Sulfonylurea-sensitive K+ channels and their probable role for the membrane potential of mouse motor nerve endings

We studied the effect of the K_ATP channel blockers tolbutamide and glibenclamide on presynaptic membrane currents in the mouse M. triangularis sterni preparation using the perineural recording technique. Both sulfonylureas blocked part of the fast K⁺ component within 2 min after application. The block was much more pronounced under glucose-free conditions and was completelyreversible by washing. Addition of glucose to glucose-free bath solution also reduced the K⁺ component. A further effect of the sulfonylureas was observed under glucose-free conditions. With a delay of 5 to 10 min, the nodal Na⁺ component began to diminish and disappeared within 30 min. This was associated with a dramatic increase in spontaneous quantal transmitter release suggesting that the block of sulfonylurea-sensitive K⁺ channels causes depolarization of motor nerve terminals and fibres thus inactivating Na⁺ channels. Tetraethylammonium (TEA) which blocks ATP-dependent K⁺ channels in high concentrations caused, under glucose-free conditions, the same delayed effect as the sulfonylureas. This delayed effect was fully reversible by washing with glucose-containing, but not with glucose-free solution. Our findings strongly suggest that K_ATP channels exist in mammalian motor nerve endings and that under hypoglycemic conditions these channels open and become essential for the maintenance of the membrane potential.     

A gene signature of inhibitory MHC receptors identifies a BDCA3(+) subset of IL-10-induced dendritic cells with reduced allostimulatory capacity in vitro

Dendritic cells (DC) are crucial gatekeepers in regulating immunity. Whereas mature immunostimulatory myeloid DC (DC(ims)) potently promote immune responses, IL-10-induced myeloid DC (DC-IL-10) counteract T cell activation. To elucidate the molecular repertoire by which DC-IL-10 secure reduced T cell activation, comparative gene expression profiling was done using Affymetrix U133A microarrays. Among the genes overexpressed in DC-IL-10, eight immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing inhibitory molecules (ILT2, ILT3, ILT4, ILT5, DCIR, PILRA, FcgammaRIIB, SLAM) were found. Phenotypic analysis of DC-IL-10 defined an ILT(high) DC subset further characterized by expression of CD14, TLR2, DC-SIGN, and CD123 and the lack of lymphocyte, monocyte/macrophage, and plasmacytoid DC markers such as CD3, CD8, and CD68. A unique feature of the ILT(high) DC subset was expression of the novel DC marker BDCA3, which was not detected on monocytes, immature DC, DC(ims), or ILT(low) DC-IL-10. While the allogeneic T cell proliferation induced by the entire DC-IL-10 population was approximately 30% of that stimulated by DC(ims), allogeneic MLR responses driven by the ILT(high) DC subset were reduced to 8% of the allostimulatory capacity of DC(ims), although secretion of the inhibitory cytokine IL-10 and other Th1/Th2-associated cytokines was similar in these cells.     

PG-M1: A New Monoclonal Antibody Directed against a Fixative-Resistant Epitope on the Macrophage-Restricted Form of the CD68 Molecule

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.     

Pulsed-field gel electrophoresis of NotI digests of leptospiral DNA: a new rapid method of serovar identification.

Fingerprints for 72 reference serovar strains of pathogenic Leptospira spp. were obtained by pulsed-field gel electrophoresis (PFGE) following NotI restriction digests of the chromosome. These strains included the serovar reference strains of serogroups Australis, Ballum, Bataviae, Grippotyphosa, Panama, Pomona, and Pyrogenes. Sixty-four serovars could be identified by a unique NotI restriction profile. The remaining serovars were differentiated by chromosomal digestion with SgrAI. These included four serovars from serogroup Australis, two serovars from serogroup Ballum, and two serovars from serogroup Bataviae. Thirteen of 18 recent clinical isolates identified by microagglutination test and cross-adsorption procedure were correctly typed by PFGE. The results indicate that PFGE, which is considerably more rapid than serology, should be useful for identification and epidemiological studies.     

Modulation of Whole-Cell Currents in Plasmodium Falciparum-Infected Human Red Blood Cells by Holding Potential and Serum

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum -infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole-cell configuration of the patch-clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole-cell recordings in malaria-infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4-fold and inward currents by approximately 2-fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.     

Daytime sleepiness during Ramadan intermittent fasting: polysomnographic and quantitative waking EEG study

During the lunar month of Ramadan, Muslims abstain from eating, drinking and smoking from sunrise to sunset. We reported previously that Ramadan provokes a shortening in nocturnal total sleep time by 40 min, an increase in sleep latency, and a decrease in slow-wave sleep (SWS) and rapid eye movement (REM) sleep duration during Ramadan. During the same study, the effects of Ramadan intermittent fasting on daytime sleepiness were also investigated in eight healthy young male subjects using a quantitative waking electroencephalograph (EEG) analysis following the multiple sleep latency test (MSLT) procedure. This procedure was combined with subjective alertness and mood ratings and was conducted during four successive experimental sessions: (1) baseline (BL) 15 days before Ramadan, (2) beginning of Ramadan (R11) on the 11th day of Ramadan, (3) end of Ramadan (R25) on the 25th day of Ramadan, (4) recovery 2 weeks after Ramadan (AR). During each session, four 20-min nap opportunities (MSLTs) were given at 10:00, 12:00, 14:00 and 16:00 h and were preceded by rectal temperature readings. Nocturnal sleep was recorded before each daytime session. Subjective daytime alertness did not change in R25 but decreased in R11 at 12:00 h, and subjective mood decreased at 16:00 h, both in R11 and R25. During the MSLT, mean sleep latency decreased by an average of 2 min in R11 (especially at 10:00 and 16:00 h) and 6 min in R25 (especially at 10:00 and 12:00 h) compared with BL. There was an increase in the daily mean of waking EEG absolute power in the theta (5.5-8.5 Hz) frequency band. Significant correlations were found between sleep latency during the MSLT and the waking EEG absolute power of the fast alpha (10.5-12.5 Hz), sigma (11.5-15.5 Hz) and beta (12.5-30 Hz) frequency bands. Sleep latency was also related to rectal temperature. In conclusion, Ramadan diurnal fasting induced an increase in subjective and objective daytime sleepiness associated with changes in diurnal rectal temperature.     

Preventive Effects of Escherichia coli Strain Nissle 1917 on Acute and Chronic Intestinal Inflammation in Two Different Murine Models of Colitis

Escherichia coli strain Nissle 1917 (EcN) is as effective in maintaining remission in ulcerative colitis as is treatment with mesalazine. This study aims to evaluate murine models of acute and chronic intestinal inflammation to study the antiinflammatory effect of EcN in vivo. Acute colitis was induced in mice with 2% dextran-sodium sulfate (DSS) in drinking water. EcN was administered from day −2 to day +7. Chronic colitis was induced by transfer of CD4⁺ CD62L⁺ T lymphocytes from BALB/c mice in SCID mice. EcN was administered three times/week from week 1 to week 8 after cell transfer. Mesenteric lymph node (MLN) cytokine secretion (of gamma interferon [IFN-γ], interleukin 5 [IL-5], IL-6, and IL-10) was measured by enzyme-linked immunosorbent assay. Histologic sections of the colon were analyzed by using a score system ranging from 0 to 4. Intestinal contents and homogenized MLN were cultured, and the number of E. coli-like colonies was determined. EcN was identified by repetitive extragenic palindromic (REP) PCR. EcN administration to DSS-treated mice reduced the secretion of proinflammatory cytokines (IFN-γ, 32,477 ± 6,377 versus 9,734 ± 1,717 [P = 0.004]; IL-6, 231 ± 35 versus 121 ± 17 [P = 0.02]) but had no effect on the mucosal inflammation. In the chronic experimental colitis of the transfer model, EcN ameliorated the intestinal inflammation (histology score, 2.7 ± 0.2 versus 1.9 ± 0.3 [P = 0.02]) and reduced the secretion of proinflammatory cytokines. Translocation of EcN and resident E. coli into MLN was observed in the chronic colitis model but not in healthy controls. Administration of EcN ameliorated acute and chronic experimental colitis by modifying proinflammatory cytokine secretion but had no influence on the acute DSS-induced colitis. In this model, preexisting colitis was necessary for translocation of EcN and resident E. coli into MLN.     

Detection of Serum Antibodies to Ovine Progressive Pneumonia Virus in Sheep by Using a Caprine Arthritis-Encephalitis Virus Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [³⁵S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.     

Isolation and propagation of enteric adenoviruses in HEp-2 cells.

Eighty-two stool samples from children with gastroenteritis in Canada, England, and Thailand which had been shown to contain adenovirus antigen (by a group-specific enzyme-linked immunosorbent assay) or adenovirus particles (by electron microscopy) or both, were tested for primary isolation of enteric adenoviruses in HEp-2 and Graham 293 cells. Graham 293 cells are known to support the replication of enteric adenovirus types (Ad40 and Ad41) on primary isolation, whereas HEp-2 cells reportedly do not. Of the 82 adenovirus isolates, 73 could be typed as Ad40 or Ad41 by type-specific monoclonal antibody enzyme-linked immunosorbent assay and by analysis of SmaI endonuclease digests. Of these 73, 30 (41%) could be isolated in HEp-2 cells, which included 43% (9/21) of those typed as Ad40 and 40% (21/52) of those typed as Ad41. On the basis of these results, the growth characteristics of adenoviruses in HEp-2 cell cultures, commonly used to distinguish enteric from nonenteric adenovirus types, are not valid for either diagnosis or epidemiological studies. For the samples studied here, use of these nondefinitive criteria would result in underestimation of the incidence of enteric adenoviruses in viral gastroenteritis.