Vesico-anal influences in healthy intact humans: Quantificated by responses in the external anal sphincter following transcranial cortical stimulation

EMG responses in the external anal sphincter (EAS), the rectus abdominis muscle (RA), and the anterior tibial muscle (TA) were recorded following single magnetic transcranial cortical stimulations (TCCS) in seven healthy volunteers. The responses in the EAS differed from the responses in the other muscles. They had comparatively long durations ranging from 1 to 2 seconds, no inhibitory periods were observed, and there was no tendency for habituation to occur following a limited number of stimuli. The responses recorded in the EAS were used as test responses in order to evaluate the excitability changes in the EAS motoneurons occurring during bladder filling. Cystometries with filling rates of 15, 50 and 200 ml/min were done. During these cystometries TCCS were applied repeatedly, with constant strength, after each 50 ml of filling up to bladder capacity. The responses following TCCS changed in a highly reproducible way during bladder filling. After 100–200 ml of filling, the responses had longer latencies, diminished sizes, and shorter durations. When the filling reached a level 50–150 ml below capacity, the responses in most subjects again became greater and the latencies shorter. The changes were believed to be physiological. It was concluded that the EAS motoneurons are under both inhibitory and facilitatory influence during bladder filling in intact healthy humans. Facilitatory influences are often observed when the bladder is filled close to capacity. At lower bladder volumes the observed influence is always inhibitory. A decrease in the EMG activity of the EAS during filling cystometry should consequently not be regarded as a pathological response.     

Dependence of saccadic eye-movements on stimulus luminance, and an effect of task

Various aspects of saccadic eye-movements are related to stimulus luminance for a small lit stimulus that steps 10 degrees horizontally in complete darkness. The relations depend on whether the stimulus is the target for a foveating saccade, or is the cue for an "anti" saccade which peripheralizes the retinal image of the cue: (1) at scotopic luminances the differences between foveating and anti saccades are diminished, largely because foveation is the more severely affected. Latencies are long amplitudes are scattered, and direction errors are not infrequent in both tasks; (2) the latency-luminance relation for foveating saccades shows an abrupt discontinuity at the perceptual rod-cone transition. Above the cone threshold corrective secondary saccades appear in greater numbers; (3) the corresponding latency transition for anti saccades is anomalous and protracted. Latency remains constant for mesopic cue luminances up to 1.0 log unit brighter than the perceptual rod-cone threshold. Direction errors are especially common in this mesopic luminance range. Mechanisms are discussed.     

Synergistic action of immunostimulatory DNA and fcgamma receptor IIB-crosslinking on B-cell phenotype and function

CpG DNA functions via the toll-like receptor-9 (TLR-9) receptor, inducing B cell proliferation and promoting immunoglobulin production. B cell responses to CpG DNA-containing immune complexes could be important in chronic autoimmunity and immune responses to bacterial components. Therefore, we investigated the potential synergy of CpG DNA-stimulation with FcgammaR clustering (CFR) on splenic B cell activity. CFR-induced splenocyte proliferation was significantly increased compared to treatment with CpG DNA alone. While the levels of interleukin-10 (IL-10) were increased in CpG DNA-treated splenocyte cultures, particularly following FcgammaRII/III-clustering, CFR treatment reduced IL-6 levels. B-cell maturation in culture was enhanced by CFR. Indeed, the frequency of IgG expressing cells after stimulation with CpG DNA was increased and was even higher after CFR stimulation. Furthermore, the frequency of plasma cell precursors was markedly increased by stimulation with CFR. Late splenic B cell subsets, transitional type 2 (T2) and mature (M) B cells, responded strongly to CpG DNA with proliferation and the response was enhanced by FcgammaR-clustering. Immature transitional type 1 (T1) B cells showed distinctly lower proliferative response to CpG DNA and very small effects of FcgammaR-clustering, despite similar expression of Fcgamma-receptors by all B cell subsets. In conclusion, these data show synergistic impact of CpG DNA and simultaneous FcgammaR-clustering on B cell proliferation and differentiation.     

Spatial EEG Synchronisation Over Sensorimotor Hand Areas in Brisk and Slow Self-Paced Index Finger Movements

The changes of spatial EEG synchronisation during brisk and slow voluntary self-paced movements of the right and left index finger were analysed in 12 right-handed and 11 left-handed subjects. EEG was recorded from the left and right sensorimotor area using 24 closely spaced electrodes. A novel measure of spatial EEG synchronisation, -complexity, was computed separately for the left and right sensorimotor area in 64 overlapping one-second epochs representing 4.5 s of the pre-movement and 3.5 s of the post-movement period. -complexity was higher, hence spatial synchronisation was lower, in slow than in brisk movements, especially in the right-handed. A sustained increase of -complexity was observed during execution of a slow movement. A decrease of -complexity which was often associated with a brief burst of spatially synchronised 10-Hz oscillations occurred at the onset of extensor muscle contraction. We suggest that increased spatial EEG synchronisation at movement onset may prevent spillover of excitation from the sensorimotor hand area to other cortical regions. During movement, the cortical neuronal assemblies subserve distinct, specialised functions manifesting in increased -complexity.     

Host response toBothrops asper snake venom

As part of the characterization of the host reactivity to the venom ofBothrops asper, we investigated the inflammatory responses in the mouse footpad model. The subcutaneously injected venom induced a rapid increase of serum IL-6 concentration, which peaked between 3 and 6 h and returned to normal values at 12 h. In contrast, serum TNF- and IL-1 were not detectable at any time point studied. A myotoxic phospholipase A₂ isoform purified from this venom, myotoxin II, was also able to induce a systemic IL-6 release when injected into the footpad. Both venom and myotoxin induced local edema and a leukocyte infiltrate accumulating in the muscle and subdermal tissue within 6 h. The infiltrate consisted predominantly of neutrophils at 6 and 24 h, but at later times, mononuclear cells also appeared. The edema, leukocyte infiltration, and IL-6 responses did not depend on the hemorrhagic activity of venom, since all three effects were seen after injection of (1) preneutralized venom, devoid of hemorrhagic activity, and (2) purified myotoxin II. Circulating platelet numbers were significantly decreased 30 min after venom injection and returned to normal after 12 h. The venom also induced a rapid inversion in the ratio of neutrophils to lymphocytes in peripheral blood, which did not normalize until 12 h later. The present observations suggest that venom, besides its cytotoxic properties, induces early hematologic and immunologic alterations. These findings may be of relevance in future treatment modalities.     

Local production of IgA- and IgM-rheumatoid factors in adult periodontal disease

The enzyme-linked immunospot assay was used to enumerate both the number and the frequency of spontaneous IgG, IgA, and IgM immunoglobulin-secreting cells and IgA- and IgM-rheumatoid factor (RF)-producing cells present in the gingivae and peripheral blood of adult periodontitis patients. Cells from 29 patients were incubated on plates coated with human IgG, Fc, or F(ab)₂ fragments and on plates coated with class-specific antihuman antibodies and secreted antibodies were subsequently visualized by means of an immunoenzymatic procedure. The data indicate that (1) IgA-RF- and IgM-RF-secreting cells are frequently present in the gingiva of adult periodontitis patients; (2) production of RF in gingivae of adult periodontitis patients occurs in the absence of demonstrable RF production by simultaneously obtained peripheral blood mononuclear cells, suggesting that local autoimmune reactions may occur in this disease; and (3) lack of correlation between IgA-RF and IgM-RF production in diseased gingiva suggests that the two RF isotypes are regulated independently of each other.     

Rapid and Sensitive Assay for Detection of Enterotoxigenic Bacteroides fragilis

Bacteroides fragilis is an obligatory anaerobic, gram-negative bacterium found among the normal intestinal flora of humans. Enterotoxigenic strains of B. fragilis (ETBF) have been associated with diarrheal diseases in humans and animals. The enterotoxin of ETBF induces fluid changes in ligated intestinal segments and cytotoxic response in HT29/C1 cells. By using a pair of monoclonal antibodies (MAbs; MAb C3 and MAb 4H8) specific for the lipopolysaccharide of B. fragilis, an assay based on immunomagnetic separation (IMS) in combination with PCR (IMS-PCR) was developed. After DNA extraction, a 294-bp fragment was amplified. The specificity of the IMS-PCR assay was evaluated by adding previously isolated and confirmed ETBF strains to normal fecal samples. All fecal samples to which ETBF strains were added were positive, showing a 100% specificity. The spiked fecal samples were also used for evaluation of the sensitivity of the assay. The detection limit was found to be ~50 CFU/g of feces. By this method 10 clinical fecal samples (5 from patients with diarrhea and 5 from healthy controls) were examined. The results of PCR were in accordance with the results of the HT29/C1 cell assay for all samples. The minimum time to retrieval of the final result by the IMS-PCR method is 36 h. The proposed IMS-PCR assay is rapid and sensitive for the direct detection of ETBF in stool samples.     

Involvement of MMPs in delayed neuronal death after global ischemia

Spatial and temporal relations between metalloproteinase (MMP-2 and MMP-9) activation and laminin degradation in gerbil hippocampus after transient cerebral ischemia has been studied. Activity of MMPs was determined by gelatin zymography in homogenates from dorsal (DP, an equivalent of CA1 sector) and abdominal (AbP, containing CA2-4 and gyrus dentatus) parts of hippocampus. A significant activation of both investigated metalloproteinases was found at 72 h of recovery. Whereas MMP-2 up-regulation did not show any spatial preferences, the increase of MMP-9 activity was observed exclusively in DP. Activation of MMP-9 at this time point correlated spatially with degradation of laminin-protein of extracellular matrix. These results show that MMP pathway may function as a component of delayed neuronal death cascade in the apoptogenic CA1 sector after transient global ischemia.     

Targeting myeloperoxidase to azurophilic granules in HL-60 cells

Myeloperoxidase (MPO) is a cationic protein and one of the major constituents of azurophilic granules in neutrophils. Here, we examined whether intracellular transport of MPO and serglycin, a chondroitin sulfate (CS)-bearing proteoglycan, is correlated. First, we examined binding of MPO to CS-Sepharose and measured an ionic interaction, which was disrupted by 200-400 mM NaCl. Next, HL-60 promyelocytes were activated with a phorbol ester, which induced an almost complete rerouting of serglycin from the granular to the secretory pathway, concomitant with a similar effect on MPO transport and secretion. We then used the membrane-permeable cross-linker dithiobis(succininmidylpropionate; DSP) after labeling HL-60 cells with [35S]methionine and [35S]cysteine for 19 h. Immunoprecipitation of MPO revealed its cross-linking to high molecular material having the appearance of a proteoglycan in sodium dodecyl sulfate-polyacrylamide gels. This assumption was confirmed by labeling HL-60 cells with [35S]sulfate for 10 min followed by DSP cross-linking and immunoprecipitation. From three granular enzymes immunoprecipitated, only the cationic MPO was cross-linked to [35S]sulfate-labeled serglycin in appreciable quantities, whereas cathepsin D or beta-N-acetylhexosaminidase was not. Thus, intracellular transport of MPO appears to be linked to that of serglycin. Extracts from high buoyant density organelles from human placenta containing MPO activity were subjected to CS-affinity chromatography. Proteins binding to CS were identified by mass spectrometry as MPO, lactoferrin, cathepsin G, and azurocidin/cationic antimicrobial protein of molecular weight 37 kDa, suggesting that serglycin may be a general transport vehicle for the cationic granular proteins of neutrophils.