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The aim of this study was to investigate the diagnostic power of CA 549, MSA and CA 15-3 in identifying breast cancer. The study included 232 patients of which 56 were healthy, 43 had benign breast cancer and 191 with other growths. The results were obtained using a specific immunoassay and using producers' cut offs. The following sensitivity and specificity of markers were found: CA 549 (sen.: 40%/spec.: 90%), MSA (sen.: 22%/spec.: 96%), and CA 15-3 (sen.: 33%/spec.: 86%). Ideal cut offs were defined with ROC curves. A significant correlation was found between CA 549, MSA, and CA 15-3. The combination of markers does not improve the clinical usefulness to identify only breast cancer. Serum tumor markers are abnormally elevated in patients with breast cancer. CA 549, MSA, CA 15-3 are useful clinical markers, good indicators of disease extent, and may have important prognostic value. This study demonstrates the role of the tumor markers in breast cancer.
Background: The ability of stroke volume variation (SVV), pulse pressure variation (PPV) and global end-diastolic volume (GEDV) for prediction of fluid responsiveness in presence of pleural effusion is unknown. The aim of the present study was to challenge the ability of SVV, PPV and GEDV to predict fluid responsiveness in a porcine model with pleural effusions.
Methods: Pigs were studied at baseline and after fluid loading with 8 ml kg−1 6% hydroxyethyl starch. After withdrawal of 8 ml kg−1 blood and induction of pleural effusion up to 50 ml kg−1 on either side, measurements at baseline and after fluid loading were repeated. Cardiac output, stroke volume, central venous pressure (CVP) and pulmonary occlusion pressure (PAOP) were obtained by pulmonary thermodilution, whereas GEDV was determined by transpulmonary thermodilution. SVV and PPV were monitored continuously by pulse contour analysis.
Results: Pleural effusion was associated with significant changes in lung compliance, peak airway pressure and stroke volume in both responders and non-responders. At baseline, SVV, PPV and GEDV reliably predicted fluid responsiveness (area under the curve 0.85 (p<0.001), 0.88 (p<0.001), 0.77 (p = 0.007). After induction of pleural effusion the ability of SVV, PPV and GEDV to predict fluid responsiveness was well preserved and also PAOP was predictive. Threshold values for SVV and PPV increased in presence of pleural effusion.
Conclusions: In this porcine model, bilateral pleural effusion did not affect the ability of SVV, PPV and GEDV to predict fluid responsiveness.
Background. The aim of this study was to show the importance of the bone marker procollagen type 1 aminoterminal propeptide (P1NP) in detecting bone metastases in women suffering from breast cancer. We furthermore investigated to what degree P1NP is correlated to the degree of bone metastases, and if P1NP is increased in patients with metastases other than bone. Patients and Methods. We analyzed 80 serum samples of women (17 premenopausal/63 postmenopausal) with breast cancer. Therefore we used a specific immunoassay “ELECSYS 2010” by Roche Diagnostics. We divided our group of patients with regard to menopausal status, sites of metastases and number of bone metastases. Results. As a result we found higher concentrations of P1NP in women with radiologically confirmed bone metastases (median: 125.75 ng/mL) in comparison to the collective without bone involvement (median: 73.61 ng/mL). However, both groups showed values above the applied cutoff values of median 27.8 ng/mL for premenopausal women and median: 37.1 ng/mL for the postmenopausal group due to the fact that all patients had cancer. Furthermore higher P1NP concentrations were found in women with more than 5 sites of bone metastases (median: 183.9 ng/mL) than in patients with only one site of bone metastases (median: 37 ng/mL). Also patients with no bone involvement but other sites of metastases showed quite high P1NP concentrations (median: 73.61 ng/mL). Conclusion. The marker of bone turnover procollagen type 1 aminoterminal propeptide can be considered as a useful tool for estimating the extent of bone involvement and for the detection of bone metastases. P1NP cannot replace conventional methods for detecting bone metastases such as radiological methods but it can help clarify unclear radiological results. This study does not take into account the change of P1NP concentration during the course of therapy.
BACKGROUND: Exsanguinating hemorrhage is the major cause of death in patients with pelvic ring disruption.
AIMS: The aim of this study was to document outcomes after the stabilization of pelvic ring injuries by a C-clamp and control of hemorrhage by pelvic packing. Physiological parameters were tested as prognostic factors.
SETTING AND DESIGN: This was a retrospective study at a level I trauma center. The study period was from January 1996 to December 2007.
MATERIALS AND METHODS: Fifty patients with pelvic ring disruption and hemorrhagic shock were analyzed. The pelvic rings were fixed by a C-clamp, and patients with ongoing hemorrhage underwent laparotomy and extra- and/or intra-peritoneal pelvic packing. Clinical parameters (heart rate, mean arterial pressure) and physiological parameters (lactate levels, hemoglobin, hematocrit) were documented at admission and at different time points during the initial treatment (1, 2, 3, 4, 6, 8, and 12h after admission).
RESULTS: Within 12 h of admission, 16 patients died (nonsurvivors) due to hemorrhagic shock (n=13) or head injuries (n=3). In this group, 12 patients underwent laparotomy with pelvic packing. Thirty-four patients survived the first 12 h (early survivors) after fixation by a C-clamp and additional packing in 23 patients. Four of these patients died 12.3±7.1 days later due to multiple organ failure (n=3) or severe head injury (n=1). The blood lactate level at admission was significantly higher in the group of nonsurvivors (7.2±0.8 mmol/L) compared to the early survivors (4.3±0.5 mmol/L, P<0.05). While hemoglobin values improved within the first 2 h in nonsurvivors, lactate levels continued to increase.
CONCLUSION: Pelvic packing in addition to the C-clamp fixation effectively controls severe hemorrhage in patients with pelvic ring disruption. Early sequential measurements of blood lactate levels can be used to estimate the severity of shock and the response to the shock treatment.
During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug-drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.
Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFNγ-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.
Despite the availability of new antifungal compounds, invasive fungal infection remains a significant cause of morbidity and mortality in children and adults undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Allogeneic HSCT recipients suffer from a long lasting defect of different arms of the immune system, which increases the risk for and deteriorates the prognosis of invasive fungal infections. In turn, advances in understanding these immune deficits have resulted in promising strategies to enhance or restore critical immune functions in allogeneic HSCT recipients. Potential approaches include the administration of granulocytes, since neutropenia is the single most important risk factor for invasive fungal infection, and preliminary clinical results suggest a benefit of adoptively transferred donor-derived antifungal T cells. In vitro data and animal studies demonstrate an antifungal effect of natural killer cells, but clinical data are lacking to date. This review summarizes and critically discusses the available data of immunotherapeutic strategies in allogeneic HSCT recipients suffering from invasive fungal infection.
To search for novel strategies to enhance the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis pathways in glioblastoma, we used the B-cell lymphoma 2/Bcl2-like 2-inhibitor ABT-737. Here we report that ABT-737 and TRAIL cooperate to induce apoptosis in several glioblastoma cell lines in a highly synergistic manner (combination index <0.1). Interestingly, the concerted action of ABT-737 and TRAIL to trigger the accumulation of truncated Bid (tBid) at mitochondrial membranes is identified as a key underlying mechanism. ABT-737 and TRAIL cooperate to cleave BH3-interacting domain death agonist (Bid) into its active fragment tBid, leading to increased accumulation of tBid at mitochondrial membranes. Coinciding with tBid accumulation, the activation of Bcl2-associated X protein (Bax), loss of mitochondrial membrane potential, release of cytochrome-c and second mitochondria-derived activator of caspase (Smac) into the cytosol and caspase activation are strongly increased in cotreated cells. Of note, knockdown of Bid significantly decreases ABT-737- and TRAIL-mediated Bax activation and apoptosis. Also, caspase-3 silencing reduces ABT-737- and TRAIL-induced Bid cleavage and apoptosis, indicating that a caspase-3-driven, mitochondrial feedback loop contributes to Bid processing. Importantly, ABT-737 profoundly enhances TRAIL-triggered apoptosis in primary cultured glioblastoma cells derived from tumor material, underlining the clinical relevance. Also, ABT-737 acts in concert with TRAIL to suppress tumor growth in an in vivo glioblastoma model. In conclusion, the rational combination of ABT-737 and TRAIL cooperates to trigger tBid mitochondrial accumulation and apoptosis. This approach presents a promising strategy for targeting the apoptosis pathways in glioblastoma, which warrants further investigation.
Immunomodulatory properties and molecular effects in inflammatory diseases of low-dose X-irradiation
(2012)
Inflammatory diseases are the result of complex and pathologically unbalanced multicellular interactions. For decades, low-dose X-irradiation therapy (LD-RT) has been clinically documented to exert an anti-inflammatory effect on benign diseases and chronic degenerative disorders. By contrast, experimental studies to confirm the effectiveness and to reveal underlying cellular and molecular mechanisms are still at their early stages. During the last decade, however, the modulation of a multitude of immunological processes by LD-RT has been explored in vitro and in vivo. These include leukocyte/endothelial cell adhesion, adhesion molecule and cytokine/chemokine expression, apoptosis induction, and mononuclear/polymorphonuclear cell metabolism and activity. Interestingly, these mechanisms display comparable dose dependences and dose-effect relationships with a maximum effect in the range between 0.3 and 0.7 Gy, already empirically identified to be most effective in the clinical routine. This review summarizes data and models exploring the mechanisms underlying the immunomodulatory properties of LD-RT that may serve as a prerequisite for further systematic analyses to optimize low-dose irradiation procedures in future clinical practice.
Signaling via the intrinsic (mitochondrial) pathway of apoptosis represents one of the critical signal transduction cascades that control the regulation of cell death. This pathway is typically altered in human cancers, thereby providing a suitable target for therapeutic intervention. Members of the Bcl-2 family of proteins as well as cell survival signaling cascades such as the PI3K/Akt/mTOR pathway are involved in the regulation of mitochondria-mediated apoptosis. Therefore, further insights into the molecular mechanisms that form the basis for the control of mitochondria-mediated apoptosis will likely open new perspectives to bypass evasion of apoptosis and treatment resistance in human cancers.
Interleukin (IL)-10 and IL-22 are key members of the IL-10 cytokine family that share characteristic properties such as defined structural features, usage of IL-10R2 as one receptor chain, and activation of signal transducer and activator of transcription (STAT)-3 as dominant signaling mode. IL-10, formerly known as cytokine synthesis inhibitory factor, is key to deactivation of monocytes/macrophages and dendritic cells. Accordingly, pre-clinical studies document its anti-inflammatory capacity. However, the outcome of clinical trials assessing the therapeutic potential of IL-10 in prototypic inflammatory disorders has been disappointing. In contrast to IL-10, IL-22 acts primarily on non-leukocytic cells, in particular epithelial cells of intestine, skin, liver, and lung. STAT3-driven proliferation, anti-apoptosis, and anti-microbial tissue protection is regarded a principal function of IL-22 at host/environment interfaces. In this hypothesis article, hidden/underappreciated pro-inflammatory characteristics of IL-10 and IL-22 are outlined and related to cellular priming by type I interferon. It is tempting to speculate that an inherent inflammatory potential of IL-10 and IL-22 confines their usage in tissue protective therapy and beyond that determines in some patients efficacy of type I interferon treatment.
Background: Candida spp. are a frequent cause of nosocomial bloodstream infections worldwide.
Objective: To evaluate the use patterns and outcomes associated with intravenous (IV) fluconazole therapy in intensive care units in Spain and Germany.
Patients and methods: The research reported here was a prospective multicenter longitudinal observational study in adult intensive care unit patients receiving IV fluconazole. Demographic, microbiologic, therapy success, length of hospital stay, adverse event, and all-cause mortality data were collected at 14 sites in Spain and five in Germany, from February 2004 to November 2005.
Results: Patients (n = 303) received prophylaxis (n = 29), empiric therapy (n = 140), preemptive therapy (n = 85), or definitive therapy (n = 49). A total of 298 patients (98.4%) were treated with IV fluconazole as first-line therapy. The treating physicians judged therapy successful in 66% of prophylactic, 55% of empiric, 45% of preemptive, and 43% of definitive group patients. In the subgroup of 152 patients with proven and specified Candida infection only, 32% suffered from Candida specified as potentially resistant to IV fluconazole. The overall mortality rate was 42%.
Conclusion: Our study informs treatment decision makers that approximately 32% of the patients with microbiological results available suffered from Candida specified as potentially resistant to IV fluconazole, highlighting the importance of appropriate therapy.
ABSTRACT: BACKGROUND: Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the 'gatekeeper' T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation.
METHODS: Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs.
RESULTS: In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity.
CONCLUSIONS: Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells.
In the current event-related potential (ERP) study, we investigated how speech rhythm impacts speech segmentation and facilitates the resolution of syntactic ambiguities in auditory sentence processing. Participants listened to syntactically ambiguous German subject- and object-first sentences that were spoken with either regular or irregular speech rhythm. Rhythmicity was established by a constant metric pattern of three unstressed syllables between two stressed ones that created rhythmic groups of constant size. Accuracy rates in a comprehension task revealed that participants understood rhythmically regular sentences better than rhythmically irregular ones. Furthermore, the mean amplitude of the P600 component was reduced in response to object-first sentences only when embedded in rhythmically regular but not rhythmically irregular context. This P600 reduction indicates facilitated processing of sentence structure possibly due to a decrease in processing costs for the less-preferred structure (object-first). Our data suggest an early and continuous use of rhythm by the syntactic parser and support language processing models assuming an interactive and incremental use of linguistic information during language processing.
Tissue plasminogen activator (tPA) is the only FDA-approved treatment for reperfusing ischemic strokes. But widespread use of tPA is still limited by fears of inadvertently administering tPA in patients with intracerebral hemorrhage (ICH). Surprisingly, however, the assumption that tPA will worsen ICH has never been biologically tested. Here, we assessed the effects of tPA in two models of ICH. In a mouse model of collagenase-induced ICH, hemorrhage volumes and neurological deficits after 24 hrs were similar in saline controls and tPA-treated mice, whereas heparin-treated mice had 3-fold larger hematomas. In a model of laser-induced vessel rupture, tPA also did not worsen hemorrhage volumes, while heparin did. tPA is known to worsen neurovascular injury by amplifying matrix metalloproteinases during cerebral ischemia. In contrast, tPA did not upregulate matrix metalloproteinases in our mouse ICH models. In summary, our experimental data do not support the assumption that intravenous tPA has a deleterious effect in acute ICH. However, due to potential species differences and the inability of models to fully capture the dynamics of human ICH, caution is warranted when considering the implications of these findings for human therapy.
The BCR/ABL fusion protein is the hallmark of Philadelphia Chromosome positive (Ph+) leukemia. The constitutive activation of the ABL-kinase in BCR/ABL cells induces the leukemic phenotype. Targeted inhibition of BCR/ABL by small molecule inhibitors reverses the transformation potential of BCR/ABL. Recently, we definitively proved that targeting the tetramerization of BCR/ABL mediated by the N-terminal coiled-coil domain (CC) using competitive peptides, representing the helix-2 of the CC, represents a valid therapeutic approach for treating Ph+ leukemia. To further develop competitive peptides for targeting BCR/ABL, we created a membrane permeable helix-2 peptide (MPH-2) by fusing the helix-2 peptide with a peptide transduction tag. In this study, we report that the MPH-2: (i) interacted with BCR/ABL in vivo; (ii) efficiently inhibited the autophosphorylation of BCR/ABL; (iii) suppressed the growth and viability of Ph+ leukemic cells; and (iv) was efficiently transduced into mononuclear cells (MNC) in an in vivo mouse model.
This study provides the first evidence that an efficient peptide transduction system facilitates the employment of competitive peptides to target the oligomerization interface of BCR/ABL in vivo.
Rho GTPases are involved in homing and mobilization of hematopoietic stem and progenitor cells due to their impact on cytoskeleton remodeling. We have previously shown that inhibition of Rho, Rac and Cdc42 clearly impairs adhesion of normal and leukemic hematopoietic progenitor cells (HPC) to fibronectin and migration in a three-dimensional stromal cell model. Here, we identified the Ras GTPase-Activating Protein SH3 Domain-Binding Protein (G3BP) as a target gene of Rho GTPases and analysed its role in regulating HPC motility. Overexpression of G3BP significantly enhanced adhesion of murine 32D HPC to fibronectin and human umbilical vein endothelial cells, increased the proportion of adherent cells in a flow chamber assay and promoted cell migration in a transwell assay and a three-dimensional stromal cell model suggesting a strong impact on the cytoskeleton. Immunofluorescent staining of G3BP-overexpressing fibroblasts revealed a Rho-like phenotype characterized by formation of actin stress fibers in contrast to the Rac-like phenotype of control fibroblasts. This is the first report implicating a role for G3BP in Rho GTPase-mediated signalling towards adhesion and migration of HPC. Our results may be of clinical importance, since G3BP was found overexpressed in human cancers.
Renal perfusion in scleroderma patients assessed by microbubble-based contrast-enhanced ultrasound
(2012)
OBJECTIVES: Renal damage is common in scleroderma. It can occur acutely or chronically. Renal reserve might already be impaired before it can be detected by laboratory findings. Microbubble-based contrast-enhanced ultrasound has been demonstrated to improve blood perfusion imaging in organs. Therefore, we conducted a study to assess renal perfusion in scleroderma patients utilizing this novel technique.
MATERIALS AND METHODOLOGY: Microbubble-based contrast agent was infused and destroyed by using high mechanical index by Siemens Sequoia (curved array, 4.5 MHz). Replenishment was recorded for 8 seconds. Regions of interests (ROI) were analyzed in renal parenchyma, interlobular artery and renal pyramid with quantitative contrast software (CUSQ 1.4, Siemens Acuson, Mountain View, California). Time to maximal Enhancement (TmE), maximal enhancement (mE) and maximal enhancement relative to maximal enhancement of the interlobular artery (mE%A) were calculated for different ROIs.
RESULTS: There was a linear correlation between the time to maximal enhancement in the parenchyma and the glomerular filtration rate. However, the other parameters did not reveal significant differences between scleroderma patients and healthy controls.
CONCLUSION: Renal perfusion of scleroderma patients including the glomerular filtration rate can be assessed using microbubble-based contrast media.
Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3rNutlin10 μM, harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3rNutlin10 μM cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3rNutlin10 μM cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3rNutlin10 μM cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3rNutlin10 μM and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.
Definition: Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are both cholestatic liver diseases. A common feature of these disorders is the accumulation of bile acids in the liver and blood, presumably because of decreased biliary secretion and reduced bile flow.
Etiology: Etiology and pathogenesis of PBC and PSC are still unknown. PBC is considered to be an autoimmune disease. Immunological mechanisms may also be involved in PSC since there is an association with ulcerative colitis and autoantibodies can be detected. Furthermore, genetic factors seem to play an important role in both diseases.
Therapy: Since the pathogenesis of both diseases is unclear, there is no definite causal treatment. However, ursodeoxycholic acid (UDCA) was shown to be highly effective. Other drugs which can be used alone or in combination with UDCA are promising and might further improve the outcome of the diseases.
Diabetic nephropathy (DN) is a major cause of end-stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down-regulated α3-integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored α3 integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin-induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN.
Interleukin (IL)-18, formerly known as interferon (IFN)-γ-inducing factor, is a crucial mediator of host defence and inflammation. Control of IL-18 bioactivity by its endogenous antagonist IL-18 binding protein (IL-18BP) is a major objective of immunoregulation. IL-18BP is strongly up-regulated by IFN-γ, thereby establishing a negative feedback mechanism detectable in cell culture and in vivo. Here we sought to investigate in D.L. Dexter (DLD) colon carcinoma cells molecular mechanisms of IL-18BP induction under the influence of IFN-γ. Mutational analysis revealed that a proximal γ-activated sequence (GAS) at the IL-18BP promoter is of pivotal importance for expression by IFN-γ-activated cells. Use of siRNA underscored the essential role of the signal transducer and activator of transcription (STAT)-1 in this process. Indeed, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis proved STAT1 binding to this particular GAS site. Maximal expression of IL-18BP was dependent on de novo protein synthesis but unaffected by silencing of interferon regulatory factor-1. Altogether, data presented herein indicate that direct action of STAT1 on the IL-18BP promoter at the proximal GAS element is key to IL-18BP expression by IFN-γ-stimulated DLD-1 colon carcinoma cells.
Histone deacetylase (HDAC) inhibitors represent a promising class of antineoplastic agents which affect tumour growth, differentiation and invasion. The effects of the HDAC inhibitor valproic acid (VPA) were tested in vitro and in vivo on pre-clinical renal cell carcinoma (RCC) models. Caki-1, KTC-26 or A498 cells were treated with various concentrations of VPA during in vitro cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and to evaluate cell cycle manipulation. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. The anti-tumoural potential of VPA combined with low-dosed interferon-α (IFN-α) was also investigated. VPA significantly and dose-dependently up-regulated histones H3 and H4 acetylation and caused growth arrest in RCC cells. VPA altered cell cycle regulating proteins, in particular CDK2, cyclin B, cyclin D3, p21 and Rb. In vivo, VPA significantly inhibited the growth of Caki-1 in subcutaneous xenografts, accompanied by a strong accumulation of p21 and bax in tissue specimens of VPA-treated animals. VPA–IFN-α combination markedly enhanced the effects of VPA monotherapy on RCC proliferation in vitro, but did not further enhance the anti-tumoural potential of VPA in vivo. VPA was found to have profound effects on RCC cell growth, lending support to the initiation of clinical testing of VPA for treating advanced RCC.
The manifestation of chronic back pain depends on structural, psychosocial, occupational and genetic influences. Heritability estimates for back pain range from 30% to 45%. Genetic influences are caused by genes affecting intervertebral disc degeneration or the immune response and genes involved in pain perception, signalling and psychological processing. This inter-individual variability which is partly due to genetic differences would require an individualized pain management to prevent the transition from acute to chronic back pain or improve the outcome. The genetic profile may help to define patients at high risk for chronic pain. We summarize genetic factors that (i) impact on intervertebral disc stability, namely Collagen IX, COL9A3, COL11A1, COL11A2, COL1A1, aggrecan (AGAN), cartilage intermediate layer protein, vitamin D receptor, metalloproteinsase-3 (MMP3), MMP9, and thrombospondin-2, (ii) modify inflammation, namely interleukin-1 (IL-1) locus genes and IL-6 and (iii) and pain signalling namely guanine triphosphate (GTP) cyclohydrolase 1, catechol-O-methyltransferase, μ opioid receptor (OPMR1), melanocortin 1 receptor (MC1R), transient receptor potential channel A1 and fatty acid amide hydrolase and analgesic drug metabolism (cytochrome P450 [CYP]2D6, CYP2C9).
Protein catabolism should be reduced and protein synthesis promoted with parenteral nutrion (PN). Amino acid (AA) solutions should always be infused with PN. Standard AA solutions are generally used, whereas specially adapted AA solutions may be required in certain conditions such as severe disorders of AA utilisation or in inborn errors of AA metabolism. An AA intake of 0.8 g/kg/day is generally recommended for adult patients with a normal metabolism, which may be increased to 1.2–1.5 g/kg/day, or to 2.0 or 2.5 g/kg/day in exceptional cases. Sufficient non-nitrogen energy sources should be added in order to assure adequate utilisation of AA. A nitrogen calorie ratio of 1:130 to 1:170 (g N/kcal) or 1:21 to 1:27 (g AA/kcal) is recommended under normal metabolic conditions. In critically ill patients glutamine should be administered parenterally if indicated in the form of peptides, for example 0.3–0.4 g glutamine dipeptide/kg body weight/day (=0.2–0.26 g glutamine/kg body weight/day). No recommendation can be made for glutamine supplementation in PN for patients with acute pancreatitis or after bone marrow transplantation (BMT), and in newborns. The application of arginine is currently not warranted as a supplement in PN in adults. N-acetyl AA are only of limited use as alternative AA sources. There is currently no indication for use of AA solutions with an increased content of glycine, branched-chain AAs (BCAA) and ornithine-α-ketoglutarate (OKG) in all patients receiving PN. AA solutions with an increased proportion of BCAA are recommended in the treatment of hepatic encephalopathy (III–IV).
There are special challenges in implementing parenteral nutrition (PN) in paediatric patients, which arises from the wide range of patients, ranging from extremely premature infants up to teenagers weighing up to and over 100 kg, and their varying substrate requirements. Age and maturity-related changes of the metabolism and fluid and nutrient requirements must be taken into consideration along with the clinical situation during which PN is applied. The indication, the procedure as well as the intake of fluid and substrates are very different to that known in PN-practice in adult patients, e.g. the fluid, nutrient and energy needs of premature infants and newborns per kg body weight are markedly higher than of older paediatric and adult patients. Premature infants <35 weeks of pregnancy and most sick term infants usually require full or partial PN. In neonates the actual amount of PN administered must be calculated (not estimated). Enteral nutrition should be gradually introduced and should replace PN as quickly as possible in order to minimise any side-effects from exposure to PN. Inadequate substrate intake in early infancy can cause long-term detrimental effects in terms of metabolic programming of the risk of illness in later life. If energy and nutrient demands in children and adolescents cannot be met through enteral nutrition, partial or total PN should be considered within 7 days or less depending on the nutritional state and clinical conditions.
Cardiovascular diseases account for more than half of total mortality before the age of 75 in industrialized countries. To develop therapies promoting the compensatory growth of blood vessels could be superior to palliative surgical surgical interventions. Therefore, much effort has been put into investigating underlying mechanisms. Depending on the initial trigger, growth of blood vessels in adult organisms proceeds via two major processes, angiogenesis and arteriogenesis. While angiogenesis is induced by hypoxia and results in new capillaries, arteriogenesis is induced by physical forces, most importantly fluid shear stress. Consequently, chronically elevated fluid shear stress was found to be the strongest trigger under experimental conditions. Arteriogenesis describes the remodelling of pre-existing arterio-arteriolar anastomoses to completely developed and functional arteries. In both growth processes, enlargement of vascular wall structures was proposed to be covered by proliferation of existing wall cells. Recently, increasing evidence emerges, implicating a pivotal role for circulating cells, above all blood monocytes, in vascular growth processes. Since it has been shown that monocytes/macrophage release a cocktail of chemokines, growth factors and proteases involved in vascular growth, their contribution seems to be of a paracrine fashion. A similar role is currently discussed for various populations of bone-marrow derived stem cells and endothelial progenitors. In contrast, the initial hypothesis that these cells -after undergoing a (trans-)differentiation- contribute by a structural integration into the growing vessel wall, is increasingly challenged.
FTY720 is a novel immunosuppressive drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. In its phosphorylated form FTY720 is a potent S1P receptor agonist. Recently it was also shown that FTY720 can reduce prostaglandin synthesis through the direct inhibition of the cytosolic phospholipase A2 (cPLA2). Since prostaglandins are important mediators of nociception, we studied the effects of FTY720 in different models of nociception. We found that intraperitoneal administration of FTY720 reduced dose-dependently the nociceptive behaviour of rats in the formalin assay. Although the antinociceptive doses of FTY720 were too low to alter the lymphocyte count, prostanoid concentrations in the plasma were dramatically reduced. Surprisingly, intrathecally administered FTY720 reduced the nociceptive behaviour in the formalin assay without altering spinal prostaglandin synthesis, indicating that additional antinociceptive mechanisms beside the inhibition of prostaglandin synthesis are involved. Accordingly, FTY720 reduced also the nociceptive behaviour in the spared nerve injury model for neuropathic pain which does not depend on prostaglandin synthesis. In this model the antinociceptive effect of FTY720 was similar to gabapentin, a commonly used drug to treat neuropathic pain. Taken together we show for the first time that FTY720 possesses antinociceptive properties and that FTY720 reduces nociceptive behaviour during neuropathic pain.
The 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes. We have previously shown that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) activates 5-LO transcription via recruitment of Sp1, Sp3 and RNA polymerase II to the proximal promoter. To identify the HDACs involved in the regulation of 5-LO promoter activity isoform-specific HDAC inhibitors were applied. 5-LO promoter activity and mRNA expression were up-regulated by the class I HDAC inhibitors apicidin and MS-275 but not by class II inhibitors. Knockdown of HDAC 1, 2 and 3 revealed that HDAC2 and HDAC3 but not HDAC1 is involved in the up-regulation of 5-LO mRNA expression. To analyse the chromatin modifications at the 5-LO promoter associated with HDAC inhibition, the time course of 5-LO mRNA induction by trichostatin A was investigated and the concomitant changes in histone modifications at the 5-LO promoter in HL-60, U937 and Mono Mac6 cells were determined. Chromatin immunoprecipitation analysis revealed that trichostatin A increases acetylation of histones H3 and H4 at the 5-LO core promoter in HL-60 and U937 cells whereas no significant changes were observed in Mono Mac6 cells. The appearance of H3 and H4 acetylation preceded the 5-LO mRNA induction whereas in all three cell lines, induction of 5-LO mRNA expression correlated with histone H3 lysine 4 trimethylation (H3K4me3), a marker for transcriptional activity of gene promoters.
