Defective postreperfusion metabolic recovery directly associates with incident delayed graft function.

Delayed graft function (DGF) following kidney transplantation affects long-term graft function and survival and is considered a manifestation of ischemia reperfusion injury. Preclinical studies characterize metabolic defects resulting from mitochondrial damage as primary driver of ischemia reperfusion injury. In a comprehensive approach that included sequential establishment of postreperfusion arteriovenous concentration differences over the human graft, metabolomic and genomic analysis in tissue biopsies taken before and after reperfusion, we tested whether the preclinical observations translate to the context of clinical DGF. This report is based on sequential studies of 66 eligible patients of which 22 experienced DGF. Grafts with no DGF immediately recovered aerobic respiration as indicated by prompt cessation of lactate release following reperfusion. In contrast, grafts with DGF failed to recover aerobic respiration and showed persistent adenosine triphosphate catabolism indicated by a significant persistently low post reperfusion tissue glucose-lactate ratio and continued significant post-reperfusion lactate and hypoxanthine release (net arteriovenous difference for lactate and hypoxanthine at 30 minutes). The metabolic data for the group with DGF point to a persistent post reperfusion mitochondrial defect, confirmed by functional (respirometry) and morphological analyses. The archetypical mitochondrial stabilizing peptide SS-31 significantly preserved mitochondrial function in human kidney biopsies following simulated ischemia reperfusion. Thus, development of DGF is preceded by a profound post-reperfusion metabolic deficit resulting from severe mitochondrial damage. Strategies aimed at preventing DGF should be focused on safeguarding a minimally required post-reperfusion metabolic competence.

The disappearing San of southeastern Africa and their genetic affinities.

Southern Africa was likely exclusively inhabited by San hunter-gatherers before ~2000 years ago. Around that time, East African groups assimilated with local San groups and gave rise to the Khoekhoe herders. Subsequently, Bantu-speaking farmers, arriving from the north (~1800 years ago), assimilated and displaced San and Khoekhoe groups, a process that intensified with the arrival of European colonists ~350 years ago. In contrast to the western parts of southern Africa, where several Khoe-San groups still live today, the eastern parts are largely populated by Bantu speakers and individuals of non-African descent. Only a few scattered groups with oral traditions of Khoe-San ancestry remain. Advances in genetic research open up new ways to understand the population history of southeastern Africa. We investigate the genomic variation of the remaining individuals from two South African groups with oral histories connecting them to eastern San groups, i.e., the San from Lake Chrissie and the Duma San of the uKhahlamba-Drakensberg. Using ~2.2 million genetic markers, combined with comparative published data sets, we show that the Lake Chrissie San have genetic ancestry from both Khoe-San (likely the ||Xegwi San) and Bantu speakers. Specifically, we found that the Lake Chrissie San are closely related to the current southern San groups (i.e., the Karretjie people). Duma San individuals, on the other hand, were genetically similar to southeastern Bantu speakers from South Africa. This study illustrates how genetic tools can be used to assess hypotheses about the ancestry of people who seemingly lost their historic roots, only recalling a vague oral tradition of their origin.

The tumor area occupied by Tbet+ cells in deeply invading cervical cancer predicts clinical outcome.

BACKGROUND: Deep invasion of the normal surrounding tissue by primary cervical cancers is a prognostic parameter for postoperative radiotherapy and relatively worse survival. However, patients with tumor-specific immunity in the blood at the time of surgery displayed a much better disease free survival. Here we analyzed if this was due to a more tumor-rejecting immune population in the tumor. METHODS: Tumor sections from a group of 58 patients with deep normal tissue-invading cervical tumors were stained for the presence of immune cells (CD45), IFNγ-producing cells (Tbet) and regulatory T cells (Foxp3) by immunohistochemistry. The slides were scanned and both the tumor area and the infiltration of the differently stained immune cells were objectively quantified using computer software. RESULTS: We found that an increased percentage of tumor occupied by CD45+ cells was strongly associated with an enhanced tumor-infiltration by Tbet+ cells and Foxp3+ cells. Furthermore, the area occupied by CD45+ immune cells, Tbet+ cells but not Foxp3+ cells within the tumor were, in addition to the lymph node status of patients, associated with a longer disease free survival and disease specific survival. Moreover, interaction analyses between these immune parameters and lymph node status indicated an independent prognostic effect of tumor infiltrating Tbet+ cells. This was confirmed in a multivariate Cox analysis. CONCLUSIONS: The area occupied by a preferentially type I oriented CD45+ cell infiltrate forms an independent prognostic factor for recurrence-free and disease-specific survival on top of the patient's lymph node status.

Reliability of proliferation assessment by Ki-67 expression in neuroendocrine neoplasms: eyeballing or image analysis?

BACKGROUND: The latest WHO classification for neuroendocrine neoplasms (NEN) of the gastrointestinal tract defines grade according to Ki-67 and mitotic indices. Some have questioned the reproducibility and thus the reliability of Ki-67 assessment. We therefore investigated the accuracy of this proliferation marker in NEN. METHODS: The Ki-67 index of tumor specimens of NEN (n = 73) was assessed by two pathologists as in routine practice with eyeballing and twice by image analysis using ImageJ freeware at different magnifications. RESULTS were correlated with overall survival. RESULTS: The intraclass correlation coefficient (ICC) between pathologists was 0.88. The ICC for the measurements using image analysis was 0.85. The ICC between all four measurements (pathologists and ImageJ) was 0.80. If the Ki-67 index was translated to grade as prescribed by the current WHO classification (<3% = grade 1, 3-20% = grade 2, >20% = grade 3), kappa was between 0.61 and 0.75. Grades based on pathologist scoring were often (16-29%) higher than grades assigned by image analysis (p < 0.001). Grade was significantly correlated with survival (p < 0.0001) irrespective of the way Ki-67 was assessed. CONCLUSION: Assessment of the Ki-67 index by eyeballing correlates remarkably well with the Ki-67 index as calculated by image analysis and is therefore an accurate parameter. Moreover, it is significantly related to survival irrespective of the method used. Yet if the Ki-67 index is translated to grade, the grade should be interpreted with caution due to values around threshold levels.

Distinct defects in collagen microarchitecture underlie vessel-wall failure in advanced abdominal aneurysms and aneurysms in Marfan syndrome.

An aneurysm of the aorta is a common pathology characterized by segmental weakening of the artery. Although it is generally accepted that the vessel-wall weakening is caused by an impaired collagen metabolism, a clear association has been demonstrated only for rare syndromes such as the vascular type Ehlers-Danlos syndrome. Here we show that vessel-wall failure in growing aneurysms of patients who have aortic abdominal aneurysm (AAA) or Marfan syndrome is not related to a collagen defect at the molecular level. On the contrary our findings indicate similar (Marfan) or even higher collagen concentrations (AAA) and increased collagen cross-linking in the aneurysms. Using 3D confocal imaging we show that the two conditions are associated with profound defects in collagen microarchitecture. Reconstructions of normal vessel wall show that adventitial collagen fibers are organized in a loose braiding of collagen ribbons. These ribbons encage the vessel, allowing the vessel to dilate easily but preventing overstretching. AAA and aneurysms in Marfan syndrome show dramatically altered collagen architectures with loss of the collagen knitting. Evaluations of the functional characteristics by atomic force microscopy showed that the wall has lost its ability to stretch easily and revealed a second defect: although vascular collagen in normal aortic wall behaves as a coherent network, in AAA and Marfan tissues it does not. As result, mechanical forces loaded on individual fibers are not distributed over the tissue. These studies demonstrate that the mechanical properties of tissue are strongly influenced by collagen microarchitecture and that perturbations in the collagen networks may lead to mechanical failure.

Growth plate regulation and osteochondroma formation: insights from tracing proteoglycans in zebrafish models and human cartilage.

Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine-PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (ß3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no gradient formation. Most chondrocytes from human osteochondromas showed normal PEI staining. However, approximately 10% of tumour chondrocytes were similar to those found in the dak mutant (e.g., lack of PEI gradients). The cells in the reduced PEI-stained areas are likely associated with loss-of-function mutations in the EXT genes, and they might contribute to tumour initiation by disrupting the gradients.

Cartilage ultrastructure in proteoglycan-deficient zebrafish mutants brings to light new candidate genes for human skeletal disorders.

Proteoglycans are molecules consisting of protein cores onto which sugar chains, i.e., glycosaminoglycans (GAGs) such as heparan or chondroitin sulphates, are attached. Proteoglycans are produced by nearly all cells, and once secreted they become a major component of the extracellular matrix. Cartilage is particularly rich in proteoglycans, and changes in the structure and composition of GAGs have been found in osteochondromas and osteoarthritis. The zebrafish (Danio rerio) exhibits fast development, a growth plate-like organization of its craniofacial skeleton and an availability of various mutants, making it a powerful model for the study of human skeletal disorders with unknown aetiology. We analysed skeletons from five zebrafish lines with known mutations in genes involved in proteoglycan synthesis: dackel (dak/ext2), lacking heparan sulphate; hi307 (ß3gat3), deficient for most GAGs; pinscher (pic/slc35b2), presenting defective sulphation of GAGs and other molecules; hi954 (uxs1), lacking Notch and most GAGs due to impaired protein xylosylation; and knypek (kny/gpc4), missing the protein core of the Glypican-4 proteoglycan. Here we show that each mutant displays different phenotypes related to: (a) cartilage morphology; (b) composition of the extracellular matrix; (c) ultrastructure of the extracellular matrix; and (d) the intracellular ultrastructure of chondrocytes, proving that sulphated GAGs orchestrate the cartilage intra- and extracellular ultrastructures. The mild phenotype of the hi307 mutant suggests that proteoglycans consisting of a protein core and a short sugar linker might suffice for proper chondrocyte stacking. Finally, knypek supports the involvement of Glypican-4 in the craniofacial phenotype of Simpson-Golabi-Behmel syndrome and suggests GPC4 as a modulator of the overgrowth phenotype that is associated with this syndrome and is primarily caused by a mutation in GPC3. Moreover, we speculate on the potential involvement of SLC35B2, ß3GAT3 and UXS1 in skeletal dysplasias. This work promotes the use of zebrafish as a model of human skeletal development and associated pathologies.

Human decidual tissue contains differentiated CD8+ effector-memory T cells with unique properties.

During pregnancy, maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Recently, CD4(+)CD25(bright) regulatory T cells have been shown to be concentrated in decidual tissue, where they are able to suppress fetus-specific and nonspecific immune responses. Decidual CD8(+) T cells are the main candidates to recognize and respond to fetal HLA-C at the fetal-maternal interface, but data on the characteristics of these cells are limited. In this study we examined the decidual and peripheral CD8(+) T cell pool for CD45RA, CCR7, CD28, and CD27 expression, using nine-color flow cytometry. Our data demonstrate that decidual CD8(+) T cells mainly consist of differentiated CD45RA(-)CCR7(-) effector-memory (EM) cells, whereas unprimed CD45RA(+)CCR7(+) naive cells are almost absent. Compared with peripheral blood EM CD8(+) T cells, the decidual EM CD8(+) T cells display a significantly reduced expression of perforin and granzyme B, which was confirmed by immunohistochemistry of decidual tissue sections. Interestingly, quantitative PCR analysis demonstrates an increased perforin and granzyme B mRNA content in decidual EM CD8(+) T cells in comparison with peripheral blood EM CD8(+) T cells. The presence of high levels of perforin and granzyme B mRNA in decidual EM T cells suggests that decidual CD8(+) T cells pursue alternative means of EM cell differentiation that may include a blockade of perforin and granzyme B mRNA translation into functional perforin and granzyme B proteins. Regulation of decidual CD8(+) T cell differentiation may play a crucial role in maternal immune tolerance to the allogeneic fetus.

Image-guided tumor resection using real-time near-infrared fluorescence in a syngeneic rat model of primary breast cancer.

Tumor involvement of resection margins is found in a large proportion of patients who undergo breast-conserving surgery. Near-infrared (NIR) fluorescence imaging is an experimental technique to visualize cancer cells during surgery. To determine the accuracy of real-time NIR fluorescence imaging in obtaining tumor-free resection margins, a protease-activatable NIR fluorescence probe and an intraoperative camera system were used in the EMR86 orthotopic syngeneic breast cancer rat model. Influence of concentration, timing and number of tumor cells were tested in the MCR86 rat breast cancer cell line. These variables were significantly associated with NIR fluorescence probe activation. Dosing and tumor size were also significantly associated with fluorescence intensity in the EMR86 rat model, whereas time of imaging was not. Real-time NIR fluorescence guidance of tumor resection resulted in a complete resection of 17 out of 17 tumors with minimal excision of normal healthy tissue (mean minimum and a mean maximum tumor-free margin of 0.2 ± 0.2 mm and 1.3 ± 0.6 mm, respectively). Moreover, the technique enabled identification of remnant tumor tissue in the surgical cavity. Histological analysis revealed that the NIR fluorescence signal was highest at the invasive tumor border and in the stromal compartment of the tumor. In conclusion, NIR fluorescence detection of breast tumor margins was successful in a rat model. This study suggests that clinical introduction of intraoperative NIR fluorescence imaging has the potential to increase the number of complete tumor resections in breast cancer patients undergoing breast-conserving surgery.

IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides.

The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.

Symptoms in patients with ulcerative colitis in remission are associated with visceral hypersensitivity and mast cell activity.

OBJECTIVE: Patients with ulcerative colitis in remission (UCR) frequently report irritable bowel syndrome (IBS)-like symptoms. Recent studies have pointed to the role of mast cells in mediating visceral hypersensitivity in IBS. We hypothesized that visceral hypersensitivity is frequently present in patients with UCR and is related to the quantity and activity of mast cells in the sigmoid mucosa. MATERIAL AND METHODS: A group of 17 controls and 19 patients with UCR were studied. Rectal compliance and perception were measured by electronic barostat. Sigmoid biopsies were taken to quantify the amount of mast cells, degranulating mast cells and mast cells in close proximity to mucosal nerve endings. RESULTS: Visceroperception significantly increased in UCR (p < 0.05) versus controls. Rectal perception correlated positively with IBS-like symptoms in UCR (r = 0.969; p < 0.05). The amount of mucosal mast cells (per 100 crypts) was significantly increased in UCR versus controls: 228 ± 20 versus 163 ± 18 (p < 0.05). In the UCR patients a higher percentage of mucosal mast cells was in close proximity to nerve endings (58 ± 4 vs. 38 ± 3% in controls; p < 0.05) or was degranulating (40 ± 7 vs. 16 ± 4% in controls; p < 0.05). There was a significant but weak correlation between quantity of mucosal mast cells and pain perception (r = 0.32; p < 0.05). CONCLUSION: Rectal hypersensitivity is associated with mucosal presence and activation of mast cells and with IBS-like symptoms in patients with UCR.

A novel undergraduate biomedical laboratory course concept in synergy with ongoing faculty research.

Optimal integration of education and ongoing faculty research in many undergraduate science programs is limited to the capstone project. Here, we aimed to develop a novel course-based undergraduate research experience (CURE) in synergy with ongoing faculty research. This 10-week course called Biomedical Research Lab is embedded in the curriculum of the undergraduate program Biomedical Sciences and grounded in the theoretical framework of research-based learning. Four groups of four students work together in a dedicated laboratory on an actual ongoing research problem of faculty. All groups work on the same research problem, albeit from different (methodological) perspectives, thereby stimulating interdependence between all participants. Students propose new research, execute the experiments, and collectively report in a single research article. According to students, the course enhanced scientific, laboratory, and academic skills. Students appreciated ownership and responsibilities of the research, laboratory teachers as role models, and they were inspired and motivated by doing authentic actual research. The course resulted in a better understanding of what doing research entails. Faculty valued the didactical experience, research output and scouting opportunities. Since topics can change per course edition, we have showcased a widely applicable pedagogy creating synergy between ongoing research and undergraduate education.

Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma.

Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining. Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture ('mosaic') of normal lining (EXT(+/-) or EXT(wt/wt)) and EXT(-/-) cells in the cartilaginous cap of osteochondromas.

Aberrant chemokine receptor expression and chemokine production by Langerhans cells underlies the pathogenesis of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterized by a clonal proliferation and retention of cells with a Langerhans cell (LC)-like phenotype at various sites within the body. The present study set out to elucidate whether aberrant expression of chemokine receptors or dysregulation of chemokine production in LCH lesions could explain abnormal retention of these cells. Immunohistochemical analysis on 13 LCH biopsies of bone, skin, and lymph node all expressed the immature dendritic cell (DC) marker CCR6 on the lesional LCs and absence of the mature DC marker CCR7. Furthermore, regardless of the tissue site, LCH lesions markedly overexpressed CCL20/MIP-3alpha, the ligand for CCR6. The lesional LCs appeared to be the source of this CCL20/MIP-3alpha production as well as other inflammatory chemokines such as CCL5/RANTES and CXCL11/I-TAC. These may explain the recruitment of eosinophils and CD4+CD45RO+ T cells commonly found in LCH lesions. The findings of this study emphasize that, despite abundant TNF-alpha, lesional LCs remain in an immature state and are induced to produce chemokines, which via autocrine and paracrine mechanisms cause not only the retention of the lesional LCs but also the recruitment and retention of other lesional cells. We postulate that the lesional LCs themselves control the persistence and progression of LCH.

Human leukocyte antigen class I, MHC class I chain-related molecule A, and CD8+/regulatory T-cell ratio: which variable determines survival of cervical cancer patients?

PURPOSE: To investigate the effect of intraepithelial tumor-infiltrating lymphocytes (ieTIL) and their ligands expressed by cervical tumor cells on the outcome of cervical cancer patients. EXPERIMENTAL DESIGN: The prognostic value of ieTILs was investigated in 115 cases of cervical cancer. T-cell subsets, CD57(+) cells, and regulatory T cells (Treg) were enumerated. The associations of these different ieTIL subtypes with human leukocyte antigen (HLA) class I and MHC class I chain-related molecule A (MICA) expression were determined in relation to clinical variables and patient survival. RESULTS: Survival analysis showed that a high number of intraepithelial Treg (FoxP3(+)), a low CD8(+)/regulatory T-cell ratio, and a weak HLA-A expression were all associated with worse survival (P=0.034, 0.025, and 0.033, respectively, log-rank test). Further stratification of patient groups based on HLA-A-MICA expression and HLA-A-MICA-CD8(+)/Treg ratio revealed an even poorer survival (P=0.005). In a multivariate Cox analysis, low CD8(+)/Treg ratio (P=0.047), weak HLA-A-MICA expression (P=0.003), and weak HLA-A-MICA expression combined with low CD8(+)/Treg ratio (P=0.002) were all found to be independent unfavorable prognostic predictors in cervical carcinoma (hazard ratios, 2.7, 4.0, and 4.9, respectively). CONCLUSION: Weak HLA-A-MICA expression combined with low CD8(+)/Treg ratio reveals a patient group with the poorest survival in cervical cancer. As a single variable, low CD8(+)/Treg ratio was a significant independent unfavorable prognostic factor.

Anti-C1q autoantibodies deposit in glomeruli but are only pathogenic in combination with glomerular C1q-containing immune complexes.

Anti-C1q autoantibodies are present in sera of patients with several autoimmune diseases, including systemic lupus erythematosus (SLE). Strikingly, in SLE the presence of anti-C1q is associated with the occurrence of nephritis. We have generated mouse anti-mouse C1q mAb's and used murine models to investigate whether anti-C1q autoantibodies actually contribute to renal pathology in glomerular immune complex disease. Administration of anti-C1q mAb JL-1, which recognizes the collagen-like region of C1q, resulted in glomerular deposition of C1q and anti-C1q autoantibodies and mild granulocyte influx, but no overt renal damage. However, combination of JL-1 with a subnephritogenic dose of C1q-fixing anti-glomerular basement membrane (anti-GBM) antibodies enhanced renal damage characterized by persistently increased levels of infiltrating granulocytes, major histological changes, and increased albuminuria. This was not observed when a non-C1q-fixing anti-GBM preparation was used. Experiments with different knockout mice showed that renal damage was dependent not only on glomerular C1q and complement activation but also on Fcgamma receptors. In conclusion, anti-C1q autoantibodies deposit in glomeruli together with C1q but induce overt renal disease only in the context of glomerular immune complex disease. This provides an explanation why anti-C1q antibodies are especially pathogenic in patients with SLE.

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70-90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, ß-cellulin, epidermal growth factor (EGF), epiregulin, heparin-binding EGF-like growth factor (HB­EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB­EGF had the highest mRNA expression and HB­EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB­EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB­EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB­EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB­EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB­EGF. Therefore, we propose an autocrine EGFR stimulation model in cervical carcinomas.

Four-color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and localization in tumor tissue sections.

Immune-cell infiltration is frequently seen within human solid tumors. A detailed phenotypic analysis of these cells may aid in the understanding of an antitumor immune response. Standard hematoxylin/eosin and conventional immunohistochemical stainings are helpful, but have major limitations in the number of markers that can be identified and localized per tissue section. Therefore, we developed a combined fluorescence and brightfield microscopic technique by using both immunofluorescence and immunogold-silver methods, thereby discriminating three different leukocyte markers plus one tumor marker simultaneously in a single section. This enabled us to study both phenotype and location of infiltrating immune cells in colorectal tumors. We used a two-step staining in which primary and secondary antibodies were selected for minimal cross-reactivity. Furthermore, the secondary fluorescent antibody conjugates were selected for minimal spectral overlap. For computer-assisted analysis the brightfield microscopy image was combined with the fluorescence microscopy images. This combination of techniques provides a powerful tool for detailed multiparameter microscopic analysis of formalin-fixed, paraffin-embedded tissue sections in general and for tumor-immune cell infiltration in particular.

Association of Lung Inflammatory Cells with Small Airways Function and Exhaled Breath Markers in Smokers - Is There a Specific Role for Mast Cells?

BACKGROUND: Smoking is associated with a mixed inflammatory infiltrate in the airways. We evaluated whether airway inflammation in smokers is related to lung function parameters and inflammatory markers in exhaled breath. METHODS: Thirty-seven smokers undergoing lung resection for primary lung cancer were assessed pre-operatively by lung function testing including single-breath-nitrogen washout test (sb-N2-test), measurement of fractional exhaled nitric oxide (FeNO) and pH/8-isoprostane in exhaled breath condensate (EBC). Lung tissue sections containing cancer-free large (LA) and small airways (SA) were stained for inflammatory cells. Mucosal (MCT) respectively connective tissue mast cells (MCTC) and interleukin-17A (IL-17A) expression by mast cells was analysed using a double-staining protocol. RESULTS: The median number of neutrophils, macrophages and mast cells infiltrating the lamina propria and adventitia of SA was higher than in LA. Both MCTC and MCT were higher in the lamina propria of SA compared to LA (MCTC: 49 vs. 27.4 cells/mm2; MCT: 162.5 vs. 35.4 cells/mm2; P<0.005 for both instances). IL-17A expression was predominantly detected in MCTC of LA. Significant correlations were found for the slope of phase III % pred. of the sb-N2-test (rs= -0.39), for the FEV1% pred. (rs= 0.37) and for FEV1/FVC ratio (rs=0.38) with MCT in SA (P<0.05 for all instances). 8-isoprostane concentration correlated with the mast cells in the SA (rs=0.44), there was no correlation for pH or FeNO with cellular distribution in SA. CONCLUSIONS: Neutrophils, macrophages and mast cells are more prominent in the SA indicating that these cells are involved in the development of small airway dysfunction in smokers. Among these cell types, the best correlation was found for mast cells with lung function parameters and inflammatory markers in exhaled breath. Furthermore, the observed predominant expression of IL-17A in mast cells warrants further investigation to elucidate their role in smoking-induced lung injury, despite the lack of correlation with lung function and exhaled breath parameters.

Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance.

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes.