Contrasting contributions of TNF from distinct cellular sources in arthritis.

OBJECTIVES: Neutralisation of tumour necrosis factor (TNF) is widely used as a therapy for rheumatoid arthritis (RA). However, this therapy is only effective in less than a half of patients and is associated with several side effects. We hypothesised that TNF may possess non-redundant protective and immunomodulatory functions in vivo that cannot be blocked without a cost. The present work aimed to identify cellular sources of protective and pathogenic TNF, and its molecular forms during autoimmune arthritis. METHODS: Mice lacking TNF expression by distinct cell types, such as myeloid cells and T or B lymphocytes, were subjected to collagen-induced arthritis (CIA) and collagen antibody-induced arthritis. Mice lacking soluble TNF production were also employed. The severity and incidence of the disease, as well as humoral and cellular responses were assessed. RESULTS: Myeloid cell-derived TNF contributes to both induction and pathogenesis of autoimmune arthritis. Conversely, T cell-derived TNF is protective during the induction phase of arthritis via limiting of interleukin-12 production by dendritic cells and by subsequent control of autoreactive memory T cell development, but is dispensable during the effector phase of arthritis. B cell-derived TNF mediates severity of CIA via control of pathogenic autoantibody production. CONCLUSIONS: Distinct TNF-producing cell types may modulate disease development through different mechanisms, suggesting that in arthritis TNF ablation from restricted cellular sources, such as myeloid cells, while preserving protective TNF functions from other cell types may be superior to pan-anti-TNF therapy.

Prognostic and Pathogenic Role of Angiopoietin-1 and -2 in Pneumonia.

RATIONALE: During pneumonia, pathogen-host interaction evokes inflammation and lung barrier dysfunction. Tie2 activation by angiopoietin-1 reduces, whereas Tie2 blockade by angiopoietin-2 increases, inflammation and permeability during sepsis. The role of angiopoietin-1/-2 in pneumonia remains unidentified. OBJECTIVES: To investigate the prognostic and pathogenic impact of angiopoietins in regulating pulmonary vascular barrier function and inflammation in bacterial pneumonia. METHODS: Serum angiopoietin levels were quantified in pneumonia patients of two independent cohorts (n = 148, n = 395). Human postmortem lung tissue, pneumolysin- or angiopoietin-2-stimulated endothelial cells, isolated perfused and ventilated mouse lungs, and mice with pneumococcal pneumonia were investigated. MEASUREMENTS AND MAIN RESULTS: In patients with pneumonia, decreased serum angiopoietin-1 and increased angiopoietin-2 levels were observed as compared with healthy subjects. Higher angiopoietin-2 serum levels were found in patients with community-acquired pneumonia who died within 28 days of diagnosis compared with survivors. Receiver operating characteristic analysis revealed improved prognostic accuracy of CURB-65 for 28-day survival, intensive care treatment, and length of hospital stay if combined with angiopoietin-2 serum levels. In vitro, pneumolysin enhanced endothelial angiopoietin-2 release, angiopoietin-2 increased endothelial permeability, and angiopoietin-1 reduced pneumolysin-evoked endothelial permeability. Ventilated and perfused lungs of mice with angiopoietin-2 knockdown showed reduced permeability on pneumolysin stimulation. Increased pulmonary angiopoietin-2 and reduced angiopoietin-1 mRNA expression were observed in Streptococcus pneumoniae-infected mice. Finally, angiopoietin-1 therapy reduced inflammation and permeability in murine pneumonia. CONCLUSIONS: These data suggest a central role of angiopoietin-1/-2 in pneumonia-evoked inflammation and permeability. Increased angiopoietin-2 serum levels predicted mortality and length of hospital stay, and angiopoietin-1 may provide a therapeutic target for severe pneumonia.

Synovitis as a concomitant disease in shoulder pathologies.

INTRODUCTION: Shoulder pathologies are often accompanied by rotator interval synovitis. This phenomenon is poorly described in the literature so far. The aim of the study was to analyze the occurrence of macroscopically visible synovial reaction in the rotator interval in patients with chronic shoulder pathologies and to perform a histopathological evaluation. MATERIALS AND METHODS: In this prospective cohort study, 167 consecutive patients undergoing arthroscopic shoulder surgery for chronic shoulder pathology were included (♀ = 45, ♂ = 122; [Formula: see text]54.5 years ± 12.8). Included patients were divided into subgroups according to the encountered chronic shoulder pathology: (1) impingement syndrome with or without bursal sided partial rotator cuff tear (RCT); (2) articular sided partial RCT; (3) full-thickness RCT; (4) RCT that involves at least two tendons; (5) shoulder instability; and (6) cartilage damage. Standardized soft tissue biopsies from the rotator interval were taken. The synovitis score of Krenn/Morawietz was used for histopathological examination. RESULTS: Extraarticular pathology (group 1) showed significantly decreased synovitis scores compared to all the other groups. Increased size of rotator cuff tears (group 4), as well as cartilage damage (group 6) showed significantly higher synovitis scores than group 3 (p < 0.05). Moreover, the synovitis score was significantly increased in patients with concomitant pathologies of the long head of the biceps (p = 0.001). CONCLUSIONS: This study suggests that chronic intra- and extraarticular shoulder diseases are very often accompanied by a histopathologically verifiable low-grade synovitis. Intraarticular pathologies seem to induce increased levels of synovitis. Furthermore, the increased size of rotator cuff tears is accompanied by a higher degree of synovitis. STUDY DESIGN: Cohort study, level of evidence, 2b.

Measuring inter-rater reliability for nominal data - which coefficients and confidence intervals are appropriate?

BACKGROUND: Reliability of measurements is a prerequisite of medical research. For nominal data, Fleiss' kappa (in the following labelled as Fleiss' K) and Krippendorff's alpha provide the highest flexibility of the available reliability measures with respect to number of raters and categories. Our aim was to investigate which measures and which confidence intervals provide the best statistical properties for the assessment of inter-rater reliability in different situations. METHODS: We performed a large simulation study to investigate the precision of the estimates for Fleiss' K and Krippendorff's alpha and to determine the empirical coverage probability of the corresponding confidence intervals (asymptotic for Fleiss' K and bootstrap for both measures). Furthermore, we compared measures and confidence intervals in a real world case study. RESULTS: Point estimates of Fleiss' K and Krippendorff's alpha did not differ from each other in all scenarios. In the case of missing data (completely at random), Krippendorff's alpha provided stable estimates, while the complete case analysis approach for Fleiss' K led to biased estimates. For shifted null hypotheses, the coverage probability of the asymptotic confidence interval for Fleiss' K was low, while the bootstrap confidence intervals for both measures provided a coverage probability close to the theoretical one. CONCLUSIONS: Fleiss' K and Krippendorff's alpha with bootstrap confidence intervals are equally suitable for the analysis of reliability of complete nominal data. The asymptotic confidence interval for Fleiss' K should not be used. In the case of missing data or data or higher than nominal order, Krippendorff's alpha is recommended. Together with this article, we provide an R-script for calculating Fleiss' K and Krippendorff's alpha and their corresponding bootstrap confidence intervals.

Prevention of liver cancer cachexia-induced cardiac wasting and heart failure.

AIMS: Symptoms of cancer cachexia (CC) include fatigue, shortness of breath, and impaired exercise capacity, which are also hallmark symptoms of heart failure (HF). Herein, we evaluate the effects of drugs commonly used to treat HF (bisoprolol, imidapril, spironolactone) on development of cardiac wasting, HF, and death in the rat hepatoma CC model (AH-130). METHODS AND RESULTS: Tumour-bearing rats showed a progressive loss of body weight and left-ventricular (LV) mass that was associated with a progressive deterioration in cardiac function. Strikingly, bisoprolol and spironolactone significantly reduced wasting of LV mass, attenuated cardiac dysfunction, and improved survival. In contrast, imidapril had no beneficial effect. Several key anabolic and catabolic pathways were dysregulated in the cachectic hearts and, in addition, we found enhanced fibrosis that was corrected by treatment with spironolactone. Finally, we found cardiac wasting and fibrotic remodelling in patients who died as a result of CC. In living cancer patients, with and without cachexia, serum levels of brain natriuretic peptide and aldosterone were elevated. CONCLUSION: Systemic effects of tumours lead not only to CC but also to cardiac wasting, associated with LV-dysfunction, fibrotic remodelling, and increased mortality. These adverse effects of the tumour on the heart and on survival can be mitigated by treatment with either the ß-blocker bisoprolol or the aldosterone antagonist spironolactone. We suggest that clinical trials employing these agents be considered to attempt to limit this devastating complication of cancer.

