Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity.

Late-onset Alzheimer's disease (AD) is a complex multifactorial disease. The greatest known risk factor for late-onset AD is the E4 allele of the apolipoprotein E (APOE), while increasing age is the greatest known non-genetic risk factor. The cell type-specific functions of neural stem cells (NSCs), in particular their stem cell plasticity, remain poorly explored in the context of AD pathology. Here, we describe a new model that employs late-onset AD patient-derived induced pluripotent stem cells (iPSCs) to generate NSCs and to examine the role played by APOE4 in the expression of aging markers such as sirtuin 1 (SIRT1) in comparison to healthy subjects carrying APOE3. The effect of aging was investigated by using iPSC-derived NSCs from old age subjects as healthy matched controls. Transcript and protein analysis revealed that genes were expressed differently in NSCs from late-onset AD patients, e.g., exhibiting reduced autophagy-related protein 7 (ATG7), phosphatase and tensin homolog (PTEN), and fibroblast growth factor 2 (FGF2). Since SIRT1 expression differed between APOE3 and APOE4 NSCs, the suppression of APOE function in NSCs also repressed the expression of SIRT1. However, the forced expression of APOE3 by plasmids did not recover differently expressed genes. The altered aging markers indicate decreased plasticity of NSCs. Our study provides a suitable in vitro model to investigate changes in human NSCs associated with aging, APOE4, and late-onset AD.

Blood Immune Cells as Biomarkers in Long-Term Surviving Patients with Advanced Non-Small-Cell Lung Cancer Undergoing a Combined Immune/Chemotherapy.

An important challenge remains in identifying the baseline characteristics of cancer patients who will mostly benefit from immune checkpoint inhibitor (ICI) therapies. Furthermore, biomarkers could help in the choice of an optimal therapy duration after a primary therapy response. In this pilot study, the time courses of four different immune cell parameters were followed in 12 patients with advanced non-small-cell lung cancer (NSCLC) undergoing ICI therapy combined with chemotherapy and surviving at least 12 months. Blood was collected at the time point of the first and third antibody administration, as well as after 12 months of patients' survival. Using multi-color flow cytometry, two suppressive markers (neutrophil/lymphocyte ratio (NLR) and the frequency of circulating HLA-DRlow monocytes), as well as two markers of an ongoing immune response (6-Sulfo LacNAc (slan)+ non-classical monocytes and dendritic cell (DC) subtypes), were determined. In most of those who survived > 12 months, a low NLR and a low number of HLA-DRlow monocytes combined with clearly detectable numbers of slan+ non-classical monocytes and of DC subtypes were seen. Two of the patients had an increase in the suppressive markers paired with a decrease in slan+ non-classical monocytes and in DC subtypes, which, in at least one patient, was the correlate of an ongoing clinical progression. Our results implicate that the NLR, specific subtypes of monocytes, and the number of blood DCs might be useful predictive biomarkers for cancer patients during long-term treatment with ICI/chemotherapy.

Monitoring Blood Immune Cells in Patients with Advanced Small Cell Lung Cancer Undergoing a Combined Immune Checkpoint Inhibitor/Chemotherapy.

In this exploratory prospective observational study on 40 small cell lung cancer (SCLC) patients treated with a combination of chemotherapy and immune checkpoint inhibitors, blood immune cells were characterized by multi-color flow cytometry at the baseline and at the third therapy cycle. The numbers of neutrophils and of T-, B-, and NK cells, as well as the frequency of HLA-DRlow monocytes, 6-SulfoLacNAc (slan)+ non-classical monocytes and circulating dendritic cell (DC) subtypes were determined. The prognostic value of the parameters was evaluated by the patient's survival analysis with overall survival (OS) as the primary endpoint. In addition, blood cell parameters from SCLC patients were compared to those from non-SCLC (NSCLC). The global median OS of patients was 10.4 ± 1.1 months. Disease progression (15% of patients) correlated with a higher baseline neutrophil/lymphocyte ratio (NLR), more HLA-DRlow monocytes, and lower NK cell and DC numbers. The risk factors for poor OS were the presence of brain/liver metastases, a baseline NLR ≥ 6.1, HLA-DRlow monocytes ≥ 21% of monocytes, slan+ non-classical monocytes < 0.12%, and/or CD1c+ myeloid DC < 0.05% of leukocytes. Lymphocytic subpopulations did not correlate with OS. When comparing biomarkers in SCLC versus NSCLC, SCLC had a higher frequency of brain/liver metastases, a higher NLR, the lowest DC frequencies, and lower NK cell numbers. Brain/liver metastases had a substantial impact on the survival of SCLC patients. At the baseline, 45% of SCLC patients, but only 24% of NSCLC patients, had between three and five risk factors. A high basal NLR, a high frequency of HLA-DRlow monocytes, and low levels of slan+ non-classical monocytes were associated with poor survival in all lung cancer histotypes. Thus, the blood immune cell signature might contribute to a better prediction of SCLC patient outcomes and may uncover the pathophysiological peculiarities of this tumor entity.

Blood Immune Cell Biomarkers in Lung Cancer Patients Undergoing Treatment with a Combination of Chemotherapy and Immune Checkpoint Blockade.

Although immune checkpoint inhibitor (ICI) therapies have improved the treatment of patients with advanced non-small cell lung cancer (NSCLC), several patients do not achieve durable clinical responses. Biomarkers for the prediction of therapy responses are urgently needed. To identify blood cell parameters correlating with patients' survival, immune cells from 90 patients with NSCLC undergoing a combination of ICI and chemotherapy were prospectively monitored. At the time point of the first and third antibody administration, complete leukocyte blood count, the percentage of HLA-DRlow monocytes, the percentage of 6-Sulfo LacNAc (slan)+CD16+ non-classical monocytes, and the number of circulating dendritic cell (DC) subtypes, as well as T-, B-, and NK cells were determined by multi-color flow cytometry in peripheral blood. The prognostic value of the immune cell parameters investigated was evaluated by patients' survival analysis, with progression-free survival (PFS) as the main criterion. A total of 67 patients (74.4%) showed a partial remission or a stable disease, and 35% of patients even survived 12 months and longer. Patients with a neutrophil-to-lymphocyte ratio (NLR) ≥6.1, a frequency of HLA-DRlow monocytes ≥22%, of slan+ non-classical monocytes <0.25% of leukocytes, and/or a sum of myeloid DC (MDC) and plasmacytoid DC (PDC) ≤0.14% of leukocytes had a poorer prognosis. The hazard ratio for PFS was 2.097 (1.208−3.640) for the NLR, 1.964 (1.046−3.688) for HLA-DRlow monocytes, 3.202 (1.712−5.99) for slan+ non-classical monocytes, and 2.596 (1.478−4.56) for the MDC/PDC sum. Patients without any of the four risk factors showed the best PFS. Furthermore, low NK cell counts correlated with shorter PFS (cutoff 200 cells/µL). Female patients had lower baseline NK cell counts and a shorter PFS. Our study confirms the usefulness of blood immune cells as biomarkers for clinical response and survival in NSCLC patients undergoing a combined ICI/chemotherapy.

High PD-L1/CD274 Expression of Monocytes and Blood Dendritic Cells Is a Risk Factor in Lung Cancer Patients Undergoing Treatment with PD1 Inhibitor Therapy.

The aim of this study was to investigate the expression of the coinhibitory molecule PD-L1/CD274 in monocytes and dendritic cells (DC) in the blood of lung cancer patients undergoing PD1 inhibitor therapy and to correlate data with patient's outcome. PD-L1/CD274 expression of monocytes, CD1c+ myeloid DC (mDC) and CD303+ plasmacytoid DC (pDC) was determined by flow cytometry in peripheral blood at immunotherapy onset. The predictive value of the PD-L1/CD274-expression data was determined by patients' survival analysis. Patients with a high PD-L1/CD274 expression of monocytes and blood DC subpopulations rarely responded to PD1 inhibitor therapy. Low PD-L1/CD274 expression of monocytes and DC correlated with prolonged progression-free survival (PFS) as well as overall survival (OS). The highest PD-L1/CD274 expression was found in CD14+HLA-DR++CD16+ intermediate monocytes. Whereas the PD-L1/CD274 expression of monocytes and DC showed a strong positive correlation, only the PD-L1/CD274 expression of DC inversely correlated with DC amounts and lymphocyte counts in peripheral blood. Our results implicate that a high PD-L1/CD274 expression of blood monocytes and DC subtypes is a risk factor for therapy response and for the survival of lung cancer patients undergoing PD1 inhibitor therapy.

Blood Immune Cell Biomarkers in Patient With Lung Cancer Undergoing Treatment With Checkpoint Blockade.

Characterization of host immune cell parameters before and during immunotherapy is expected to identify predictive biomarkers for clinical outcome. We prospectively monitored blood immune cells from 35 patients with advanced non-small cell lung cancer undergoing checkpoint inhibitor monotherapy. The aim was to identify parameters correlating with better/worse outcome. Peripheral blood was serially collected before each infusion at the onset and at cycle 3 and 5 of immunotherapy. A complete leukocyte blood count, the lymphocytic subpopulations and the percentages of both HLA-DR monocytes and dendritic cells (DC) were monitored. Disease control was defined as partial/complete response and stable disease on computed tomography scan according to RECIST 1.1. The predictive value of the immune cell parameters investigated was evaluated by patients' survival analysis. Forty percent of patients showed a clinical response, and the global median overall survival was 7.0 months (95% confidence interval: 3.5-10.5). Patients with an initial neutrophil-to-lymphocyte ratio (NLR) ≥5.2, and/or an amount of HLA-DR monocytes ≥11% and/or a total DC level ≤0.4% of leukocytes did rarely respond to PD-1 inhibitor therapy. Otherwise, the immunotherapy-induced decrease of the neutrophil-to-lymphocyte ratio and/or HLA-DR monocytes and the increase of total DC frequencies were correlated with improved therapy response and prolonged overall survival. Blood values in the third cycle of immunotherapy did already reflect the effects observed. On the basis of the 3 immune cell parameters identified we created 3 different variants of scores that enable to stratify patients into groups of risk/therapy response. Our results warrant further investigation in larger prospective clinical trials for validation.

Identification of patient-specific and tumor-shared T cell receptor sequences in renal cell carcinoma patients.

A major requirement for cancer immunotherapy is the development of biomarkers for prognosis and for monitoring therapy response. In an attempt to evaluate the immune response of renal cell carcinoma (RCC) patients, tumor lesions and / or blood samples from 12 RCC patients underwent deep T cell receptor (TCR) sequencing. Despite the low number of samples, different TCR distribution patterns could be detected. Most of the RCC patients presented "patient-specific" TCR sequences, and those clonotypes were present at higher frequency in tumor lesions suggesting a specific extravasation from the blood. Comparison among the tumor samples revealed also "patient-shared" TCR patterns. Indeed, a central core of 16 different TCRs were shared by 3 patients, whereas other 6 patients shared between 4 and 6 TCR sequences, with two sub-groups sharing 12 to 17 different clonotypes. The relative frequencies of shared clonotypes were very different varying from < 1% to a maximum of 37% of the total TCR repertoire. These data confirm the presence of tumor-specific TCR within the cancer tissue and suggest the existence of shared epitopes among different patients that might be used as targets for tumor immunotherapy.

Immune signature of tumor infiltrating immune cells in renal cancer.

Tumor-associated immune cells have been discussed as an essential factor for the prediction of the outcome of tumor patients. Lymphocyte-specific genes are associated with a favorable prognosis in colorectal cancer but with poor survival in renal cell carcinoma (RCC). Flow cytometric analyses combined with immunohistochemistry were performed to study the phenotypic profiles of tumor infiltrating lymphocytes (TIL) and the frequency of T cells and macrophages in RCC lesions. Data were correlated with clinicopathological parameters and survival of patients. Comparing oncocytoma and clear cell (cc)RCC, T cell numbers as well as activation-associated T cell markers were higher in ccRCC, whereas the frequency of NK cells was higher in oncocytoma. An intratumoral increase of T cell numbers was found with higher tumor grades (G1:G2:G3/4 = 1:3:4). Tumor-associated macrophages slightly increased with dedifferentiation, although the macrophage-to-T cell ratio was highest in G1 tumor lesions. A high expression of CD57 was found in T cells of early tumor grades, whereas T cells in dedifferentiated RCC lesions expressed higher levels of CD69 and CTLA4. TIL composition did not differ between older (>70 y) and younger (<58 y) patients. Enhanced patients' survival was associated with a higher percentage of tumor infiltrating NK cells and Th1 markers, e.g. HLA-DR+ and CXCR3+ T cells, whereas a high number of T cells, especially with high CD69 expression correlated with a worse prognosis of patients. Our results suggest that immunomonitoring of RCC patients might represent a useful tool for the prediction of the outcome of RCC patients.

Interleukin-1 potently contributes to 25-hydroxycholesterol-induced synergistic cytokine production in smooth muscle cell-monocyte interactions.

OBJECTIVES: Inflammation is essential for atherogenesis. Cholesterol, a cardiovascular risk factor, may activate inflammation in the vessel wall during this process. Cytokine-mediated interactions of human monocytes with vascular smooth muscle cells (SMCs) may perpetuate this process. METHODS: We investigated the capacity of the cholesterol metabolite 25-hydroxycholesterol to induce inflammatory mediators in cocultures of freshly isolated monocytes with SMCs. We determined the role of interleukin-(IL)-1 in this interaction using qPCR, bioassays, ELISA and western blot. Cocultures with SMC to monocyte ratios from 1:4 to 1:20 were tested. RESULTS: In separate SMC and monocyte cultures (monocultures) 25-hydroxycholesterol only poorly activated IL-1, IL-6 and MCP-1 production, whereas LPS stimulated much higher cytokine levels than unstimulated cultures. In contrast, cocultures of SMCs and monocytes stimulated with 25-hydroxycholesterol produced hundredfold higher cytokine levels than the corresponding monocultures. Blocking experiments with IL-1-receptor antagonist showed that IL-1 decisively contributed to the 25-hydroxycholesterol-induced synergistic IL-6 and MCP-1 production. The presence of intracellular IL-1ß precursor, released mature IL-1ß, and caspase-1 p10 indicated that the inflammasome was involved in this process. Determination of IL-1-mRNA in Transwell experiments indicated that the monocytes are the major source of IL-1, which subsequently activates the SMCs, the primary source of IL-6 in the coculture. CONCLUSION: Taken together, these interactions between local vessel wall cells and invading monocytes may multiply cholesterol-triggered inflammation in the vessel wall, and IL-1 may play a key role in this process. The data also indicate that lower cholesterol levels than expected from monocultures may suffice to initiate inflammation in the tissue.

Granulocyte-to-dendritic cell-ratio as marker for the immune monitoring in patients with renal cell carcinoma.

BACKGROUND: Neutrophil-to-lymphocyte ratio (NLR) has been shown to be a predictor of patients' outcome for several types of malignancies. FINDINGS: Using multicolor flow cytometry we searched for predictive markers to monitor blood immune cells in patients with renal cell carcinoma undergoing surgery of the primary tumor. Due to a high standard deviation, pre-surgery NLR values did not significantly differ between tumor patients and the control group. In contrast, the granulocyte-to-dendritic cell (DC) ratio revealed significant higher values in tumor patients. Whereas NLR values did not differ between patients with early stage tumors and locally advanced tumors, the granulocyte/DC ratio was significantly different in these groups. Additionally, comparison of both ratios before and after tumor resection in the two groups "open surgery" and "laparoscopy" could demonstrate the suitability of granulocyte/DC ratio as a marker for immune monitoring. CONCLUSIONS: Granulocyte/DC ratio may serve as a new putative biomarker for the immune monitoring of tumor patients.

Reduced immunosuppressive properties of axitinib in comparison with other tyrosine kinase inhibitors.

The multikinase inhibitors sunitinib, sorafenib, and axitinib have an impact not only on tumor growth and angiogenesis, but also on the activity and function of immune effector cells. In this study, a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. Tyrosine kinase inhibitor treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib, axitinib did not affect the mitochondrial membrane potential (Δψm) but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl-1), leading to an irreversible arrest in the G2/M cell cycle phase and delayed apoptosis. Furthermore, the sorafenib-mediated suppression of immune effector cells, in particular the reduction of the CD8(+) T cell subset along with the down-regulation of key immune cell markers such as chemokine CC motif receptor 7 (CCR7), CD26, CD69, CD25, and CXCR3, was not observed in axitinib-treated immune effector cells. Therefore, axitinib rather than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy.

Hypoxia supports reprogramming of mesenchymal stromal cells via induction of embryonic stem cell-specific microRNA-302 cluster and pluripotency-associated genes.

Pluripotency is characterized by specific transcription factors such as OCT4, NANOG, and SOX2, but also by pluripotency-associated microRNAs (miRs). Somatic cells can be reprogrammed by forced expression of these factors leading to induced pluripotent stem cells (iPSCs) with characteristics similar to embryonic stem cells (ESCs). However, current reprogramming strategies are commonly based on viral delivery of the pluripotency-associated factors, which affects the integrity of the genome and impedes the use of such cells in any clinical application. In an effort to establish nonviral, nonintegrating reprogramming strategies, we examined the influence of hypoxia on the expression of pluripotency-associated factors and the ESC-specific miR-302 cluster in primary and immortalized mesenchymal stromal cells (MSCs). The combination of hypoxia and fibroblast growth factor 2 (FGF2) treatments led to the induction of OCT4 and NANOG in an immortalized cell line L87 and primary MSCs, accompanied with increased doubling rates and decreased senescence. Most importantly, the endogenous ECS-specific cluster miR-302 was induced upon hypoxic culture and FGF2 supplementation. Hypoxia also improved reprogramming of MSCs via episomal expression of pluripotency factors. Thus, our data illustrate that hypoxia in combination with FGF2 supplementation efficiently facilitates reprogramming of MSCs.

Promoter methylation of aminopeptidase N/CD13 in malignant melanoma.

Aminopeptidase N (APN)/CD13 as ubiquitously expressed membrane peptidase exerts important functions in diverse cellular processes, such as proliferation, migration and differentiation. Previously, a role of APN in the invasiveness of melanoma cells has been demonstrated, but the underlying molecular mechanisms controlling APN expression are not understood. The present study demonstrates that lack of APN expression in primary and established melanoma cells was directly associated with a high-grade DNA methylation status of the myeloid APN promoter. Demethylation by 5-aza-2'-desoxycytidine not only induced constitutive and cytokine-regulated APN protein expression but also resulted in an increased APN-dependent migration of melanoma cells. Furthermore, its heterogeneous expression was inversely correlated to the expression of melanocytic marker proteins in established as well as in short-term cultured human melanoma cells. Staining of tissue microarrays generated from a large series of melanoma samples and control tissues demonstrated a higher APN expression in primary melanoma lesions when compared with nevi and metastases, which was neither associated with clinico-pathological parameters nor with the patients' outcome. Thus, the heterogeneous APN expression pattern in melanoma cells is epigenetically controlled and directly associated with an altered migration capacity but not of clinical significance in our study group.

Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows.

Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study, we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPARγ2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 µM) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 µM) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.

Regulatory T cells with reduced repressor capacities are extensively amplified in pulmonary sarcoid lesions and sustain granuloma formation.

Sarcoidosis can evolve into a chronic disease with persistent granulomas accompanied by progressive fibrosis. While an unlimited inflammatory response suggests an impaired immune control in sarcoid lesions, it stands in contrast to the massive infiltration with CD4(+)CD25(high)FoxP3(+) regulatory T cells. We here revealed that those Treg cells in affected lung lesions were mainly derived from activated natural Treg cells with GARP (LRRC32)-positive phenotype but exhibited reduced repressor capacities despite high IL-10 and TGF-beta 1 levels. The repressive capacity of blood Treg cells, in contrast, was not impaired compared to age-matched healthy donors. Treg derived cells in granuloma lesions have undergone extensive rounds of amplifications indicated by shortened telomeres compared to blood Treg cells of the same patient. Lesional Treg derived cells moreover secreted pro-inflammatory cytokines including IL-4 which sustains granuloma formation through fibroblast amplification and the activation of mast cells, the latter indicated by the expression of membrane-bound oncostatin M.

Monitoring peri-operative immune suppression in renal cancer patients.

The aim of this study was the identification of surrogate markers of host immunity in renal cell carcinoma (RCC) patients. Using 4-color flow cytometry the immunophenotype of blood immune cells of RCC patients was compared to that of healthy volunteers and correlated with staging and grading of patients. Furthermore, the time course of these immune markers was compared in RCC patients undergoing either open surgery or laparoscopy. Compared to the healthy control group, blood of RCC patients contained more granulocytes and higher percentages of CTLA4+CD8+ T lymphocytes, but reduced numbers of dendritic cells (DCs) and of CD28+ CD8+ T cells. Tumor progression was associated with a higher white blood cell count, a reduced frequency of blood DCs and increased numbers of CD57+ T and NK cells. Monocytes of patients with advanced RCC showed a reduced HLA-DR surface expression associated with higher aminopeptidase N(APN)/CD13 expression. Tumor surgery caused an increase of granulocytes and a decrease of all lymphocytic and DC subpopulations within 24 h, whereas the number of HLA-DR low monocytes was up-regulated. As demonstrated by time kinetic analysis, laparoscopic intervention caused a more moderate immunosuppression and an enhanced restoration of immune activity than open surgery. These results suggest that the composition and the phenotype of innate immune cells reflect well the differences in the cellular immunity of RCC patients associated with tumor disease as well as surgery. The monocytic HLA-DR intensity represents a suitable marker for monitoring tumor stage and surgery-associated immunosuppression in RCC patients.

Trefoil factor 3 is induced during degenerative and inflammatory joint disease, activates matrix metalloproteinases, and enhances apoptosis of articular cartilage chondrocytes.

OBJECTIVE: Trefoil factor 3 (TFF3, also known as intestinal trefoil factor) is a member of a family of protease-resistant peptides containing a highly conserved motif with 6 cysteine residues. Recent studies have shown that TFF3 is expressed in injured cornea, where it plays a role in corneal wound healing, but not in healthy cornea. Since cartilage and cornea have similar matrix properties, we undertook the present study to investigate whether TFF3 could induce anabolic functions in diseased articular cartilage. METHODS: We used reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry to measure the expression of TFF3 in healthy articular cartilage, osteoarthritis (OA)-affected articular cartilage, and septic arthritis-affected articular cartilage and to assess the effects of cytokines, bacterial products, and bacterial supernatants on TFF3 production. The effects of TFF3 on matrix metalloproteinase (MMP) production were measured by enzyme-linked immunosorbent assay, and effects on chondrocyte apoptosis were studied by caspase assay and annexin V assay. RESULTS: Trefoil factors were not expressed in healthy human articular cartilage, but expression of TFF3 was highly up-regulated in the cartilage of patients with OA. These findings were confirmed in animal models of OA and septic arthritis, as well as in tumor necrosis factor alpha- and interleukin-1beta-treated primary human articular chondrocytes, revealing induction of Tff3/TFF3 under inflammatory conditions. Application of the recombinant TFF3 protein to cultured chondrocytes resulted in increased production of cartilage-degrading MMPs and increased chondrocyte apoptosis. CONCLUSION: In this study using articular cartilage as a model, we demonstrated that TFF3 supports catabolic functions in diseased articular cartilage. These findings widen our knowledge of the functional spectrum of TFF peptides and demonstrate that TFF3 is a multifunctional trefoil factor with the ability to link inflammation with tissue remodeling processes in articular cartilage. Moreover, our data suggest that TFF3 is a factor in the pathogenesis of OA and septic arthritis.

Aminopeptidase N (APN)/CD13-dependent CXCR4 downregulation is associated with diminished cell migration, proliferation and invasion.

Aminopeptidase N (APN/CD13) is a 150 kDa membrane-bound ubiquitously expressed protease with a broad functional repertoire. It hydrolyzes small peptide mediators, modulates cell motility and adhesion to extracellular matrix and also acts as a viral receptor. In order to dissect the function of enzymatically active and inactive APN/CD13, substitutions of different enzymatic active amino acid residues were generated by site-directed mutagenesis and stably transfected into human embryonic kidney cells. All APN variants analyzed exhibited a complete loss of enzymatic activity, whereas wild type APN transfectants exerted a strong aminopeptidase-specific activity. Furthermore, wild type APN expression was associated with a significant decrease in proliferation, migration and also reduced anchorage-independent growth when compared to enzymatically inactive APN variants and controls. This appeared to be due to a downregulated mRNA and protein expression of the chemokine receptor CXCR4 and an inhibition of the stromal cell-derived factor (SDF)-1alpha/CXCL12-mediated migration. Thus, high APN enzyme activity may antagonize the cellular properties regulated by the CXCR4/SDF-1alpha system in embryonic kidney cells.

CD13 and CD10 expression of granulocytes as markers for the functioning of the immune system: quantification of the expression of membrane molecules using 1:1 labeled monoclonal antibodies and flow cytometry.

HLA-DR expression on monocytes as a marker for the functioning of the immune system is known to be severely depressed in immunodeficiency. Up to now, other markers for the function of the immune system are scarce. In the peripheral blood of patients with open heart surgery the expression of the membrane peptidases neprilysin/CD10 and aminopeptidase N/CD13, was determined on granulocytes in comparison to the monocytic HLA-DR expression. We used the QuantiBRITE flow cytometry system, which yields an absolute antigen expression value (antibodies bound per cell) and may be useful in standardizing surface antigen expression analysis. This system makes use of a highly purified phycoerythrin-labeled antibody with a 1:1 fluorochrome-to-protein ratio, and multilevel calibrated beads with known absolute phycoerythrin fluorescence. Our results show that both membrane peptidases on granulocytes show a similar time-course of expression after heart surgery as do HLA-DR molecules on monocytes, with a decrease from days one to three and a subsequent recovery to normal values. In future analyses a possible relationship between the immunodeficiency of patients and a diminished expression of both membrane peptidases on granulocytes has to be investigated.