Molecular mechanism of action and pharmacokinetic properties of methotrexate.

Since its discovery in 1945, methotrexate has become a standard therapy for number of diseases, including oncological, inflammatory and pulmonary ones. Major physiological interactions of methotrexate include folate pathway, adenosine, prostaglandins, leukotrienes and cytokines. Methotrexate is used in treatment of pulmonary sarcoidosis as a second line therapy and is drug of choice in patients who are not candidates for corticosteroid therapy, with recommended starting weekly dose of 5-15 mg. Number of studies dealt with methotrexate use in rheumatoid arthritis and oncological patients. Authors are conducting research on oral methotrexate use and pharmacokinetics in chronic sarcoidosis patients and have performed literature research to better understand molecular mechanisms of methotrexate action as well as high level pharmacokinetic considerations. Polyglutamation of methotrexate affects its pharmacokinetic and pharmacodynamic properties and prolongs its effect. Bile excretion plays significant role due to extensive enterohepatic recirculation, although majority of methotrexate is excreted through urine. Better understanding of its pharmacokinetic properties in sarcoidosis patients warrant optimizing therapy when corticosteroids are contraindicated in these patients.

Potentials of human bile acids and their salts in pharmaceutical nano delivery and formulations adjuvants.

In the last decade, the attention of the scientific community has been focused on bile acids and their salts as systems for the transportation of drugs; specifically their role as carriers and integration into nanomedicine. Bile acids can play a critical role as drug carriers in the form of chemical conjugates, complexation, mixed micelles formation as well as stabilized bile acid liposomes (bilosomes). The unique molecular structure and interaction of these amphiphilic-steroidal compounds make them an interesting subject of research. This review is based on literature research in order to emphasize the importance of bile acids and their salts as absorption modulators in order to improve therapeutic potentials of low bioavailability drugs.

Increased expression of αv integrin as a regulator of fibrosis in Dupuytren's nodules.

Although Dupuytren's contracture is characterized by increased transforming growth factor-ß1 (TGF-ß1) and fibrosis in the palmar fascia, the relationship between TGF-ß1 and integrins, which are considered to be related to fibrosis, remains unclear. We investigated the involvement of TGF-ß1 and integrins in the pathological palmar fascia of Dupuytren's contracture. Seven patients underwent partial fasciectomy for treatment of this disease. The nodule and cord were isolated from the fascial tissues of the patients. Control fasciae were obtained from seven patients with carpal tunnel syndrome. Immunohistochemical analysis was performed to detect the fibrosis marker α-smooth muscle actin and integrins in the fascial tissues. The expression of TGF-ß1 and integrins was assessed by real-time polymerase chain reaction. The results suggest that nodules may be areas involved in activation of fibrosis in the fascia, associated with increased expression of TGF-ß1 and αv integrin. Thus, αv integrin may contribute to fibrosis in Dupuytren's contracture by activating TGF-ß1. LEVEL OF EVIDENCE: IV.

Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell-related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group-namely, the tolerant recipients-were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS-independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross-validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

Docking-based preliminary study on the interactions of bile acids with drugs at the transporter level in intestinal bacteria.

OBJECTIVE: The aim of this study was to estimate the binding-affinities of different bile acids towards drug transporters in Lactobacillus acidophilus and Bifidobacterium longum in order to predict the influence of bile acids and probiotics interactions on drug pharmacokinetics. MATERIALS AND METHODS: In order to study interactions of bile acids with transporters of intestinal bacteria, molecular-docking step was performed, using SwissDock web-service. For the purpose of comparison, two natural bile acids, cholic acid (CA) and deoxycholic acid (DCA), and one semi-synthetic bile acid, 12-monoketocholic acid (MKC), were studied in parallel. The free-binding energy was used as the main criterion for ranking ligands. RESULTS: Studied bile acids exhibited different binding affinities towards bacterial transporters with MKC showing the most prominent effect. For the majority of studied transporters, the estimated affinities of bile acids decreased in the following order: MKC-CA-DCA. Namely, 38.7% of examined transport proteins gave the lowest free-binding energy with MKC. The weak inverse relationship between number of hydrogen bonds and estimated free-binding energies was revealed. CONCLUSIONS: The predominant effect of MKC for the majority of studied transport proteins suggests that keto group at carbon 12 of the steroid core has a significant influence on the properties of MKC and consequently, on interactions with membrane transporters. Present findings might have a role in the prediction of potential influence of bile acids and probiotics on drug pharmacokinetics.

Phenotypic characteristics associated with reduced short physical performance battery score in COPD.

BACKGROUND: The Short Physical Performance Battery (SPPB) is commonly used in gerontology, but its determinants have not been previously evaluated in COPD. In particular, it is unknown whether pulmonary aspects of COPD would limit the value of SPPB as an assessment tool of lower limb function. METHODS: In 109 patients with COPD, we measured SPPB score, spirometry, 6-min walk distance, quadriceps strength, rectus femoris cross-sectional area, fat-free mass, physical activity, health status, and Medical Research Council dyspnea score. In a subset of 31 patients with COPD, a vastus lateralis biopsy was performed, and the biopsy specimen was examined to evaluate the structural muscle characteristics associated with SPPB score. The phenotypic characteristics of patients stratified according to SPPB were determined. RESULTS: Quadriceps strength and 6-min walk distance were the only independent predictors of SPPB score in a multivariate regression model. Furthermore, while age, dyspnea, and health status were also univariate predictors of SPPB score, FEV 1 was not. Stratification by reduced SPPB score identified patients with locomotor muscle atrophy and increasing impairment in strength, exercise capacity, and daily physical activity. Patients with mild or major impairment defined as an SPPB score < 10 had a higher proportion of type 2 fibers (71% [14] vs 58% [15], P = .04). CONCLUSIONS: The SPPB is a valid and simple assessment tool that may detect a phenotype with functional impairment, loss of muscle mass, and structural muscle abnormality in stable patients with COPD.

Les electrisations : a propos de 35 cas

Introduction: L'electrisation est rare mais grave. Elle met en jeu le pronostic fonctionnel et vital. Quelles sont les populations les plus affectees; les prejudices qu'elle provoquent et les principales mesures de traitement a appliquer ? Les objectifs etaient d'identifier la population exposee et de rappeler les principes du traitement chirurgical ainsi que les mesures preventives.Materiel et methode : il s 'agissait d'une etude retrospective et analytique realisee de janvier 2011 a decembre 2013; chez des patients; age et sexe confondus; ayant presente une electrisation et dont les dossiers etaient exploitables Resultats et discussion : trente cinq patients; d'une moyenne d'age de 26 ans avec une predominance masculine; ont ete colliges. Les electrisations etaient secondaires a des accidents de travail (48;6) ou domestiques (37;1) dont la majorite etait due a un courant de bas voltage. La surface cutanee brulee moyenne etait de 10;6 de la SCT. Les brulures etaient profondes et siegeaient principalement aux membres superieurs. Le traitement chirurgical etait constitue d'incisions de decharges; d'aponevrotomies; de debridements ou d'excisions iteratifs; d'amputations et de greffes de peau mince. Cinq patients sont decedes. La duree moyenne d'hospitalisation etait de 66 jours.Un retard et une prise en charge inadaptee aggravent le pronostic fonctionnel et vital. La prevention demeure un moyen fondamental pour remedier aux electrisations

Osteopontin Deficiency Suppresses Tumor Necrosis Factor-α-Induced Apoptosis in Chondrocytes.

OBJECTIVE: Apoptosis of chondrocytes in articular cartilage has been observed in rheumatoid arthritis patients. However, molecules involved in such chondrocyte apoptosis in arthritic joints have not been fully understood. We previously observed that apoptosis of chondrocytes is enhanced in a murine arthritis model induced by injection with anti-type II collagen antibodies and lipopolysaccharide (mAbs/LPS), and osteopontin (OPN) deficiency suppresses chondrocyte apoptosis in this arthritis model in vivo. To understand how OPN deficiency renders resistance against chondrocyte apoptosis, we examined the cellular basis for this protection. DESIGN: Chondrocytes were prepared from wild-type and OPN-deficient mouse ribs, and tumor necrosis factor (TNF)-α-induced cell death was examined based on lactate dehydrogenase (LDH) release assay and TUNEL assay. RESULTS: TNF-α treatment induced LDH release in wild-type chondrocytes, while OPN deficiency suppressed such LDH release in the cultures of these cells. TNF-α-induced increase in the number of TUNEL-positive cells was observed in wild-type chondrocytes, while OPN deficiency in chondrocytes suppressed the TNF-α induction of TUNEL-positive cells. OPN deficiency suppressed TNF-α-induced increase in caspase-3 activity in chondrocytes in culture. Furthermore, OPN overexpression in chondrocytes enhanced TNF-α-induced apoptosis. CONCLUSION: These results indicated that the presence of OPN in chondrocytes is involved in the susceptibility of these cells to TNF-α-induced apoptosis.

Osteopontin is involved in migration of eosinophils in asthma.

BACKGROUND: Osteopontin (OPN) is an extracellular matrix protein with a wide range of functions, and is involved in various inflammatory diseases. However, the role of OPN in eosinophilic airway inflammation is unclear. OBJECTIVE: To elucidate the role of OPN in eosinophilic airway inflammation. METHODS: OPN protein levels in induced sputum from asthmatic patients and healthy controls were measured. Eosinophil migration assays were performed in the presence or absence of OPN, a blocking antibody (Ab) recognizing its integrin-binding domain (2K1) and an anti-integrin alpha 4 Ab (P1H4). In the mouse asthma model, the levels of eosinophilia were examined in bronchoalveolar lavage fluids (BALFs) from ovalbumin (OVA)-sensitized and -challenged mice with or without administration of an Ab (M5) corresponding to human 2K1. RESULTS: Levels of OPN in induced sputum were significantly higher in asthmatic patients when compared with healthy controls. In addition, levels of OPN were correlated with the percentage of sputum eosinophils. OPN induced significant migration of human eosinophils and this effect was inhibited by 2K1 and P1H4. M5 significantly attenuated OVA-induced eosinophilia in BALFs. CONCLUSION: These results indicate that OPN plays a role in the migration of eosinophils into the airways and may be involved in the pathogenesis of asthma.

WITHDRAWN: P.P.2 04 Chronological observation of glucose intolerance in myotonic dystrophy.

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Tumor lysis syndrome and acute renal failure--an increasing spectrum of presentations.

Tumor lysis syndrome has historically been associated with hyperuricemia and uric acid crystal deposition. We present three cases of tumor lysis syndrome resulting in renal failure in the context of normouricema, highlighting the spectrum of clinical presentations and mechanisms of renal damage. Two cases occurred following the treatment of hematological malignancies and were associated with hyperphosphatemia; the third resulted from ischemic necrosis following transarterial chemoembolization of a hepatic tumor. We also discuss the role of renal biopsy in the investigation of tumor lysis syndrome.

Bioavailability and hypoglycemic activity of the semisynthetic bile acid salt, sodium 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholanate, in healthy and diabetic rats.

Previous studies in our laboratory have shown that the semisynthetic bile acid derivative, sodium 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholanate (MKC), has hypoglycemic activity. The aim of this study was to investigate the relationship between the pharmacokinetics and hypoglycemic activity of MKC in healthy and diabetic rats. Groups of healthy and alloxan-induced diabetic rats were dosed intravenously (i.v.) and orally with MKC (4 mg/kg). Blood samples were taken before administration of the dose and at 20, 40, 60, 80, 120, 150, 180, 210 and 240 minutes post-dose. MKC serum concentration was measured by HPLC, and pharmacokinetic parameters determined using the WinNonlin program. The absolute bioavailability of MKC was found to be low in healthy and diabetic rats (29 and 23% respectively) and was not significantly different between the two groups. Mean residence time (MRT), volume of distribution (Vd) and half-life (t1/2) of MKC after oral administration were significantly lower in diabetic than in healthy rats (21, 31 and 29% respectively). After the i.v. dose, the change in blood glucose concentration was not significant in either healthy or diabetic rats. After the oral dose, the decrease in blood glucose concentration was significant, reaching a maximum decrease from baseline of 24% in healthy rats and 15% in diabetic rats. The results suggest that a first-pass effect is crucial for the hypoglycemic activity of MKC, indicating that a metabolite of MKC and/or interference with metabolism and glucose transport is responsible.

High plasma concentrations of osteopontin in patients with interstitial pneumonia.

Osteopontin (OPN) produced by alveolar macrophages functions as a fibrogenic cytokine in the development of bleomycin (BLM)-induced murine pulmonary fibrosis, and OPN mRNA is expressed on lung tissues from patients with idiopathic pulmonary fibrosis (IPF). The present study investigates plasma OPN levels in human interstitial pneumonia (IP) and their relationships with disease severity by analyzing the correlation between plasma OPN concentrations and pulmonary functions. The concentrations of OPN in plasma were measured in 17 patients with IP, in 9 with sarcoidosis and in 20 healthy controls using an antigen-capture enzyme-linked immunosorbent assay. The concentrations of OPN in plasma were significantly higher in IP patients than in those with sarcoidosis or in controls. Based on a Receiver Operating Characteristic curve analysis, cut-off points between 300 and 380 ng/ml discriminated between IP and control subjects with 100% sensitivity and 100% specificity. In such case, the sensitivity for sarcoidosis decreased (55.5-33.3%) in cut-offs with 100% specificity. Plasma OPN levels inversely and closely correlated with arterial oxygen tension (PaO2) in patients with IP. Immunohistochemically, OPN was localized predominantly in macrophages and airway epithelium. These findings suggest that plasma OPN levels were found to be associated with the presence of IP, and that OPN play an important role in the development of IP.

Osteopontin is involved in the initiation of cutaneous contact hypersensitivity by inducing Langerhans and dendritic cell migration to lymph nodes.

Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and alphav integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.

Lymphoepithelioma-like carcinoma of rectum: possible relation with EBV.

Lymphoepithelioma-like carcinoma (LEC) of the colon is very rare. Here we report a case of LEC originating in the rectum that was closely associated with Epstein-Barr virus (EBV) infection. The histologic and immunohistologic features, namely, poorly differentiated adenocarcinoma with lymphoid stroma, showed this tumor to be an LEC. The EBV genome was detected by PCR using DNA obtained from tumor tissue sections. Immunohistochemically, EBV-determined nuclear antigen 2 was detected in the tumor cells, and in situ hybridization using EBV-encoded small RNAs probe showed positive labeling in some tumor cells together with a few stromal lymphoid cells. There are some reports of LEC cases that originated in the colon; however, a relation with EBV was not demonstrated. We report here a case of LEC of the rectum demonstrating a possible relation with EBV.

Hereditary predisposition to low interleukin-10 production in children with extended oligoarticular juvenile idiopathic arthritis.

OBJECTIVE: To determine whether children with extended oligoarticular juvenile idiopathic arthritis (JIA) produce less of the anti-inflammatory cytokine interleukin-10 (IL-10) than those with persistent oligoarticular JIA. METHODS: We measured IL-10 production in the parents of children with oligoarticular or extended oligoarticular JIA, from whole-blood cultures stimulated with lipopolysaccharide. RESULTS: IL-10 production was lower in the parents of children with extended oligoarticular JIA compared with those of children with oligoarticular JIA (P=0.034). There was an increase in the percentage of ATA-containing genotypes (i.e. genotypes ATA/ATA, ATA/ACC or ATA/GCC) in the parents of children with extended oligoarticular JIA compared with healthy controls (P<0.02) but not in the parents of children with oligoarticular JIA. CONCLUSIONS: As approximately 84% of the variation in IL-10 production is thought to be genetically regulated, these results suggest that stimulated IL-10 production would be lower in children with extended oligoarticular JIA. Because IL-10 is an anti-inflammatory cytokine, this may partly explain why this group of children has more severe disease.

Role of osteopontin in the pathogenesis of bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is initiated by migration, adhesion, and proliferation of fibroblasts. Osteopontin (OPN) is one of the cytokines produced by activated macrophages and mediates various functions, including cell attachment and migration, by interacting with alphav integrin. In this study, we have investigated the role of OPN in the pathogenesis of pulmonary fibrosis. We developed a mouse model for pulmonary fibrosis by intratracheal instillation of bleomycin (BLM). OPN was strongly expressed in alveolar macrophages accumulating in the fibrotic area of the lung. OPN messenger RNA (mRNA) in the lung was notably induced by BLM instillation, and the development of the fibrotic process was associated with an increase in the expression of OPN mRNA and protein. In vitro, recombinant OPN enhanced migration, adhesion, and platelet-derived growth factor (PDGF)-mediated DNA synthesis of murine fibroblast cell line NIH3T3. These effects of OPN on fibroblasts were significantly suppressed by addition of antimouse alphav integrin monoclonal antibody (RMV-7). Furthermore, treatment of mice with RMV-7 repressed the extent of pulmonary fibrosis in this model. Conclusively, these data suggest that OPN produced by alveolar macrophages functions as a fibrogenic cytokine that promotes migration, adhesion, and proliferation of fibroblasts in the development of BLM-induced pulmonary fibrosis.

Reduced urinary excretion of intact osteopontin in patients with IgA nephropathy.

Osteopontin (OPN) is a phosphoprotein secreted by many cells of epithelial, mesenchymal, and hematopoietic origin. In the kidney, OPN is expressed in the renal tubules and collecting ducts and is excreted into the urine. A pathophysiologic role for urinary OPN has not been established. In this study, urinary excretion of OPN was analyzed in patients with primary glomerular diseases, including immunoglobulin A nephropathy (IgAN; n = 32), minimal change nephrotic syndrome (MCNS; n = 16), and membranous nephropathy (MN; n = 18). Compared with normal controls (n = 20), mean +/- SD of urinary OPN in IgAN patients was decreased significantly (21.4 +/- 6.2 versus 11.6 +/- 9.6 mg/g creatinine, P: < 0.001). In contrast, the levels of urinary OPN in patients with MCNS or MN did not differ significantly from normal values. Immunoblot analysis showed that OPN is present as a 55- to 60-kd molecule in normal urine. A 34-kd fragment of OPN was the major immunoreactive band in samples from IgAN patients. This fragment also was detectable in the urine from some patients with MCNS or MN but was absent in normal subjects. OPN has a thrombin-cleavage site near its central portion. Thrombin treatment of the urine from normal controls could result in 34-kd OPN fragments. Although the underlying mechanisms remain to be determined, these data provide evidence that secretion or processing (or both) of urinary OPN is altered in patients with IgAN.

Prevention of lethal acute graft-versus-host disease in mice by oral administration of T helper 1 inhibitor, TAK-603.

Acute graft-versus-host diseases (GVHD) is a major cause of morbidity and mortality in patients undergoing allogeneic bone marrow transplantation (BMT). T helper 1 (Th1)-type cytokines such as interferon-gamma or tumor necrosis factor-alpha have been implicated in the pathogenesis of acute GVHD. TAK-603 is a new quinoline derivative, which is now in clinical trials for use as a disease-modifying antirheumatic drug. In preclinical studies, it inhibited delayed-type hypersensitivity, but not Arthus-type reaction, in mice, and selectively suppressed Th1 cytokine production. Thus, the present study was designed to investigate whether the Th1 inhibitor (TAK-603) ameliorates lethal acute GVHD in a mouse model. Administration of TAK-603 into BALB/c mice given 10 Gy total body irradiation followed by transplantation of bone marrow and spleen cells from C57BL/6 mice markedly reduced the mortality in association with minimal signs of GVHD pathology in the liver, intestine, and skin. TAK-603 reduced not only the production of Th1-type cytokines, but also the proportion of Th1 cells in CD4(+) helper T cells in this GVHD mouse model. These results suggest that TAK-603 could be a potent therapeutic agent for acute lethal GVHD.