via the monocyte, (c) the loss of inhibition in the presence of the monosaccharide L-rhamnose, (d) the elaboration by cells irradiated with 500 ro 2,000 rad, and (e) molecular weights of 60,000--90,000. Furthermore, the suppression by this mediator appears to be specific for B cell immunoglobulin production in that CTC-SISS B has no effect on T cell proliferation to mitogens, antigens, an allogeneic cells or on T cell-mediated cytotoxicity. These data indicate that one possible mechanism of suppressor T cell inhibition of human immunoglobulin production is via the generation of a lectinlike suppressor lymphokine that interacts with defined saccharide determinants on the cell surface of either the B cell or monocyte.

The human OX40/gp34 system directly mediates adhesion of activated T cells to vascular endothelial cells.

Fresh leukemic cells from patients with adult T cell leukemia (ATL) and some ATL-derived T cell lines show adhesion to human umbilical vein endothelial cells (HUVECs) mainly through E-selectin, but a proportion of this binding remains unaffected by the addition of combinations of antibodies against known adhesion molecules. By immunizing mice with one of such cell lines, we established monoclonal antibodies (mAbs), termed 131 and 315, that recognize a single cell surface antigen (Ag) and inhibit the remaining pathway of the adhesion. These mAbs did not react with normal resting peripheral blood mononuclear cells (PBMC) or most of the cell lines tested except for two other human T cell leukemia virus type I (HTLV-I)-infected T cell lines. After stimulation with phytohemagglutinin (PHA), PBMC expressed Ag 131/315 transiently, indicating that these mAbs define a T cell activation Ag. Western blotting and immunoprecipitation revealed that Ag 131/315 has an apparent molecular mass of 50 kD. Expression cloning was done by transient expression in COS-7 cells and immunological selection to isolate a cDNA clone encoding Ag 131/315. Sequence analysis of the cDNA indicated that it is identical to human OX40, a member of the tumor necrosis factor/nerve growth factor receptor family. We then found that gp34, the ligand of OX40, was expressed on HUVECs and other types of vascular endothelial cells. Furthermore, it was shown that the adhesion of CD4+ cells of PHA-stimulated PBMC to unstimulated HUVECs was considerably inhibited by either 131 or 315. Finally, OX40 transfectants of Kit 225, a human interleukin 2-dependent T cell line, were bound specifically to gp34 transfectants of MMCE, a mouse epithelial cell line, and this binding was blocked by either 315 or 5A8, an anti-gp34 mAb. These results indicate that the OX40/gp34 system directly mediates adhesion of activated T cells or OX40+-transformed T cells to vascular endothelial cells.

Origin of human T-lymphotrophic virus I-positive T cell lines in adult T cell leukemia. Analysis of T cell receptor gene rearrangement.

Using the clone-specific rearrangement of the T cell receptor gene as the genetic marker of the clonotype, we analyzed the clonal origin of the interleukin 2 (IL-2)-dependent human T-lymphotrophic virus I (HTLV-I)-positive T cell lines established from various adult T cell leukemia (ATL) patients. From a patient with chronic ATL, whose leukemic cells proliferated in vitro in response to IL-2, we repeatedly established leukemic T cell clones having the same rearrangement profile of the T beta chain gene as the leukemic cells. By contrast, established cell lines from acute ATL patients had different beta chain gene rearrangements from those of the leukemic cells. These HTLV-I+ T cell lines might not be the direct progeny of the leukemic cells, but that of T cells infected either in vivo or in vitro. These IL-2-reactive nonleukemic T cells might have been selected in vitro, because their leukemic cells failed to respond to IL-2, despite the expression of IL-2 receptor. The analysis of the T cell receptor gene rearrangement may give a new approach for the elucidation of the mechanism of leukemogenesis and the origin of the HTLV-I+ T cell lines in ATL.

Portal vein thrombosis treated using danaparoid sodium and antithrombin III.

A 45-year-old man under treatment for liver cirrhosis (LC) due to chronic hepatitis C and hemophilia A was seen in our emergency room because of a 10-kg weight gain in the previous week due to ascites. Portal vein thrombosis (PVT) was detected with computer tomography (CT) and ultrasonographic (US). Danaparoid sodium (DS) and antithrombin III (AT III) were administrated and doppler US images showed improvement of portal venous blood flow. DS or AT III may be safe and alternative therapies for PVT.

Changes in mechanical properties of poly-l-lactic acid mini-plate under functional load simulating sagittal splitting ramus osteotomy.

The purpose of this study was to investigate how the characteristics of a poly-l-lactic acid mini-plate changed with dynamic loading in an environment with hydrolytic degradation. A mandible osteosynthesis model was prepared with specimen poly-l-lactic acid mini-plates to simulate sagittal splitting ramus osteotomy. The model was then subjected to dynamic loading, and changes in specimen shape and surface quality were observed. Specimen bending strength was then measured, and degree of hydrolytic degradation estimated. Neither dynamic loading nor degree of load clearly affected degree of hydrolytic degradation. The specimens maintained their original shape and bending strength for up to 4 weeks with dynamic loading of 40 N or less in an environment with hydrolytic degradation. At 8 weeks, under the same conditions, the specimens showed cracks or fractures, or both, together with a clear decrease in bending strength. The results suggest that dynamic loading causes cracking in a poly-l-lactic acid mini-plate, and that growth of these cracks decreases bending strength over time, leading to fatigue fracture.

Close association between interleukin 2 receptor mRNA expression and human T cell leukemia/lymphoma virus type I viral RNA expression in short-term cultured leukemic cells from adult T cell leukemia patients.

Human T cell leukemia/lymphoma (T-lymphotropic) virus type I (HTLV-I) infection has been considered to be closely associated with the leukemogenesis of adult T cell leukemia (ATL), in which interleukin 2 (IL-2) receptors are abnormally expressed. In this study, however, Southern blot analysis revealed no gross rearrangement or obvious amplification of the IL-2 receptor gene in ATL leukemic cells, indicating that abnormal IL-2 receptor expression in ATL is not due to the structural change of its gene. Hence, we studied the expression of the IL-2 receptor and HTLV-I at the RNA level during short-term cultures of leukemic cells from 9 ATL patients. Cytoplasmic dot hybridization and Northern hybridization revealed that fresh leukemic cells from seven of nine patients expressed a small amount of IL-2 receptor mRNA but HTLV-I RNA was undetectable in all cases. After cultures for up to 7 d, both IL-2 receptor mRNA and HTLV-I RNA (including pX message) expression concomitantly increased, whereas the amounts of other cellular genes, except for beta-actin, did not. The increases in their RNA expression were inhibited by early addition (within 12 h after the beginning of the culture) of cycloheximide, indicating that these increases are mediated by newly synthesized protein(s). These results strongly suggested that IL-2 receptor expression is closely associated with HTLV-I expression in leukemic cells from ATL patients.

Immunopathophysiological aspects of an emerging neonatal infectious disease induced by a bacterial superantigen.

We recently discovered an emerging neonatal infectious disease, neonatal toxic shock syndrome-like (TSS-like) exanthematous disease (NTED), which is induced by a superantigen, TSS toxin-1 (TSST-1), produced by methicillin-resistant Staphylococcus aureus (MRSA). Here, we analyzed the activation and the response of TSST-1-reactive Vss2(+) T cells in NTED patients during the acute and recovery phases and in asymptomatic infants exposed to MRSA. In the acute phase, Vss2(+) T cells were anergic to stimulation with TSST-1 and underwent marked expansion, but by 2 months after disease onset, their numbers had declined to about 10% of the control level. Although the percentage of Vss2(+) T cells in the ten asymptomatic neonatal MRSA carriers was within the control range, these individuals could be divided into two groups on the basis of Vss2(+) T-cell activation. Vss2(+)CD4(+) T cells from three of these infants (Group 1) highly expressed CD45RO and were anergic to TSST-1, whereas in the other seven asymptomatic neonatal MRSA carriers (Group 2), these cells expressed CD45RO at the control level and were highly responsive to stimulation with TSST-1. The serum anti-TSST-1 IgG Ab titer was negligible in the four NTED patients in the acute phase and the three asymptomatic neonatal MRSA carriers in Group 1, but it was high in the seven asymptomatic carriers in Group 2. We suggest that maternally derived anti-TSST-1 IgGs helps to suppress T-cell activation by TSST-1 and protects infants from developing NTED.

Interleukin-2 receptor (Tac antigen) expressed on adult T cell leukemia cells.

We studied the expression of the interleukin-2 (IL-2) receptor and the proliferative response to exogenous IL-2 of peripheral blood leukemic cells from patients with adult T cell leukemia (ATL) in order to see whether IL-2 receptor expressed on ATL cells is different from normal IL-2 receptor and whether it plays a role in the neoplastic growth in ATL. Peripheral blood leukemic cells from 42 patients with ATL examined expressed IL-2 receptors that were detected by anti-Tac monoclonal antibody when examined immediately after the separation of cells or after the culture for 24 or 48 h. The number of anti-Tac binding sites ranged from 3,100 to 11,400 in fresh cells and from 3,600 to 96,000/cell in short-term cultured leukemic cells, whereas phytohemagglutinin-P (PHA-P)-stimulated normal T cells exhibited 6,900-35,000 anti-Tac binding sites per cell. ATL-derived and human T cell leukemia/lymphoma virus, type I (HTLV-I)-infected cell lines such as MT-1 and Hut102 expressed a much higher number of anti-Tac binding sites. Leukemic cells from 15 patients with ATL examined showed no or very poor proliferative response to various concentrations of immunoaffinity-purified IL-2, although they expressed Tac antigen (Ag). Radiolabeled IL-2 binding experiments demonstrated that ATL leukemic cells could bind IL-2, and they expressed both high and low affinity IL-2 receptors, although the number of high affinity IL-2 receptor was much less than that of low affinity IL-2 receptor and that of anti-Tac binding sites. In contrast, leukemic T cells from a patient with T cell chronic lymphocytic leukemia (CLL), in whom HTLV-I infection was not demonstrated, responded as well as PHA-P-stimulated normal T cells, and their IL-2 receptors, unlike ATL cells, were modulated (down regulated) by anti-Tac antibody. No differences were noted between ATL cells and normal activated T cells in one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the IL-2 receptor. Thus, leukemic cells in ATL spontaneously and continuously express IL-2 receptor, which appears to be abnormally regulated and unresponsive to IL-2. These results, taken together with those on normal IL-2 receptors on HTLV-I-negative T-CLL cells, suggest that abnormal expression of the IL-2 receptor in ATL is closely associated with HTLV-I infection and may play a role in the neoplastic growth of ATL cells.

Synthesis of pyrophosphate-containing compounds that stimulate Vgamma2Vdelta2 T cells: application to cancer immunotherapy.

Human Vgamma2Vdelta2 T cells recognize nonpeptide antigens, such as isoprenoid pyrophosphomonoester intermediates, alkylamine compounds, and bisphosphonate drugs, as well as some tumor cells. Although attempts have been made to derive novel cancer immunotherapies based on the discovery of these unconventional antigens, effective therapies remain to be developed. Here, we synthesized a series of pyrophosphate-containing compounds and examined the chemical requirements for the recognition of pyrophosphomonoester antigens by gammadelta T cells. The structural analysis clearly demonstrated that a proximal methylene moiety plays a crucial role in the stimulatory activity of the antigens. For optimal gammadelta T cell proliferation, we find that the use of human serum albumin was preferred and that pyrophosphomonoesters were superior to nitrogen-containing bisphosphonate compounds. Using these techniques, we have successfully expanded gammadelta T cells from healthy donors as well as from cancer patients using one of the most active compounds, 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP). The resulting expanded gammadelta T cells exhibited potent, cytotoxic activity against a wide variety of tumor cell lines. Even gammadelta T cells from a patient with advanced liver carcinoma efficiently responded to 2M3B1PP and exhibited strong cytotoxic activity against tumor cells. The pretreatment of tumor cells with nonpeptide antigens was essential for efficient cytotoxicity via TCR-gammadelta. The present study suggests a novel strategy for cancer immunotherapy using synthetic small pyrophosphate-containing compounds and nitrogen-containing bisphosphonates.

Effects of electrical stimulation of the raphe area on the micturition reflex in cats.

The raphe nucleus has a variety of physiological functions, including emotion, regulation of skeletal muscle motoneurons, spinal transmission of nociceptive signals, sleep, respiration, gastric motility, and cardiovascular function. Recent evidence has shown that centrally administered serotonin has modulatory effects on micturition function, and that decreased brain serotonin might underlie depression and an overactive bladder. We applied high-frequency stimulation (HFS; 0.2-ms duration, 100 Hz) in the raphe nucleus and the adjacent midline area in 20 supracollicular decerebrate cats, which mostly elicited inhibition of the micturition reflex. The effective amplitude of the electrical stimulation for evoking inhibitory responses was less than 50 muA. We also examined single neuronal activities in the raphe nucleus in response to isovolumetric spontaneous micturition reflexes. In total, 79 neurons were recorded in the raphe nucleus that were related to urinary storage/micturition cycles. Of the neurons recorded, the most common were tonic storage neurons (48%), followed by tonic micturition neurons (28%), phasic storage neurons (18%), and phasic micturition neurons (6%). In addition to the tonic/phasic as well as storage/micturition classification, the neurons showed diverse discharge patterns: augmenting, constant and decrementing, with the constant discharge pattern being most common. Among neurons in the raphe nucleus, the neurons with a decrementing discharge pattern were concentrated in the rostral portion, whereas the augmenting and constant neurons existed diffusely. The storage and micturition neurons were intermingled in the rostral portion, whereas they were separate in the caudal portion. In conclusion, the results of the present study indicate that HFS of the raphe area inhibits the micturition reflex and that there are micturition-related neuronal firings in the raphe area in cats, suggesting that the raphe nucleus is involved in neural control of micturition.

Molecular anatomy of BCL6 translocations revealed by long-distance polymerase chain reaction-based assays.

BCL6 translocations involve not only immunoglobulin (IG) genes but also a number of non-IG loci as partners. Junctional sequences of three IG/BCL6 translocations were readily obtained by long-distance PCR. In cases where partner loci were not determined, we developed a long-distance inverse PCR method, which amplifies unknown fragments flanked by known BCL6 sequences. Using these two long-distance PCR-based approaches, we cloned junctional areas of BCL6 translocations from a total of 58 cases of B-cell tumors. Nucleotide sequencing and database searches revealed that 30 cases involved IGs as partners: IG heavy chain gene in 22, IG kappa light chain gene in 1, and IG lambda light chain gene in 7. In contrast, 23 cases affected non-IG loci, including the H4 histone gene, heat shock protein genes HSP89alpha and HSP90beta, and PIM-1 proto-oncogene. On der(3) chromosomes, complete sets of the promoters of these partner genes replaced that of BCL6 in the same transcriptional orientation. These results suggest that BCL6 gene affected by the translocation is transcriptionally activated by a variety of stimuli, including cell cycle control, changes in the physical environment, and response to cytokines. Break points on BCL6 occurred within the major translocation cluster, and we identified a 120-bp hyper-cluster region a short distance from the 3' end of exon 1. Gel mobility-shift assay suggested the presence of a protein(s) that bound to this particular region.