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In the present study, we were interested in the decline over time of anti SARS-CoV-2 antibodies and SARS-CoV-2 specific T-cell responses after two doses of mRNA vaccines in total and by age group and comorbidity. The second goal was to suggest an immunological correlate for protection on an individual basis and to describe the probability of protection over time after second vaccination.We analysed blood samples from 228 residents (median age 83.8 years) and from 273 Health Care Workers (HCW; median age 49.7 years) of five nursing homes and one home for the elderly with assisted living support. Participants had received two vaccinations. The blood samples were analysed for SARS-CoV-2 specific antibody and T-cell responses. We compared outcomes in the HCW and residents in the respective institutions. No breakthrough infections occurred during the study period. The initial immune responses in the younger participants were about 30 % higher than in the older ones. Over time, all parameters dropped continuously in all groups within the maximum observation period of 232 days. Comorbidities such as coronary heart disease or diabetes mellitus reduced the initial immune responses, regardless of age. In contrast to an almost linear decline in antibody levels, we observed that the interferon-gamma response remained at a constant level between about day 120 and 180, only to decline further thereafter.Based on our data, we propose on an individual level a correlate of protection: Persons who have a neutralizing capacity of 75 % (which would correspond to approx. 200 BAU/ml) and an interferon-gamma response above 200 mIU/ml should be considered to be protected resp. sufficiently immunized.

Microorganisms constitute 70 percent of the biomass on Planet Earth. Comparatively few species are adapted to colonize human surfaces and form a complex Meta-Organism with manyfold mutual benefits. Occasionally, microorganisms may overcome the barriers of the skin and mucosal surfaces and may multiply locally or in multiple sites inside the body. This process is called infection. Infections can be caused by bacteria, viruses, parasites, helminths, and fungi. Immediately after infection, numerous defense mechanisms of the immune system are activated to combat replication of the microbes. There is a balance between microorganism and human defense mechanisms, which may lead to either asymptomatic infection or result in a wide spectrum of symptoms from mild to severe disease and even death. The most important factors in the diagnosis of infectious diseases are a careful history, physical examination and the appropriate collection of body fluids and tissues. Laboratory diagnosis requires between 2 and 72 hours. Wherever possible, antibiotics should only be used when sufficient evidence of efficacy is available. Then, however, they should be used as early as possible and in high doses. In addition to everyday hygiene measures, vaccination is the most effective measure to prevent infectious diseases.

Background The treatment of psoriasis has been revolutionized by the development of biologic therapies. However, the pathogenesis of psoriasis, in particular the role of the cutaneous microbiome, remains incompletely understood. Moreover, skin microbiome studies have relied heavily on 16S rRNA sequencing data in the absence of bacterial culture. Objectives To characterize and compare the cutaneous microbiome in 20 healthy controls and 23 patients with psoriasis using metagenomic analyses and to determine changes in the microbiome during treatment. Methods Swabs from lesional and nonlesional skin from patients with psoriasis, and from controls matched for site and skin microenvironment, were analysed using both 16S rRNA sequencing and traditional culture combined with mass spectrometry (MALDI-TOF) in a prospective study. Results Psoriasis was associated with an increased abundance of Firmicutes and a corresponding reduction in Actinobacteria, most marked in lesional skin, and at least partially reversed during systemic treatment. Shifts in bacterial community composition in lesional sites were reflected in similar changes in culturable bacteria, although changes in the microbiota over repeated swabbing were detectable only with sequencing. The composition of the microbial communities varied by skin site and microenvironment. Prevotella and Staphylococcus were significantly associated with lesional skin, and Anaerococcus and Propionibacterium with nonlesional skin. There were no significant differences in the amount of bacteria cultured from the skin of healthy controls and patients with psoriasis. Conclusions Shifts in the cutaneous microbiome in psoriasis, particularly during treatment, may shed new light on the pathogenesis of the disease and may be clinically exploited to predict treatment response. What's already known about this topic? Alterations in the composition of the cutaneous microbiome have been described in psoriasis, although methodological differences in study design prevent direct comparison of results. To date, most cutaneous microbiome studies have focused on 16S rRNA sequencing data, including both living and dead bacteria. What does this study add? This prospective observational study confirms that changes in the composition of the cutaneous microbiome, detected by 16S rRNA sequencing, are consistent with those identified by bacterial culture and mass spectrometry. The changes in the microbiome during antipsoriasis therapy should be further investigated to determine whether these represent potential novel biomarkers of treatment response. What is the translational message? Characterization of cutaneous microbiota may ultimately move into the clinic to help facilitate treatment selection, not only by optimizing currently available treatments, but also by identifying new therapeutic targets.

Polymorphonuclear Neutrophils (PMNs) are metabolically highly active phagocytes, present in abundant numbers in the circulation. These active cells take the onus of clearing invading pathogens by crowding at inflammatory sites in huge numbers. Though PMNs are extremely short living and die upon spontaneous apoptosis, extended lifespan has been observed among those cells arrive at the inflammation sites or tackle intracellular infections or face any microbial challenges. The delay/inhibition of spontaneous apoptosis of these short-living cells at the inflammatory core rather helps in combating pathogens. Like many candidates, type-1 interferons (type-1 IFNs) is a group of cytokines predominant at the inflammation site. Although there are some isolated reports, a systematic study is still lacking which addresses the impact of the predominant type of interferon on the spontaneous apoptosis of neutrophils. Here in, we have observed that exposure of these IFNs (IFN-β, IFN-α & IFN-ω etc) on human neutrophils prevents the degradation of the Bfl1, an important anti-apoptotic partner in the apoptotic cascade. Treatment showed a significant reduction in the release of cytochrome-C in the cytosol, a critical regulator in the intrinsic apoptotic pathway. We also noticed a reduction in the conversion of procaspase -3 to active caspase-3, a crucial executioner caspase towards initiation of apoptosis. Taken together our results show that exposure to interferon interferes with apoptotic pathways of neutrophils and thereby delay its spontaneous apoptosis. These findings would help us further deciphering specific roles if these inflammatory agents are causing any immune-metabolomic changes on PMNs at the inflammatory and infection core.

Lymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN-γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN-γ-induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN-γ-induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN-γ-induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor-2α (eIF2α) and down-regulation of the vesicle-associated membrane protein-associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN-γ-induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER-related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN-γ-induced persistent infection.

Chlamydia trachomatis is the most prevalent cause of preventable blindness worldwide and a major reason for infectious infertility in females. Several bacterial factors have been implicated in the pathogenesis of C. trachomatis . Combining structural and mutational analysis, we have shown that the proteolytic function of CT441 depends on a conserved Ser/Lys/Gln catalytic triad and a functional substrate-binding site within a flexible PDZ ( p ostsynaptic density of 95 kDa, d iscs large, and z onula occludens) domain. Previously, it has been suggested that CT441 is involved in modulating estrogen signaling responses of the host cell. Our results show that although in vitro CT441 exhibits proteolytic activity against SRAP1, a coactivator of estrogen receptor α, CT441-mediated SRAP1 degradation is not observed during the intracellular developmental cycle before host cells are lysed and infectious chlamydiae are released. Most compellingly, we have newly identified a chaperone activity of CT441, indicating a role of CT441 in prokaryotic protein quality control processes.

With several million microbes per cm2 of skin, the task of mapping the physiological cutaneous microbiome is enormous. Indeed, the reliance on bacterial culture to identify cutaneous bacterial communities has led to a systematic under-appreciation of cutaneous microbial diversity, potentially limiting our understanding of common inflammatory skin diseases including psoriasis. However, based heavily on developments in molecular biology and bioinformatics, including next generation sequencing, the last decade has witnessed a marked increase in our understanding of the extent and composition of the cutaneous microbiome. It is already clear that skin-specific (skin site and skin microenvironment), individual-specific (hygiene, sex, age, and hormonal status), disease-specific (atopic eczema, acne) and genetic factors can all influence the cutaneous microbiome, albeit to varying and, as yet ill-defined, extents. This review briefly describes the process of 16S ribosomal RNA sequencing, before charting our current understanding of the cutaneous microbiome in health and the alterations (dysbiosis) associated with chronic inflammatory diseases with particular reference to psoriasis. The possibility and clinical relevance of intra-individual cross-talk between the various microbiomes is discussed and potential mechanisms underpinning the interactions between resident skin flora and the immune system are highlighted. We outline how the power of microbiome studies can be harnessed to provide new insights into disease pathogenesis and treatment selection. Ultimately, in the age of personalized medicine, the integration of cutaneous microbiome signatures and comprehensive disease and drug response endotypes will herald a novel approach in the clinical management of chronic, multi-system inflammatory diseases. This article is protected by copyright. All rights reserved.

Transmission of bacteria from inanimate surfaces in healthcare associated environments is an important source of hospital acquired infections. A number of commercially available medical devices promise to fulfill antibacterial activity to reduce environmental contamination. Under usual ambient air conditions of hospital rooms no condensation of humidity is expected on inanimate surfaces. In contrast, current standardized methods for the analysis of antibacterial activity of solid surfaces in general use mostly planktonic bacterial cells which are kept in thin liquid or agarose layers on tested surfaces. Therefore, we developed a touch transfer assay modeling fingerprint transmission to investigate the antibacterial activity of surfaces in a dry state. We suggest the newly developed touch transfer assay as a new additional tool for the assessment of potential antimicrobial surfaces prior utilization in hospital environments.

Transmission of bacteria from inanimate surfaces in healthcare associated environments is an important source of hospital acquired infections. A number of commercially available medical devices promise to fulfill antibacterial activity to reduce environmental contamination. Under usual ambient air conditions of hospital rooms no condensation of humidity is expected on inanimate surfaces. In contrast, current standardized methods for the analysis of antibacterial activity of solid surfaces in general use mostly planktonic bacterial cells which are kept in thin liquid or agarose layers on tested surfaces. Therefore, we developed a touch transfer assay modeling fingerprint transmission to investigate the antibacterial activity of surfaces in a dry state. We suggest the newly developed touch transfer assay as a new additional tool for the assessment of potential antimicrobial surfaces prior utilization in hospital environments.

Canonical neutrophil antimicrobial effector mechanisms, such as degranulation, production of reactive oxygen species, and release of neutrophil extracellular traps (NETs), can result in severe pathology. Activation of neutrophils through immune complexes (ICs) plays a central role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report that immobilized ICs (iICs), which are hallmarks of several autoimmune diseases, induce the release of NETs from primary human neutrophils. The iIC-induced NET formation was found to require production of reactive oxygen species by NADPH oxidase and myeloperoxidase and to be mediated by FcγRIIIb. Blocking of the β2 integrin macrophage-1 Ag but not lymphocyte function–associated Ag-1 abolished iIC-induced NET formation. This suggests that FcγRIIIb signals in association with macrophage-1 Ag. As intracellular signaling pathways involved in iIC-induced NET formation we identified the tyrosine kinase Src/Syk pathway, which downstream regulates the PI3K/Akt, p38 MAPK, and ERK1/2 pathways. To our knowledge, the present study shows for the first time that iICs induce NET formation. Thus, we conclude that NETs contribute to pathology in autoimmune inflammatory disorders associated with surface-bound ICs.

Surveillanceberichte zu Krankheitserregern mit speziellen Resistenzen und Multiresistenzen sowie zum Verbrauch von Antibiotika sind fur viele medizinische Einrichtungen in Deutschland nunmehr gesetzlich vorgeschrieben. Zum angemessenen Umfang und zur aussagekraftigen Form dieser Berichte fehlen bisher jedoch praxisorientierte Empfehlungen. Dieses Konsensuspapier entstand auf der Basis der Erfahrungen aus funf deutschen Universitatskliniken im Umgang mit infektionsepidemiologischen Daten und daraus resultierenden Moglichkeiten zur Berichterstellung. Das Konsensuspapier gibt zu den bestehenden gesetzlichen Vorgaben erganzende Empfehlungen fur die Erstellung der geforderten Surveillanceberichte. Dabei wird auch die Relevanz von Aussagen zur Haufigkeit und Qualitat mikrobiologischer Untersuchungen einbezogen. Ferner werden Vorschlage zur Standardisierung der Daten-Analysen gemacht, mit dem Ziel der regionalen und nationalen Vergleichbarkeit der Ergebnisse auf hohem Qualitatsniveau, wie bei der seit langerem etablierten, standardisierten nationalen Erfassung nosokomialer Infektionen. Das Konsensuspapier stellt dar, in welcher Form die gesetzliche Berichtspflicht aussagekraftig und standardisiert umgesetzt werden kann, so dass die Ableitung von geeigneten Praventionsmasnahmen und deren Umsetzung erleichtert wird.

