Early postoperative CRP predicts major complications following cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC).

Objectives: Cytoreductive surgery (CRS) and heated intraperitoneal chemotherapy (HIPEC) is associated with significant postoperative complications. Early detection of at-risk patients may lead to improved outcomes. The role of C-reactive protein (CRP) in predicting postoperative complications has only been recently investigated. Methods: Postoperative complications were categorized according to Clavien-Dindo classification and further divided into minor (Grade <3) and major complications (Grade ≥3A). Absolute CRP counts (mg/L) on postoperative days (POD) 1-7, and proportional change in CRP was compared and the area under (AUC) receiver operating characteristics (ROC) curve was calculated. Univariate and multivariate analysis was performed. Significant findings were externally validated. Results: Twenty-five percent of patients experienced one or more major complications. A CRP level of ≥106 mg/L on POD 2 and 65.5 mg/L on POD 4 were significantly associated with an increased risk of major complications with an AUC of 0.658 and 0.672, respectively. The proportional increase in CRP between POD 1 and 4 (ΔCRP POD 1/4) at a cut-off of 30 % had the best AUC of 0.744 and was the only independent risk factor for major complications (p<0.0001) on multivariate analysis. ∆CRP had an AUC of 0.716 (p=0.002) when validated in an independent database. Conclusions: CRP can be used in a variety of ways to predict major complications after CRS and HIPEC. However, the ∆CRP POD 1/4>30 % is the best indicator of major complications. Serial CRP measurements in the early postoperative period may lead to early detection of patients at risk of major complications allowing for alternative management strategies to improve outcomes.

Meeting actual benchmarks for short- and long-term outcomes after cytoreductive surgery for peritoneal surface malignancy at a newly established academic treatment center.

BACKGROUND: Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy is a curative treatment for selected patients with peritoneal surface malignancy. Reaching actual outcomes benchmarks is challenging given the complex nature of peritoneal surface malignancy surgery. The aim of this study was to assess how the benchmarks for morbidity and oncologic outcome can be reached at a newly established program for cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. METHODS: Building on existing institutional experience in complex abdominal surgery and interdisciplinary ovarian cancer treatment, a peritoneal surface malignancy center for cytoreductive surgery and hyperthermic intraperitoneal chemotherapy was established at the Medical University of Vienna using a structured mentoring process. This is a retrospective analysis of the first 100 consecutive patients. Morbidity and mortality were assessed using the Clavien-Dindo classification, and oncologic outcomes using overall survival. RESULTS: Major morbidity and mortality were 26% and 3%, and median overall survival was 49.0 months. In patients with colorectal peritoneal metastases, the median overall survival was 35.1 months (all colorectal peritoneal metastases patients) and 48.8 months in the subgroup with Peritoneal Surface Disease Severity Score ≤3. No median overall survival could be calculated in patients with low-grade appendiceal mucinous neoplasms, appendiceal adenocarcinoma, or peritoneal mesothelioma due to >50% of patients being alive at the end of follow-up. CONCLUSION: We show that the current morbidity and oncological outcomes benchmarks can be reached within the first 100 cases of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy at a newly established peritoneal surface malignancy center. Previous institutional experience in complex abdominal surgery and a structured mentoring process are key factors in achieving this goal.

Metastatic colorectal carcinoma-associated fibroblasts have immunosuppressive properties related to increased IGFBP2 expression.

Fibroblasts are the most abundant stromal constituents of the tumour microenvironment in primary as well as metastatic colorectal cancer (CRC). Their supportive effect on tumour cells is well established. There is growing evidence that stromal fibroblasts also modulate the immune microenvironment in tumours. Here, we demonstrate a difference in fibroblast-mediated immune modulation between primary CRC and peritoneal metastasis. Cancer-associated fibroblasts (CAFs) were isolated from primary cancer and from peritoneal metastases (MAFs) from a total of 17 patients. The ectoenzyme CD38 was consistently expressed on the surface of all MAFs, while it was absent from CAFs. Furthermore, MAFs secreted higher levels of IGFBP2, CXCL2, CXCL6, CXCL12, PDGF-AA, FGFb, and IL-6. This was associated with a decreased activation of macrophages and a suppression of CD25 expression and proliferation of co-cultivated T-cells. Downregulation of IGFBP2 abolished these immunosuppressive effects of MAFs. Taken together, these results show that MAFs contribute to an immunosuppressive tumour microenvironment in CRC metastases by modulating the phenotype of immune cells through an IGFBP2-dependent mechanism.

Tumour cell apoptosis modulates the colorectal cancer immune microenvironment via interleukin-8-dependent neutrophil recruitment.

Sporadic apoptosis of tumour cells is a commonly observed feature of colorectal cancer (CRC) and strongly correlates with adverse patient prognosis. The uptake of apoptotic cell debris by neutrophils induces a non-inflammatory, pro-regenerative, and hence potentially pro-tumorigenic phenotype. In this study, we therefore sought to investigate the impact of apoptotic CRC cells on neutrophils and its consequence on other immune cells of the tumour microenvironment. Apoptosis induced by combined TNFα-treatment and UV-C irradiation, as well as various chemotherapeutic agents, led to a substantial release of neutrophil-attracting chemokines, most importantly interleukin-8 (IL-8), in both primary patient-derived and established CRC cells. Accordingly, conditioned media of apoptotic tumour cells selectively stimulated chemotaxis of neutrophils, but not T cells or monocytes. Notably, caspase-inhibition partially reduced IL-8 secretion, suggesting that caspase activity might be required for apoptosis-induced IL-8 release. Moreover, apoptotic tumour cell-conditioned media considerably prolonged neutrophil lifespan and induced an activated CD66bhighCD11bhighCD62Llow phenotype, comparable to that of tumour-associated neutrophils in CRC patients, as assessed by flow cytometry of dissociated CRC tissues. Immunohistochemical analyses of 35 CRC patients further revealed a preferential accumulation of neutrophils at sites of apoptotic tumour cells defined by the expression of epithelial cell-specific caspase-cleaved cytokeratin-18. The same areas were also highly infiltrated by macrophages, while T cells were virtually absent. Notably, neutrophils induced an M2-like CD86lowCD163+CD206+ phenotype in co-cultured monocyte-derived macrophages and suppressed LPS-induced pro-inflammatory cytokine release. In an in vitro transwell model, IL-8 blockade efficiently prevented neutrophil-induced anti-inflammatory macrophage polarisation by inhibiting neutrophil migration towards IL-8 gradients generated by apoptotic CRC cells. To conclude, our data suggest that apoptotic cancer cells release chemotactic factors that attract neutrophils into the tumour, where their interaction with neighbouring macrophages might promote an immunologically unfavourable tumour microenvironment. This effect may contribute to tumour recurrence after chemotherapy-induced apoptosis.

Protein Signatures of Remodeled Airways in Transplanted Lungs with Bronchiolitis Obliterans Syndrome Obtained Using Laser-Capture Microdissection.

Bronchiolitis obliterans syndrome, a common form of chronic lung allograft dysfunction, is the major limitation to long-term survival after lung transplantation. The histologic correlate is progressive, fibrotic occlusion of small airways, obliterative bronchiolitis lesions, which ultimately lead to organ failure. The molecular composition of these lesions is unknown. In this sutdy, the protein composition of the lesions in explanted lungs from four end-stage bronchiolitis obliterans syndrome patients was analyzed using laser-capture microdissection and optimized sample preparation protocols for mass spectrometry. Immunohistochemistry and immunofluorescence were used to determine the spatial distribution of commonly identified proteins on the tissue level, and protein signatures for 14 obliterative bronchiolitis lesions were established. A set of 39 proteins, identified in >75% of lesions, included distinct structural proteins (collagen types IV and VI) and cellular components (actins, vimentin, and tryptase). Each respective lesion exhibited a unique composition of proteins (on average, n = 66 proteins), thereby mirroring the morphologic variation of the lesions. Antibody-based staining confirmed these mass spectrometry-based findings. The 14 analyzed obliterative bronchiolitis lesions showed variations in their protein content, but also common features. This study provides molecular and morphologic insights into the development of chronic rejection after lung transplantation. The protein patterns in the lesions were correlated to pathways of extracellular matrix organization, tissue development, and wound healing processes.

Crosstalk between Mast Cells and Lung Fibroblasts Is Modified by Alveolar Extracellular Matrix and Influences Epithelial Migration.

Mast cells play an important role in asthma, however, the interactions between mast cells, fibroblasts and epithelial cells in idiopathic pulmonary fibrosis (IPF) are less known. The objectives were to investigate the effect of mast cells on fibroblast activity and migration of epithelial cells. Lung fibroblasts from IPF patients and healthy individuals were co-cultured with LAD2 mast cells or stimulated with the proteases tryptase and chymase. Human lung fibroblasts and mast cells were cultured on cell culture plastic plates or decellularized human lung tissue (scaffolds) to create a more physiological milieu by providing an alveolar extracellular matrix. Released mediators were analyzed and evaluated for effects on epithelial cell migration. Tryptase increased vascular endothelial growth factor (VEGF) release from fibroblasts, whereas co-culture with mast cells increased IL-6 and hepatocyte growth factor (HGF). Culture in scaffolds increased the release of VEGF compared to culture on plastic. Migration of epithelial cells was reduced by IL-6, while HGF and conditioned media from scaffold cultures promoted migration. In conclusion, mast cells and tryptase increased fibroblast release of mediators that influenced epithelial migration. These data indicate a role of mast cells and tryptase in the interplay between fibroblasts, epithelial cells and the alveolar extracellular matrix in health and lung disease.

Loss of T cells expressing CD27 at the site of active tuberculosis - A prospective diagnostic study.

The lack of a rapid and reliable diagnostic test for active tuberculosis is still a burden to the control of the infection. The accumulation of Mycobacterium tuberculosis (MTB)-specific CD4+ T cells at the site of infection and the increase of MTB-specific CD27- cells seem to be characteristic for active tuberculosis. We evaluated CD27 expression of non-stimulated T cells at the site of infection compared to peripheral blood of seventy-two patients (n = 72) presenting with symptoms of active MTB-infection. Twenty patients (n = 20, 27.8%) were actually confirmed to have active tuberculosis. Overall, a significant increase of terminally differentiated CD27- CD4+ T cells at the site of disease was noted when compared to peripheral blood (<0.001). However, the loss of CD27 at the site of disease was not restricted to active tuberculosis (p = 0.253). The CD27 expression profile of tuberculosis patients was only discriminative to patients with malignancy.

VEGF synthesis and VEGF receptor 2 expression in patients with bronchiolitis obliterans syndrome after lung transplantation.

OBJECTIVES: Chronic lung allograft dysfunction including Bronchiolitis obliterans syndrome (BOS) is common after lung transplantation. Histologically, BOS is recognized as fibrotic lesions with accumulated extracellular matrix (ECM) in small airways. Lung fibroblasts are major producers of ECM and vascular endothelial growth factor (VEGF). In this study we hypothesize that VEGF is involved in BOS development after lung transplantation. METHODS: We investigated the effect of profibrotic transforming growth factor (TGF-ß) on VEGF synthesis in lung fibroblasts isolated from distal lung tissue biopsies taken from patients at 3, 6 and 12 months after lung transplantation (n = 14). Co-expression of VEGF receptor (VEGFR) 2 and collagen marker prolyl4-hydroxylase (p4OH) were analyzed in lung tissue from patients with BOS (n = 11). RESULTS: VEGF synthesis from distal derived lung fibroblasts were significantly lower 3 months after lung transplantation (168.6 ± 133.7; n = 7) compared to non-transplanted subjects (451.8 ± 185.9; n = 9; p = 0.0033) and increased over time at 6 months (584.1 ± 264.9; n = 9; p = 0.0033) and 12 months (451.1 ± 207.5; n = 8; p = 0.0065) post transplantation. TGF-ß significantly induced VEGF synthesis at all time points. At 12 months post transplantation there was significantly less VEGF synthesis after TGF-ß stimulation in fibroblasts obtained from BOS patients (1170 ± 450.2; n = 4) compared to patients without any chronic rejection process (1980 ± 417.9; n = 4; p < 0.039). The numbers of cells expressing VEGFR2/p4OH were increased in patients with BOS (33.2 ± 10.9; n = 11) compared to control subjects (10.1 ± 9.9; n = 11; p < 0.001). CONCLUSIONS: Our results support that changes in VEGF/VEGFR2 axis could be involved in BOS development and marker of poor outcome.

Restoration of intestinal continuity after stoma formation for Crohn's disease in the era of biological therapy : A retrospective cohort study.

BACKGROUND: The rate of restoration of intestinal continuity after colonic resection and stoma creation in patients with Crohn's disease has not been well-documented in the era of biologics. Thus, the incidence of restoration of intestinal continuity since the introduction of biological drugs was assessed. METHODS: Consecutive patients (n = 43) who underwent colonic resection with ileostomy or colostomy formation for Crohn's disease at a single tertiary referral center between 2002 and 2014 were identified. Data from individual chart review were analyzed retrospectively. Patients were personally contacted for follow-up. RESULTS: Of the 43 patients 8 (18.4%) had a proctectomy leaving 35 patients (81.4%) with the rectum preserved. Of the 30 patients qualifying for final analysis restoration of bowel continuity was finally achieved in 10 patients (33.3%). Permanent stoma rates were comparable in the group of patients with and without biological therapy after surgery (64.3% vs. 60%). The median follow-up period was 7 years (range 3-15 years). Of the patients 20 suffered from perianal disease involvement (66.7%), which was associated with a higher rate of permanent stoma (n = 16/20, 80%) in contrast to patients without perianal disease (n = 4/10, 40%, p = 0.045). CONCLUSION: The overall incidence of stoma formation was low for patients with Crohn's disease; however, once a stoma is created the chance of ending up with a permanent stoma is high even in the era of biologics. Despite the use of new therapeutic agents perianal disease increases the risk of a permanent stoma.

Surgical Complexity and Outcome During the Implementation Phase of a Robotic Colorectal Surgery Program-A Retrospective Cohort Study.

BACKGROUND: Robotic surgery holds particular promise for complex oncologic colorectal resections, as it can overcome many limitations of the laparoscopic approach. However, similar to the situation in laparoscopic surgery, appropriate case selection (simple vs. complex) with respect to the actual robotic expertise of the team may be a critical determinant of outcome. The present study aimed to analyze the clinical outcome after robotic colorectal surgery over time based on the complexity of the surgical procedure. METHODS: All robotic colorectal resections (n = 85) performed at the Department of Surgery, Medical University of Vienna, between the beginning of the program in April 2015 until December 2019 were retrospectively analyzed. To compare surgical outcome over time, the cohort was divided into 2 time periods based on case sequence (period 1: patients 1-43, period 2: patients 44-85). Cases were assigned a complexity level (I-IV) according to the type of resection, severity of disease, sex and body mass index (BMI). Postoperative complications were classified using the Clavien-Dindo classification. RESULTS: In total, 47 rectal resections (55.3%), 22 partial colectomies (25.8%), 14 abdomino-perineal resections (16.5%) and 2 proctocolectomies (2.4%) were performed. Of these, 69.4% (n = 59) were oncologic cases. The overall rate of major complications (Clavien Dindo III-V) was 16.5%. Complex cases (complexity levels III and IV) were more often followed by major complications than cases with a low to medium complexity level (I and II; 25.0 vs. 5.4%, p = 0.016). Furthermore, the rate of major complications decreased over time from 25.6% (period 1) to 7.1% (period 2, p = 0.038). Of note, the drop in major complications was associated with a learning effect, which was particularly pronounced in complex cases as well as a reduction of case complexity from 67.5% to 45.2% in the second period (p = 0.039). CONCLUSIONS: The risk of major complications after robotic colorectal surgery increases significantly with escalating case complexity (levels III and IV), particularly during the initial phase of a new colorectal robotic surgery program. Before robotic proficiency has been achieved, it is therefore advisable to limit robotic colorectal resection to cases with complexity levels I and II in order to keep major complication rates at a minimum.

Comparison of Normal and Metaplastic Epithelium in Patients with Stable versus Persistently Symptomatic Severe Asthma Using Laser-Capture Microdissection and Data-Independent Acquisition-Mass Spectrometry.

A proportion of patients with severe asthma (SA) show poor responses to traditional asthma medications; however, it remains unknown why some patients remain persistently symptomatic. Our objective was to explore the use of laser-capture microdissection of specific epithelial structures combined with quantitative data-independent acquisition mass spectrometry to elucidate differences in protein composition in patients with SA with varying symptom control. Unbiased label-free quantitative proteome analyses were performed on laser-capture-microdissected areas of specific epithelial structures from patients with SA with varying degrees of symptom control. A total of 1993 stable SA and 1652 symptomatic SA proteins in normal epithelium and 1458 stable SA and 1647 symptomatic SA proteins in metaplastic epithelium were quantified. When comparing proteome profiles based on symptom control, 33 proteins in patients with stable SA (≥twofold change; P ≤ 0.05) and 13 proteins in patients with persistently symptomatic SA (≥twofold change; P ≤ 0.05) were enriched significantly. When comparing proteome profiles based on epithelial status, 21 proteins in normal epithelium (≥twofold change; P ≤ 0.05) and 6 proteins in metaplastic epithelium (≥twofold change; P ≤ 0.05) were enriched significantly. New treatment strategies are needed for patients with severe asthma and exploratory studies of unbiased nature such as this may help when searching for new mechanisms and potential targets involved in the disease pathology.

Matrisome Properties of Scaffolds Direct Fibroblasts in Idiopathic Pulmonary Fibrosis.

In idiopathic pulmonary fibrosis (IPF) structural properties of the extracellular matrix (ECM) are altered and influence cellular responses through cell-matrix interactions. Scaffolds (decellularized tissue) derived from subpleural healthy and IPF lungs were examined regarding biomechanical properties and ECM composition of proteins (the matrisome). Scaffolds were repopulated with healthy fibroblasts cultured under static stretch with heavy isotope amino acids (SILAC), to examine newly synthesized proteins over time. IPF scaffolds were characterized by increased tissue density, stiffness, ultimate force, and differential expressions of matrisome proteins compared to healthy scaffolds. Collagens, proteoglycans, and ECM glycoproteins were increased in IPF scaffolds, however while specific basement membrane (BM) proteins such as laminins and collagen IV were decreased, nidogen-2 was also increased. Findings were confirmed with histology, clearly showing a disorganized BM. Fibroblasts produced scaffold-specific proteins mimicking preexisting scaffold composition, where 11 out of 20 BM proteins were differentially expressed, along with increased periostin and proteoglycans production. We demonstrate how matrisome changes affect fibroblast activity using novel approaches to study temporal differences, where IPF scaffolds support a disorganized BM and upregulation of disease-associated proteins. These matrix-directed cellular responses emphasize the IPF matrisome and specifically the BM components as important factors for disease progression.

Correction: The proportion of alveolar type 1 cells decreases in murine hypoplastic congenital diaphragmatic hernia lungs.

[This corrects the article DOI: 10.1371/journal.pone.0214793.].

The proportion of alveolar type 1 cells decreases in murine hypoplastic congenital diaphragmatic hernia lungs.

BACKGROUND: Pulmonary hypoplasia, characterized by incomplete alveolar development, remains a major cause of mortality and morbidity in congenital diaphragmatic hernia. Recently demonstrated to differentiate from a common bipotent progenitor during development, the two cell types that line the alveoli type 1 and type 2 alveolar cells have shown to alter their relative ratio in congenital diaphragmatic hernia lungs. OBJECTIVE: We used the nitrofen/bisdiamine mouse model to induce congenital diaphragmatic hernia and accurately assess the status of alveolar epithelial cell differentiation in relation to the common bipotent progenitors. STUDY DESIGN: Pregnant Swiss mice were gavage-fed with nitrofen/bisdiamine or vehicle at embryonic day 8.5. The administered dose was optimized by assessing the survival, congenital diaphragmatic hernia and facial abnormality rates of the exposed mouse pups. NanoCT was performed on embryonic day 11.5 and 16.5 to assess the embryonic and early canalicular stages of lung development. At embryonic day 17.5 corresponding to late canalicular stage, congenital diaphragmatic hernia lungs were characterized by measuring the lung weight/body weight ratio, morphometry, epithelial cell marker gene expression levels and alveolar cell type quantification. RESULTS: Nitrofen/bisdiamine associated congenital diaphragmatic hernia lungs showed delayed development, hypoplasia with morphologic immaturity and thickened alveolar walls. Expression levels of distal epithelial progenitor marker Id2 increased, alveolar type 1 cell markers Pdpn and Hopx decreased, while type 2 cell markers pro-SPC and Muc1 remained constant during the canalicular stage. The number of Pdpn+ type 1 alveolar cells also decreased in congenital diaphragmatic hernia lungs. CONCLUSION: The mouse nitrofen/bisdiamine model is a potential model allowing the study of congenital diaphragmatic hernia lung development from early stages using a wide array of methods. Based on this model, the alveolar epithelium showed a decrease in the number of alveolar type 1 cell in congenital diaphragmatic hernia lungs while type 2 cell population remains unchanged.

Delta albumin is a better prognostic marker for complications following laparoscopic intestinal resection for Crohn's disease than albumin alone - A retrospective cohort study.

PURPOSE: Little is known about the perioperative dynamic of albumin and its effect on surgical outcome in Crohn's disease. Thus, we aimed to assess postoperative changes of albumin levels and their potentially predictive role for complications after laparoscopic intestinal resections. METHODS: We identified 182 patients who underwent laparoscopic intestinal resection for symptomatic Crohn´s disease between 2000 and 2014 for this retrospective cohort study. Pre- and postoperative serum albumin levels (within 4 days) were recorded retrospectively and proportional postoperative reduction (delta (Δ) albumin) was calculated. Complications were defined according to the Clavien-Dindo classification. Univariate and multivariate analysis describing an eventful postoperative course were conducted. RESULTS: Complications were found in 22.5% (n = 41), 6% (n = 11) developed major complications defined as Clavien Dindo III-V and 16.5% (n = 30) had minor complications (Clavien Dindo I-II). The median Δ albumin was 22.75% (range: -18.46-47.14%). Delta albumin was found to be significantly higher in patients who developed complications after surgery (p = 0.03). Notably, neither preoperative (p = 0.28) nor postoperative albumin levels (p = 0.41) taken as absolute numerical values correlated with an eventful course following intestinal resection. In the multivariate analysis, based on a cut-off of 24.27%, Δ albumin remained an independent factor for surgical complications (p = 0.04, OR 2.232) next to conversion rate (p<0.001, OR 5.577) and the presence of an inflammatory mass (p = 0.003, OR 0.280). CONCLUSION: Δ albumin is a better prognostic marker for an eventful postoperative course after laparoscopic surgery in patients with Crohn's disease in comparison to albumin alone.

Quantifying extracellular matrix turnover in human lung scaffold cultures.

Remodelling of the extracellular matrix is accomplished by altering the balance between matrix macromolecule production and degradation. However, it is not well understood how cells balance production of new matrix molecules and degradation of existing ones during tissue remodelling and regeneration. In this study, we used decellularized lung scaffolds repopulated with allogenic lung fibroblasts cultured with stable isotope labelled amino acids to quantify the balance between matrix production and degradation at a proteome-wide scale. Specific temporal dynamics of different matrisome proteins were found to correspond to the proliferative activity of the repopulating cells and the degree of extracellular deposition. The remodeling of the scaffold was characterized by an initial phase with cell proliferation and high production of cell adhesion proteins such as emilin-1 and fibronectin. Extended culture time resulted in increased levels of core matrisome proteins. In a comparison with monolayer cultures on plastic, culture in lung scaffolds lead to a pronounced accumulation of proteoglycans, such as versican and decorin, resulting in regeneration of an extracellular matrix with greater resemblance to native lung tissue compared to standard monolayer cultures. Collectively, the study presents a promising technique for increasing the understanding of cell- extracellular matrix interactions under healthy and diseased conditions.

A case report of septic shock syndrome caused by S. pneumoniae in an immunocompromised patient despite of vaccination.

BACKGROUND AND CASE PRESENTATION: We report a case of septic shock syndrome caused by Streptococcus pneumoniae in a patient who had undergone splenectomy due to an autoimmune lymphoproliferative syndrome (ALPS), which is characterized as a dysfunction of immunoregulation. Although the patient was vaccinated with a conjugated polysaccharide vaccine after the splenectomy, he was still susceptible to S. pneumoniae infection, because the isolated serovar (24F), a serovar long thought to be apathogenic, is not covered by any vaccine currently approved, neither a conjugated nor an unconjugated polysaccharide one. CONCLUSIONS: This case demonstrates that, due to presence of different serovars, also infections with bacteria against which patients are vaccinated have to be considered as differential diagnosis. Although vaccine development has extended the coverage of S. pneumoniae from 7 to 23 serovars within recent years, there is still demand for novel vaccines which can provide broader protection also against so-thought "apathogenic" strains, especially for groups at high risk.

Early extracellular matrix changes are associated with later development of bronchiolitis obliterans syndrome after lung transplantation.

BACKGROUND: Chronic lung allograft dysfunction in the form of bronchiolitis obliterans syndrome (BOS) is the main cause of death beyond 1-year post-lung transplantation. The disease-initiating triggers as well as the molecular changes leading to fibrotic alterations in the transplanted lung are largely unknown. The aim of this study was to identify potential early changes in the extracellular matrix (ECM) in different compartments of the transplanted lung prior to the development of BOS. METHODS: Transbronchial biopsies from a cohort of 58 lung transplantation patients at the Copenhagen University hospital between 2005 and 2006, with or without development of BOS in a 5-year follow-up, were obtained 3 and 12 months after transplantation. Biopsies were assessed for total collagen, collagen type IV and biglycan in the alveolar and small airway compartments using Masson's Trichrome staining and immunohistochemistry. RESULTS: A time-specific and compartment-specific pattern of ECM changes was detected. Alveolar total collagen (p=0.0190) and small airway biglycan (p=0.0199) increased between 3 and 12 months after transplantation in patients developing BOS, while collagen type IV (p=0.0124) increased in patients without BOS. Patients with early-onset BOS mirrored this increase. Patients developing grade 3 BOS showed distinct ECM changes already at 3 months. Patients with BOS with treated acute rejections displayed reduced alveolar total collagen (p=0.0501) and small airway biglycan (p=0.0485) at 3 months. CONCLUSIONS: Patients with future BOS displayed distinct ECM changes compared with patients without BOS. Our data indicate an involvement of alveolar and small airway compartments in post-transplantation changes in the development of BOS.

5-HT2B receptor antagonists attenuate myofibroblast differentiation and subsequent fibrotic responses in vitro and in vivo.

Pulmonary fibrosis is characterized by excessive accumulation of connective tissue, along with activated extracellular matrix (ECM)-producing cells, myofibroblasts. The pathological mechanisms are not well known, however serotonin (5-HT) and 5-HT class 2 (5-HT2) receptors have been associated with fibrosis. The aim of the present study was to investigate the role of 5-HT2B receptors in fibrosis, using small molecular 5-HT2B receptor antagonists EXT5 and EXT9, with slightly different receptor affinity. Myofibroblast differentiation [production of alpha-smooth muscle actin (α-SMA)] and ECM synthesis were quantified in vitro, and the effects of the receptor antagonists were evaluated. Pulmonary fibrosis was also modeled in mice by subcutaneous bleomycin administrations (under light isoflurane anesthesia), and the effects of receptor antagonists on tissue density, collagen-producing cells, myofibroblasts and decorin expression were investigated. In addition, cytokine expression was analyzed in serum. Lung fibroblasts displayed an increased α-SMA (P < 0.05) and total proteoglycan production (P < 0.01) when cultured with TGF-ß1 together with 5-HT, which were significantly reduced with both receptor antagonists. Following treatment with EXT5 or EXT9, tissue density, expression of decorin, number of collagen-producing cells, and myofibroblasts were significantly decreased in vivo compared to bleomycin-treated mice. Receptor antagonization also significantly reduced systemic levels of TNF-α and IL-1ß, indicating a role in systemic inflammation. In conclusion, 5-HT2B receptor antagonists have potential to prevent myofibroblast differentiation, in vitro and in vivo, with subsequent effect on matrix deposition. The attenuating effects of 5-HT2B receptor antagonists on fibrotic tissue remodeling suggest these receptors as novel targets for the treatment of pulmonary fibrosis.

Quantitative proteomics at different depths in human articular cartilage reveals unique patterns of protein distribution.

The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease.