A co-stimulatory signal through ICAM-beta2 integrin-binding potentiates neutrophil phagocytosis.

The beta2 integrin LFA-1 (lymphocyte function associated antigen; CD11a/CD18) is the common ligand for the intercellular adhesion molecules (ICAMs). Integrins support cell function by providing co-stimulatory second signals that are a precondition for full cell activation first described for ICAM-1-binding to LFA-1 in lymphocytes. Integrins can also serve to activate functions associated with distinct subunits of other integrins. In addition to LFA-1, neutrophils express the beta2 integrin Mac-1 (CD11b/CD18; CR3) that apparently contains multiple sites that bind invading microbes directly or through surface-fixed C3, resulting in activation of the phagocyte function. Expression of the LFA-1 counter-receptor ICAM-1 on endothelial cells occurs only at the site of inflammation. Therefore, in neutrophils, ICAM-1 ligand binding could, as with lymphocytes, also play a part as a co-stimulatory signal to induce full phagocytotic function. We show that in neutrophils, the LFA-1 ligand interaction is the stimulatory signal to express full phagocytotic activation. This is best demonstrated by the rapid association of Streptococcus pyogenes with neutrophils, followed by ingestion, strong oxidative-burst induction and enhanced killing of these bacteria, which are well-known for their resistance to human neutrophil defense. These findings may contribute to the development of therapeutic strategies targeting the modulation of ICAM-1-leukocyte interaction.

Simultaneous measurement of biopolymer-mediated Mac-1 up-regulation and adherence of neutrophils: a novel flow cytometric approach for predicting initial inflammatory interaction with foreign materials.

Implantation of any medical device normally causes an inflammatory cell interaction with the foreign material. In vitro cell activation of human neutrophils (Mac-1 upregulation) has been taken as one measure to assess the attributable risk of inflammation due to biopolymers before their clinical application. Mac-1 expression has generally been measured by flow cytometric assays, whereas quantification of neutrophil adhesion to the biopolymer surfaces has been performed by separate and time-consuming assays, e.g. microscopically by differential cell counting. However, due to an increasing number of surface-modified novel biopolymers entering clinical usage, effective testing of their inflammatory potential is now mandatory. To facilitate these analyses, we have developed a novel flow cytometric assay permitting simultaneous measurement of biopolymer-mediated neutrophil activation and adhesion. The biopolymers were used as beads (diameter 25+/-10 microm), and were demonstrated to be non-phagocytosable and non-fluorescent before being co-incubated with whole human blood (range of ratio granulocytes/beads from 5:1 to 1:1). Besides flow cytometric measurement of Mac-1 up-regulated neutrophils as fluorescing events, a fluorescence of the bead population indicates the adherence of activated neutrophils to the biopolymer surface.After establishing this assay, we evaluated it by comparing six different biopolymers. We observed high levels of Mac-1 expression (>70% of positive control) accompanied by increased adhesiveness (>60% of neutrophils) for polyurethane (PUR), polymethylmetacrylate (PMMA), and poly-DL-lactide (PDLLA) beads. Low Mac-1 expression levels (<10%) accompanied by a low percentage of adhering neutrophils (<10%) were observed for polyethylene (PE), polyisoprene (PI), and silicone (SI) beads.

The rationale and method for constructing internal control DNA used in pertussis polymerase chain reaction.

The inclusion of an appropriate internal control DNA in polymerase chain reaction (PCR) is a rapid and simple method for the detection of PCR failure. Two PCR coamplification internal control DNAs (ICD I and ICD II) with the same primer-binding sequences as the target DNA for the detection of Bordetella pertussis and Bordetella parapertussis were produced using an overlap extension technique and a PCR MIMIC construction kit, respectively. The ICD II was further evaluated in a prospective clinical study in 360 patients with a clinical diagnosis of pertussis. From 360 nasopharyngeal swabs the internal control was positive in 318 (88%) samples, but was negative in 42 (12%). After phenol-chloroform extraction an additional 10 internal controls became positive. For the detection of PCR failure, the use of internal control DNA is highly recommended for PCR-based identification of B. pertussis and B. parapertussis organisms from nasopharyngeal swabs and aspirates.

Phagocytosis, oxidative burst, and killing of Candida dubliniensis and Candida albicans by human neutrophils.

Candida dubliniensis is a phylogenetically closely related species to Candida albicans. So far virtually nothing is known about the virulence factors of C. dubliniensis. Cell surface hydrophobicity (CSH) plays a critical role in adhesion of microorganisms to phagocytic cells; hydrophobic cells of C. albicans have been reported to be less sensitive to phagocytic killing than hydrophilic cells. C. dubliniensis displays CSH at 37 degrees C in contrast to C. albicans. To elucidate this issue, we determined levels of phagocytosis, oxidative burst and killing by human neutrophils of C. dubliniensis (n=10) compared to C. albicans (n=10) both cultured at 37 degrees C. Obtained test results revealed no statistically significant differences between these two yeast species for the level of phagocytosis (77.3 vs. 76.2% after 60 min), evoked oxidative burst (64.5 vs. 67.3% after 30 min) and killing (72.7 vs. 73.1% after 240 min). Therefore, human neutrophils can be considered to be equally efficient against these two yeast species.

Remission of IgA nephropathy following treatment of cytomegalovirus infection with ganciclovir.

Following the detection of cytomegalovirus antigen in mesangial cells of some patients with IgA nephropathy, an important role of human cytomegalovirus in the pathogenesis of IgA nephropathy has been discussed. We studied a case of IgA nephropathy with rapid deterioration of renal function associated with cytomegalovirus infection. Following an infection of the upper respiratory tract, a 57-year-old woman developed with hematuria and acute renal failure. The histological diagnosis of IgA nephropathy was established and renal function transiently improved during immunosuppressive therapy. However, the ensuing clinical course was complicated by severe bleeding from intestinal ulcera, thrombocytopenia, pneumonia and relapse of renal failure. The histological investigation of colonic mucosa showed characteristic "owl's eye" cells leading to the diagnosis of cytomegalovirus disease as the cause of intestinal bleeding. Immunosuppression was stopped and treatment with ganciclovir started. Pneumonia as well as intestinal bleeding disappeared and, of particular note, renal function improved considerably. Following discontinuation of antiviral therapy CMV-disease reoccurred and renal function deteriorated again. The patient was restarted on ganciclovir therapy and, again, serum creatinine fell quickly. This impressive and reproducible clinical improvement of renal insufficiency under antiviral therapy with ganciclovir provides some evidence for an important role of cytomegalovirus in the pathogenesis of this case of IgA nephropathy.

Phenotyping of Staphylococcus aureus reveals a new virulent ST398 lineage.

Staphylococcus aureus sequence type (ST)398, which is commonly found as a colonization strain in pig farming, is emerging more frequently as the cause of human infections. In this study, we analysed ST398 of porcine and human origin at the genetic, protein and immunogenic levels. Although genetic analysis of the genes encoding the major virulence factors revealed the presence of the same genes in all strains studied, the results demonstrated spa type crossing alterations in adhesion abilities in addition to a strongly enhanced lysis activity directly linked to impaired clearance attributable to polymorphonuclear leukocytes (PMNs). This change in virulence pattern indicates high heterogenicity in the ST398 pool that is not based on a different genetic make-up, but probably on variations in the genetic regulation systems. These modifications, which are tightly connected to pathogenicity, cannot be detected by conventional diagnostic methods.

Electrically stimulated cardiomyocytes: evaluation of shortening, oxygen consumption and tracer uptake.

Progress in the utilisation of the model "isolated cardiac myocytes" was achieved by designing a special stimulation chamber. This chamber allows eliciting of contraction of suspended myocytes by electrical stimulation. Oxygen consumption was thereby linearly enhanced dependent on the rate of stimulation. Different extent of shortening was observed indicating different inotropic state. Considering the myocytes to behave as a spring (with small delta L) a correlate to work performed was obtained which allowed evaluation of changes in ATP/O-ratios. Uptake of 3-O-methyl-D-glucose was dependent on the mechanical and therefore metabolic activity of the cells. The results show that transfer of glucose across the myocardial sarcolemma is adapted to the metabolic demand by affinity variation and not by recruitment of the transporters.

[Not Available]. / Anerkannte Impfschäden in der Bundesrepublik Deutschland 1990-1999.

The decrease in infectious diseases preventable by immunisation and the absence of complications caused by these diseases leads to an increased awareness of vaccine-associated adverse events. The analysis of a survey of the vaccine injury compensation data from the German Bundesländer shows the decrease in accepted and demanded compensation from 1991 to 1999. From 1976 to 1990 1139 of 4569 demands were accepted, whereas from 1991 to 1999, acceptance of only 389 of 2543 demands was reported. In all, 38% of the accepted compensations refer to the smallpox vaccine which is not longer recommended by the STIKO (Permanent Vaccination Commission in Germany) since immunisation against smallpox was stopped in the 1980s. Regional differences show that process elements of the German healthcare system as well as political and social reasons express most of the differences in rates and prevalence of vaccine associated adverse events. Epidemiological questions and questions of causality cannot be answered by the analysis of data collected in vaccine injury programs. Valid analysis needs a register of individual documented cases of vaccine adverse events. The surveillance of adverse events following immunisation will make progress by the analysis of data reported according to the Protection against Infection Act (IfSG) and by further surveillance systems that should be implemented in the near future. A centralised commission with expert opinion concerning causality could increase transparency and homogeneity within judgement and documentation of vaccine associated adverse events.

M-related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes.

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsonophagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M-related proteins, Mrp and Enn, which have been described as IgG- and IgA-binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M-untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony-forming units, all mutant strains with the exception of the mrp4 mutant exhibited reduced multiplication factors as compared to the isogenic wild-type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM-labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild-type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis-sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co-incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.

Impact of M49, Mrp, Enn, and C5a peptidase proteins on colonization of the mouse oral mucosa by Streptococcus pyogenes.

Resistance to phagocytosis is a hallmark of virulent Streptococcus pyogenes (group A streptococcus). Surface-bound C5a peptidase reduces recruitment of phagocytes to the site of infection, and hyaluronic acid capsules and/or the M protein limit the uptake of streptococci. In this study the relative impact of M and M-like proteins and the C5a peptidase on the virulence of a serotype M49 strain was assessed. The capacities of isogenic strains with an insertion mutation in emm49; with a deletion mutation in scpA49 (C5a peptidase gene); and with a deletion that removes all three M-like genes, mrp49, emm49, and enn49, to colonize mice and resist phagocytosis were compared. Experiments confirmed results obtained in an earlier study, which showed that the M49 protein was not required for in vitro resistance to phagocytosis, and also showed that the M protein was not required for colonization of mice. Failure to produce all three M-like proteins, M49, Mrp, and Enn49, significantly reduced the ability of these streptococci to resist phagocytosis in vitro but did not significantly alter the persistence of streptococci on the oral mucosa. In vitro experiments indicate that M+ streptococci are phagocytized by polymorphonuclear leukocytes that have been activated with phorbol-12-myristate 13-acetate or recombinant human C5a. This observation may explain the finding that expression of M49 protein is not essential for short-term colonization of the mouse oral mucosa.

Rapid identification of Candida glabrata by using a dipstick to detect trehalase-generated glucose.

Candida glabrata is a yeast frequently isolated from human specimens. Based upon its well-known ability to rapidly hydrolyze trehalose, we have developed a novel and cost-effective test incubating one yeast colony emulsified in 50 microl of citrate buffer (0.1 M [pH 5. 0]) containing 4% (wt/vol) trehalose for 3 h at 37 degrees C. Trehalase-generated glucose is detected with a commercially available dipstick (range, 1.0 to 50 g/liter). For evaluation, consecutive clinical isolates and several reference strains of C. glabrata (n = 160), C. albicans (n = 120), and other yeast species with potential ability for utilization of trehalose (C. dubliniensis, n = 11; C. famata, n = 15; C. guilliermondii, n = 5; C. lusitaniae, n = 16; C. parapsilosis, n = 20; C. tropicalis, n = 34; C. viswanathii, n = 5; Pichia angusta, n = 2; C. zeylanoides, n = 2; Saccharomyces cerevisiae, n = 16; C. neoformans, n = 7) were tested. Identification of C. glabrata is achieved within 3 h, with a specificity of 99.1% and a sensitivity of 98.8% when grown on Sabouraud dextrose agar supplemented with 4% glucose.

Relation of suppressor activity to lymphocyte subsets, in vitro IL-2R expression, and biopsy results in cardiac transplant patients.

Nonspecific suppressor activity of peripheral blood mononuclear cells (PBMC) was determined from 8 patients preoperatively and 22 patients subsequent to heart transplantation. Whereas no correlation was found between any defined lymphocyte subsets, in vitro interleukin-2 (IL-2) production, and IL-2R expression, or biopsy-proven rejection and suppressor cell activity when data obtained on the same day as the suppressor assay were analysed, the phytohemagglutinin--but not the concanavalin A induced, in vitro IL-2 production--was significantly enhanced (P < 0.02) in patients with evidence of concomitant rejection. In contrast, a significant correlation between diminished, nonspecific suppression and rejection was found when the results of biopsies performed up to 2 (P < 0.0075) and 4 (P < 0.0033) weeks after the investigation of suppressor cell activity were compared. We conclude that periodic determination of the suppressor functional status may be useful to discriminate between patients at low or high risk for graft rejection after heart transplantation.

Isolation of catalase-negative Listeria monocytogenes strains from listeriosis patients and their rapid identification by anti-p60 antibodies and/or PCR.

Two catalase-negative Listeria monocytogenes serovar 1/2b strains were isolated from listeriosis patients in 1995 in Germany. The infections appeared in individuals from different cities at different seasons and were caused by L. monocytogenes strains of different clonal types. In particular, the catalase reaction of one strain isolated from blood was consistently negative, whereas this reaction was only reversibly blocked when the strain was freshly isolated from ascitic fluid. After subculturing, the catalase-positive reaction was restored. Initially, identification of these isolates was difficult to achieve not only because of the lack of a catalase reaction, which generally distinguishes L. monocytogenes from other morphologically similar pathogenic gram-positive bacteria, but also because other routinely used biochemical tests such as CAMP and the commercial API test gave unclear results. However, rapid and unequivocal identification of these strains was possible by analyzing secretions of the p60 protein in culture supernatants by enzyme-linked immunosorbent assay or Western blot (immunoblot) analysis with our recently developed Listeria- and L. monocytogenes-specific anti-p60 antibodies. Additionally, the identifications were confirmed by Listeria- and L. monocytogenes-specific PCR analyses with primers derived from the iap, hly, and prfA genes. Immunoanalyses also allowed for the differentiation of these two strains, whereas no differentiation was possible by PCR when the internal, variable repetitive iap gene portion was analyzed. However, size variations of the PCR products comprising these gene portions which were obtained from a number of L. monocytogenes strains belonging to the same serotypes indicated that this type of PCR is not only useful for specific identifications but may be used in parallel as an additional marker for epidemiological studies. In conclusion, the data suggest that catalase production should not be taken as a strict criterion for the identification of listeriae. Furthermore, at least the infection caused by the stably catalase-negative strain supports the notion that catalase does not seem to be necessary for the intracellular growth of L. monocytogenes.

[Effect of premedication and barbiturate hypnotics on motor evoked potentials induced by magnetic cortex stimulation]. / Einfluss von Prämedikation und Barbiturathypnotika auf motorisch evozierte Potentiale mittels magnetischer Kortexstimulation.

OBJECTIVE: The aim of this study was to investigate the influence of the premedication and the influence of the two barbiturates methohexital and thiopental on magnetically evoked compound muscle action potentials (magnet MEP) in humans. METHODS: 40 Patients (ASA-PS I-II) undergoing lumbar nucleotomy were included in this study after obtaining written informed consent. The study was approved by the local ethical committee. All patients were premedicated with 0.5 mg atropine, 25 mg promethazine and 50 mg pethidine. For induction of anaesthesia patients randomly received methohexital or thiopental by continuous infusion with increasing infusion rates every 15 seconds up to a minimal anaesthesia level in 15 minutes. Transcranial magnetic stimulation was delivered by the magstim 200 magnetic stimulator. Magnetic MEPs were recorded from the surface of the short abductor pollices muscle. MEP-examination was performed preoperatively, after premedication and every two minutes during the induction of anaesthesia. Every other two minutes the patients level of consciousness were assessed and documented. Statistical calculations were performed with the U-test. RESULTS: No statistical differences were found for the mean induction time in the two groups. No statistical difference in amplitude and latency could be observed between the preoperative values and the values measured after premedication. During anaesthesia induction the amplitude decreased in both groups. In 25 of the 40 cases, the MEP disappeared completely before the patients fell asleep. The thiopental group showed a significantly lower incidence of MEP preservation (20%) compared to methohexital (50%). CONCLUSIONS: Premedication with atropine, promethazine and pethidine has no influence on magnetic MEP. Methohexital allows the highest incidence of successful MEP recordings with sufficient anaesthesia. A success rate of only 50% even in cases without motorpathway affection makes the application of magnetic MEP an unreliable tool for intraoperative monitoring.

Measuring resistance to phagocytosis of group A and G streptococci: comparison of direct bactericidal assay and flow cytometry.

M protein is thought to contribute to the ability of non-opsonized group A and group G streptococci (GAS and GGS, respectively) to resist phagocytosis by polymorphonuclear leukocytes. In previous studies, correlation between M protein expression and phagocytosis was determined by incubating these pathogens in human blood and comparing colony-forming bacterial counts prior to and after exposure to blood (direct bactericidal assay; DBA). Here, we report the application of flow cytometry to measure GAS and GGS resistance to phagocytosis. The results of the assays were in complete agreement with those from DBAs. Nevertheless, flow cytometry was regarded as superior to DBA because of its speed and potential uses for quantitative studies. In addition, the use of anti-CD11b monoclonal antibody for granulocyte staining guaranteed a non-compromized granulocyte function. The optimized protocol for flow cytometry presented here could be utilized to directly measure the involvement of individual protein types in bacterial resistance to phagocytosis.

Neisseria meningitidis serogroup B peritonitis associated with continuous ambulatory peritoneal dialysis.

A case of Neisseria meningitidis peritonitis in a 41-year-old female undergoing continuous ambulatory peritoneal dialysis for end-stage renal failure due to insulin-dependent diabetes mellitus is reported. The bacterial strain was serosubtyped as 4:P1.15, a rarely encountered type. Surprisingly, the minimal inhibitory concentration of vancomycin for the isolate was low (16 mg/l). This may have contributed to the complete recovery of the patient.

Endophthalmitis due to Microbacterium species: case report and review of microbacterium infections.

Microbacterium species (formerly CDC [Centers for Disease Control and Prevention] coryneform group A-4 and A-5 bacteria) are widely distributed in the environment and rarely cause infections in humans. We present a case of endophthalmitis due to Microbacterium species that occurred after accidental trauma and review the literature on microbacterium infections. If the infected tissue or medical device is removed and antimicrobial therapy (preferably with beta-lactams or glycopeptides) is instituted, the prognosis is usually favorable for patients with microbacterium infections.

M or M-like protein gene polymorphisms in human group G streptococci.

Many group G streptococci (GGS) isolated from infected humans (but not from animal sources) express M or M-like proteins with biological, immunochemical, and genetic features similar to those of group A streptococci (GAS). To further elucidate the recently proposed M-like protein gene (emmL gene) polymorphisms in GGS, Southern blots of genomic DNAs from 38 epidemiologically unrelated GGS strains isolated from human specimens and 12 GGS strains recovered from animal sources were hybridized with oligonucleotide probes designed to specifically detect GAS M class I and M class II M protein (emm) genes. All human-associated GGS strains showed DNA homology to the GAS M class I emm gene probe, whereas no hybridization was found with DNA from any of the animal-associated strains. The emmL genes from all human isolates were amplified by PCR, and the complete sequence of the emmL gene of the Rebecca Lancefield grouping strain D166B was determined. Again, this gene exhibited the structural features typical for emm genes of M class I GAS. The 5' regions of the PCR-amplified emmL genes of the remaining 37 human GGS strains were sequenced. This region showed a sequence diversity similar to that known for GAS emm genes. When strains whose N-terminal emmL gene sequences showed a homology of > 95% were defined as belonging to one genetic type, 30 strains were segregated into six distinct genetic types, whereas the remaining 8 strains each exhibited a unique emmL gene sequence. A high degree of homology between the N-terminal emmL gene segments of six GGS strains and the corresponding regions of either the emm12 or the emm57 gene of GAS was found, suggesting a horizontal gene transfer between strains of these species of beta-hemolytic streptococci. Besides a further understanding of the evolution of GGS emmL genes, the observed emmL gene polymorphisms in GGS could provide the basis for a molecular subspecies delineation of strains and offers the potential of typing GGS for epidemiological purposes.