Generation and functional characterization of anti-human and anti-mouse IL-36R antagonist monoclonal antibodies

Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, β and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.     

Ion cyclotron resonance as a tool in regenerative medicine

The identification of suitable stem cell cultures and differentiating conditions that are free of xenogenic growth supplements is an important step in finding the clinical applicability of cell therapy in two important fields of human medicine: heart failure and bone remodeling, growth and repair. We recently demonstrated the possibility of obtaining cardiac stem cells (CSCs) from human endomyocardial biopsy specimens. CSCs self-assemble into multi-cellular clusters known as cardiospheres (CSps) that engraft and partially regenerate infarcted myocardium. CSps and cardiosphere-derived-cells (CDCs) were exposed for five days in an incubator regulated for temperature, humidity, and CO(2) inside a solenoid system. This system was placed in a magnetically shielded room. The cells were exposed simultaneously to a static magnetic field (MF) and a parallel low-alternating frequency MF, close to the cyclotron frequency corresponding to the charge/mass ratio of the Ca(++) ion. In this exposure condition, CSps and CDCs modulate their differentiation turning on cardiogenesis and turning off vasculogenesis. Cardiac markers such as troponin I (TnI) and myosin heavy chain (MHC) were up-regulated. Conversely, angiogenic markers such as vascular endothelial growth factor (VEGF) and kinase domain receptor (KDR) were down-regulated as evidenced by immunocytochemistry. Exposure to the 7 Hz calcium ion cyclotron resonance (ICR) frequency can modulate the cardiogenic vs. angiogenic differentiation process of ex vivo expanded CSCs. This may pave the way for novel approaches in tissue engineering and cell therapy. With regard to bone remodeling, it has been suggested that bone marrow-derived mesenchymal stem cells (MSC) may be considered as a potential therapeutic tool. Using the Ca(++)-dependent specific differentiation potential of the ELF-MF 7 Hz ICR, we show here that exposure of human MSC to these same MF conditions enhanced the expression of osteoblast differentiation markers such as alkaline phosphatase, osteocalcin, and osteopontin, as analyzed by real-time quantitative PCR, without affecting cell proliferation. As expected, while the differentiation marker factors were up regulated, the ICR electromagnetic field down regulated osteoprotegerin gene expression, a critical regulator of postnatal skeletal development and homeostasis in humans as well as mice.     

Elucidation of an alternate isoleucine biosynthesis pathway in Geobacter sulfurreducens

The central metabolic model for Geobacter sulfurreducens included a single pathway for the biosynthesis of isoleucine that was analogous to that of Escherichia coli, in which the isoleucine precursor 2-oxobutanoate is generated from threonine. 13C labeling studies performed in G. sulfurreducens indicated that this pathway accounted for a minor fraction of isoleucine biosynthesis and that the majority of isoleucine was instead derived from acetyl-coenzyme A and pyruvate, possibly via the citramalate pathway. Genes encoding citramalate synthase (GSU1798), which catalyzes the first dedicated step in the citramalate pathway, and threonine ammonia-lyase (GSU0486), which catalyzes the conversion of threonine to 2-oxobutanoate, were identified and knocked out. Mutants lacking both of these enzymes were auxotrophs for isoleucine, whereas single mutants were capable of growth in the absence of isoleucine. Biochemical characterization of the single mutants revealed deficiencies in citramalate synthase and threonine ammonia-lyase activity. Thus, in G. sulfurreducens, 2-oxobutanoate can be synthesized either from citramalate or threonine, with the former being the main pathway for isoleucine biosynthesis. The citramalate synthase of G. sulfurreducens constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases, which contains representatives from a wide variety of microorganisms.     

Cytotoxicity of methoctramine and methoctramine-related polyamines

Methoctramine and its analogues are polymethylene tetramines that selectively bind to a variety of receptor sites. Although these compounds are widely used as pharmacological tools for receptor characterization, the toxicological properties of these polyamine-based structures are largely unknown. We have evaluated the cytotoxic effects of methoctramine and related symmetrical analogues differing in polymethylene chain length between the inner nitrogens against a panel of cell lines. Methoctramine caused cell death only at high micromolar concentrations, whereas its pharmacological action is exerted at nanomolar level. Increasing the spacing between the inner nitrogen atoms resulted in a significative increase in cytotoxicity. In particular, an elevated cytotoxicity is associated to a methylene chain length of 12 units dividing the inner amine functions (compound 5). H9c2 cardiomyoblasts were the most sensitive cells, followed by SH-SY5Y neuroblastoma, whereas HL60 leukaemia cells were much more resistant. Methoctramine and related compounds down-regulated ornithine decarboxylase, the first enzyme of polyamine biosynthesis even at non-toxic concentration. Further, methoctramine and compound 5 caused a limited up-regulation of spermine/spermidine N-acetyltransferase, suggesting that interference in polyamine metabolism is not a primary mechanism of toxicity. Methoctramine and its analogues bound to DNA with a higher affinity than spermine, but the correlation with their toxic effect was poor. The highly toxic compound 5 killed the cells in the absence of caspase activation and caused an increase in p53 expression and ERK1/2 phosphorylation. Compound 5 was directly oxidized by cell homogenates producing hydrogen peroxide and its toxic effect was partially subdued by the inhibition of its uptake, by the NMDA ligand MK-801, and by the antioxidant N-acetylcysteine, suggesting that compound 5 can act at different cellular levels and lead to oxidative stress.     

Bioconversion of soy isoflavones daidzin and daidzein by <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains

Twenty-two strains of Bifidobacterium, representative of eight major species of human origin, were screened for their ability to transform the isoflavones daidzin and daidzein. Most of the strains released the aglycone from daidzin and 12 gave yields higher than 90%. The kinetics of growth, daidzin consumption, and daidzein production indicated that the hydrolytic activity occurred during the growth. The supernatant of the majority of the strains did not release the aglycone from daidzin, suggesting that cell-associated β-glucosidases (β-Glu) are mainly responsible for the metabolism of soybean glyco-conjugates. Cell-associated β-Glu was mainly intracellular and significantly varied among the species and the strains. The lack of β-Glu was correlated with the inability to hydrolyze daidzin. Although S-equol production by anaerobic intestinal bacteria has been established, information on S-equol-producing bifidobacteria is contradictory. In this study, 22 bifidobacteria failed to transform daidzein into reduced metabolites under all the experimental conditions, excluding any role in the reductive pathway of daidzein toward the production of S-equol. These results suggest that selected probiotic strains of Bifidobacterium can be used to speed up the release of daidzein, improving its bioavailability for absorption by colonic mucosa and/or biotransformation to S-equol by other intestinal microorganisms.     

The process of thermodialysis in bioremediation of waters polluted by endocrine disruptors

Endocrine disruptors are chemicals able to induce adverse effects into wildlife and humans owing to their ability of interfering with the endocrine system. Bisphenol A (BPA) has been chosen as model of endocrine disruptors. To reduce the BPA pollution in waters we proposed the employment of the process of thermodialysis. Two different catalytic membranes have been prepared by covalently immobilizing laccase (from Trametes versicolor) by means of a diazotation process or tyrosinase (from mushroom) by condensation. The support was a nylon membrane. The bioremediation power of both catalytic membranes has been analysed under isothermal and non-isothermal conditions.The advantages in using non-isothermal bioreactors were discussed in terms of reduction of the bioremediation times.     

PKR activity is required for acute leukemic cell maintenance and growth: A role for PKR‐mediated phosphatase activity to regulate GSK‐3 phosphorylation

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p‐T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant‐negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time‐dependent augmentation of AKT S473 and GSK‐3α S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK‐3α was not dependent on PI3K activity. PKR inhibition augmented levels of p‐S473 AKT and p‐S21/9 GSK‐3α/β in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK‐3α or β phosphorylation in the presence of the AKT inhibitor, A443654. Pre‐treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2α, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2α phosphorylation. The effects of PKR inhibition on AKT and GSK‐3 phosphorylation were found to be, in part, PP2A‐dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK‐3. J. Cell. Physiol. 221: 232–241, 2009. © 2009 Wiley‐Liss, Inc     

NMR assignments of the DNA binding domain of Ml4 protein from Mesorhizobium loti

Ml4 protein from Mesorhizobium loti has a 58% sequence identity with the Ros protein from Agrobacterium tumefaciens that contains a prokaryotic Cys₂His₂ zinc finger domain. Interestingly, Ml4 is a zinc-lacking protein that does not contain the Cys₂His₂ motif and is able to bind the Ros DNA target sequence with high affinity. Here we report the ¹H, ¹⁵N and ¹³C NMR assignments of the Ml4 protein DNA binding domain (residue 52–151), as an important step toward elucidating at a molecular level how this prokaryotic domain can overcome the metal requirement for proper folding and DNA-binding activity.     

Role of Dicer1 in thyroid cell proliferation and differentiation

DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation.