Regional analysis of inflammation and contractile function in reperfused acute myocardial infarction by in vivo 19F cardiovascular magnetic resonance in pigs.

Inflammatory cell infiltration is central to healing after acute myocardial infarction (AMI). The relation of regional inflammation to edema, infarct size (IS), microvascular obstruction (MVO), intramyocardial hemorrhage (IMH), and regional and global LV function is not clear. Here we noninvasively characterized regional inflammation and contractile function in reperfused AMI in pigs using fluorine (19F) cardiovascular magnetic resonance (CMR). Adult anesthetized pigs underwent left anterior descending coronary artery instrumentation with either 90 min occlusion (n = 17) or without occlusion (sham, n = 5). After 3 days, in surviving animals a perfluorooctyl bromide nanoemulsion was infused intravenously to label monocytes/macrophages. At day 6, in vivo 1H-CMR was performed with cine, T2 and T2* weighted imaging, T2 and T1 mapping, perfusion and late gadolinium enhancement followed by 19F-CMR. Pigs were sacrificed for subsequent ex vivo scans and histology. Edema extent was 35 ± 8% and IS was 22 ± 6% of LV mass. Six of ten surviving AMI animals displayed both MVO and IMH (3.3 ± 1.6% and 1.9 ± 0.8% of LV mass). The 19F signal, reflecting the presence and density of monocytes/macrophages, was consistently smaller than edema volume or IS and not apparent in remote areas. The 19F signal-to-noise ratio (SNR) > 8 in the infarct border zone was associated with impaired remote systolic wall thickening. A whole heart value of 19F integral (19F SNR × milliliter) > 200 was related to initial LV remodeling independently of edema, IS, MVO, and IMH. Thus, 19F-CMR quantitatively characterizes regional inflammation after AMI and its relation to edema, IS, MVO, IMH and regional and global LV function and remodeling.

Genomic and phenotypic diversity of Clostridium difficile during long-term sequential recurrences of infection.

Infection with the emerging pathogen Clostridioides (Clostridium) difficile might lead to colonization of the gastrointestinal tract of humans and mammals eventually resulting in antibiotic-associated diarrhea, which can be mild to possibly life-threatening. Recurrences after antibiotic treatment have been described in 15-30% of the cases and are either caused by the original (relapse) or by new strains (reinfection). In this study, we describe a patient with ongoing recurrent C. difficile infections over 13 months. During this time, ten C. difficile strains of six different ribotypes could be isolated that were further characterized by phenotypic and genomic analyses including motility and sporulation assays, growth fitness and antibiotic susceptibility as well as whole-genome sequencing. PCR ribotyping of the isolates confirmed that the recurrences were a mixture of relapses and reinfections. One recurrence was due to a mixed infection with three different strains of two different ribotypes. Furthermore, genomes were sequenced and multi-locus sequence typing (MLST) was carried out, which identified the strains as members of sequence types (STs) 10, 11, 14 and 76. Comparison of the genomes of isolates of the same ST originating from recurrent CDI (relapses) indicated little within-patient microevolution and some concurrent within-patient diversity of closely related strains. Isolates of ribotype 126 that are binary toxin positive differed from other ribotypes in various phenotypic aspects including motility, sporulation behavior and cell morphology. Ribotype 126 is genetically related to ribotype 078 that has been associated with increased virulence. Isolates of the ribotype 126 exhibited elongated cells and a chaining phenotype, which was confirmed by membrane staining and scanning electron microscopy. Furthermore, this strain exhibits a sinking behavior in liquid medium in stationary growth phase. Taken together, our observation has proven multiple CDI recurrences that were based on a mixture of relapses and reinfections.

Multilocus sequence typing and CTX-M characterization of ESBL-producing E. coli: a prospective single-centre study in Lower Saxony, Germany.

The increasing prevalence of extended-spectrum ß-lactamase (ESBL)-producing Gram-negative bacteria is a serious threat for current healthcare settings. In this study we investigated the molecular epidemiology of ESBL-producing E. coli at the University Medical Center Göttingen in Lower Saxony, Germany. All E. coli isolates with an ESBL phenotype were collected during a 6-month period in 2014. Multilocus sequence typing and CTX-M characterization were performed on 160 isolates. Of the ESBL-producing isolates 95·6% were CTX-M positive. Compared to recent Germany-wide studies, we found CTX-M-1 to occur in higher frequency than CTX-M-15 (44·4% vs. 34·4%). CTX-M-14 and CTX-M-27 were detected at 9·4% and 5·0%, respectively. The globally dominant sequence type (ST) 131, which is often associated with CTX-M-15, occurred at a relatively low rate of 24%. Major non-ST131 sequence types were ST101 (5%), ST58 (5%), ST10 (4·4%), ST38 (4·4%), ST410 (3·8%) and ST453 (3·1%). Several of these major sequence types were previously shown to be associated with livestock farming. Together, our study indicates that E. coli lineage distribution in individual healthcare settings can significantly differ from average numbers obtained in nationwide studies.

Improved diagnosis of idiopathic giant cell myocarditis and cardiac sarcoidosis by myocardial gene expression profiling.

AIMS: Improvement of clinical diagnostics of idiopathic giant cell myocarditis (IGCM) and cardiac sarcoidosis (CS), two frequently fatal human myocardial diseases. Currently, IGCM and CS are diagnosed based on differential patterns of inflammatory cell infiltration and non-caseating granulomas in histological sections of endomyocardial biopsies (EMBs), after heart explantation or postmortem. We report on a method for improved differential diagnosis by myocardial gene expression profiling in EMBs. METHODS AND RESULTS: We examined gene expression profiles in EMBs from 10 patients with histopathologically proven IGCM, 10 with CS, 18 with active myocarditis (MCA), and 80 inflammation-free control subjects by quantitative RT-QPCR. We identified distinct differential profiles that allowed a clear discrimination of tissues harbouring giant cells (IGCS, CS) from those with MCA or inflammation-free controls. The expression levels of genes coding for cytokines or chemokines (CCL20, IFNB1, IL6, IL17D; P < 0.05), cellular receptors (ADIPOR2, CCR5, CCR6, TLR4, TLR8; P < 0.05), and proteins involved in the mitochondrial energy metabolism (CPT1, CYB, DHODH; P < 0.05) were deregulated in 2- to 300-fold, respectively. Bioinformatic analyses and correlation of the gene expression data with immunohistochemical findings provided novel information regarding the differential cellular and molecular pathomechanisms in IGCM, CS, and MCA. CONCLUSION: Myocardial gene expression profiling is a reliable method to predict the presence of multinuclear giant cells in the myocardium, even without a direct histological proof, in single small EMB sections, and thus to reduce the risk of sampling errors. This profiling also facilitates the discrimination between IGCM and CS, as two different clinical entities that require immediate and tailored differential therapy.

[Incidence of Candidaemia Following Abdominal Surgery in German Hospitals]. / Inzidenz der Candidämie nach gastrointestinal-chirurgischen Eingriffen in deutschen Akutkrankenhäusern.

BACKGROUND: Invasive Candida infections following abdominal surgery represent a significant medical problem. This study initiates a benchmarking project to pinpoint the current role and epidemiology of candidemia in this patient group in German hospitals. MATERIAL AND METHODS: During the year 2010 data derived from 47 704 abdominal surgery cases in hospitals from Germany were analysed in order to determine benchmarking incidences for candidemia. RESULTS AND CONCLUSION: In 20.3 % of all recognised bloodstream infections Candida spp. were identified as the responsible organisms. If related to all abdominal surgery cases analysed in this study, a candidemia-benchmarking incidence of 0.15 % (95 % CI: 0.10-0.21 %) was determined. In patients who required intensive care after surgery the incidence of candidemia was found to be 0.89 % (95 % CI: 0.57-1.38 %). The incidence increased to 3.13 % (95 % CI: 2.09-4.66 %) in patients who received blood culture diagnosis. The German National Reference Centre of Systemic Mycosis provides hospital specific data for participants of this study to enable benchmarking and infection control (www.nrz-mykosen.de/gastrointestinalchirurgie).

Seroprevalence of campylobacteriosis and relevant post-infectious sequelae.

Post-infectious sequelea such as Guillain Barré syndrome (GBS), reactive arthritis (RA), and inflammatory bowel disease (IBD) may arise as a consequence of acute Campylobacter-enteritis (AE). However, reliable seroprevalence data of Campylobacter-associated sequelae has not been established. The objectives of this study were, first, to identify the most specific and sensitive test antigen in an optimized ELISA assay for diagnosing a previous Campylobacter-infection and, second, to compare the prevalence of anti-Campylobacter antibodies in cohorts of healthy blood donors (BD), AE, GBS, RA, and IBD patients with antibodies against known GBS, RA and IBD triggering pathogens. Optimized ELISAs of single and combined Campylobacter-proteins OMP18 and P39 as antigens were prepared and sera from AE, GBS, RA and IBD patients and BD were tested for Campylobcter-specific IgA and IgG antibodies. The results were compared with MIKROGEN™-recomLine Campylobacter IgA/IgG and whole cell lysate-immunoblot. Antibodies specific for Helicobacter pylori, Mycoplasma pneumoniae, Yersinia enterocolitica, and Borrelia afzelii were tested with commercial immunoblots. ROC plot analysis revealed AUC maxima in the combination of OMP18 and P39 for IgA and in the P39-antigen for IgG. As a result, 34-49 % GBS cases, 44-62 % RA cases and 23-40 % IBD cases were associated with Campylobacter-infection. These data show that Campylobcater-seropositivity in these patient groups is significantly higher than other triggering pathogens suggesting that it plays an important role in development of GBS and RA, and supports the hypothesis that recurrent acute campylobacteriosis triggers IBD.

[Fever of unknown origin in 74-year-old multimorbid man]. / Fieber unklarer Genese bei einem 74-jährigen multimorbiden Patienten.

A 74-year-old multimorbid man was admitted with fever of unknown origin. Over time the fever ceased spontaneously. The patient developed signs of a right heart failure without evidence of a primarily cardiac pathogenesis and died of acute right heart failure. Miliary tuberculosis that had lead to pulmonary artery hypertension was diagnosed at autopsy.

The German national registry for primary immunodeficiencies (PID).

In 2009, a federally funded clinical and research consortium (PID-NET, http://www.pid-net.org) established the first national registry for primary immunodeficiencies (PID) in Germany. The registry contains clinical and genetic information on PID patients and is set up within the framework of the existing European Database for Primary Immunodeficiencies, run by the European Society for Primary Immunodeficiencies. Following the example of other national registries, a central data entry clerk has been employed to support data entry at the participating centres. Regulations for ethics approvals have presented a major challenge for participation of individual centres and have led to a delay in data entry in some cases. Data on 630 patients, entered into the European registry between 2004 and 2009, were incorporated into the national registry. From April 2009 to March 2012, the number of contributing centres increased from seven to 21 and 738 additional patients were reported, leading to a total number of 1368 patients, of whom 1232 were alive. The age distribution of living patients differs significantly by gender, with twice as many males than females among children, but 15% more women than men in the age group 30 years and older. The diagnostic delay between onset of symptoms and diagnosis has decreased for some PID over the past 20 years, but remains particularly high at a median of 4 years in common variable immunodeficiency (CVID), the most prevalent PID.

A distinct subgroup of cardiomyopathy patients characterized by transcriptionally active cardiotropic erythrovirus and altered cardiac gene expression.

Recent studies have detected erythrovirus genomes in the hearts of cardiomyopathy and cardiac transplant patients. Assessment of the functional status of viruses may provide clinically important information beyond detection of the viral genomes. Here, we report transcriptional activation of cardiotropic erythrovirus to be associated with strongly altered myocardial gene expression in a distinct subgroup of cardiomyopathy patients. Endomyocardial biopsies (EMBs) from 415 consecutive cardiac erythrovirus (B19V)-positive patients with clinically suspected cardiomyopathy were screened for virus-encoded VP1/VP2 mRNA indicating transcriptional activation of the virus, and correlated with cardiac host gene expression patterns in transcriptionally active versus latent infections, and in virus-free control hearts. Transcriptional activity was detected in baseline biopsies of only 66/415 patients (15.9 %) harbouring erythrovirus. At the molecular level, significant differences between cardiac B19V-positive patients with transcriptionally active versus latent virus were revealed by expression profiling of EMBs. Importantly, latent B19V infection was indistinguishable from controls. Genes involved encode proteins of antiviral immune response, B19V receptor complex, and mitochondrial energy metabolism. Thus, functional mapping of erythrovirus allows definition of a subgroup of B19V-infected cardiomyopathy patients characterized by virus-encoded VP1/VP2 transcripts and anomalous host myocardial transcriptomes. Cardiac B19V reactivation from latency, as reported here for the first time, is a key factor required for erythrovirus to induce altered cardiac gene expression in a subgroup of cardiomyopathy patients. Virus genome detection is insufficient to assess pathogenic potential, but additional transcriptional mapping should be incorporated into future pathogenetic and therapeutic studies both in cardiology and transplantation medicine.

miRNA as activity markers in Parvo B19 associated heart disease.

Parvovirus B19 is a frequent virus detected in endomyocardial biopsies of patients with clinically suspected myocarditis or dilated cardiomyopathy (DCM). Viruses often cause a more symptomatic disease with increased tissue injury if they become reactivated. A disease-specific differential expression of microRNAs (miRNAs) has been described in the regulation of replicating viruses. Analyzing patients with latent and reactivated B19V infection, we found 29 differentially regulated miRNAs and, in order to test whether predicted genes are differentially expressed, selected mRNAs were tested by TaqMan-QPCR.

Identification of Toxoplasma gondii SUB1 antigen as a marker for acute infection by use of an innovative evaluation method.

By the separation of Toxoplasma lysate using two-dimensional gel electrophoresis and its analysis with human serum samples and mass spectrometry, the subtilisin-like protein (SUB1) was identified to be a potential marker for acute toxoplasmosis. Following expression of the C-terminal domain of SUB1 in Escherichia coli, it was tested in a line blot assay using a total of 80 human serum samples. Two computer programs based on different evaluation strategies were used for judgment of the line blot results: (i) a time-dependent method with a predefined cutoff value and (ii) a fixed-time-point method with a calculated cutoff. Thereby, SUB1 was proven to be rather reactive with specific immunoglobulin A (IgA), IgM, and IgG of patients with an acute infection. This finding makes this antigen an attractive candidate for improving diagnosis of toxoplasmosis and demonstrates that not only the selection of respective antigens but also the evaluation method chosen are important for the evaluation of new diagnostic markers.

[Campylobacteriosis of man : livestock as reservoir for Campylobacter species]. / Die Campylobacteriose des Menschen : Nutztiere als Reservoir für Campylobacter-Arten.

Over the last few years, infections with Campylobacter have significantly increased in Europe and Germany and these bacteria have even surpassed Salmonella as the most prevalent bacteria, causing gastroenteritis. Especially contamination during the handling and consumption of meat products seems to be the most important risk factor which plays a prominent role for transmission to man. In addition, contact with pets and other animals, drinking raw or improperly pasteurized milk, and the tenacity of Campylobacter in different environments, especially water, have also to be considered for an adequate risk assessment. Besides gastroenteritis, arthralgia, and Guillain-Barré syndrome are important clinical complications of Campylobacter infections in man. At the same time, it is mostly unclear why the course of infection in man and in reservoir animals differs significantly, especially as only a few classical bacterial virulence factors have been identified so far. For these reasons, the development of efficient prevention strategies is of utmost importance in order to control campylobacteriosis.

Diversity of the diploid sequence type of Candida albicans clinical isolates from a tertiary-care hospital in Mwanza, Tanzania.

Geographical strain variations of Candida albicans causing different clinical conditions in susceptible individuals have been reported. In this study, the distribution of diploid sequence type of C. albicans was investigated in Mwanza, Tanzania. A total of 64 C. albicans were selected on the basis of their antifungal susceptibility patterns, followed by multilocus sequence typing (MLST) to establish the circulating sequence types (STs). Forty-eight MLST were obtained out of 64 isolates amounting to 75% population structure differences. Out of these STs, 27 (56.3%) were new diploid ST types. C. albicans isolates with new ST were more diverse than isolates with known STs (27/29, 93.1% vs. 21/35, 60%, p 0.002). In conclusion, C. albicans from clinical specimens were highly diverse, with more than half of the detected diploid ST not previously reported in the MLST database, thus confirming the genetic differences of C. albicans from different geographical regions.

Control of Toxoplasma gondii infection by athymic LEW-Whn rnu rats.

In immunocompetent rats and humans infection with Toxoplasma gondii remains mostly without overt clinical symptoms, but can be fatal, if the T-cell response is impaired. For a better understanding of the lack of control of T. gondii infection under immunosuppressed conditions, congenitally athymic rats were used as the experimental model. Whereas athymic F344-Whn(rnu) (F344 nude) rats die from a generalized infection during the first 3 weeks after peritoneal inoculation with 10(6) tachyzoites of T. gondii strain NTE, LEW-Whn(rnu) (LEW nude) rats and euthymic LEW rats infected with a 10-fold higher number of parasites developed chronic infection. To identify underlying mechanisms of LEW rats resistance to T. gondii infection and to investigate a possible contribution of residual T-cells to LEW-Whn(rnu) rat resistance, we characterized the immune response of LEW rats by determination of cellularity and composition of lymphocyte population, antigen-specific IgG2b response as well as assays of antigen-specific proliferation and production of IL-2, IFN-gamma and TNF-alpha. As only euthymic LEW rats developed production of antigen-specific IgG and cellular in vitro responses, these results strongly suggest that the genetic background of LEW rats permits a control of the infection independent of an adaptive immune response.

Anodic cell-protein deposition on inverse inkjet printed micro structured gold surfaces.

The transformation of fibrinogen into fibrin is biologically activated in a complex multi-step process known as the coagulation cascade. This transformation can also be triggered by anodic surfaces. It has been suggested that this mechanism is a result of an electron transfer from the anode to the fibrinogen molecule resulting in the formation of fibrin. In this study we used this pathway to simultaneously deposit vital cells (fibroblasts and keratinocytes) and fibrin on micro structured gold electrodes. The electrodes were produced using a novel inverse inkjet-printing technology in combination with subsequent gold-sputtering, resulting in minimal structure-sizes of 35 microm (+/-6 microm). Cell deposition and fibrin-coagulation were found to occur on the anode only, following exactly the micro structured electrode surface. Successful deposition was limited by the minimal voltage (0.8 V) needed for the formation of fibrin and the maximum voltage (1.85 V) resulting in the deterioration of the Au-electrodes due to electrolysis and possible damaging of the deposited cells due to the formation of molecular chlorine. Furthermore, it was demonstrated that this technique is suitable to co-cultivate different cell types in a layered fashion. Subsequent to the electrically mediated anodic cell-protein deposition, cells were cultivated for up to 4 days and then characterized by vital fluorescence staining, methyl violet-staining and scanning electron microscopy. Cell-vitality was found to be dependent on the experimental setup; in this study non-vital cells were only observed, when sequentially depositing two different cell types. Finally, the coagulation mechanism was studied using HPLC, SDS-gel-chromatography and ATR/FTIR.

Perspectives for the tissue engineering of cartilage from a biological and biomaterial point of view.

Tissue engineering has become a fast growing interdisciplinary branch of research at the interface between life and engineering sciences with important clinical end-points. In this context the regeneration of articular cartilage represents an exciting challenge since hyaline cartilage has a limited capacity for self-repair. Today the use of different scaffold materials combined with in vitro expanded chondrocytes and signalling molecules poses great hopes for an optimal treatment of articular cartilage defects. However, until today the optimal construct of scaffolds, cells and signalling molecules has not yet been found. Since repair and regeneration recapitulate in part ontogenetic processes, the present paper summarizes the regulative mechanisms of endochondral ossification in the growth plate of the long bones to identify possible new signalling molecules for the improvement of tissue engineering-based solutions in the treatment of cartilage defects. The growth plate represents a highly organized structure of chondrocytes and extracellular matrix components in distinguishable proliferation and differentiation stages. It is regulated by various paracrine and hormonal factors. In a second part we present actual trends in scaffold design based on synthetic polymers and natural polymers, stressing their potential use in the regeneration of cartilage defects from the point of view of bioactivity and biocompatibility. In conclusion, both new signalling molecules from basic research and innovative scaffold materials with variable physico-chemical properties open up new and interesting perspectives for the research in optimized tissue engineeredbased therapeutic strategies to treat cartilage defects.

Active secretion of S100B from astrocytes during metabolic stress.

In patients suffering from cerebrovascular diseases and traumatic brain damage, increases in serum levels of protein S100B are positively correlated with the severity of the insult. Since high concentrations of S100B have been shown to exert neurotoxic effects, the objective of this study was to characterize the regulatory mechanisms underlying control of S100B release from astrocytes. To that end, we analyzed the kinetics and amount of S100B release in correlation with regulation of S100B gene expression in an in vitro ischemia model. Astrocyte cultures were treated with combined oxygen, serum and glucose deprivation, serum and glucose deprivation or hypoxia alone for 6, 12 and 24 h, respectively. While oxygen, serum and glucose deprivation triggered the most rapid release of S100B, serum and glucose deprivation provoked comparable levels of released S100B at the later time points. In contrast to oxygen, serum and glucose deprivation and serum and glucose deprivation, hypoxia alone elicited only marginal increases in secreted S100B. Parallel analysis of extracellular lactate dehydrogenase and the number of viable cells revealed only moderate cell death in the cultures, indicating that S100B was actively secreted during in vitro ischemia. Interestingly, S100B mRNA expression was potently downregulated after 12 and 24 h of oxygen, serum and glucose deprivation, and prolonged oxygen, serum and glucose deprivation for 48 h was associated with a significant reduction of S100B release at later time intervals, whereas lactate dehydrogenase levels remained constant. Our data suggest that secretion of S100B during the glial response to metabolic injury is an early and active process.

Apoptosis and its modulation during infection with Toxoplasma gondii: molecular mechanisms and role in pathogenesis.

Infection with the obligate intracellular protozoan Toxoplasma gondii leads to lifelong persistence of the parasite in its mammalian hosts including humans. Apoptosis plays crucial roles in the interaction between the host and the parasite. This includes innate and adaptive defense mechanisms to restrict intracellular parasite replication as well as regulatory functions to modulate the host's immune response. Not surprisingly, however, T. gondii also extensively modifies apoptosis of its own host cell or of uninfected bystander cells. After infection, apoptosis is triggered in T lymphocytes and other leukocytes, thereby leading to suppressed immune responses to the parasite. T cell apoptosis may be largely mediated by Fas engagement but also occurs independently of Fas under certain conditions. Depending on the magnitude of T cell apoptosis, it is either associated with unrestricted parasite replication and severe pathology or facilitates a stable parasite-host-interaction. However, T. gondii has also evolved strategies to inhibit host cell apoptosis. Apoptosis is blocked by indirect mechanisms in uninfected bystander cells, thereby modulating the inflammatory response to the parasite. In contrast, inhibition of apoptosis in infected host cells by direct interference with apoptosis-signaling cascades is thought to facilitate the intracellular development of T. gondii. Blockade of apoptosis by intracellular parasites may be achieved by different means including interference with the caspase cascade, increased expression of antiapoptotic molecules by infected host cells, and a decreased activity of the poly(ADP-ribose) polymerase. The intriguing dual activity of T. gondii to both promote and inhibit apoptosis requires a tight regulation to promote a stable parasite host-interaction and establishment of persistent toxoplasmosis.

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections.

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 microm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + trade mark dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.

Induction of interleukin-8 in human neutrophils after MHC class II cross-linking with superantigens.

Neutrophils have been shown to express major histocompatibility complex class II (MHC II) after stimulation. However, reports concerning the functional effect of MCH II expression are still lacking. In our hands, granulocyte-monocyte colony-stimulating factor (GM-CSF) alone and in combination with interferon (IFN)-gamma, but not IFN-gamma or interleukin (IL)-3, induced a significant level of expression of human leukocyte antigen DR on neutrophils. The addition of staphylococcal enterotoxin E to neutrophils resulted in a significant increase in IL-8 production only after prestimulation with GM-CSF alone or in combination with IFN-gamma but had no effect on neutrophils preincubated with IFN-gamma alone or IL-3. Staphylococcal enterotoxin A, another bivalent superantigen, also stimulated production of IL-8 by preincubated polymorphonuclear neutrophils, whereas staphylococcal enterotoxin A mutants that are not able to cross-link MHC II molecules failed to induce IL-8 production. Taken together, our results clearly demonstrate that after induction of MHC II, neutrophils are able to respond to MHC II-specific stimulation. These findings support the ideas that the induced MHC II complex is completely functional and that neutrophils may be able to present antigens.