Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates

A modified multiplex PCR method for detection of nine Staphylococcus aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and one form of immunoreactive toxic shock syndrome toxin based on a previously published method (S. R. Monday and G. A. Bohach, J. Clin. Microbiol. 37:3411-3414, 1999) has been developed. The modified PCR protocol seems robust and gives reliable results.     

Acute cytomegalovirus infection modulates the intestinal microbiota and targets intestinal epithelial cells

Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to assess the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes coincided with high-titer MCMV replication in the colon, crypt hyperplasia, increased colonic pro-inflammatory cytokine levels, and a transient increase in the expression of the antimicrobial protein Regenerating islet-derived protein 3 gamma (Reg3γ). Further analyses revealed that murine and human intestinal epithelial cell lines, as well as primary intestinal crypt cells and organoids represent direct targets of CMV infection causing increased cell death. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV transiently induces colitis in immunocompetent hosts by altering the intestinal homeostasis.     

Mechanistic insight of interleukin-9 induced osteoclastogenesis

Interleukin (IL)-9 is an emerging player in the pathogenesis of various chronic inflammatory diseases including bone disorders like rheumatoid arthritis (RA) and psoriatic arthritis. Recently, IL-9 was shown to enhance the osteoclast formation and their function in RA. However, the mechanisms by which IL-9 influences osteoclastogenesis are not known. Therefore, in this study we aimed to unravel the direct and indirect ways by which IL-9 can influence osteoclast formation. We used mouse bone marrow precursor cells for checking the effect of IL-9 on osteoclast differentiation and its function. Next, IL-9 induced signalling pathway were checked in the process of osteoclastogenesis. T cells play an important role in enhancing osteoclastogenesis in inflammatory conditions. We used splenic T cells to understand the impact of IL-9 on the functions of T effector (Teff) and regulatory T (Treg) cells. Furthermore, the effect of IL-9 mediated modulation of the T cell response on osteoclasts was checked using a coculture model of T cells with osteoclast precursors. We showed that IL-9 enhanced osteoclast formation and its function. We found that IL-9 activates STAT3, P38 MAPK, ERK1/2, NFκB and we hypothesize that it mediates the effect on osteoclastogenesis by accelerating mitochondrial biogenesis. Additionally, IL-9 was observed to facilitate the functions of pro-osteoclastogenic IL-17 producing T cells, but inhibits the function of anti-osteoclastogenic Treg cells. Our observations suggest that IL-9 can influence osteoclastogenesis directly by modulating the signalling cascade in the precursor cells; indirectly by enhancing IL-17 producing T cells and by reducing the functions of Treg cells.     

Prednicarbate Versus Conventional Topical Glucocorticoids: Pharmacodynamic Characterization In Vitro

Purpose. Pharmacodynamic characterization of topical glucocorticoids as prednicarbate (PC), its metabolites prednisolone 17-ethylcarbonate (PEC) and prednisolone (PD), betamethasone 17-valerate (BMV), beta-methasone (BM) and desoximetasone (DM) by evaluating their effects on epidermal and dermal cells. Synopsis of pharmacokinetic and pharmacodynamic studies, possibly explaining the improved benefit-risk ratio of prednicarbate. Methods. Isolated foreskin keratinocytes were used to investigate the influence on epidermal inflammatory processes, dermal fibroblasts of the same origin to study antiproliferative activities of glucocorticoids. Interleukins were measured by ELISA-assay, the influence on II-l-production also on mRNA-level by RNAse protection assay. Proliferation was assessed by ³H thymidine incorporation and biodegradation by HPLC/UV-absorption. Cell viability was controlled by MTT assay. Results. In keratinocytes, inflammation was induced by TNF, resulting in an increased II- l synthesis. This cytokine was particularly suppressed by PC and BMV, whereas PEC, PD, DM and BM were less potent (p 0.05). Since, however, the double ester PC is rapidly degraded in keratinocytes, a RNAse-protection assay of II-1 mRNA was performed allowing short incubation times and thus minimizing biodegradation effects. In agreement with the previous experiment, the antiinflammatory potency of native PC was confirmed. In fibroblasts, II-l and II-6 synthesis indicate proliferation and inflammation respectively. Whereas PC inhibited II- l and II-6 production in fibroblasts to a minor extent only, it was strongly reduced by the conventional glucocorticoids and PEC (p 0.05). The minor unwanted effect of PC on fibroblasts was also reflected by its low influence on cell proliferation as assayed by ³H thymindine incorporation. More pronounced antiproliferative features were observed with BM, PEC and espectially BMV. Conclusions. Correlating antiphlogistic effects in keratinocytes (suppression of II-l) with antiproliferative effects in fibroblasts (suppression of II-l and II-6), the improved benefit–risk ratio of PC compared to conventional glucocorticoids does not result only from distinct drug metabolism in the skin but also from a specific influence on the cytokine network.     

Cationic amino acid fluxes beyond the proximal convoluted tubule of rat kidney

To investigate the fluxes of cationic amino acids beyond the proximal convolution, we micropunctured and microperfused superficial tubules of male Wistar rats in vivo et situ. In free-flow micropuncture experiments, the concentrations of endogenous L-arginine⁺, [Arg], and of intravenously infused L-homoarginine⁺, [HoArg], were determined by HPLC. Fluorescein isothiocyanatelabeled inulin was detected on-line in the same tubular fluid samples. To determine undirectional fluxes, radiolabeled Arg and inulin were (1) microperfused through short loops of Henle and (2) microinfused into different tubule segments to measure urinary recovery of the radiolabel. At a mean [Arg]_plasma of 116 mol/l, [Arg] was 9.3 mol/l in the late proximal tubule (LPT), and 35.6 mol/l in the early distal tubule (EDT) corresponding to fractional deliveries (FD) of 0.055 in LPT and 0.078 in EDT. Fractional urinary excretion (FE) of Arg was 0.00033 (P<0.05 vs FD_EDT). Infusion of HoArg (2.5 or 7.5 mol/min) led to respective mean [HoArg]_plasma values of 1.44 and 3.73 mmol/l, and resulted in respective FD_LPT values for HoArg of 0.23 and 0.53, respective FD_EDT values of 0.29 and 0.41, and finally, respective FE values for HoArg of 0.25 and 0.58. When short loops of Henle were microperfused with 1 or 50 mmol/l [¹⁴C]Arg (+[³H]inulin), fractional recovery (FR) of ¹⁴C (relative to inulin) in the EDT was 0.13 and 0.36, respectively. During microinfusion of radiolabeled Arg (1 or 50 mmol/l) and inulin into LPT, the urinary FR of the radiolabel was 0.14, or 0.59, respectively. If 0.007, 1 or 50 mmol/l radiolabeled Arg were microinfused into EDT, the respective urinary FR of the radioactivity was 1.02, 1.10, or 1.01. Microperfusion of microinfusion of 1 mmol/l [¹⁴C]Arg plus 50 mmol/l HoArg resulted in a FR_EDT of ¹⁴C of 0.43 (loop, perfusion) and an FE for ¹⁴C of 0.69. Five conclusions can be drawn. First, cationic amino acids can enter and leave the lumen of short loops of Henle through specific carrier(s) at high rates, although, secondly, net transport is small or absent. Thus, medullary tubule cells can be supplied with Arg from the lumen of short loops of Henle for urea and nitric oxide production. Thirdly, the distal convolution of superficial nephrons and the collecting duct are not permeable to Arg. Thus, fourthly, the difference between FD_EDT and urinary FE of Arg must be explained by an inter-nephron heterogeneity between deep and superficial nephrons. Finally, the process responsible for the different Arg handling in deep nephrons is not accessible to HoArg or, if so, it is saturated at millimolar concentrations.     

Cationic-amphiphilic arpromidine-derived guanidines and a histamine trifluoromethyl-toluidide derivative may activate pertussis toxin-sensitive G-proteins by a receptor-independent mechanism

Formyl peptides activate superoxide anion (O₂ ^–) formation in human neutrophils and in HL-60 cells via pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins), and histamine (HA) mediates inhibition of O₂ ^– formation via H₂-receptors. We have studied the effects of lipophilic arpromidine-derived guanidines, which are potent, full H₂-receptor agonists in the guinea pig atrium, on O₂ ^– formation and on activation of G-proteins in HL-60 membranes and on purified G-proteins. We have also studied the effects of a HA trifluoromethyl-toluidide derivative (HTMT), a cationic-amphiphilic HA derivative which activates O₂ ^– formation in HL-60 cells through a mechanism which is independent of known HA receptor subtypes, on G-protein activation. Guanidines, at concentrations, up to 30 mol/l inhibited and, at concentrations above 30 mol/l, enhanced formyl peptide-induce O₂ ^– formation in neutrophils. In HL-60 cells, guanidines per se activated O₂ ^– formation. The stimulatory effects of guanidines on O₂ ^– formation were not inhibited by H₁- or H₂-receptor antagonists. In HL-60 membranes, guanidines and HTMT, activated high-affinity GTPase in a PTX-sensitive manner. These substances also increased GTP hydrolysis effected by transducin and G_i/G_o-proteins. Our data suggest that lipophilic guanidines and HTMT may act as receptor-independent activators of PTX-sensitive G-proteins, resulting in stimulation of O ₂ ^– formation.     

Increased cation transport inmdr1-gene-expressing K562 cells

Cation-transport properties were compared in a human leukemic cell line (K562) and its vincristineselected,mdr1-gene-expressing sublines (K562/Vcr30 and K562/Vcr150) by the capacity of the cells to accumulate the potassium analogue thallium (²⁰¹Tl). Determination of the time course of thallium accumulation in the absence and presence of ouabain, an inhibitor of sodium-potassium adenosine triphosphatase (ATPase), showed that the initial (at 20 min) rate of ouabain-resistant uptake was about 70% higher in the K562/Vcr30 cells than in the parental line. The maximal rate (V_max) of ouabain-resistant uptake was 78 mmol/h for K562 cells and 115 mmol/h for K562/Vcr30 cells, and the Michaelis constant (K _m) was 0.37 and 0.18 mmol, respectively. Bumetanide (50 M), a specific inhibitor of ouabain-resistant Na–K–Cl cotransport, inhibited the elevated²⁰¹TI uptake in K562/Vcr150 cells but had no effect on cellular vincristine accumulation. Incubation with different multidrug resistance (MDR)-reversing agents (verapamil as well as cyclosporin A and its analogue PSC833) had no significant effect on²⁰¹Tl uptake. Membrane depolarization by an elevation of the potassium concentration in the incubation medium did not affect vincristine accumulation in any cell line, which indicated that the changed drug-transport properties inmdr1-gene-expressing cells were not due to membrane hyperpolarization. It was concluded that P-glycoprotein-positive cells have a more efficient ouabain-resistant cation-transport mechanism than to cells without P-glycoprotein. A functional relationship between this phenomenon and MDR was not identified.     

Non-radioactive hybridization in microwells using enzyme linked immune sorbent assay for detection of RT-PCR-Am-plified CK19- and CEA-mRNA

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Einfluß verschiedener niedriger Droperidol-Dosierungen auf postoperative Angst, innere Anspannung, allgemeine Befindlichkeit und PONV

Background: Droperidol even in low doses such as 0,5?mg to 1,25?mg can increase postoperative anxiety and state of tension. The aim of this study was to determine whether these side effects occur frequently following low-dose droperidol and to see whether these are dose related. Methods: 184 female in- and outpatients ASA grade 1 and 2 undergoing gynaecological laparoscopy were recruited to this prospective, double-blind study. General anaesthesia was standardized (induction with thiopentone, fentanyl 2?µg/kg and vecuronium 0,1?mg/kg, tracheal intubation, maintainance with enflurane in N₂O/O₂). Patients were randomly allocated to receive saline (n=45), 0,625?mg (n=46), 1,25?mg (n=47) or 2,5?mg (n=46) droperidol i.v. 10 minutes before the end of surgery. 1, 3, 6, and 24 hours postoperatively, the patients’ anxiety, state of tension and overall mood was evaluated using two psychological questionnaires which had been tested for the perioperative period (Erlanger anxiety and tension-scale / BSKE-EWL-test). Sedation was evaluated by the staff of the recovery room. In addition, postoperative nausea and vomiting (PONV) was assessed using a 100?mm visual analogue scale and by counting the episodes of retching or vomiting. PONV was then rated over the whole observation period as none, mild, moderate or severe using a fixed scoring algorithm. Statistical analysis was performed using the ANOVA and the chi²-test. Results: The patients did not differ with regard to biometric data, duration of surgery and anaesthesia. The postoperative scores for anxiety, state of tension and overall mood were not different between the groups at any observation time (Fig.?1: anxiety and tension: P=0,5687; figure 2: overall mood: P=0,0647). Quality of sleep in the first night after surgery was the same in all groups (Table?2 and 3). Sedation was not significantly different (Table?4; P=0,0704). Furthermore, duration of stay in the recovery room did not differ (P=0,4353). On the other hand, three patients from the 2,5?mg droperidol group had to stay unexpectedly on the ward overnight, because they had been too much sedated to be discharged at home. This was not the case with any patient from the other groups. Compared to placebo, PONV over the whole 24?h observation period was significantly reduced by droperidol (Fig.?3; P=0,0338): completely free from PONV: placebo: 41,3%, 0,625?mg droperidol: 67,4%, 1,25?mg droperidol: 53,2%, 2,5?mg droperidol: 71,7%. Also the severity of PONV was reduced. Conclusion: In gynaecological laparoscopy under general anaesthesia with tracheal intubation, we recommend droperidol 0,625?mg in the prevention of PONV, as it reduces PONV as well as 2,5?mg with no severe sedation in this dosage. Psychological side effects did not occur more frequently after droperidol compared to placebo in any of the investigated dosages.     

Physicochemical Characterization of Protein-Free Low Density Lipoprotein Models and Influence of Drug Loading

Purpose. Drug free and drug loaded protein-free low density lipoprotein (LDL) models consisting mainly of phospholipids, cholesterol, cholesterol esters, and triglycerides in ratios found for physiological LDL have been prepared. Their physicochemical characteristics were compared with those of physiological LDL. Methods. Different characterization methods were used: photon correlation spectroscopy, transmission electron microscopy, X-ray solution scattering, and ¹H nuclear magnetic resonance spectroscopy (NMR). Results. Particle sizes are highly dependent on the preparation method and in particular on the homogenization conditions. Electron microscopy indicates that the size distributions of model systems are much broader than those of physiological LDL. The X-ray solution scattering patterns of the model systems display a temperature dependent maximum near 3.8 nm similar to that found in the patterns of physiological LDL. NMR indicates a comparable mobility of the lipid molecules in model particles and in physiological LDL. The influence of drug loading is similar to that found earlier for physiological LDL. In particular, the incorporation of the anti-cancer drug WB 4291 seems to have a fluidizing effect on the lipids in the core region of the particles. Conclusions. The preparation method of LDL model systems is of crucial importance as only the solvent evaporation method yielded systems in the size range of physiological LDL with acceptable high lipid concentrations. The fluidizing influence of temperature and drug incorporation (WB 4291) may be a disadvantage in drug targeting.