Implications of BCRP modulation on PTZ-induced seizures in mice: Role of ko143 and metformin as adjuvants to lamotrigine.

Blood-brain barrier (BBB) efflux transporters' overexpression hinders antiepileptic drug brain entry. Breast cancer resistance protein (BCRP) is a major BBB efflux transporter. In the present work, BCRP's role as a mechanism that might contribute to drug-resistant epilepsy (DRE) in a mouse model of acute seizures was studied with further assessment of the effect of its inhibition by ko143 and metformin (MET) on lamotrigine (LTG) bioavailability and efficacy. 42 male mice divided into 6 groups: G1: Normal control, G2: LTG-injected healthy mice: LTG 20 mg/kg i.p., G3: Acute seizures (A.S) mice: Pentylenetetrazole (PTZ) 50 mg/kg i.p., G4: LTG-treated A.S mice: LTG 20 mg/kg + PTZ 50 mg/kg i.p., G5: Ko143 + LTG treated A.S mice: Ko143 15 mg/kg i.p. before LTG + PTZ, G6: MET + LTG treated A.S mice: MET 200 mg/kg i.p. before LTG + PTZ. Seizures severity, serum, brain LTG, and brain BCRP were assessed. PTZ group experienced the highest seizure frequency and brain BCRP expression. Ko143 and MET groups showed a significant decrease in brain BCRP with subsequent improvement in brain LTG level and better seizure control. BCRP has a significant role in epilepsy resistance and its inhibition with ko143 or MET adds value to DRE management.

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl3) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.

TTC39B destabilizes retinoblastoma protein promoting hepatic lipogenesis in a sex-specific fashion.

BACKGROUND & AIMS: Molecular mechanisms underlying the different susceptibility of men and women to non-alcoholic fatty liver disease (NAFLD) are poorly understood. The TTC39B locus encodes a scaffolding protein, associates with gynecological disorders and its deletion protects mice from diet-induced steatohepatitis. This study aimed to elucidate the molecular mechanisms linking TTC39B (T39) to the expression of lipogenic genes and to explore sex-specific effects. METHODS: Co-expression in HEK293A cells validated the novel T39/pRb interaction predicted by a protein-protein interaction algorithm. T39 was knocked down using an antisense oligonucleotide (ASO) in mice with dietary NAFLD and a genetic deficiency of pRb or its downstream effector E2F1, as well as in primary human hepatocytes. RESULTS: T39 interacts with pRb via its C-terminal TPR domain and promotes its proteasomal degradation. In female mice, T39 deficiency reduces the mRNA of lipogenic genes, especially Pnpla3, in a pRb- and E2F1-dependent manner. In contrast, in male mice, T39 deficiency results in a much smaller reduction in lipogenic gene expression that is independent of pRb/E2F1. T39 also interacts with VAPB via an N-terminal FFAT motif and stabilizes the interaction of VAPB with SCAP. Ovariectomy abolishes the effect of T39 knockdown on the hepatic pRb/E2F1/Pnpla3 axis. In both sexes T39 knockdown reduces SCAP independently of pRb. In primary human hepatocytes, T39 knockdown reduces expression of PNPLA3 and other lipogenic genes in women but not men. CONCLUSIONS: We have uncovered a conserved sexual dimorphism in the regulation of hepatic lipogenic genes, with effects of T39 mediated through pRb/E2F1 in females and VAPB/SCAP in both sexes. T39 inhibition could be a novel strategy to downregulate PNPLA3 and treat NAFLD in women. LAY SUMMARY: In females, the protein TTC39B degrades a tumor suppressor in the liver to promote the synthesis of new fat and the expression of a major genetic risk factor for non-alcoholic fatty liver disease. TTC39B is a potential therapeutic target for non-alcoholic fatty liver disease, especially in women.

Newly developed pseudogout arthritis after therapy with MAGE-A4 directed TCR T cells responded to treatment with tocilizumab.

With durable cancer responses, genetically modified cell therapies are being implemented in various cancers. However, these immune effector cell therapies can cause toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Pseudogout arthritis is an inflammatory arthritis induced by deposition of calcium pyrophosphate dihydrate crystals. Here, we report a case of pseudogout arthritis in a patient treated with MAGE-A4 directed T cell receptor T cells, for fallopian tube cancer. The patient developed CRS and ICANS 7 days after infusion of the T cells. Concurrently, the patient newly developed sudden onset of left knee arthritis. Synovial fluid analyses revealed the presence of calcium pyrophosphate dihydrate crystal. Notably, the pseudogout arthritis was resolved with tocilizumab, which was administered for the treatment of CRS and ICANS. Immunoprofiling of the synovial fluid showed that the proportion of inflammatory interleukin 17 (IL-17)-producing CD4+ T (Th17) cells and amount of IL-6 were notably increased, suggesting a potential role of Th17 cells in pseudogout arthritis after T-cell therapy. To the best of our knowledge, this is the first reported case of pseudogout arthritis after cell therapy. Clinicians, especially hematologists, oncologists and rheumatologists, should be aware that pseudogout arthritis can be associated with CRS/ICANS.

Recent advances of highly selective CDK4/6 inhibitors in breast cancer.

Uncontrolled cell division is the hallmark of cancers. Full understanding of cell cycle regulation would contribute to promising cancer therapies. In particular, cyclin-dependent kinases 4/6 (CDK4/6), which are pivotal drivers of cell proliferation by combination with cyclin D, draw more and more attention. Subsequently, extensive studies were carried out to explore drugs inhibiting CDK4/6 and assess the efficacy and safety of these drugs in cancer, especially breast cancer. Due to the insuperable adverse events and the less activity observed in vivo, the drug development of the initial pan-CDK inhibitor flavopiridol was consequently discontinued, and then highly specific inhibitors were extensively researched and developed, including palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219). Food and Drug Administration has approved palbociclib and ribociclib for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer, and recent clinical trial data suggest that palbociclib significantly improved clinical outcome when combined with letrozole or fulvestrant. Besides, the favorable effects of abemaciclib on prolonging survival of breast cancer patients have also been observed in clinical trials both for single-agent and combination strategy. In this review, we outline the preclinical and clinical advancement of these three orally bioavailable and highly selective CDK4/6 inhibitors in breast cancer.

Non-clinical safety evaluation of single and repeated intramuscular administrations of MAGE-A3 Cancer Immunotherapeutic in rabbits and cynomolgus monkeys.

The MAGE-A3 recombinant protein combined with AS15 immunostimulant (MAGE-A3 Cancer Immunotherapeutic) is under development by GlaxoSmithKline for the treatment of lung cancer and melanoma. We performed non-clinical safety studies evaluating potential local and systemic toxic effects induced by MAGE-A3 Cancer Immunotherapeutic in rabbits (study 1) and cynomolgus monkeys (study 2). Animals were allocated to two groups to receive a single (rabbits) or 25 repeated (every 2 weeks) injections (monkeys) of MAGE-A3 Cancer Immunotherapeutic (treatment groups) or saline (control groups). All rabbits were sacrificed 3 days post-injection and monkeys 3 days following last injection (3/5 per gender per group) or after a 3-month treatment-free period (2/5 per gender per group). Local and systemic reactions and MAGE-A3-specific immune responses (monkeys) were assessed. Macroscopic and microscopic (for rabbits, injection site only) post-mortem examinations were performed on all animals. No systemic toxicity or unscheduled mortalities were recorded. Single (rabbits) and repeated (monkeys; up to four times at the same site) injections were well tolerated. Following five to seven repeated injections, limb circumferences increased up to 26% (5 h post-injection), but returned to normal after 1-8 days. Three days after the last injection, enlargements of iliac, popliteal, axillary and inguinal lymph nodes, and increased incidence or severity of mononuclear inflammatory cell infiltrates was observed in injected muscles of treated monkeys. No treatment-related macroscopic findings were recorded after the treatment-free period. MAGE-A3-specific antibody and T-cell responses were raised in all treated monkeys, confirming test item exposure. Single or repeated intramuscular injections of MAGE-A3 Cancer Immunotherapeutic were well tolerated in rabbits and monkeys.

Independent oncogenic and therapeutic significance of phosphatase PRL-3 in FLT3-ITD-negative acute myeloid leukemia.

BACKGROUND: Internal tandem duplication of FMS-like tyrosine kinase (FLT3-ITD) is well known to be involved in acute myeloid leukemia (AML) progression, but FLT3-ITD-negative AML cases account for 70% to 80% of AML, and the mechanisms underlying their pathology remain unclear. This study identifies protein tyrosine phophatase PRL-3 as a key mediator of FLT3-ITD-negative AML. METHODS: A total of 112 FLT3-ITD-negative AML patients were sampled between 2010 and 2013, and the occurrence of PRL-3 hyperexpression in FLT3-ITD-negative AML was evaluated by multivariate probit regression analysis. Overexpression or depletion of endogenous PRL-3 expression with the specific small interfering RNAs was performed to investigate the role of PRL-3 in AML progression. Xenograft models were also used to confirm the oncogenic role of PRL-3. RESULTS: Compared to healthy donors, PRL-3 is upregulated more than 3-fold in 40.2% of FLT3-ITD-negative AML patients. PRL-3 expression level is adversely correlated to the overall survival of the AML patients, and the AML relapses accompany with re-upregulation of PRL-3. Mechanistically, aberrant PRL-3 expression promoted cell cycle progression and enhanced the antiapoptotic machinery of AML cells to drug cytotoxicity through downregulation of p21 and upregulation of Cyclin D1 and CDK2 and activation of STAT5 and AKT. Depletion of endogenous PRL-3 sensitizes AML cells to therapeutic drugs, concomitant with apoptosis by upregulation of cleaved PARP (poly ADP ribose polymerase) and apoptosis-related caspases. Xenograft assays further confirmed PRL-3's oncogenic role in leukemogenesis. CONCLUSIONS: Our results demonstrated that PRL-3 is a novel independent crucial player in both FLT3-ITD-positive and FLT3-ITD-negative AML and could be a potential therapeutic target.

Tumor-induced osteomalacia associated with a maxillofacial tumor producing fibroblast growth factor 23: report of a case and review of the literature.

Tumor-induced osteomalacia (TIO) is a rare acquired paraneoplastic disease characterized by renal phosphate wasting and hypophosphatemia. Recently, it was reported that tumors associated with TIO produce fibroblast growth factor (FGF) 23, identified as the last member of the FGF family and of which excessive action causes several hypophosphatemic diseases whereas deficient FGF23 activity results in hyperphosphatemic tumoral calcinosis. In this case, although it was difficult to locate the associated tumor, an abnormal mass in the left maxilla was detected by imaging. The tumor was removed by partial resection of the left maxillary alveolar region. Thereafter, serum level of FGF23 rapidly decreased, hypophosphatemia improved, and the clinical symptoms greatly improved. Histopathologic diagnosis of the tumor was phosphaturic mesenchymal tumor, mixed connective tissue variant. Immunohistochemical findings confirmed that the removed tumor produced FGF23. These results indicate that development of osteomalacia in this patient was related to the maxillary tumor, which overexpressed FGF23.

MAGE A3 antigen-specific cancer immunotherapeutic.

Non-small-cell lung cancer (NSCLC) and melanoma are devastating diseases with high rates of recurrence. Current clinical recommendations include postoperative adjuvant chemotherapy in stages II and IIIA NSCLC, while there is a debate regarding its clinical benefit in stage IB. Recent Phase II trials have demonstrated a clinical benefit by postoperative vaccine with melanoma-specific antigen A3 (MAGE A3) in NSCLC and in stage IV melanoma. These trials have led to the current Phase III trials. MAGE A3 is a tumor-specific shared antigen that is frequently expressed in lung cancer and melanoma, as well as in few other tumors. Its level is associated with disease burden and with prognosis, while normal tissues do not express it, except the testis and the placenta. This review will summarize the recent developments and clinical experience with the MAGE A3 vaccine.

Tumor necrosis factor alpha inhibitors and methotrexate: implications for deployed personnel.

Rheumatic diseases such as rheumatoid and psoriatic arthritis are often diagnosed in younger age groups and are, therefore, likely to be encountered in active-duty military populations. These patients are increasingly being treated with disease modifying anti-rheumatic drugs (DMARDs) and biologic agents such as tumor necrosis factor alpha (TNF-alpha) inhibitors. While these classes of drugs have revolutionized the treatment of rheumatic diseases, they are also associated with serious potential adverse effects. At present, there are no published guidelines for the routine monitoring of laboratory parameters in patients receiving anti-TNF therapy. Currently, no official consensus among military physicians exists regarding duty and geographic limitations for patients receiving these types of therapy. Major adverse effects of these agents are reviewed in this article. A survey of U.S. Air Force, Army, and Navy rheumatologists was performed. The results of laboratory monitoring and operational deployment recommendations are reported. The majority of U.S. military rheumatologists do not recommend deployment of patients while taking methotrexate or TNF-alpha inhibitors.

[Clinical experience with TNF-alpha inhibitors in rheumatoid arthritis]. / Klinisk erfaring med TNF-alpha-hemmere ved revmatoid artritt.

BACKGROUND: Infliximab and etanercept, both tumour necrosis factor-alpha inhibitors, are proven to be effective in patients with rheumatoid arthritis in randomised controlled trials. MATERIAL AND METHODS: Patients with active rheumatoid arthritis were treated with infliximab (n = 29) or etanercept (n = 24) in clinical hospital practice. They were examined before and during treatment. All patients had tried at least one DMARD before. Details of disease activity were monitored by measuring tender and swollen joints, global and pain patient visual analogue scales, Disease Activity Index Score (DAS 28), the Modified Health Assessment Questionnaire, blood and urine samples, and adverse effects. The patients were monitored regularly for two years or until they stopped treatment. RESULTS: In the infliximab group we observed statistically significantly better values for all the registered variables after 6 weeks. At the other times of registration the variables were varying a lot; however, DAS 28 scores after baseline were all within the limits of moderate effect. In the etanercept group we observed statistically significantly better values for all the variables except for erythrocyte sedimentation rate after 6 weeks. At the other times of registration all the variables had significantly better values. Adverse effects were reported in 9 patients in the infliximab group and in 5 in the etanercept group, but no serious adverse effects were reported. 18 patients in the infliximab group (61%) and 10 in the etanercept group (42%) had stopped treatment within two years, either because of adverse effects or lack of effect. CONCLUSION: In this open study of patients with active rheumatoid arthritis, most experienced a rapid effect of infliximab, but a varying effect later on. In the etanercept group the patients experienced both a rapid and sustained effect among those who tolerated the medication. Compared to what several others have reported, a large number of patients stopped treatment; this may reflect limited experience.

Phase 1/2 study of subcutaneous and intradermal immunization with a recombinant MAGE-3 protein in patients with detectable metastatic melanoma.

The purpose of this phase 1/2 study was to evaluate toxicity, tumor evolution and immunologic response following administration of a fixed dose of a recombinant MAGE-3 protein by subcutaneous and intradermal routes in the absence of immunologic adjuvant. Thirty-two patients with detectable metastatic melanoma expressing gene MAGE-3 were included and 30 received at least one injection with a fixed dose of a ProtD-MAGE-3 fusion protein. The immunization schedule included 6 intradermal and subcutaneous injections at 3-week intervals. Afterward, patients without major tumor progression who required other treatments received additional vaccinations at increasing time intervals. The vaccine was generally well tolerated. Among the 26 patients who received at least 4 vaccinations, we observed 1 partial response and 4 mixed responses. For these 5 responding patients, time to progression varied from 3.5 to 51+ months. An anti-MAGE-3 CD4 T-lymphocyte response was detected in 1 out of the 5 responding patients. The majority of patients had no anti-MAGE-3 antibody response. The clinical and immunologic responses generated by the vaccine are rather limited. Nevertheless, given the potential antitumor efficacy and the very mild toxicity of vaccinations, further studies combining MAGE proteins and/or peptides with potent immunologic adjuvants are warranted, not only in metastatic melanoma, but also in the adjuvant setting.

Persistent clinical response to the anti-TNF-alpha antibody infliximab in patients with ankylosing spondylitis over 3 years.

OBJECTIVE: Infliximab, a monoclonal antibody against tumour necrosis factor alpha (TNF-alpha), is approved in Europe for the treatment of patients with active ankylosing spondylitis (AS) who have responded inadequately to conventional therapy. This report provides analyses from a 3-yr extension study, as a follow-up to both the 1- and 2-yr open label extensions of the original 3-month randomized controlled trial of infliximab in patients with AS. METHODS: Of the 49 patients with AS who completed the second year of the study, 46 continued treatment with infliximab 5 mg/kg every 6 weeks up to week 156. The Bath AS Disease Activity Index (BASDAI), the Bath AS Functional Index, the Bath AS Metrology Index, patient's and physician's global assessments, quality of life (Short Form-36), C-reactive protein (CRP) and erythrocyte sedimentation rate were assessed throughout the study period. RESULTS: The improvement of signs and symptoms observed in the majority of the patients during the first and second year was sustained throughout the third year of the study. Forty-three patients (62% of the 69 patients enrolled at baseline and 93% of the patients who started the third year) completed week 156. In the intention-to-treat analysis, an ASAS '5 out of 6' and ASAS 40% response was seen by 46% and 50% of the patients, respectively. The scores for other efficacy assessments were similar to the values observed at weeks 54 and 102. Median CRP levels remained low (1.5 mg/l at week 156). There were no relevant side-effects and no discontinuation because of drug-related adverse events during the third year of the study. CONCLUSIONS: Patients with AS receiving infliximab for 3 yr showed a durable clinical response without loss of efficacy. Long-term infliximab treatment was well tolerated by patients in this study.

[Increased risk of infection with biological immunomodifying antirheumatic agents. Clear guidelines are necessary as shown by case reports]. / Okad risk för infektion med biologiska immunmodifierande antireumatika.

Several potent immunosuppressive drugs have become available in the new millennium for patients with rheumatologic diseases, Crohn's disease and other autoimmune disorders. Five patient cases from Växjö central hospital (uptake area 178 000 individuals) with Listeria meningitis, Pneumocystis jiroveci and tuberculosis pneumonia, Listeria sepsis, Legionella pneumonia and E coli sepsis are described. A doubled risk for infections has previously been observed for RA patients, as compared to healthy individuals. There is clearly an increased risk of tuberculosis (depending on the actual and historic environmental prevalence) for patients on TNF antagonists, and therefore tuberculosis screening is now mandatory before start of therapy. Since TNF has a central role in the immune defence, an increased risk of opportunistic infections like listeriosis. mycobacteriosis, and invasive fungal infections has been established. Eight hospitals in southern Sweden participate in a register for the use of TNF blockers in rheumatologic diseases (South Swedish Arthritis Treatment Group, SSATG). Guidelines for screening and treatment of latent and active tuberculosis, possible prophylactic antibiotic treatment for endocarditis and vaccination programs for patients on TNF antagonists are discussed.

[TNF blockade in rheumatoid arthritis can cause severe fibrosing alveolitis. Six case reports]. / TNF-blockad vid reumatoid artrit kan ge svår fibrotiserande alveolit. Sex fallbeskrivningar.

TNF-blockade has been increasingly used in the treatment of rheumatoid arthritis (RA). However, the safety is unclear and an increased risk of both tuberculosis and other infections has been identified. Recently severe fibrosing alveolitis has also been reported in RA-patients treated with TNF-blockade. We report a further six RA patients, who during treatment with infliximab or etanercept developed fulminant lung fibrosis with alveolitis. For four of the patients, the fibrosing alveolitis was fatal. All patients were RF positive and above 60 years and five had mild fibrosis associated with RA before TNF-blockade treatment. Duration of TNF-blockade treatment was for three patients only two months and for the other three, 20-51 months. Age above 60 years and previous lung fibrosis appear to be risk factors for developing fibrosing alveolitis in RA patients treated with TNF-blockade.

[Measures for tuberculosis in compromised hosts--mainly on chemoprophylaxis].

As for the measures for tuberculosis in Japan, BCG inoculation and chemoprophylaxis have been done with emphasis placed on children and young people. Since, however, about 90% are older than 30 years and more than 50% are older than 60 years among the new TB patients, measures, particularly chemoprophylaxis aiming at the middle-old aged people are needed in the future. We discuss the method to select cases for chemoprophylaxis as to the cases of diabetes, collagen diseases and lung cancer administered corticosteroid preparations as well as the cases of Crohn's disease and rheumatoid arthritis administered anti-TNF-alpha among compromised hosts. In diabetics, chemoprophylaxis is necessary for those who show healing of TB despite there being no history of TB treatment. Where a corticosteroid preparation, more than 10 mg in terms of prednisolone is administered over a long period of time for collagen disease and lung cancer, chemoprophylaxis is necessary for those who show healing of TB despite there being no history of TB treatment and those who are suspected of having TB infection by a tuberculin test. In the cases of Crohn's disease and rheumatoid arthritis administered anti-TNF-alpha, chemoprophylaxis is necessary for those who show healing of TB despite those who are suspected of having TB infection by a tuberculin test. The administration period of INH as chemoprophylaxis should preferably be set at 9 months instead of 6 months hitherto used.

Two phase I studies of low dose recombinant human IL-12 with Melan-A and influenza peptides in subjects with advanced malignant melanoma.

Preclinical studies have shown that low dose IL-12 can potentiate cytotoxic lymphocyte responses. Since previous trials have demonstrated significant toxicity from high dose recombinant human IL-12 (rhIL-12), we sought to determine an optimal biological dose for rhIL-12 at lower doses when combined with peptide antigens. Two studies were undertaken. The rhIL-12 was administered at doses of 0 (placebo), 10, 30 and 100 ng/kg, subcutaneously in one study and intravenously in the other. Apart from IL-12 dosing, the studies were identical. Subjects had evaluable stage III or IV melanoma which expressed Melan-A by RT-PCR or immunohistochemistry. Melan-A (26-35) (EAAGIGILTV) and influenza matrix (58-66) (GILGFVFTL) peptides were administered intradermally on weeks 1, 2, 3, 4 and 9. Twenty-eight subjects were enrolled, of whom 24 were evaluable for clinical and immunological responses. Therapy was well tolerated, the main adverse event being influenza-like symptoms. Immunological monitoring included the evaluation of cutaneous reactions and assays for antigen-specific T-cells. Clinical responses included a complete response in a subject with small volume subcutaneous disease, a partial response in a subject with hepatic metastases, and mixed responses in pulmonary, pleural and nodal disease. Biopsies of accessible tumors showed infiltration with CD4+ and CD8+ lymphocytes capable of lysing Melan-A peptide-pulsed targets in vitro. No clear dose-dependent effect of rhIL-12 could be determined. The rhIL-12 given either s.c. or i.v. was well tolerated at doses of 10-100 ng/kg. Clinical and immunological activity has been observed in this study where peptides were administered either with or without low dose rhIL-12.