Does intraperitoneal ropivacaine reduce postoperative inflammation? A prospective, double-blind, placebo-controlled pilot study.

BACKGROUND: Postoperative inflammation is a common consequence of surgery and the ensuing stress response. Local anesthetics have anti-inflammatory properties. The primary aim of this study was to evaluate if LA administrated intraperitoneally perioperatively might inhibit expression of inflammatory cytokines. METHODS: This was a, randomized, double blind, placebo-controlled study (ClinicalTrial.gov reg no: NCT02256228) in patients undergoing surgery for ovarian cancer. Patients were randomized to receive: intraperitoneal ropivacaine (Group IPLA) or saline (Group P) perioperatively. Except for study drug, patients were treated similarly. At the end of surgery, a multi-port catheter was inserted intraperitoneally, and ropivacaine 2 mg/mL or 0.9% saline, 10 mL was injected intermittently every other hour during 72 hours postoperatively. Systemic expression of cytokines and plasma ropivacaine were determined before and 6, 24, and 48 hours after surgery. Stress response was measured by serum glucose, cortisol, and insulin. RESULTS: Forty patients were recruited, 20 in each group. There was no statistical significant difference in systemic cytokine between the groups at any time point. Serum cortisol was significantly lower in the IPLA group at 6 hours, median 103 nmol/L (IQR 53-250) compared to placebo, median 440 nmol/L (IQR 115-885), P = 0.023. Serum glucose and insulin were similar between the groups. Total and free serum concentrations of ropivacaine were well below toxic concentrations. CONCLUSION: In this small study, perioperative intraperitoneal ropivacaine did not reduce the systemic inflammatory response associated with major abdominal surgery. Total and free ropivacaine concentrations were below known toxic concentrations in humans.

Antieosinophil Antibodies Alone or in Combination with Antineutrophil Cytoplasmic Antibodies (ANCA) Detected in Different Autoimmune Conditions.

Circulating antieosinophil antibodies (AEOSA) have been associated with various autoimmune conditions affecting the liver, kidneys, lungs, and joints but are not part of routine clinical diagnostics. While analyzing human sera for antineutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence (IIF) on granulocytes, 0.8% of analyzed samples were found to be reactive with eosinophils. Our aim was to determine the diagnostic relevance and antigenic specificity of AEOSA. AEOSA were seen either in combination with an myeloperoxidase (MPO)-positive p-ANCA (44%; AEOSA+/ANCA+) or on their own (56%; AEOSA+/ANCA-). AEOSA/ANCA positivity was seen in patients with thyroid disease (44%) or vasculitis (31%), while AEOSA+/ANCA- pattern was more common in patients with autoimmune disorders of the gastrointestinal tract and/or liver. Eosinophil peroxidase (EPX) was the main target recognized in 66% of the AEOSA+ sera by enzyme-linked immunosorbent assay (ELISA). Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) were also identified as target antigens but less frequently and only in combination with EPX. In conclusion, we confirmed that EPX is a major target of AEOSA, illustrating the high antigenic potential of EPX. Our results also demonstrate the presence of concomitant AEOSA/ANCA positivity in a defined patient group. Further research should aim to elucidate the association of AEOSA with autoimmunity.

The neutrophil subset defined by CD177 expression is preferentially recruited to gingival crevicular fluid in periodontitis.

In recent years, the concept of distinct subpopulations of human neutrophils has attracted much attention. One bona fide subset marker, exclusively expressed by a proportion of circulating neutrophils in a given individual, and therefore dividing neutrophils in two distinct subpopulations, is the glycoprotein CD177. CD177 is expressed on the plasma and granule membranes of 0-100% of circulating neutrophils depending on the donor. Several in vitro studies have linked CD177 to neutrophil transmigration, yet very few have looked at the role of CD177 for tissue recruitment in vivo. We investigate whether the CD177+ and CD177- neutrophil subsets differ in their propensity to migrate to both aseptic- and microbe-triggered inflamed human tissues. Microbe-triggered neutrophil migration was evaluated in samples of gingival crevicular fluid (GCF) from patients with periodontitis, whereas neutrophil migration to aseptic inflammation was evaluated in synovial fluid from patients with inflammatory arthritis, as well as in exudate from experimental skin chambers applied on healthy donors. We found that the proportion of CD177+ neutrophils was significantly higher in GCF from patients with periodontitis, as compared to blood from the same individuals. Such accumulation of CD177+ neutrophils was not seen in the two models of aseptic inflammation. Moreover, the proportion of CD177+ neutrophils in circulation was significantly higher in the periodontitis patient group, as compared to healthy donors. Our data indicate that the CD177+ neutrophil subset is preferentially recruited to the gingival crevice of periodontitis patients, and may imply that this subtype is of particular importance for situations of microbe-driven inflammation.

Getting under the skin: the immunopathogenesis of Streptococcus pyogenes deep tissue infections.

Streptococcus pyogenes can cause a variety of diseases in immunocompetent individuals, from pharyngotonsillitis to life-threatening invasive diseases, such as streptococcal toxic shock syndrome, and rapidly progressing deep-tissue infections, such as necrotizing fasciitis. Necrotizing fasciitis is often seen in combination with streptococcal toxic shock syndrome, which further increases morbidity and mortality. We review here the host-pathogen interactions in the tissue milieu and discuss the use of intravenous immunoglobulin as potential adjunctive therapy in these life-threatening infections.

Determination of Subset-Restricted Anti-neutrophil Cytoplasmic Antibodies (ANCA) by Immunofluorescence Cytochemistry.

Neutrophils have long been considered a homogeneous cell type where all circulating cells of a particular individual express the same proteins. Lately, however, this view is changing and distinct neutrophil subsets, defined by the presence or absence of different proteins, are being increasingly recognized. At least two separate protein markers, CD177 and Olfactomedin-4 (OLFM4) are known to be expressed by some, but not all, circulating neutrophils of a given individual. We recently described the existence of subset-restricted serum autoantibodies targeting OLFM4; these were discovered during clinical testing for anti-neutrophil cytoplasmic antibodies (ANCAs). ANCA testing is part of the clinical examinations routinely carried out to support diagnosis of suspected autoimmune conditions, especially vasculitis. Positive sera typically react with all neutrophils from a single donor, whereas subset-restricted ANCA sera (such as those containing anti-OLFM4 antibodies) only react with a fraction of neutrophils. Described in this chapter is an indirect immunofluorescence (IIF) approach to test human sera for the presence of subset-restricted ANCA as well as instructions for costaining experiments using sera and purified antibodies directed against established subset markers.

Exocrine pancreatic function is preserved in systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) has been suggested to cause exocrine pancreatic dysfunction. However, a case-control-based autopsy study failed to associate systemic sclerosis with any pancreatic histopathology. The primary objective of this study was to examine the exocrine pancreatic function in consecutive SSc patients in relation to an age- and sex-matched control group. A secondary objective was to relate exocrine pancreatic function to radiological, laboratory, and clinical SSc characteristics. METHODS: One hundred twelve consecutive patients fulfilling the 2013 American Congress of Rheumatology/European League Against Rheumatism criteria for SSc and 52 control subjects were matched for sex and age. Exocrine pancreatic function was assessed by ELISA-based measurement of fecal elastase, and levels ≤ 200 µg/g were considered pathological, i.e., representing exocrine pancreatic insufficiency. Patients were characterized regarding SSc manifestations including gastrointestinal and hepatobiliary function, by use of laboratory and clinical examinations. Pancreas parenchyma characteristics were evaluated by high-resolution computer tomography (HRCT). RESULTS: A similar proportion of subjects exhibited pathological levels of fecal elastase among SSc patients (6/112; 5.4%) and control subjects (3/52; 5.8%). Patients with fecal elastase ≤ 200 µg/g did not differ from other SSc patients with respect to laboratory and clinical characteristics, including malnutrition. SSc subjects with low levels of fecal elastase displayed significantly lower pancreas attenuation on HRCT examinations compared to the control subjects. CONCLUSIONS: In this study encompassing 112 consecutive SSc patients and 52 matched control subjects, we were unable to associate systemic sclerosis with clinically significant exocrine pancreatic dysfunction.

Cathelicidin LL-37 in severe Streptococcus pyogenes soft tissue infections in humans.

Severe soft tissue infections, such as necrotizing fasciitis and severe cellulitis, caused by group A streptococci (GAS) are rapidly progressing life-threatening infections characterized by massive bacterial loads in the tissue even late after the onset of infection. Antimicrobial peptides are important components of the innate host defense, and cathelicidins have been shown to protect against murine necrotic skin infections caused by GAS. However, it has been demonstrated that the streptococcal cysteine protease SpeB proteolytically inactivates the human cathelicidin LL-37 in vitro. Here we have investigated the expression of LL-37 and its interaction with GAS and SpeB during acute severe soft tissue infections by analyses of patient tissue biopsy specimens. The results showed large amounts of LL-37, both the proform (hCAP18) and the mature peptide, in the tissue. Confocal microscopy identified neutrophils as the main source of the peptide. A distinct colocalization between the bacteria and LL-37 could be noted, and bacterial loads showed positive correlation to the LL-37 levels. Areas with high LL-37 levels coincided with areas with large amounts of SpeB. Confocal microscopy confirmed strong colocalization of GAS, SpeB, and LL-37 at the bacterial surface. Taken together, the findings of this study provide in vivo support of the hypothesis that SpeB-mediated inactivation of LL-37 at the streptococcal surface represents a bacterial resistance mechanism at the infected tissue site in patients with severe GAS tissue infections.

Viable group A streptococci in macrophages during acute soft tissue infection.

BACKGROUND: Group A streptococcal severe soft tissue infections, such as necrotizing fasciitis, are rapidly progressive infections associated with high mortality. Group A streptococcus is typically considered an extracellular pathogen, but has been shown to reside intracellularly in host cells. METHODS AND FINDINGS: We characterized in vivo interactions between group A streptococci (GAS) and cells involved in innate immune responses, using human biopsies (n = 70) collected from 17 patients with soft tissue infections. Immunostaining and in situ image analysis revealed high amounts of bacteria in the biopsies, even in those collected after prolonged antibiotic therapy. Viability of the streptococci was assessed by use of a bacterial viability stain, which demonstrated viable bacteria in 74% of the biopsies. GAS were present both extracellularly and intracellularly within phagocytic cells, primarily within macrophages. Intracellular GAS were predominantly noted in biopsies from newly involved tissue characterized by lower inflammation and bacterial load, whereas purely extracellular GAS or a combination of intra- and extracellular GAS dominated in severely inflamed tissue. The latter tissue was also associated with a significantly increased amount of the cysteine protease streptococcal pyrogenic exotoxin SpeB. In vitro studies confirmed that macrophages serve as reservoirs for viable GAS, and infection with a speB-deletion mutant produced significantly lower frequencies of cells with viable GAS following infection as compared to the wild-type bacteria. CONCLUSIONS: This is the first study to demonstrate that GAS survive intracellularly in macrophages during acute invasive infections. This intracellular presence may have evolved as a mechanism to avoid antibiotic eradication, which may explain our finding that high bacterial load is present even in tissue collected after prolonged intravenous antibiotic therapy. This new insight into the pathogenesis of streptococcal soft tissue infections highlights a need for alternative therapeutic strategies.

Osteoblasts promote castration-resistant prostate cancer by altering intratumoral steroidogenesis.

The skeleton is the preferred site for prostate cancer (PC) metastasis leading to incurable castration-resistant disease. The increased expression of genes encoding steroidogenic enzymes found in bone metastatic tissue from patients suggests that up-regulated steroidogenesis might contribute to tumor growth at the metastatic site. Because of the overall sclerotic phenotype, we hypothesize that osteoblasts regulate the intratumoral steroidogenesis of castration resistant prostate cancer (CRPC) in bone. We here show that osteoblasts alter the steroidogenic transcription program in CRPC cells, closely mimicking the gene expression pattern described in CRPC. Osteoblast-stimulated LNCaP-19 cells displayed an increased expression of genes encoding for steroidogenic enzymes (CYP11A1, HSD3B1, and AKR1C3), estrogen signaling-related genes (CYP19A1, and ESR2), and genes for DHT-inactivating enzymes (UGT2B7, UGT2B15, and UGT2B17). The observed osteoblast-induced effect was exclusive to osteogenic CRPC cells (LNCaP-19) in contrast to osteolytic PC-3 and androgen-dependent LNCaP cells. The altered steroid enzymatic pattern was specific for the intratibial tumors and verified by immunohistochemistry in tissue specimens from LNCaP-19 xenograft tumors. Additionally, the overall steroidogenic effect was reflected by corresponding levels of progesterone and testosterone in serum from castrated mice with intratibial xenografts. A bi-directional interplay was demonstrated since both proliferation and Esr2 expression of osteoblasts were induced by CRPC cells in steroid-depleted conditions. Together, our results demonstrate that osteoblasts are important mediators of the intratumoral steroidogenesis of CRPC and for castration-resistant growth in bone. Targeting osteoblasts may therefore be important in the development of new therapeutic approaches.

Olfactomedin-4 autoantibodies give unusual c-ANCA staining patterns with reactivity to a subpopulation of neutrophils.

Testing for the presence of ANCAs in circulation is part of the clinical examinations routinely performed upon suspected autoimmune disorders, mainly vasculitis. The autoantibodies are typically directed toward neutrophil MPO or PR3. These are major granule-localized proteins, and similar to all hitherto-described ANCA antigens, they are expressed by all neutrophils, and ANCA-containing sera thus give rise to uniform reactivity toward all neutrophils in a sample. In this paper, we describe sera from 2 unrelated patients with diffuse inflammatory symptoms that gave rise to peculiar c-ANCA patterns, only reacting with a subpopulation (roughly 30%) of human neutrophils. By immunoblotting, both sera reacted to the same antigen, which was expressed in intracellular granules. The antigen could be released to the extracellular milieu through secretion but also through the formation of NETs. Neutrophils have long been considered a homogenous cell population, but it is becoming increasingly clear that distinct subpopulations, defined by the presence or absence of certain proteins, exist. One such marker that defines a neutrophil subset is the granule protein OLFM4. The unusual, subset-restricted c-ANCA sera reacted only with OLFM4-positive neutrophils, and MS analysis revealed that the autoantigen was, in fact, OLFM4. These data describe for the first time a c-ANCA pattern reactive to only a subpopulation of neutrophils and identify the granule protein OLFM4 as a novel autoantigen.

HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes.

Extracellular High Mobility Group Box 1 (HMGB1) has been associated with acute and chronic inflammatory conditions. However, little is known about HMGB1 in necrotizing bacterial infections. We hypothesized that the local HMGB1 response is excessive in severe soft tissue infections (STIs), which are characterized by necrosis and hyperinflammation. To explore this, tissue biopsies were collected from patients with varying severity of Streptococcus pyogenes skin and STIs, including erysipelas, cellulitis, and necrotizing fasciitis. Tissue sections were immunostained for HMGB1, S. pyogenes, and inflammatory cell infiltrates and results quantified by acquired computerized image analysis (ACIA). HMGB1 expression increased in parallel to disease severity and was significantly higher in necrotizing fasciitis than in erysipelas (p = 0.0023). Confocal microscopy of sections co-stained for HMGB1 and cell markers revealed both extracellular and cytoplasmic HMGB1, the latter of which was found predominantly in macrophages. To further verify macrophages as main source of activation triggered HMGB1 release, human macrophages were infected with clinical S. pyogenes isolates. The results demonstrated infection triggered release of HMGB1. Dual staining's visualized HMGB1 in areas close to, but not overlapping, with neutrophils, indicating a potential chemotactic role. In vitro transmigration experiments showed a chemotactic effect of HMGB1 on neutrophils. The data furthermore provided in vivo support that HGMB1 may form immunostimulatory complexes with IL-1ß. Taken together, the findings provide the first in vivo evidence that HMGB1 is abundant at the local site of severe bacterial STIs and its levels correlated to severity of infections; hence, indicating its potential value as a biomarker for tissue pathology.

Erysipelas caused by group A streptococcus activates the contact system and induces the release of heparin-binding protein.

Bacterial skin infections, such as erysipelas or cellulitis, are characterized by fever and a painful erythematous rash. Despite the high prevalence of these infections, little is known about the underlying pathogenic mechanisms. This is partly due to the fact that a bacterial diagnosis is often difficult to attain. To gain insight into the pathogenesis of erysipelas, we investigated the samples obtained from infected and noninfected areas of skin from 12 patients with erysipelas. Bacterial cultures, detection of specific streptococcal antibodies in convalescent sera, and immunohistochemical analyses of biopsies indicated group A streptococcal etiology in 11 of the 12 patients. Also, electron micrographs of erythematous skin confirmed the presence of group A streptococcal cells and showed a limited solubilization of the surface-attached M protein. Degradation of high-molecular-weight kininogen and upregulation of the bradykinin-1 receptor in inflamed tissues indicated activation of the contact system in 11 patients. Analyses of release of the vasoactive heparin-binding protein (HBP) showed increased levels in the infected as compared with the noninfected areas. The results suggest that group A streptococci induce contact activation and HBP release during skin infection, which likely contribute to the symptoms seen in erysipelas: fever, pain, erythema, and edema.

Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation.

These days it has been increasingly recognized that mast cells (MCs) are critical components of host defense against pathogens. In this study, we have provided the first evidence that MCs can kill bacteria by entrapping them in extracellular structures similar to the extracellular traps described for neutrophils (NETs). We took advantage of the ability of MCs to kill the human pathogen Streptococcus pyogenes by a phagocytosis-independent mechanism in order to characterize the extracellular antimicrobial activity of MCs. Close contact of bacteria and MCs was required for full antimicrobial activity. Immunofluorescence and electron microscopy revealed that S pyogenes was entrapped by extracellular structures produced by MCs (MCETs), which are composed of DNA, histones, tryptase, and the antimicrobial peptide LL-37. Disruption of MCETs significantly reduced the antimicrobial effect of MCs, suggesting that intact extracellular webs are critical for effective inhibition of bacterial growth. Similar to NETs, production of MCETs was mediated by a reactive oxygen species (ROS)-dependent cell death mechanism accompanied by disruption of the nuclear envelope, which can be induced after stimulation of MCs with phorbol-12-myristate-13-acetate (PMA), H(2)O(2), or bacterial pathogens. Our study provides the first experimental evidence of antimicrobial extracellular traps formation by an immune cell population other than neutrophils.

Release of SpeA from Streptococcus pyogenes after exposure to penicillin: dependency on dose and inhibition by clindamycin.

The amount and time course of SpeA release from group A streptococci (GAS) was studied at different starting inoculate after exposure to different doses of penicillin, clindamycin or a combination of the 2. The release was related to the bacterial concentration and killing rate. A clinical GAS strain was exposed to benzylpenicillin, 2 and 1000 x MIC, clindamycin, 2 and 32 x MIC, or combinations of the 2. Samples for viable counts and SpeA analyses were drawn before and after the addition of antibiotics and at 3, 6 and 24 h. The SpeA release was higher at low than at high concentrations of penicillin and the combination (both, p<0.05). The addition of clindamycin to penicillin reduced SpeA production at both concentrations (p<0.01). Most SpeA was released before 3 h, and for penicillin and the combination, the amount correlated to the number of killed bacteria during this period (r=0.50; p<0.05). A positive correlation was found between the inoculum size and the SpeA concentration at time zero (r=0.54; p<0.05). The SpeA concentration was dependent on the initial number of bacteria, the class of antibiotic, the dose of penicillin and the killing rate.

Successful management of severe group A streptococcal soft tissue infections using an aggressive medical regimen including intravenous polyspecific immunoglobulin together with a conservative surgical approach.

Intravenous polyspecific immunoglobulin G (IVIG) has been reported to be efficacious as adjunctive therapy in patients with toxic shock syndrome caused by a group A streptococci (GAS). GAS is also an important cause of necrotizing fasciitis, for which an early and extensive surgical intervention is currently advocated. Here we report on the use of an aggressive medical regimen including high-dose IVIG together with a conservative surgical approach in severe GAS soft tissue infection. We describe 7 patients with severe soft tissue infection caused by GAS, who all were treated with effective antimicrobials and high-dose IVIG. Surgery was either not performed or only limited exploration was carried out. Six of the patients had toxic shock syndrome. All patients survived. Immunostaining of tissue biopsies from 2 of the patients revealed high levels of GAS, superantigen and pro-inflammatory cytokines initially, which were dramatically reduced in a repeat biopsy of the initial operative site collected from 1 of the patients 66 h post-IVIG administration. The study suggests that the use of a medical regimen including IVIG in patients with severe GAS soft tissue infections may allow an initial non-operative or minimally invasive approach, which can limit the need to perform immediate wide debridements and amputations in unstable patients.