Histopathologic evaluation of passive stabilizers in shoulder instability.

BACKGROUND: The macroscopic pathomorphology in recurrent shoulder instability has been described. However, less is known regarding the histopathologic details of the affected structures. This study evaluates different histopathologic stages of shoulder instability by assessing biopsy specimens of static stabilizers for possible correlations with clinical parameters. Our hypothesis was that clinical parameters of shoulder instability correlate with histopathologic findings. MATERIALS AND METHODS: Passive shoulder stabilizers (labrum, anterior bundle of the inferior glenohumeral ligament) were biopsied during arthroscopic shoulder stabilization. Samples were submitted to immunohistochemistry, in situ hybridization, and blinded evaluation. Clinical data, comprising age (<30 years or ≥30 years), total number of dislocations (1, 2-3, or >3), and period since initial dislocation (<6 months, 6 months to 6 years, or >6 years), were tested for statistical correlation with the following histopathologic parameters: inflammation, lipomatous changes, vascular proliferation, tissue fragmentation, and cellularity. RESULTS: Standardized biopsies were performed in 30 consecutive patients (4 women and 26 men; mean age, 32.6 years) with anterior shoulder instability. Microscopic evaluation showed only small variations in histologic changes among all samples. Only limited variations in cell density, matrix swelling, and collagen fiber disruptions were found. Immunohistochemical analysis showed a similar expression of decorin in all samples. Clinical parameters (age, total number of dislocations, and period since initial dislocation) were statistically independent from histopathologic parameters (inflammation, lipomatous changes, vascular proliferation, tissue fragmentation, and cellularity). No correlation was found in patients with 1 dislocation versus those with more than 1 dislocation. CONCLUSIONS: In contrast to macroscopic findings among different grades of shoulder instability, this study detected no correlation between clinical items (age, total number of dislocations, and period since initial dislocation) and histopathologic parameters. These clinical items seem to be independent from the tissue status of static stabilizers of the shoulder.

In-vitro-induced Th17 cells fail to induce inflammation in vivo and show an impaired migration into inflamed sites.

Recently, IL-17 produced by Th17 cells was described as pro-inflammatory cytokine with an eminent role in autoimmune diseases, e.g. rheumatoid arthritis. A lack of IL-17 leads to amelioration of collagen-induced arthritis. IL-17 induction in naïve CD4(+) T cells depends on IL-6 and TGF-beta and is enhanced by IL-23. The in vivo inflammatory potential of in vitro-primed Th17 cells however, remains unclear. Here, we show that, although IL-17 neutralisation results in amelioration of murine OVA-induced arthritis, in vitro-primed Th17 cells cannot exacerbate arthritic symptoms after adoptive transfer. Furthermore, Th17 cells cannot induce an inflammatory delayed type hypersensitivity reaction because they fail to migrate into inflamed sites, possibly due to the lack of CXCR3 expression. Also, re-isolated Th17 cells acquired IFN-gamma expression, indicating instability of the Th17 phenotype. Taken together, the data show that IL-6, TGF-beta and IL-23 might not provide sufficient signals to induce "fully qualified" Th17 cells.

Prevalence of lymph nodes in the parametrium of radical vaginal trachelectomy (RVT) specimen.

OBJECTIVE: In order to evaluate radicality in fertility preserving surgery in women with early invasive cervical cancer we analyzed the parametrium of specimens of patients treated by radical vaginal trachelectomy for the presence of lymph nodes. We tried to identify morphologic factors associated with the presence of parametrial lymph nodes. METHODS: We analyzed surgical specimens of 112 patients who underwent radical trachelectomy between June 2004 and April 2009 at the Department of Gynecologic Oncology at Charité Campus Benjamin Franklin and Campus Mitte. All parametrial tissue was step sectioned and a total of 1878H&E stained histological sections were analyzed. RESULTS: In 8 patients (7.1%) a total of 13 lymph nodes were detected. Five lymph nodes in four patients had been primarily detected by routine histological examination. In one of these patients (0.9%) a 2mm lymph node metastasis was found. Serial sectioning revealed additional seven lymph nodes in four patients. The thickness of parametrium correlated significantly with the presence of lymph nodes in the parametrium. CONCLUSION: The presence of small lymph nodes in the parametrium of specimens of radical trachelectomy is low. In patients with early-stage cervical cancer, the incidence of metastasis is less than 1%. Preoperative assessment of the volume of the parametrium may indicate which patients need parametrial resection.

Association of mannose-binding lectin-2 gene polymorphism with the development of hepatitis C-induced hepatocellular carcinoma.

BACKGROUND: Development of end-stage liver and graft disease is suspected to be partially determined by the individual genetic background. Mannose-binding lectin (MBL) is an important immunomodulatory factor, which is supposed to be involved in complement activation and oncogenesis. Genetic polymorphisms of MBL-2 alter MBL functionality. The aim of our study was to determine the prevalence of MBL-2 polymorphism (rs7096206) in hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) based on histological analysis of explanted livers in patients undergoing liver transplantation (LT). METHODS: One hundred and seventy-seven patients, who underwent LT for HCV-induced liver disease, were genotyped for MBL-2 by TaqMan genotyping assay. Sixty-two patients with histologically confirmed HCC were compared with 115 patients without HCC. MBL-2 genotypes were corelated with the growth patern, tumour size and pretransplant α-fetoprotein (AFP) level of HCC patients. RESULTS: The prevalence of GG/GC genotypes was significantly higher among HCC patients compared with tumour-free explanted livers (P = 0.004; odds ratio 2.5; 1.3-4.8). GG/GC genotype group was significantly associated with the size of HCC (P = 0.022), higher pretransplant AFP level (P = 0.010) and bilobar tumour growth (P = 0.038). Furthermore, CC genotype was found to be significantly more frequent in AFP-negative HCCs (P = 0.002). CONCLUSION: Mannose-binding lectin-2 polymorphism seems to be involved in the development of pretransplant HCV-induced HCC and should be further investigated as potential risk factor for HCV-associated carcinogenesis.

Proof of SARS-CoV-2 genomes in endomyocardial biopsy with latency after acute infection.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has reached pandemic levels. Cardiovascular complications in COVID-19 have been reported frequently, however evidence for a causal relationship has not been established. This report describes the detection of SARS-CoV-2 viral genomes in a patient with symptoms of heart failure, in whom endomyocardial biopsy was investigated following a latency period of 4 weeks after the onset of pulmonary symptoms. The viral infection was accompanied by myocardial inflammation indicating an infection of the heart muscle.

Regulatory T cells control the transition from acute into chronic inflammation in glucose-6-phosphate isomerase-induced arthritis.

OBJECTIVES: Glucose-6-phosphate isomerase (G6PI)-induced arthritis is a spontaneously remitting experimental arthritis model. It was hypothesised that regulatory T cells (Tregs) are involved in remission and their role in G6PI-induced arthritis was investigated. METHODS: Tregs were depleted by injection of anti-CD25 before immunisation of DBA/1 mice with G6PI. The severity of arthritis was assessed clinically and histologically and the number and function of G6PI-specific T helper (Th) cells were determined by flow cytometry. Th cells and monocytes/macrophages were depleted using anti-CD4 or clodronate-containing liposomes. RESULTS: Injection of anti-CD25 depleted Tregs transiently. Normal numbers of Tregs were restored 5 weeks after G6PI immunisation. Whereas arthritis started to resolve in control mice 3 weeks after immunisation with G6PI, severe arthritis was still present in the anti-CD25-treated mice 12 weeks after immunisation. The most striking ex vivo correlate of non-remitting arthritis was a strong increase in G6PI-specific Th cells 3 days after G6PI immunisation. This difference between treated and control mice declined at later time points. Depletion of CD4 cells ameliorated arthritis in controls but not in anti-CD25-treated mice. In contrast, clodronate-containing liposomes were an effective treatment in both groups. CONCLUSIONS: Tregs control the transition from acute self-limiting to non-remitting destructive G6PI-induced arthritis already in the preclinical disease stage. Once established, non-remitting destructive arthritis is not controlled by restoration of normal Treg numbers. These findings question the rationale of therapeutic approaches augmenting Treg number or function in established arthritis.

Inducible costimulator (ICOS) blockade inhibits accumulation of polyfunctional T helper 1/T helper 17 cells and mitigates autoimmune arthritis.

OBJECTIVES: Inducible costimulator (ICOS) and its ligand (ICOSL) regulate T and B cell responses. Glucose-6-phosphate isomerase (G6PI)-induced arthritis requires T and B lymphocytes. It was hypothesised that blocking ICOS/ICOSL interactions ameliorates G6PI-induced arthritis and reduces G6PI-specific B and T lymphocyte responses. METHODS: DBA/1 mice were injected with a blocking, non-depleting anti-ICOSL monoclonal antibodies (mAbs) during the induction or effector phase of G6PI-induced arthritis. G6PI-specific antibody responses were measured by ELISA. G6PI-specific T helper (Th) cell responses were assayed by polychromatic flow cytometry. RESULTS: Transient blockade of ICOS/ICOSL interactions profoundly reduced the severity of G6PI-induced arthritis. ELISA and proliferation assays showed no clear ex vivo correlates of protection. Polychromatic flow cytometry revealed two major findings: the absolute number of G6PI-specific Th cells was markedly diminished in secondary lymphatic organs from mice with blocked ICOS/ICOSL interactions. Within the pool of G6PI-specific Th cells the frequency of interleukin 17 (IL17), interferon gamma or tumour necrosis factor alpha producers or polyfunctional Th cells (expressing two or more of these cytokines) was higher in treated than in control mice. CONCLUSIONS: ICOS costimulation is not mandatory for the differentiation of Th1 or Th17 cells. Instead, the lack of ICOS costimulation results in reduced survival of G6PI-specific Th cells irrespective of their functional differentiation. This study demonstrates that a thorough examination of the quantity and the quality of antigen-specific immune responses is useful to determine ex vivo correlates of efficacy for immunomodulating treatments.

Pathological processing techniques and final diagnosis of breast cancer sentinel lymph nodes.

BACKGROUND: Recommendations for intraoperative and postoperative breast sentinel lymph node (SLN) processing differ widely. Micrometastases and isolated tumor cells (ITC) have recently been proposed as prognostically and therapeutically relevant. We compared 3 SLN protocols with regard to intraoperative and postoperative diagnosis. MATERIALS AND METHODS: SLN in cohort I (270 patients) were intraoperatively assessed by stereomicroscopy. Intraoperative frozen section (IFS) was used only in stereomicroscopically suspicious SLN. In cohort II (197 patients), all SLN were examined with only 1 IFS. Final SLN workup in cohorts I and II consisted of complete step sectioning with immunohistochemistry. In cohort III (268 patients) 2 or more IFS were performed followed by 3 step sections and immunohistochemistry. RESULTS: pN1 stages were significantly higher in cohorts I and II (33.3% and 34.0% respectively) than in cohort III (24.6%). Intraoperative false negativity for the detection of metastases (pN1) ranged from 54.4% (cohort I) and 35.8% (cohort II) to 21.2% (cohort III). In contrast, ITC were detected significantly more frequently in cohort I (9.3%) and cohort II (14.7%) than in cohort III (1.9%). CONCLUSIONS: Higher rates of SLN metastases and ITC in cohort I/II compared to cohort III suggest that IFS may result in tissue loss thus increasing the risk of missing metastases. Sparse IFS but complete postoperative SLN workup with step sectioning and immunohistochemistry provides more accurate information regarding minimal disease in SLN, but often results in delayed axillary lymph node dissection. This is important for preoperative patient information and recommendations in SLN processing protocols.

Quantitative determination of the diagnostic accuracy of the synovitis score and its components.

AIMS: To assess the diagnostic accuracy of a three-component synovitis score and to determine the relative contribution of each of its components to its overall discriminatory power. METHODS AND RESULTS: The synovitis score was determined in 666 synovial specimens: normal synovium, n = 33; post-traumatic arthropathy (PtA), n = 29; osteoarthritis (OA), n = 221; psoriatic arthritis (PsA), n = 42; and rheumatoid arthritis (RA), n = 341. The discriminatory abilities of the score and its components were quantified with binary and multicategory receiver operating characteristic (ROC) analysis. The score differentiated all arthropathies accurately from normal tissue (area under the ROC curve, AUC: 0.87-0.98) and RA from OA or PtA (AUC: 0.85 for both), but could not distinguish well within pairs of inflammatory or degenerative arthropathies. AUCs of the intimal hyperplasia and stromal cellularity components correlated with the AUCs of the complete score markedly more strongly (r = 0.94 and 0.91, respectively) than the inflammatory infiltration component (r = 0.60). Multicategory ROC analysis ranked the score several-fold higher than any of its components, and the components in the order stromal cellularity>intimal hyperplasia>infiltration. CONCLUSION: Combining three distinct histological parameters into a three-component score produces greatly increased overall diagnostic power. The discriminatory ability of the score stems more from measuring proliferative than infiltrative aspects of synovitis.

Simvastatin attenuates ventilator-induced lung injury in mice.

INTRODUCTION: Mechanical ventilation (MV) is a life saving intervention in acute respiratory failure without alternative. However, particularly in pre-injured lungs, even protective ventilation strategies may evoke ventilator-induced lung injury (VILI), which is characterized by pulmonary inflammation and vascular leakage. Adjuvant pharmacologic strategies in addition to lung protective ventilation to attenuate VILI are lacking. Simvastatin exhibited anti-inflammatory and endothelial barrier stabilizing properties in vitro and in vivo. METHODS: Mice were ventilated (12 ml/kg; six hours) and subjected to simvastatin (20 mg/kg) or sham treatment. Pulmonary microvascular leakage, oxygenation, pulmonary and systemic neutrophil and monocyte counts and cytokine release in lung and blood plasma were assessed. Further, lung tissue was analyzed by electron microscopy. RESULTS: Mechanical ventilation induced VILI, displayed by increased pulmonary microvascular leakage and endothelial injury, pulmonary recruitment of neutrophils and Gr-1high monocytes, and by liberation of inflammatory cytokines in the lungs. Further, VILI associated systemic inflammation characterized by blood leukocytosis and elevated plasma cytokines was observed. Simvastatin treatment limited pulmonary endothelial injury, attenuated pulmonary hyperpermeability, prevented the recruitment of leukocytes to the lung, reduced pulmonary cytokine levels and improved oxygenation in mechanically ventilated mice. CONCLUSIONS: High-dose simvastatin attenuated VILI in mice by reducing MV-induced pulmonary inflammation and hyperpermeability.

Validity of frozen sections for analysis of periprosthetic loosening membranes.

UNLABELLED: Clinical findings and blood parameters often are inconclusive in patients with periprosthetic joint infections. Among the accepted criteria for diagnosis, histologic analysis of debrided tissue can detect infection in most cases but does not allow intraoperative decision making. We evaluated the validity of intraoperative frozen sections for detection of prosthetic infections. The results from frozen and permanent sections of periprosthetic membranes of 64 consecutive patients who underwent exchange procedures after hip arthroplasty were compared using the histopathologic consensus classification of Morawietz et al. Blood parameters (erythrocyte sedimentation rate, leukocyte count, C-reactive protein) and culture results of preoperatively aspirated joint fluid and intraoperative tissue samples were correlated with the histologic results. In 50 patients (78.1%), agreement was found between the frozen and permanent sections. Two patients (3.1%) revealed a discrepancy between the two histologic methods. In 12 patients (18.8%), a diagnosis was not possible based on the frozen sections because the tissue samples were not representative enough for definite classification. For the analyzable cases (n = 52), the sensitivity of frozen-section histologic analysis was 86.6%, specificity 100%, and accuracy 96.2%. Our data support a recommendation for use of intraoperative frozen sections for diagnosis of septic versus aseptic loosening in revision hip surgery. LEVEL OF EVIDENCE: Level II, diagnostic study. See the Guidelines for Authors for a complete description of levels of evidence.