Angiogenesis is essential for tumor growth and progression. It has been demonstrated that tumor growth beyond a size 1 to 2 mm3 requires the induction of new vessels. Angiogenesis is regulated by several endogenous stimulators and inhibitors of endothelial cell migration, proliferation and tube formation. Under physiological conditions these mediators of endothelial cell growth are in balance and vessel growth is limited. In fact, within the angiogenic balance endothelial cell turnover is sufficient to maintain a functional vascular wall but does not allow vessel growth. Tumor growth an progression has successfully been correlated to the serum concentration of angiogenic mediators. Furthermore, the vascular density of tumor tissues could be correlated to the clinical course of the disease in several tumor entities. Within the last years several new mediators of endothelial cell growth have been isolated e.g. angiopoietin 1, angiopoietin 2, midkine, pleiotropin, leptin and maspin. In this review we discuss the mechanisms leading to tumor angiogenesis and describe some of the newer mediators of endothelial cell stimulation and inhibition.
Although motor tasks at most times do not require much attention, there are findings that attention can alter neuronal activity not only in higher motor areas but also within the primary sensorimotor cortex. However, these findings are equivocal as attention effects were investigated only in either the dominant or the nondominant hand; attention was operationalized either as concentration (i.e., attention directed to motor task) or as distraction (i.e., attention directed away from motor task), the complexity of motor tasks varied and almost no left-handers were studied. Therefore, in this study, both right- and left-handers were investigated with an externally paced button press task in which subjects typed with the index finger of the dominant, nondominant, or both hands. We introduced four different attention levels: attention-modulation-free, distraction (counting backward), concentration on the moving finger, and divided concentration during bimanual movement. We found that distraction reduced neuronal activity in both contra- and ipsilateral primary sensorimotor cortex when the nondominant hand was tapping in both handedness groups. At the same time, distraction activated the dorsal frontoparietal attention network and deactivated the ventral default network. We conclude that difficulty and training status of both the motor and cognitive task, as well as usage of the dominant versus the nondominant hand, are crucial for the presence and magnitude of attention effects on sensorimotor cortex activity. In the case of a very simple button press task, attention modulation is seen for the nondominant hand under distraction and in both handedness groups.
Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice
(2007)
Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E2 synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE2 synthase-1 (mPGES-1) isomerizes COX-2-derived PGH2 to PGE2. Here, we evaluated the effect of mPGES-1-deficiency on the noci-ceptive behavior in various models of nociception that depend on PGE2 synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE2 synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE2 synthesis to PGD2, PGF2α and 6-keto-PGF1α (stable metabolite of PGI2). Since the latter prostaglandins serve also as mediators of noci-ception they may compensate the loss of PGE2 synthesis in mPGES-1-deficient mice.
ADAM15 belongs to a family of transmembrane multi-domain proteins implicated in proteolysis, cell–cell and cell–matrix interactions in various disease conditions. In osteoarthritis (OA), ADAM15 is up-regulated in the chondrocytes already at early stages of cartilage degeneration where it seems to exert homeostatic effects likely associated with its ability to enhance integrin-mediated chondrocyte adhesion to the surrounding collagen matrix. The aim of our present study was, therefore, to characterize functional domains of ADAM15 involved in collagen II (CII) interaction and to analyse associated outside-in signalling events. Accordingly, ADAM15 and respective deletion mutants were stably transfected into the chondrocyte cell line T/C28a4. Transfected cells were adhered to CII and phosphoproteins analysed by Western blotting. Co-immunoprecipitation served to identify protein binding to ADAM15. Our results elucidate the prodomain as critical for the capacity of ADAM15 to enhance CII adhesion, thereby identifying for the first time a cell-adhesive role of a metalloproteinase prodomain. Moreover, the cytoplasmic tail of ADAM15 confers a modulatory effect on the autophosphorylation site Y397 of the focal adhesion kinase (FAK) during chondrocyte–collagen interaction. In conclusion, the newly uncovered impact of ADAM15 on signalling events that arise from chondrocyte interactions with its collagen matrix might contribute to the elucidation of the mechanism underlying its proposed chondroprotective role in degenerative cartilage disease.
Progranulin haploinsufficiency is associated with frontotemporal dementia in humans. Deficiency of progranulin led to exaggerated inflammation and premature aging in mice. The role of progranulin in adaptations to nerve injury and neuropathic pain are still unknown. Here we found that progranulin is up-regulated after injury of the sciatic nerve in the mouse ipsilateral dorsal root ganglia and spinal cord, most prominently in the microglia surrounding injured motor neurons. Progranulin knockdown by continuous intrathecal spinal delivery of small interfering RNA after sciatic nerve injury intensified neuropathic pain-like behaviour and delayed the recovery of motor functions. Compared to wild-type mice, progranulin-deficient mice developed more intense nociceptive hypersensitivity after nerve injury. The differences escalated with aging. Knockdown of progranulin reduced the survival of dissociated primary neurons and neurite outgrowth, whereas addition of recombinant progranulin rescued primary dorsal root ganglia neurons from cell death induced by nerve growth factor withdrawal. Thus, up-regulation of progranulin after neuronal injury may reduce neuropathic pain and help motor function recovery, at least in part, by promoting survival of injured neurons and supporting regrowth. A deficiency in this mechanism may increase the risk for injury-associated chronic pain.
New histone deacetylase inhibitors as potential therapeutic tools for advanced prostate carcinoma
(2008)
The anti-epileptic drug valproic acid is also under trial as an anti-cancer agent due to its histone deacetylase (HDAC) inhibitory properties. However, the effects of valproic acid (VPA) are limited and concentrations required for exerting anti-neoplastic effects in vitro may not be reached in tumour patients. In this study, we tested in vitro and in vivo effects of two VPA-derivatives (ACS2, ACS33) on pre-clinical prostate cancer models. PC3 and DU-145 prostate tumour cell lines were treated with various concentrations of ACS2 or ACS33 to perform in vitro cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and to evaluate tumour cell adhesion to endothelial cell monolayers. Analysis of acetylated histones H3 and H4 protein expression was performed by western blotting. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. Tumour sections were assessed by immunohistochemistry for histone H3 acetylation and proliferation. ACS2 and ACS33 significantly up-regulated histone H3 and H4 acetylation in prostate cancer cell lines. In micromolar concentrations both compounds exerted growth arrest in PC3 and DU-145 cells and prevented tumour cell attachment to endothelium. In vivo, ACS33 inhibited the growth of PC3 in subcutaneous xenografts. Immunohistochemistry and western blotting confirmed increased histone H3 acetylation and reduced proliferation. ACS2 and ACS33 represent novel VPA derivatives with superior anti-tumoural activities, compared to the mother compound. This investigation lends support to the clinical testing of ACS2 or ACS33 for the treatment of prostate cancer.
Ubiquitylation in immune disorders and cancer: from molecular mechanisms to therapeutic implications
(2012)
Conjugation of ubiquitin to proteins (ubiquitylation) has emerged to be one of the most crucial post-translational modifications controlling virtually all cellular processes. What was once regarded as a mere signal for protein degradation has turned out to be a major regulator of molecular signalling networks. Deregulation of ubiquitin signalling is closely associated with various human pathologies. Here, we summarize the current knowledge of ubiquitin signalling in immune deficiencies and cancer as well as the available therapeutic strategies targeting the ubiquitin system in combating these pathogenic conditions.
Aging is a complex process that is linked to an increased incidence of major diseases such as cardiovascular and neurodegenerative disease, but also cancer and immune disorders. MicroRNAs (miRNAs) are small non-coding RNAs, which post-transcriptionally control gene expression by inhibiting translation or inducing degradation of targeted mRNAs. MiRNAs target up to hundreds of mRNAs, thereby modulating gene expression patterns. Many miRNAs appear to be dysregulated during cellular senescence, aging and disease. However, only few miRNAs have been so far linked to age-related changes in cellular and organ functions. The present article will discuss these findings, specifically focusing on the cardiovascular and neurological systems.
Background and Aims: The Roadmap concept is a therapeutic framework in chronic hepatitis B for the intensification of nucleoside analogue monotherapy based on early virologic response. The efficacy and safety of this approach applied to telbivudine treatment has not been investigated.
Methods: A multinational, phase IV, single-arm open-label study (ClinicalTrials.gov ID NCT00651209) was undertaken in HBeAg-positive, nucleoside-naive adult patients with chronic hepatitis B. Patients received telbivudine (600 mg once-daily) for 24 weeks, after which those with undetectable serum HBV DNA (<300 copies/mL) continued to receive telbivudine alone while those with detectable DNA received telbivudine plus tenofovir (300 mg once-daily). Outcomes were assessed at Week 52.
Results: 105 patients commenced telbivudine monotherapy, of whom 100 were included in the efficacy analysis. Fifty-five (55%) had undetectable HBV DNA at Week 24 and continued telbivudine monotherapy; 45 (45%) received tenofovir intensification. At Week 52, the overall proportion of undetectable HBV DNA was 93% (93/100) by last-observation-carried-forward analysis (100% monotherapy group, 84% intensification group) and no virologic breakthroughs had occurred. ALT normalization occurred in 77% (87% monotherapy, 64% intensification), HBeAg clearance in 43% (65% monotherapy, 16% intensification), and HBeAg seroconversion in 39% (62% monotherapy, 11% intensification). Six patients had HBsAg clearance. Myalgia was more common in the monotherapy group (19% versus 7%). No decrease in the mean glomerular filtration rate occurred in either treatment group at Week 52.
Conclusions: Telbivudine therapy with tenofovir intensification at Week 24, where indicated by the Roadmap strategy, appears effective and well tolerated for the treatment of chronic hepatitis B.
Trial Registration: ClinicalTrials.gov NCT00651209
Background: Osteoinductive bone substitutes are defined by their ability to induce new bone formation even at heterotopic implantation sites. The present study was designed to analyze the potential osteoinductivity of two different bone substitute materials in caprine muscle tissue.
Materials and methods: One gram each of either a porous beta-tricalcium phosphate (β-TCP) or an hydroxyapatite/silicon dioxide (HA/SiO2)-based nanocrystalline bone substitute material was implanted in several muscle pouches of goats. The biomaterials were explanted at 29, 91 and 181 days after implantation. Conventional histology and special histochemical stains were performed to detect osteoblast precursor cells as well as mineralized and unmineralized bone matrix.
Results: Both materials underwent cellular degradation in which tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells and TRAP-negative multinucleated giant cells were involved. The ß-TCP was completely resorbed within the observation period, whereas some granules of the HA-groups were still detectable after 180 days. Neither osteoblasts, osteoblast precursor cells nor extracellular bone matrix were found within the implantation bed of any of the analyzed biomaterials at any of the observed time points.
Conclusions: This study showed that ß-TCP underwent a faster degradation than the HA-based material. The lack of osteoinductivity for both materials might be due to their granular shape, as osteoinductivity in goat muscle has been mainly attributed to cylindrical or disc-shaped bone substitute materials. This hypothesis however requires further investigation to systematically analyze various materials with comparable characteristics in the same experimental setting.
Background: Regulatory T cells (Treg) expressing the transcription factor forkhead-box protein P3 (Foxp3) have been identified to counteract anti-tumor immune responses during tumor progression. Besides, Foxp3 presentation by cancer cells itself may also allow them to evade from effector T-cell responses, resulting in a survival benefit of the tumor. For colorectal cancer (CRC) the clinical relevance of Foxp3 has not been evaluated in detail. Therefore the aim of this study was to study its impact in colorectal cancer (CRC).
Methods and Findings: Gene and protein analysis of tumor tissues from patients with CRC was performed to quantify the expression of Foxp3 in tumor infiltrating Treg and colon cancer cells. The results were correlated with clinicopathological parameters and patients overall survival. Serial morphological analysis demonstrated Foxp3 to be expressed in cancer cells. High Foxp3 expression of the cancer cells was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in overall patient survival.
Conclusions: Our findings strongly suggest that Foxp3 expression mediated by cancer cells rather than by Treg cells contribute to disease progression.
BACKGROUND: The growing body of data on prevalence of complementary and alternative medicine (CAM) usage means there is a need to standardize measurement on an international level. An international team has published a questionnaire0020 (I-CAM-Q), but no validation has yet been provided. The aim of the present study was to provide a German measurement instrument for CAM usage (I-CAM-G) which closely resembles the original English version, and to assess it's performance in two potential samples for measuring CAM usage.
METHODS: The English I-CAM-Q questionnaire was translated into German, and adapted slightly. The resulting I-CAM-G questionnaire was then pre-tested on N=16 healthy volunteers, and 12 cognitive interviews were carried out. The questionnaire was employed in a sample of breast cancer patients (N=92, paper and pencil), and a sample from the general population (N=210, internet survey). Descriptive analyses of items and missing data, as well as results from the cognitive interviews, are presented in this paper.
RESULTS: The translated questionnaire had to be adapted to be consistent with the German health care system. All items were comprehensible, whereby some items were unambiguous (e.g. CAM use yes/no, helpfulness), while others gave rise to ambiguous answers (e.g. reasons for CAM use), or high rates of missing data (e.g. number of times the CAM modality had been used during the last 3 months). 78% of the breast cancer patients and up to 85% of a sample of the general population had used some form of CAM.
CONCLUSIONS: Following methodologically sound and comprehensive translation, adaptation and assessment processes using recognized translation procedures, cognitive interviews, and studying the performance of the questionnaire in two samples, we arrived at a German questionnaire for measuring CAM use which is comparable with the international (English) version. The questionnaire appropriately measures CAM use, with some items being more appropriate than others. We recommend the development of a short version.
Background: Group-based social skills training (SST) has repeatedly been recommended as treatment of choice in high-functioning autism spectrum disorder (HFASD). To date, no sufficiently powered randomised controlled trial has been performed to establish efficacy and safety of SST in children and adolescents with HFASD. In this randomised, multi-centre, controlled trial with 220 children and adolescents with HFASD it is hypothesized, that add-on group-based SST using the 12 weeks manualised SOSTA–FRA program will result in improved social responsiveness (measured by the parent rated social responsiveness scale, SRS) compared to treatment as usual (TAU). It is further expected, that parent and self reported anxiety and depressive symptoms will decline and pro-social behaviour will increase in the treatment group. A neurophysiological study in the Frankfurt HFASD subgroup will be performed pre- and post treatment to assess changes in neural function induced by SST versus TAU.
Methods/design: The SOSTA – net trial is designed as a prospective, randomised, multi-centre, controlled trial with two parallel groups. The primary outcome is change in SRS score directly after the intervention and at 3 months follow-up. Several secondary outcome measures are also obtained. The target sample consists of 220 individuals with ASD, included at the six study centres.
Discussion: This study is currently one of the largest trials on SST in children and adolescents with HFASD worldwide. Compared to recent randomised controlled studies, our study shows several advantages with regard to in- and exclusion criteria, study methods, and the therapeutic approach chosen, which can be easily implemented in non-university-based clinical settings.
Trial registration: ISRCTN94863788 – SOSTA – net: Group-based social skills training in children and adolescents with high functioning autism spectrum disorder.
Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues.
The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.
Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta-cells in the pancreas. Recruitment of inflammatory cells is prerequisite to beta-cell-injury. The junctional adhesion molecule (JAM) family proteins JAM-B and JAM–C are involved in polarized leukocyte transendothelial migration and are expressed by vascular endothelial cells of peripheral tissue and high endothelial venules in lympoid organs. Blocking of JAM-C efficiently attenuated cerulean-induced pancreatitis, rheumatoid arthritis or inflammation induced by ischemia and reperfusion in mice. In order to investigate the influence of JAM-C on trafficking and transmigration of antigen-specific, autoaggressive T-cells, we used transgenic mice that express a protein of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen in the β-cells of the islets of Langerhans under the rat insulin promoter (RIP). Such RIP-LCMV mice turn diabetic after infection with LCMV. We found that upon LCMV-infection JAM-C protein was upregulated around the islets in RIP-LCMV mice. JAM-C expression correlated with islet infiltration and functional beta-cell impairment. Blockade with a neutralizing anti-JAM-C antibody reduced the T1D incidence. However, JAM-C overexpression on endothelial cells did not accelerate diabetes in the RIP-LCMV model. In summary, our data suggest that JAM-C might be involved in the final steps of trafficking and transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans.
Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.
Targeted drugs have significantly improved the therapeutic options for advanced renal cell carcinoma (RCC). However, resistance often develops, negating the benefit of these agents. In the present study, the molecular mechanisms of acquired resistance towards the histone deacetylase (HDAC) inhibitor valproic acid (VPA) in a RCC in vivo model were investigated. NMRI:nu/nu mice were transplanted with Caki-1 RCC cells and then treated with VPA (200 mg/kg/day). Controls remained untreated. Based on tumor growth dynamics, the mice were divided into “responders” and “non-responders” to VPA. Histone H3 and H4 acetylation and expression of cell signaling and cell cycle regulating proteins in the RCC mouse tumors were evaluated by Western blotting. Tumor growth of VPA responders was significantly diminished, whereas that of VPA non-responders even exceeded control values. Cdk1, 2 and 4 proteins were strongly enhanced in the non-responders. Importantly, Akt expression and activity were massively up-regulated in the tumors of the VPA non-responders. Chronic application (12 weeks) of VPA to Caki-1 cells in vitro evoked a distinct elevation of Akt activity and cancer cells no longer responded with cell growth reduction, compared to the short 2 week treatment. We assume that chronic use of an HDAC-inhibitor is associated with (re)-activation of Akt, which may be involved in resistance development. Consequently, combined blockade of both HDAC and Akt may delay or prevent drug resistance in RCC.