The central oxygen sensitive transcription factor HIF-1α has been implicated in the differentiation of nTreg and Th17 cells and to orchestrate metabolic changes of activated T cells. However, data on the functional relevance of HIF-1α and Hox, in general, for nTreg-suppressive activity and T cell function in primary human cells are still missing. Therefore, we analyzed the effect of Hox and HIF-1α on human Tres, nTreg, and Th17 cells. Under Hox, nTreg-mediated suppression of Tres proliferation, CD25 expression, and secretion of IFN-γ were significantly reduced, whereas expression levels of VEGF, TNF-α, and IL-10 were significantly increased. In contrast to observations in mice, Th17 lineage commitment, as determined by RORγt expression, was not affected by activation or inhibition of HIF-1α expression using DMOG or YC-1 treatment, respectively. Nevertheless, the secretion of IL-17A was increased by DMOG and reduced by YC-1 under Th17-skewing conditions in a dose- dependent manner. In conclusion, Hox and HIF-1α substantially influence human T cell-mediated immune responses by modulation of nTreg-suppressive function and IL-17A secretion by Th17 cells.

Neutrophil extracellular traps (NETs) have been suggested to play a pathophysiological role in several autoimmune diseases. Since NET-formation in response to several biological and chemical stimuli is mostly ROS dependent, in theory any substance that inhibits or scavenges ROS could prevent ROS-dependent NET release. Therefore, in the present comprehensive study, several antioxidative substances were assessed for their capacity to inhibit NET formation of primary human neutrophilsin vitro. We could show that the flavonoids (−)-epicatechin, (+)-catechin hydrate, and rutin trihydrate as well as vitamin C and the pharmacological substancesN-acetyl-L-cysteine and 5-aminosalicylic acid inhibited PMA induced ROS production and NET formation. Therefore, a broad spectrum of antioxidative substances that reduce ROS production of primary human neutrophils also inhibits ROS-dependent NET formation. It is tempting to speculate that such antioxidants can have beneficial therapeutic effects in diseases associated with ROS-dependent NET formation.

Anaplasma phagocytophilum , a Gram-negative, obligate intracellular bacterium infects primarily neutrophil granulocytes. Infection with A. phagocytophilum leads to inhibition of neutrophil apoptosis and consequently contributes to the longevity of the host cells. Previous studies demonstrated that the infection inhibits the executionary apoptotic machinery in neutrophils. However, little attempt has been made to explore which survival signals are modulated by the pathogen. The aim of the present study was to clarify whether the phosphatidylinositol 3-kinase (PI3K)/Akt and NF-κB signaling pathways, which are considered as important survival pathways in neutrophils, are involved in A. phagocytophilum -induced apoptosis delay. Our data show that infection of neutrophils with A. phagocytophilum activates the PI3K/Akt pathway and suggest that this pathway, which in turn maintains the expression of the antiapoptotic protein Mcl-1, contributes to the infection-induced apoptosis delay. In addition, the PI3K/Akt pathway is involved in the activation of NF-κB in A. phagocytophilum -infected neutrophils. Activation of NF-κB leads to the release of interleukin-8 (IL-8) from infected neutrophils, which, in an autocrine manner, delays neutrophil apoptosis. In addition, enhanced expression of the antiapoptotic protein cIAP2 was observed in A. phagocytophilum -infected neutrophils. Taken together, the data indicate that upstream of the apoptotic cascade, signaling via the PI3K/Akt pathway plays a major role for apoptosis delay in A. phagocytophilum -infected neutrophils.

Summary Symptoms of diseases such as rheumatoid arthritis, which is T helper 1 (Th1) dependent, and asthma, which is T helper 2 (Th2) dependent, are influenced by diurnal rhythms and natural regulatory T cells (nTreg). However, the mechanisms responsible for the diurnal rhythm of disease activity have not been identified and it is unclear whether nTreg activity is diurnal rhythm-dependent. We therefore investigated whether a 24-hr diurnal cycle affected the ability of various helper T-cell populations to generate immunomodulatory and pro-inflammatory cytokines, as well as its suppression by nTreg cells. Using a within-subject crossover design, sleep versus continuous wakefulness was compared over a 24-hr period in healthy young volunteers under defined environmental conditions. Venous blood was drawn periodically every 5 hr and the function of T cells was explored in vitro. We demonstrated that interleukin (IL)-2, interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and IL-10 secretion by naive CD4+ T cells follows a diurnal rhythm. Furthermore, multiple regression analysis, as well as subsequent in vitro experiments, suggested that serum levels of cortisol and prolactin are part of the underlying mechanism. Additionally, we observed that nTreg suppressed the secretion of IFN-γ, IL-2 and TNF-α, but not the secretion of IL-4, IL-6, IL-10 and IL-17A. However, the abrogation of IL-2 release was reversed upon inhibiting CD25 on nTreg. Highly purified nTreg secreted IL-6, IL-10 and IL-17A, but not IL-2, IL-4, IFN-γ or TNF-α. Taken together, our results demonstrate that hormones and nTreg modulate the diurnal rhythm of T helper cell activity.

The lack of sufficient amounts of sleep is a hallmark of modern living, and it is commonly perceived that in the long run this makes us sick. An increasing amount of scientific data indicate that sleep deprivation has detrimental effects on immune function. Conversely, immune responses feedback on sleep phase and architecture. Several studies have investigated the impact of short-term sleep deprivation on different immune parameters, whereas only a few studies have addressed the influence of sleep restriction on the immune system. In many cases, sleep deprivation and restriction impair immune responses by disrupting circadian rhythms at the level of immune cells, which might be a consequence of disrupted endocrine and physiological circadian rhythms. Little is known about the mechanisms underlying the circadian regulation of immunity, but recent studies have suggested that local as well as central circadian clocks drive the rhythms of immune function. In this review, we present a mechanistic model which proposes that sleep (through soluble factors and body temperature) primes immune cells on the one hand, and, on the other hand, provides a timing signal for hematopoietic circadian clocks. We hypothesize that chronic sleep disruption desynchronizes these clocks and, through this mechanism, deregulates immune responses.

Epidermolysis bullosa acquisita is a prototypical organ-specific autoimmune disease caused by autoantibodies against type VII collagen of the dermal-epidermal junction. Although mechanisms of autoantibody-induced blister formation were extensively characterized, the initiation of autoantibody production in autoimmune blistering diseases is still poorly defined. In the current study, we addressed the role of T cells for the production of blister-inducing autoantibodies in mice immunized with type VII collagen. To detect autoreactive type VII collagen-specific T cells, lymph node cells from immunized SJL mice were stimulated in vitro with recombinant Ag, and their proliferation was measured by radioactive thymidine incorporation and flow cytometry analysis of CFSE-labeled cells. Interestingly, using synthetic peptides of the immunogen, partly different T and B cell epitopes in mice immunized with type VII collagen were demonstrated. In contrast to wild-type mice, immunization with type VII collagen of SJL athymic nude mice lacking T cells did not induce an autoimmune response and blistering phenotype. Importantly, SJL nude mice repleted with T cells from immunized wild-type mice showed a robust and durable autoantibody production resulting in subepidermal blistering disease in the recipients. Our present results demonstrate that T cells are required for the initiation of autoimmunity against type VII collagen in experimental epidermolysis bullosa acquisita and provide a basis for developing T cell-directed immunomodulatory strategies for this and related autoimmune diseases.

Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of acute inflammation where they die in large numbers. Because release of toxic substances from dead neutrophils can propagate the inflammatory response leading to tissue destruction, clearance of dying inflammatory neutrophils has a critical function in the resolution of the inflammatory response. Apoptotic neutrophils are phagocytosed primarily by macrophages, provided these cells are present in adequate numbers. However, macrophages are rare at sites of acute inflammation, whereas the number of neutrophils can be extremely high. In the current study, in vitro experiments with human neutrophils were carried out to investigate whether neutrophils can ingest apoptotic neutrophils. We show that naïve granulocytes isolated from venous blood have a limited capacity to phagocytose apoptotic cells. However, exposure to activating stimuli such as LPS, GM-CSF and/or IFN-γ results in enhanced phagocytosis of apoptotic cells. The efficient uptake of apoptotic cells by neutrophils was found to depend on the presence of heat labile serum factors. Importantly, the contact to or uptake of apoptotic cells inhibited neutrophil functions such as respiratory burst and the release of the proinflammatory cytokines TNF-α and interferon-inducible protein-10. Contact to apoptotic cells, however, induced the secretion of IL-8 and growth-related oncogene-α, which was independent of NF-κB and p38 MAPK but involved C5a and the ERK1/2 pathway. The data suggest that activated neutrophils participate in the clearance of apoptotic cells. In addition, because apoptotic cells inhibit proinflammatory functions of neutrophils, uptake of apoptotic cells by neutrophils contributes to the resolution of inflammation.

A number of immunological functions are dependent on circadian rhythms and regular sleep. This has impact on the type and magnitude of immune responses following antigenic challenge, for example in vaccination. Little is known about the underlying mechanisms. One possibility may be the circadian and sleep-dependent modulation of CD4(+)CD25(-) T cell responses by CD4(+)CD25(+) natural regulatory T cells (nT(reg)). In a variety of studies, nT(reg) have been shown to regulate T cell responses negatively. Thus, we investigated the influence of sleep and circadian rhythm on the number and function of nT(reg) as well as on the function of CD4(+)CD25(-) T cells. Seven healthy young men were examined under defined conditions on two occasions, i.e. during sleep and sleep deprivation. Venous blood was drawn periodically; numbers of nT(reg), suppressive activity of nT(reg), interleukin-2 production and proliferation of CD4(+)CD25(-) T cells were explored in vitro. nT(reg) counts revealed a significant circadian rhythm with highest levels during the night (mean 95 nT(reg)/microl) and lowest levels during the day (mean 55 nT(reg)/microl). During normal sleep, the suppressive activity of nT(reg) was highest at 02.00 h and somewhat lower at 15.00 h. Surprisingly, almost no suppressive activity was present at 07.00 h. Deprivation of sleep abrogated this rhythm. CD4(+)CD25(-) T cell proliferation was dampened significantly by sleep deprivation. This is the first study in human cells to show that nT(reg) number and function follow a rhythm across the 24-h period. Furthermore, sleep deprivation severely disturbs the functional rhythm of nT(reg) and CD4(+)CD25(-) T cells.

Polymorphonuclear neutrophil granulocytes (PMN) are primary antimicrobial effector cells of the innate immune system and serve to destroy invading pathogens. Although most ingested microorganisms are killed readily inside PMN, several obligate or facultative intracellular pathogens survive even in this hostile environment. Extension of the life span of neutrophils is a general escape mechanism of pathogens residing in PMN. However, after 2-4 days, even infected neutrophils become apoptotic and are phagocytosed by macrophages. Since microbes entering macrophages via the uptake of infected apoptotic PMN may survive and multiply in macrophages, apoptotic neutrophils can serve as "Trojan horses" for certain pathogens. Interfering with activating signaling pathways appears to be another potent mechanism by which intracellular microorganisms suppress cellular activation in neutrophils. In addition to provide a short overview of the topic, the present review aims to summarize our own findings regarding the interaction between human neutrophils and intracellular pathogens as well as regarding the disease promoting role of apoptotic cells after infection with Leishmania major.

Six weeks after a holiday trip to Yugoslavia, a previously well 48-year-old man developed a reddish-livid, firm nodule, 0.5 cm in diameter, on the proximal joint of the right thumb. A similar nodule appeared nearby, as well as over the left patella. Eleven additional nodules occurred over the next 4 months. His general health remained good and physical examination merely noted the liver edge palpable 3 cm below the costal margin. The histology of one of the nodules showed a tuberculoid, plasma-rich inflammatory reaction reminiscent of leishmaniasis or brucellosis. No organisms were seen. Immunohistochemistry of a frozen section demonstrated Leishmania-associated antigens, and the Western-blot test was characteristic for leishmaniasis. No systemic treatment was undertaken because of likely alcoholic toxic liver damage. On local treatment with paromomycin-containing ointment the cutaneous nodules healed without scar within 3 months.

Chlamydiaceae are obligate intracellular bacteria that cause endemic trachoma, sexually transmitted diseases and respiratory infections. The course of the diseases is determined by local inflammatory immune responses and the propensity of the pathogen to replicate within infected host cells. Both features require energy which is inseparably coupled to oxygen availability in the microenvironment. Hypoxia-inducible factor-1 (HIF-1) regulates crucial genes involved in the adaptation to low oxygen concentrations, cell metabolism and the innate immune response. Here we report that Chlamydia pneumoniae directly interferes with host cell HIF-1alpha regulation in a biphasic manner. In hypoxia, C. pneumoniae infection had an additive effect on HIF-1alpha stabilization resulting in enhanced glucose uptake during the early phase of infection. During the late phase of intracellular chlamydial replication, host cell adaptation to hypoxia was actively silenced by pathogen-induced HIF-1alpha degradation. HIF-1alpha was targeted by the chlamydial protease-like activity factor, which was secreted into the cytoplasm of infected cells. Direct interference with HIF-1alpha stabilization was essential for efficient C. pneumoniae replication in hypoxia and highlights a novel strategy of adaptive pathogen-host interaction in chlamydial diseases.

The Chlamydia pneumoniae bacteriophage was first identified in isolate AR-39. Its relevance for chlamydial biology and pathogenicity remains unknown. In this study, a collection of 36 C. pneumoniae isolates was screened and the phage was detected in eight. As the positive isolates differed by several polymorphisms, they presumably belonged to different genetic lineages. It was investigated whether different genotypes of the phage also existed and whether they could be assigned to chlamydial genotypes as evidence of coevolution. Sequencing of >3000 bp of the 4524 bp phage genome revealed complete identity to the published sequences. Thus, it was hypothesized that the genetic conservation was related to easy transmissibility of the phage between C. pneumoniae isolates. Cocultivation of phage positive and negative isolates followed by cloning and identification of different C. pneumoniae genotypes demonstrated for the first time transmissibility of the bacteriophage from one isolate to the other. These observations indicate that the phage is capable of infecting C. pneumoniae isolates of different genetic backgrounds and suggest that all C. pneumoniae strains might be susceptible. The successful in vitro infection of C. pneumoniae with the phage provides the basis for studying its pathogenetic relevance in isolates of identical genetic background and provides a potential tool for genetic manipulation of C. pneumoniae.

Professional phagocytes like polymorphonuclear neutrophil granulocytes (PMN) and macrophages (MF) kill pathogens as the first line of defense. These cells possess numerous effector mechanisms to eliminate a threat at first contact. However, several microorganisms still manage to evade phagocytic killing, survive and retain infectivity. Some pathogens have developed strategies to silently infect their preferred host phagocytes. The best example of an immune silencing phagocytosis process is the uptake of apoptotic cells. Immune responses are suppressed by the recognition of phosphatidylserine (PS) on the outer leaflet of their plasma membrane. Taking Leishmania major as a prototypic intracellular pathogen, we showed that these organisms can use the apoptotic "eat me" signal PS to silently enter PMN. PS-positive and apoptotic parasites, in an altruistic way, enable the intracellular survival of the viable parasites. Subsequently these pathogens again use PS exposition, now on infected PMN, to silently invade their definitive host cells, the MF. In this review, we will focus on L. major evasion strategies and discuss other pathogens and their use of the apoptotic "eat me" signal PS to establish infection.

Neutrophils harbor a number of preformed effector proteins that allow for immediate antimicrobial functions without the need for time-consuming de novo synthesis. Evidence indicates that neutrophils also contain preformed cytokines, including interleukin (IL)-1ra, CXCL8 and CXCL2. In the search for additional preformed cytokines, a cytokine array analysis identified IL-16 and macrophage migration inhibitory factor (MIF) as preformed cytokines in lysates from human primary neutrophils. Both IL-16 and MIF are unconventional cytokines because they lack a signal sequence. Using confocal immunofluorescence microscopy as well as western blot analysis of subcellular fractions, IL-16 and MIF were found to be stored in the cytosol rather than in the granules of human neutrophils, which implies an unconventional secretion mechanism for both cytokines. IL-16 is synthesized and stored as a precursor (pre-IL-16). We present evidence that the processing of pre-IL-16 to the biologically active IL-16C is mediated by caspase-3 and occurs during both spontaneous and UV-induced apoptosis of human neutrophils. Although IL-16 processing occurs during apoptosis, IL-16C and MIF release was observed only during secondary necrosis of neutrophils. Screening a panel of microbial substances and proinflammatory cytokines did not identify a stimulus that induced the release of IL-16C and MIF independent of secondary necrosis. The data presented here suggest that IL-16 and MIF are neutrophil-derived inflammatory mediators released under conditions of insufficient clearance of apoptotic neutrophils, as typically occurs at sites of infection and autoimmunity.

The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates the metabolic adaptation of cells to hypoxia and T-helper cell fate. However, HIF-1α regulation in CD4(+) T cells (T cells) remains elusive. Here we observed that depletion of oxygen (O2⩽2%) alone was not sufficient to induce HIF-1α expression in T cells. However, when hypoxic T cells were stimulated, HIF-1α was expressed and this was dependent on nuclear factor-κB- and nuclear factor of activated T cell (NFAT)-mediated transcriptional upregulation of Hif-1α mRNA. HIF-1α upregulation could be blocked by drugs inhibiting NF-κB, NFAT or mammalian target of rapamycin precluding CD4(+) T-cell stimulation or translation in T cells, as well as by blocking transcription. CD3, CD28, phorbol-12-myristat-13-acetat (PMA) or ionomycin-stimulated T cells did not express HIF-1α under normoxic conditions. In conclusion, regulation of HIF-1α expression in CD4(+) T cells in hypoxia gravely relies on its transcriptional upregulation and subsequent enhanced protein stabilization.

Background. Shiga toxin-producing, enteroaggregative Escheri-chia coli was responsible for the 2011 outbreak of haemolyticuraemicsyndrome(HUS).Thepresentsingle-centre,observation-alstudydescribesthe1-yearcourseofthediseasewithanemphasisonkidney function. Outcomedataafter1 yearareassociatedwithtreatment and patient characteristics at onset of HUS.Methods. Patients were treated according to a standardizedapproach of supportive care, including a limited number ofplasmapheresis. On top of this treatment, patients with severeHUS (n=35) received eculizumab, a humanized anti-C5monoclonal antibody inhibiting terminal complement activa-tion. The per-protocol decision—to start or omit an extendedtherapy with eculizumab accompanied by azithromycin—separated the patients into two groups and marked Day 0 ofthe prospective study. Standardized visits assessed the patients’well-being, kidney function, neurological symptoms, haemato-logical changes and blood pressure.Results. Fifty-six patients were regularly seen during the follow-up. All patients had survived without end-stage renal disease.Young(er)agealleviatedrestoringkidneyfunctionafteracutekid-ney injuryeven in severe HUS. After 1 year, kidney function wasaffected with proteinuria [26.7%; 95% confidence interval (CI)13.8–39.6], increased serum creatinine (4.4%, CI 0.0–10.4), in-creased cystatin C (46.7%, CI 32.1 –61.3) and reduced (

Dynasilan is a fluoroalkylsilan that is able to interact with surface active centres on intraocular lenses (IOL), offering a new way for surface modification of different IOL materials. The purpose of this in vitro study was to investigate the influence of this new surface modification on the adherence of two typical endophthalmitis causing bacteria (Staphylococcus epidermidis, Propionibacterium acnes).In a pilot experiment, the effect of Dynasilan coating on the adherence of S. epidermidis was tested on glass slides. Forty-two Dynasilan-modified and 42 unmodified IOL (14 PMMA, 14 silicone and 14 hydrogel) were incubated at 37 degrees C in brain heart infusion broth (10(8) CFU/ml) with either S. epidermidis for 24 h or with P. acnes for 1 h. Subsequently, the adherent bacteria were resuspended using ultrasonification at 35 kHz for 3x45 s. After dilution series and incubation at 37 degrees C on Petri dishes for 24 h and 3 days, respectively, the colonies were counted.In the pilot experiment, a markedly lower number of adherent S. epidermidis was observed on Dynasilan-modified glass slides. Of all IOL materials incubated with S. epidermidis, those modified with Dynasilan showed a lower mean number of adherent bacteria (mean 1.37x10(7); SD 2.37x10(7)) than those untreated (2.43x10(7); SD 3.04x10(7)). IOLs incubated with P. acnes showed a significantly lower mean number of adherent bacteria of 2.51x10(4) (SD 2.71x10(4)) on Dynasilan-modified IOLs versus 6.27x10(4) (SD 7.70x10(4)) on untreated IOLs.The presented in vitro results indicate that Dynasilan surface modification is able to reduce the adherence of S. epidermidis and P. acnes on all IOL materials tested. Further studies regarding the stability of this modification and its biocompatibility must be performed.

Macrophages (MF) are the final host cells for multiplication of the intracellular parasite Leishmania major (L. major). However, polymorphonuclear neutrophil granulocytes (PMN), not MF, are the first leukocytes that migrate to the site of infection and encounter the parasites. Our previous studies indicated that PMN phagocytose but do not kill L. major. Upon infection with Leishmania, apoptosis of human PMN is delayed and takes 2 days to occur. Infected PMN were found to secrete high levels of the chemokine MIP-1β, which attracts MF. In this study, we investigated whether MF can ingest parasite-infected PMN. We observed that MF readily phagocytosed infected apoptotic PMN. Leishmania internalized by this indirect way survived and multiplied in MF. Moreover, ingestion of apoptotic infected PMN resulted in release of the anti-inflammatory cytokine TGF-β by MF. These data indicate that Leishmania can misuse granulocytes as a “Trojan horse” to enter their final host cells “silently” and unrecognized.

Chlamydia pneumoniae, a major cause of community-acquired pneumonia, primarily infects the respiratory tract. Chronic infection of nonrespiratory sites, such as the vascular wall, the brain or blood monocytes, requires evasion from the lungs and spreading via the bloodstream. The cell types involved in dissemination are insufficiently characterised. In this study, New Zealand White rabbits were infected intratracheally with C. pneumoniae, and lung manifestation and systemic dissemination were monitored by polymerase chain reaction and immunohistochemistry. Infection of the lungs was characterised by an early phase dominated by granulocytes and a late phase dominated by alveolar macrophages (AM). Granulocytes, AM and alveolar epithelial cells acted as host cells for chlamydiae, which remained detectable for up to 8 weeks. AM transported the pathogen to the peribronchiolar lymphatic tissue, and subsequently C. pneumoniae entered the spleen and the aorta via dissemination by peripheral blood monocytes. In conclusion, Chlamydia pneumoniae-infected alveolar macrophages transmigrate through the mucosal barrier, and give the pathogen access to the lymphatic system and the systemic circulation. Infected peripheral blood monocytes are the vector system within the bloodstream and transmit the infection to the vascular wall. This is the first description of granulocytes acting as a reservoir for Chlamydia pneumoniae early in infection.

To report the successful induction of remission with the monoclonal anti-CD20 antibody rituximab in a patient with hepatitis C virus (HCV) associated cryoglobulinaemic vasculitis and a non-Hodgkin's lymphoma (NHL) resistant to previously advocated conventional treatments.The patient was a 45 year old woman with HCV associated cryoglobulinaemic vasculitis, with purpura, arthralgia, constitutional symptoms, and a polyneuropathy. A malignant NHL was found as underlying lymphoproliferative disease. At this stage the disease was refractory to interferon alpha2b and ribavirin and to subsequent immunosuppressive treatment with cyclophosphamide. Six rituximab infusions targeting the CD20 antigen on cells of the B cell lineage induced remission of the vasculitis. Bone marrow biopsy disclosed absence of the NHL. Remission has subsequently been maintained and HCV eliminated with the new pegylated interferon alpha2b and ribavirin for nearly one year.Transition of the underlying "benign" lymphoproliferative disease to a malignant lymphoma may result in difficult to treat HCV associated cryoglobulinaemic vasculitis. Rituximab offers a new possibility for inducing remission in refractory HCV associated cryoglobulinaemic vasculitis and the lymphoproliferative disorder. After remission, HCV may subsequently be eliminated with pegylated interferon alpha2b and ribavirin.

A 59-year-old female patient with a history of malignant lymphoma presented with symptoms of septicaemia. The skin of the extremities showed bullous, necrotizing plaques. Blood culture revealed Vibrio vulnificus as the causative organism. The infection was most likely acquired while swimming in the unusually warm Baltic Sea through inadvertent swallowing of sea water. The disease is rare in Europe. It is discussed in view of its typical clinical and histological picture.

Wegener's granulomatosis (WG) is an autoimmune disease of as yet unknown etiology. To date it has remained obscure what causes WG or determines disease progression. Case reports suggest that viral infections such as cytomegalovirus (CMV) reactivation may contribute to disease flares. In this study we found a skewing of the phenotype of CMV-specific CD8+tet(ramer)+ T-cells in WG. A marked proportion of these cells displayed a late differentiated "effector memory" T-cell phenotype with decreased expression of CD28 and CD62L, and heterogeneous CD27 expression, features which were also seen in CD8+tet- T-cells in WG, but not in controls. Our results might reflect profound generalized changes in the CD8+ T-cell compartment also affecting virus-specific T-cell responses in WG.

Polymorphonuclear neutrophil granulocytes (PMNs) possess numerous effector mechanisms to kill ingested pathogens as the first line of defence. However, several microorganisms evade intracellular killing in neutrophils, survive and retain infectivity. There is increasing evidence that several pathogens even multiply within neutrophils. Taking Leishmania major as a prototypic intracellular pathogen, we suggest an evasion strategy that includes the manipulation of PMNs in such a way that the pathogens are able to use the granulocytes as host cells. The ability to survive and maintain infectivity in PMNs subsequently enables these organisms to establish productive infection. These organisms can use granulocytes as Trojan horses before they enter their definitive host cells, the macrophages.

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.

Transmission of bacteria from inanimate surfaces in healthcare associated environments is an important source of hospital acquired infections. A number of commercially available medical devices promise to fulfill antibacterial activity to reduce environmental contamination. In this study we developed a touch transfer assay modeling fingerprint transmission to investigate the antibacterial activity of surfaces, with confirmed antibacterial activity by a modified ISO 22196 (JIS Z 2801) assay to test such surfaces under more realistic conditions. Bacteria were taken up from a dry standardized primary contaminated surface (PCS) with disinfected fingers or fingers covered with sterile and moistened cotton gloves. Subsequently, bacteria were transferred by pressing on secondary contaminated surfaces (SCS) with or without potential antibacterial activity and the relative reduction rate was determined after 24 h. A stable transmission rate between PCS and SCS was observed using moistened sterile gloves. A copper containing alloy displayed at least a tenfold reduction of the bacterial load consistently reaching less than 2.5 cfu/cm2. In contrast, no significant reduction of bacterial contamination by silver containing surfaces and matured pure silver was observed in the touch transfer assay. With the touch transfer assay we successfully established a new reproducible method modeling cross contamination. Using the new method we were able to demonstrate that several surfaces with confirmed antimicrobial activity in a modified ISO 22196 (JIS Z 2801) assay lacked effectiveness under defined ambient conditions. This data indicate that liquid based assays like the ISO 22196 should be critically reviewed before claiming antibacterial activity for surfaces in the setting of contamination of dry surfaces by contact to the human skin. We suggest the newly developed touch transfer assay as a new additional tool for the assessment of potential antimicrobial surfaces prior utilization in hospital environments.

The obligatory intracellular bacterium Chlamydia pneumoniae (C. pneumoniae) can survive and multiply in neutrophil granulocytes. Since neutrophils are short living cells, inhibition of neutrophil apoptosis appears to play a major role in the productive infection of neutrophils by C. pneumoniae. In the present study, we have investigated which survival pathways and which events of the apoptotic process are modulated in C. pneumoniae-infected neutrophils. All infection experiments were carried out using primary human neutrophils in vitro. We show that infection with C. pneumoniae activates PI3K/Akt as well as the ERK1/2 and p38 MAP kinases and present evidence that activation of the PI3K/Akt and ERK1/2 pathways are essential to initiate the apoptosis delay in C. pneumoniae-infected neutrophils. Both the PI3K/Akt and ERK1/2 pathways are involved in the maintained expression of the anti-apoptotic protein Mcl-1. In addition, we also showed that the PI3K/Akt pathway leads to the activation of NF-κB-dependent release of IL-8 by infected neutrophils. Infection with C. pneumoniae activates the PI3K/Akt and ERK1/2 MAPK survival pathways in neutrophils, induces the NF-κB dependent release of IL-8 and leads to the maintenance of Mcl-1 expression in neutrophils.

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN-γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN-γ-induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN-γ-induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN-γ-induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor-2α (eIF2α) and down-regulation of the vesicle-associated membrane protein-associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN-γ-induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER-related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN-γ-induced persistent infection.

Background —Recovery of the intracellular bacterium Chlamydia pneumoniae from atherosclerotic plaques has initiated large studies on antimicrobial therapy in coronary artery disease. The basic concept that antibiotic therapy may eliminate and prevent vascular infection was evaluated in vitro and in vivo by examining the antibiotic susceptibility of C pneumoniae in circulating human monocytes, which are thought to transport chlamydiae from the respiratory tract to the vascular wall. Methods and Results —Blood monocytes (CD14+) from 2 healthy volunteers were obtained before and after oral treatment with azithromycin or rifampin and then inoculated with a vascular C pneumoniae strain and continuously cultured in the presence of the respective antibiotic. Progress of infection and chlamydial viability was assessed by immunogold-labeling and detection of C pneumoniae –specific mRNA transcripts. Circulating monocytes from patients undergoing treatment with experimental azithromycin for coronary artery disease were examined for C pneumoniae infection by cell culture. Antibiotics did not inhibit chlamydial growth within monocytes. Electron microscopy showed development of chlamydial inclusion bodies. Reverse transcription–polymerase chain reaction demonstrated continuous synthesis of chlamydial mRNA for 10 days without lysis of the monocytes. The in vivo presence of viable pathogen not eliminated by azithromycin was shown by cultural recovery of C pneumoniae from the circulating monocytes of 2 patients with coronary artery disease. Conclusions — C pneumoniae uses monocytes as a transport system for systemic dissemination and enters a persistent state not covered by an otherwise effective antichlamydial treatment. Prevention of vascular infection by antichlamydial treatment may be problematic: circulating monocytes carrying a pathogen with reduced antimicrobial susceptibility might initiate reinfection or promote atherosclerosis by the release of proinflammatory mediators.

Recovery of viable Chlamydia pneumoniae from atheromas of coronary heart diseases patients has initiated pilot studies to eradicate C. pneumoniae from vascular tissue by antibiotic treatment. To provide data for the selection of effective antibiotics, we investigated the in vitro activity of anti-chlamydial antibiotics to eliminate vascular strains of C. pneumoniae from coronary endothelial and smooth muscle cells, celltypes that are involved in the pathogenesis of atherosclerosis.

The obligate intracellular bacterium Chlamydia pneumoniae has been implicated in the pathogenesis of atherosclerosis since viable pathogen has been recovered from plaques. Chlamydiae are epithelial pathogens notorious for causing persistent infection. Atherosclerosis, however, is a chronic inflammatory disease involving mesenchymal cells of the vascular wall. A bacterial contribution to atherosclerosis appears more relevant if the resident mesenchymal cells of the vascular wall that constitute the plaque can support chlamydial infection continuously. Therefore we inoculated immortalized and primary mesenchymal cells with a vascular and a respiratory Chlamydia pneumoniae isolate. Primary human coronary artery endothelial and smooth muscle cells, primary human embryonic fibroblasts as well as the immortalized cell lines were permissive for continuous growth of both strains. Thus, the resident vascular cells that produce the atheromatous plaque can acquire permanent productive. Chlamydia pneumoniae infection. Immortalized monocytic cells and peripheral blood monocytes also supported chlamydial growth, though productive infection ceased after 5 passages. Monocytes/macrophages are not resident cells of the vascular wall but have an active role in plaque formation. Systemic circulation and transendothelial migration makes them a potential vector system for chlamydial distribution. These findings add further plausibility to the hypothesis of a chronic infectious component in the multifactorial condition of atherosclerosis. Further studies must precisely define chlamydial target cells in vivo and differentiate infection in resident cells of the vascular wall from a presence limited to migrating macrophages. Endovascular infection might provide an explanation for unclear phenomena of atherogenesis like mesenchymal cell proliferation and its distinct inflammatory component.

Susceptibility of mice to Leishmania major is associated with an insufficient NK cell-mediated innate immune response. We analyzed the expression of NK cell-activating chemokines in vivo during the first days of infection in resistant and susceptible mice. The mRNA expression of gamma interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and lymphotactin was upregulated 1 day after infection in the draining lymph nodes of resistant C57BL/6 mice but not in those of susceptible BALB/c mice. In vivo local treatment of BALB/c mice with recombinant IP-10 shortly after infection resulted in an enhanced NK cell activity in the draining lymph node. The data suggest that although the recruitment of NK cells is normal in susceptible mice, the lack of NK cell-activating chemokines is a factor resulting in a suboptimal NK cell-mediated defense.

The CD14 myelomonocytic differentiation antigen plays a major role in acute Gram-negative infections with Escherichia coli; however, its role in chronic infections has not yet been analyzed. To address this question, we studied the role of CD14 in a chronic abscess-forming peritonitis, induced by Bacteroides fragilis. B. fragilis (3x10(8) CFU/ml) were resuspended in a liquid nutrient agar and injected into the peritoneal cavity of CD14-deficient (CD 14-/-) and normal C57BL/6J (CD 14+/+) mice, respectively. After 3 days there was a severe phlegmonous intra-abdominal inflammation in both groups. After 7 days an abscess-forming peritonitis developed and by 14 days the infectious foci were compartimentalized. These observations were indistinguishable between CD14-/- and CD14+/+ mice. Although no differences were seen in abscess formation, CD14-/- mice were able to clear B. fragilis more efficiently from the blood than CD14+/+ mice. After 3, 7, and 14 days blood cultures were B. fragilis positive in 11% (1/9), 20% (2/10), and 0% (0/9) in CD14-/-compared with 90% (9/10), 78% (7/9), and 20% (2/10) in CD14+/+ mice, respectively (P

Single-nucleotide polymorphisms (SNPs) are targets to discriminate intraspecies diversity of bacteria and to correlate a genotype with a potential pathotype. Quantification of polygenotypic populations supports this task for in vitro and in vivo applications. We present a novel assay capable of quantifying mixtures of two genotypes differing by only one SNP.

Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases, for example, psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to the effects on lymphocytes and dendritic cells. Here, we focused on the effects of dimethylfumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps, and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates—in a stimulus-dependent manner-neutrophil activation using the phosphoinositide 3-kinase/Akt-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita, an organ-specific autoimmune disease caused by autoantibodies to type VII collagen, were employed. In the presence of DMF, blistering induced by injection of anti-type VII collagen antibodies into mice was significantly impaired. DMF treatment of mice with clinically already-manifested epidermolysis bullosa acquisita led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases.