Native extracellular matrix probes to target patient- and tissue-specific cell-microenvironment interactions by force spectroscopy.

Atomic Force Microscopy (AFM) is successfully used for the quantitative investigation of the cellular mechanosensing of the microenvironment. To this purpose, several force spectroscopy approaches aim at measuring the adhesive forces between two living cells and also between a cell and an appropriate reproduction of the extracellular matrix (ECM), typically exploiting tips suitably functionalised with single components (e.g. collagen, fibronectin) of the ECM. However, these probes only poorly reproduce the complexity of the native cellular microenvironment and consequently of the biological interactions. We developed a novel approach to produce AFM probes that faithfully retain the structural and biochemical complexity of the ECM; this was achieved by attaching to an AFM cantilever a micrometric slice of native decellularised ECM, which was cut by laser microdissection. We demonstrate that these probes preserve the morphological, mechanical, and chemical heterogeneity of the ECM. Native ECM probes can be used in force spectroscopy experiments aimed at targeting cell-microenvironment interactions. Here, we demonstrate the feasibility of dissecting mechanotransductive cell-ECM interactions in the 10 pN range. As proof-of-principle, we tested a rat bladder ECM probe against the AY-27 rat bladder cancer cell line. On the one hand, we obtained reproducible results using different probes derived from the same ECM regions; on the other hand, we detected differences in the adhesion patterns of distinct bladder ECM regions (submucosa, detrusor, and adventitia), in line with the disparities in composition and biophysical properties of these ECM regions. Our results demonstrate that native ECM probes, produced from patient-specific regions of organs and tissues, can be used to investigate cell-microenvironment interactions and early mechanotransductive processes by force spectroscopy. This opens new possibilities in the field of personalised medicine.

Inflammatory profile discriminates clinical subtypes in LRRK2-associated Parkinson's disease.

BACKGROUND AND PURPOSE: The presentation of Parkinson's disease patients with mutations in the LRRK2 gene (PDLRRK2 ) is highly variable, suggesting a strong influence of modifying factors. In this context, inflammation is a potential candidate inducing clinical subtypes. METHODS: An extensive battery of peripheral inflammatory markers was measured in human serum in a multicentre cohort of 142 PDLRRK2 patients from the MJFF LRRK2 Consortium, stratified by three different subtypes as recently proposed for idiopathic Parkinson's disease: diffuse/malignant, intermediate and mainly pure motor. RESULTS: Patients classified as diffuse/malignant presented with the highest levels of the pro-inflammatory proteins interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1) and macrophage inflammatory protein 1-ß (MIP-1-ß) paralleled by high levels of the neurotrophic protein brain-derived neurotrophic factor (BDNF). It was also possible to distinguish the clinical subtypes based on their inflammatory profile by using discriminant and area under the receiver operating characteristic curve analysis. CONCLUSIONS: Inflammation seems to be associated with the presence of a specific clinical subtype in PDLRRK2 that is characterized by a broad and more severely affected spectrum of motor and non-motor symptoms. The pro-inflammatory metabolites IL-8, MCP-1 and MIP-1-ß as well as BDNF are interesting candidates to be included in biomarker panels that aim to differentiate subtypes in PDLRRK2 and predict progression.

Differential chemokine expression in chronic GVHD of the conjunctiva.

In chronic GVHD after BMT, the conjunctiva represents a target organ. GVHD can lead to severe inflammation and dry-eye syndrome (sicca syndrome). The molecular mechanisms are largely unknown. We examined the expression of chemokines in the conjunctiva in cases of chronic GVHD. In this study, we included 10 patients with chronic GVHD and 10 healthy controls. Clinical data were collected and tear film analysis and conjunctival cytology were carried out. Conjunctival biopsies were taken from all participants. Gene expression profiles of chemokines and their corresponding receptors were evaluated by means of quantitative real-time PCR. Chemokine protein expression was analysed by immunohistochemical analyses. Expressions of the Th1-associated chemokines, chemokine (C-X-C motif) ligand (CXCL) 9 (Mig), CXCL10 (IP-10), and their receptor chemokine (C-X-C motif) receptor 3 (CXCR3) were significantly increased in GVHD patients. Immunohistochemical analysis confirmed marked expression of the inflammatory CXCR3 ligands. A total of six patients had a moderate or severe sicca syndrome. Impression cytology revealed a mild keratinisation, moderate keratinisation or severe squamous metaplasia in three patients, respectively. Chronic GVHD of the conjunctiva is characterised by the expression of Th1-associated chemokines. Taken together, our results confirm that the conjunctiva is a target organ in this T cell-mediated process and add to molecular understanding of conjunctival GVHD.

Clinicopathological significance of MMP-2 and its specific inhibitor TIMP-2 in gastric cancer.

Matrix metalloproteinases (MMPs) can degrade type IV collagen of extracellular matrices and basal membranes and thus play a key role in the migration of malignant cells. In vivo, MMPs are inhibited by tissue inhibitors of metalloproteinases (TIMPs). Since in a previous study we showed that the expression of MMP-2 correlates with clinicopathological parameters in gastric cancer, we have now investigated a possible correlation of MMP-2 and TIMP-2 expression with survival in gastric cancer, as well as the possible association of TIMP-2 with clinicopathological parameters. Tissue samples were obtained from 116 gastric cancer patients who underwent gastrectomy with extended lymphadenectomy. MMP-2 and TIMP-2 expression was analysed using immunohistochemical staining and was graded semiquantitatively (score 0 - 3). High epithelial MMP-2 immunoreactivity was significantly associated with tumor stage and poor survival using the Kaplan-Meier log-rank statistical method (log-rank statistics). However, using Cox regression analysis, high epithelial MMP-2 immunoreactivity was not an independent prognostic factor. TIMP-2 showed no association with survival in gastric cancer, but the intensity of TIMP-2 staining in tumor cells correlated significantly with tumor differentiation based on the WHO and Lauren and Ming classifications, as well as with presence of distant metastasis. Our results show that high epithelial MMP-2 expression in gastric cancer is associated with poor survival, although it is not an independent prognostic factor, and that aggressive forms of gastric cancer are associated with low TIMP-2 expression.

[Neurolymphomatosis. Subacute sensorimotor polyneuropathy as a first sign of non-Hodgkin's B cell lymphoma]. / Neurolymphomatose. Subakute sensomotorische Polyneuropathie als Erstmanifestation eines Non-Hodgkin-B-Zell-Lymphoms.

We report on a 65-year-old patient with subacute painful sensorimotor polyneuropathy with distal leg muscle palsy and initially presenting with bilateral leg edema. Electrophysiologic testing showed an axonal lesion pattern with acute denervation. Nerve biopsy demonstrated neurolymphomatosis as an initial manifestation of a non-Hodgkin's B cell lymphoma.

Recurrent finding of the FIP1L1-PDGFRA fusion gene in eosinophilia-associated acute myeloid leukemia and lymphoblastic T-cell lymphoma.

The FIP1L1-PDGFRA fusion gene has been described in patients with eosinophilia-associated myeloproliferative disorders (Eos-MPD). Here, we report on seven FIP1L1-PDGFRA-positive patients who presented with acute myeloid leukemia (AML, n=5) or lymphoblastic T-cell non-Hodgkin-lymphoma (n=2) in conjunction with AML or Eos-MPD. All patients were male, the median age was 58 years (range, 40-66). AML patients were negative for common mutations of FLT3, NRAS, NPM1, KIT, MLL and JAK2; one patient revealed a splice mutation of RUNX1 exon 7. Patients were treated with imatinib (100 mg, n=5; 400 mg, n=2) either as monotherapy (n=2), as maintenance treatment after intensive chemotherapy (n=3) or in overt relapse 43 and 72 months, respectively, after primary diagnosis and treatment of FIP1L1-PDGFRA-positive disease (n=2). All patients are alive, disease-free and in complete hematologic and complete molecular remission after a median time of 20 months (range, 9-36) on imatinib. The median time to achievement of complete molecular remission was 6 months (range, 1-14). We conclude that all eosinophilia-associated hematological malignancies should be screened for the presence of the FIP1L1-PDGFRA fusion gene as they are excellent candidates for treatment with tyrosine kinase inhibitors even if they present with an aggressive phenotype such as AML.

DNA damage transiently increases TRF2 mRNA expression and telomerase activity.

Telomerase activity transiently increases when HL60 cells are treated with the topoisomerase II inhibitor etoposide. A quantitative assessment revealed that telomerase is activated by etoposide treatment in a number of cell lines and that the increase is reversible after withdrawal of etoposide from the cell culture. Telomerase activation correlated with the occurrence of DNA damage but not with cell cycle arrest. We did not detect any transcriptional upregulation of hTERT mRNA, suggesting a post-transcriptional mechanism of telomerase activation. Furthermore, the mRNA expression of the telomere binding protein TRF2 was upregulated early and reversibly after etoposide treatment. TRF1 mRNA expression levels were unchanged after DNA damage, but increased when the cells accumulated in the G2/M phase. The data show that the telosome reacts after DNA damage by upregulating telomerase activity and TRF2 expression in malignant cells. It has previously been shown that overexpression of TRF2 can repress senescence signals arising from critically shortened telomeres. We show here that TRF2 is upregulated by undirected DNA damage that also affects the telomeric DNA. These data suggest that upregulation of telomerase activity and TRF2 expression might act as antiapoptotic mechanisms in the DNA-damage response of malignant cells.

[Recurrent sanguineous and mucous diarrhea and spasms in a 46-year-old woman--manifestation of endometriosis in the colon sigmoideum]. / Rezidivierende schleimig-blutige Diarrhöen und Tenesmen bei einer 46-jährigen Frau - Manifestation einer Endometriose im Colon sigmoideum.

Intestinal endometriosis is the most frequent extragenital manifestation of this disease. Sometimes patients even present with acute bowel obstruction. We report on a 46-year-old woman complaining about recurrent sanguineous and mucous diarrhea and spasms for several years. Colonoscopy showed a stenosis in the sigmoid colon without macroscopically visible alterations of the mucosa. Computertomography, ultrasound and barium contrast enema did not provide us with further information about the origin of the stenosis. Biopsies out of the mucosa at the stenosis showed typical endometriosis tissue. After starting a conservative therapy with GnRH-agonist gosereline the patient became completely free of symptoms. The coincidence of endometriosis and M. Crohn has to be taken into consideration. Therapy planning should include a close co-operation with gynaecologists and surgeons to transfer the patient to surgical intervention when needed.

Genetic determinants of IL-6 expression levels do not influence bone loss in inflammatory bowel disease.

Bone loss in inflammatory bowel disease (IBD) is presumed to be mediated by inflammation. Increased levels of the multifunctional cytokine IL-6 in inflammatory diseases have been proposed to be the link in such "inflammation-mediated osteopenia." A recently described G/C polymorphism with an effect on transcription rate and plasma levels of IL-6 suggests a genetically determined difference in the degree of the IL-6 response to stressful stimuli between individuals. This study aimed to assess the frequency of genotypes and haplotypes of the G/C polymorphism of IL-6 in IBD patients. A further aim was to assess whether carriage of the potentially protective CC genotype is favorable with respect to the development of bone disease in IBD. The IL-6 polymorphism was typed in 105 IBD patients and 113 healthy controls. Bone mineral density was evaluated at baseline and after a prospective 2-year-follow-up. The favorable CC genotype with decreased IL-6 release was not underrepresented in IBD patients compared to healthy controls. Carriage of this genotype was not protective with respect to the development of bone disease, either for the bone mineral density at baseline or for the prospectively observed bone loss. Within the subgroup of patients who did not receive steroids during follow-up, the prospectively observed bone loss was even slightly higher in CC carriers, but differences did not reach significance. Genetically determined differences in the degree of the IL-6 response to stressful stimuli are no major predictors for the degree of bone disease in IBD patients.

Genetic factors determine extent of bone loss in inflammatory bowel disease.

BACKGROUND & AIMS: Although bone loss and osteoporosis are well-known long-term sequelae of inflammatory bowel disease (IBD), the risk factors for increased bone loss have not been identified. Balances of pro- and anti-inflammatory cytokines influence mechanisms of both chronic inflammation and bone resorption. The aim of this study was to identify genetic risk factors for rapid bone loss in IBD patients as a model of disease- and inflammation-associated bone loss. METHODS: Multiple clinical parameters, biochemical markers of bone metabolism (vitamin D, parathyroid hormone, N-terminal telopeptide of type-I collagen, desoxypyridinoline, bone alkaline phosphatase), and bone mineral density were prospectively assessed in 83 IBD patients over 1.6+/-0.3 years. Eighty-six healthy bone marrow donors served as controls for allelotyping. The allele status of the interleukin 1 receptor antagonist (IL-1ra), IL-6, heat shock protein 70-2 (hsp 70-2), and heat shock protein 70-hom (hsp hom) genes was typed and correlated with clinical course of IBD and extent of bone loss. RESULTS: The extent of bone loss was not correlated to clinical severity of disease or application of corticosteroids. Noncarriage of the 240-base pair allele of the IL-1ra gene and carriage of the 130-base pair allele of IL-6 were independently associated with increased bone loss. Genetic variations of the hsp genes were not associated with degree of bone loss. The combined presence of the named risk factors was significantly associated with increasing bone loss. CONCLUSIONS: Genetic variations in the IL-6 and IL-1ra gene identify IBD patients at risk for increased bone loss.

Bone loss in long-term survivors after transplantation of hematopoietic stem cells: a prospective study.

Organ transplantation is associated with relevant bone loss. Bone loss of up to 20% of pretransplant bone mineral density (BMD) values within the first year after kidney, liver, heart and lung transplantation has been reported. Patients undergoing transplantation of hematopoietic stem cells provide an interesting model to study transplantation-induced bone loss, especially because most patients do not have preexisting bone disease. A longitudinal study was performed in 81 patients undergoing bone marrow or peripheral blood stem cell transplantation. BMD was determined by dual-energy X-ray absorptiometry before transplantation, at discharge from the hospital, and at 6 and 12 months after transplantation in all 81 patients. In 35 patients BMD was re-evaluated 24 months after transplantation. Vitamin D and parathyroid hormone, bone alkaline phosphatase as a marker of bone formation, and N-terminal telopeptide of type I collagen as a marker of bone resorption were assessed before transplantation and in the short-term follow-up 14 and 28 days after transplantation. The majority of patients (72%) showed normal BMD before transplantation. However, lower BMD was observed in patients who had received high-dose cytoreductive chemotherapy before transplantation compared with those who had received no chemotherapy or only hydroxyurea. Despite supplementation with elemental calcium (1000 mg/day) and vitamin D (1000 IU/day), the mean rate of bone loss during the first year was 7.2 +/- 6.3% at the lumbar spine, 11.9 +/- 8.1% at the femoral neck and 3.8 +/- 2.5% at the total body compartment. Evaluation of the pattern of bone loss during the first year demonstrated that the amount of bone loss was largest within the first 40 days after transplantation and small during the second half of the first year after transplantation. The majority of patients showed vitamin D deficiency and secondary hyperparathyroidism. Bone formation was normal before and after transplantation, whereas bone resorption was dramatically increased before and after transplantation. Exposure to glucocorticoids was associated with higher bone loss at spine and femoral neck but not at the total body compartment. Our data demonstrate rapid bone loss in patients undergoing transplantation of hematopoietic stem cells. Bone turnover is characterized by biochemical uncoupling of bone resorption and bone formation, changes interestingly pre-existing before transplantation. The observed alterations in bone mass and metabolism emphasize the importance of clinical trials with antiresorptive agents to prevent and treat post-transplantation osteoporosis in this group of patients.

Transforming growth factor-beta and hepatocyte growth factor plasma levels in patients with inflammatory bowel disease.

OBJECTIVE: An increased mucosal expression of transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF) has been reported in patients with active inflammatory bowel diseases (IBD) and in proximity to injured gastric and intestinal mucosal surfaces. The aim of this study was to measure systemic concentrations of TGF-beta and HGF and to assess their potential value to predict disease activity or severity of inflammation in patients with inflammatory bowel diseases. DESIGN AND METHODS: Plasma HGF and TGF-beta1 peptide levels were determined in 29 patients with ulcerative colitis, 45 patients with Crohn's disease and 28 healthy controls using commercial ELISA assays. Peptide levels were correlated with disease activity indices and various laboratory parameters. RESULTS: HGF and TGF-beta1 plasma levels were detected in all control and IBD subjects. Although a tendency towards increased HGF and TGF-beta1 peptide levels in IBD patients was observed, differences between groups were not significant In ulcerative colitis patients HGF plasma levels positively correlated with white blood cell counts and negatively correlated with serum albumin concentrations and haematocrit. In Crohn's disease patients, a positive correlation between TGF-beta and platelet count was observed. CONCLUSIONS: HGF and TGF-beta1 plasma concentrations are not significantly different in IBD and healthy control subjects. Stratification of IBD patients according to disease activity did not reveal any substantial differences, suggesting that HGF and TGF-beta plasma levels have no value in the assessment of disease activity or severity of inflammation in patients with IBD.

Bone loss in patients with inflammatory bowel disease is less than expected: a follow-up study.

BACKGROUND: In spite of the accumulating evidence of an increased prevalence of osteopenia and osteoporosis in patients with inflammatory bowel diseases (IBD), the time course of bone loss is not well described, and there is little knowledge about factors indicating an increased risk of rapid bone loss. METHODS: We conducted a follow-up study in 80 IBD patients (45 men and 25 premenopausal and 10 postmenopausal women), 19 with ulcerative colitis and 61 with Crohn disease, with a mean follow-up time of 568 +/- 60 days, to assess bone loss, risk factors of rapid bone loss, and value of bone markers to predict bone loss. Bone mineral density was measured by dual-energy X-ray absorptiometry, bone formation by bone alkaline phosphatase (BAP), and bone resorption by N-terminal telopeptide of type-I collagen (NTX) and free deoxypyridinoline (DPD). RESULTS: Bone density changes per year were 0.46% +/- 3% at the spine, 0.06% +/- 5.1% at the femoral neck, -1.1% +/- 7.7% at the triangle of Ward, and -0.52% +/- 1.86% at total body level. Type and duration of disease, sex, age, and level of NTX, DPD, and BAP at base line did not show significant differences between patients who lost and those who did not lose bone mass. Bone loss was significantly higher in patients with (n = 28) than in those without steroids (n = 52) at the femoral neck and Ward triangle but not at the spine and total body. CONCLUSIONS: Change in bone mass in IBD patients during short-term follow-up is low on average, but there is great heterogeneity within the population, which cannot be explained by the use of steroids alone. Bone loss cannot be predicted by analysis of bone markers.

Reduced bone mineral density and unbalanced bone metabolism in patients with inflammatory bowel disease.

Patients with chronic inflammatory bowel diseases (IBD) are at increased risk to develop osteopenia and osteoporosis. New parameters for the assessment of bone formation and especially bone resorption have significantly improved the diagnostic procedures to characterize bone metabolism. Biochemical characterization of bone turnover in IBD patients may provide important information about the pathogenesis of osteoporosis in this patient population. A cross-sectional study was performed. One hundred forty-nine patients (77 men, 52 premenopausal, and 20 postmenopausal women) with IBD (104 with Crohn's disease [CD] and 45 with ulcerative colitis [UC]) underwent clinical, osteodensitometric, and metabolic bone assessment. Bone mineral density was determined by dual energy X-ray absorptiometry. Bone formation (bone alkaline phosphatase), bone resorption (N-terminal telopeptide of type-I collagen, free desoxypyridinoline, total pyridinoline, and desoxypyridinoline), vitamin D, and parathyroid hormone were assessed. Thirty-six percent of patients with CD and 32% with UC showed osteopenia, 15% with CD and 7% with UC showed osteoporosis. Bone resorption was significantly increased in IBD patients compared to normal controls, whereas bone formation did not show a compensatory increase. Bone formation was even more suppressed in the subset of patients currently treated with corticosteroids. Our data confirm the high prevalence of osteopenia and osteoporosis reported in IBD patients. Furthermore, we provide evidence for an increased bone resorption accompanied by low bone formation in IBD patients. This imbalance of bone metabolism is likely to be one of the reasons for increased bone loss in IBD patients.

Low T3-syndrome and nutritional status as prognostic factors in patients undergoing bone marrow transplantation.

Bone marrow transplantation is known to be associated with considerable morbidity and mortality. The aim of this study was to determine the influence of nutritional status and development of sick euthyroid syndrome as prognostic factors for outcome after BMT. In 100 patients who underwent transplantation the following parameters were assessed before and at day 14 and 28 after transplantation: anthropometric data (body weight, body mass index, body composition, grip strength), rapid turnover proteins transferrin and prealbumin, T4, T3, free T4, reverse T3, thyroid-stimulating hormone and thyroglobulin. Following bone marrow transplantation, 22 patients died in the short-term follow-up (group A) before day 140 after BMT, 21 patients died during further follow-up between days 140 and 365 (group B) and 57 patients survived longer than 365 days (group C). All patients experienced a significant decrease of transferrin and T3, accompanied by an increase of rT3 and rT3/T3 ratio at day 14 after BMT. At day 28 after BMT, patients in group C showed recovery from these changes with an increase of transferrin and a fall in rT3 and the rT3/T3-ratio, which was not seen in patients who died during further follow-up (groups A and B). The observed changes were independent of other prognostically relevant factors (type of disease, HLA-match, immunosuppression). Impaired nutritional status and development of a sick euthyroid syndrome, without tendency to recovery, are associated with a higher probability of fatal outcome after bone marrow transplantation and have prognostic relevance in this group of patients.

[Unusual primary cutaneous localized amyloidosis]. / Ungewöhnliche primär kutan lokalisierte Amyloidose.

The case of a 41-year-old female patient with an unusual type of primary localized cutaneous amyloidosis is reported. Clinical examination revealed lesions typical for macular cutaneous amyloidosis, while histology and histochemistry, in contrast, revealed the presence of nodular amyloidosis of the skin with deposition of lambda light chain amyloid.

[Localized Sweet syndrome in urethro-prostatitis caused by Ureaplasma]. / Lokalisiertes Sweet-Syndrom bei Urethro-Prostatitis durch Ureaplasmen.

A 36-year-old patient with localized Sweet's syndrome and concomitant Ureaplasma urealyticum infection is presented. In addition to infectious diseases, malignant neoplasms are also associated with this dermatosis.