Sodium Accumulation in Infected Cells and Ion Transporters Mistargeting in Nodules of Medicago truncatula: Two Ugly Items That Hinder Coping with Salt Stress Effects

The maintenance of intracellular nitrogen-fixing bacteria causes changes in proteins’ location and in gene expression that may be detrimental to the host cell fitness. We hypothesized that the nodule’s high vulnerability toward salt stress might be due to alterations in mechanisms involved in the exclusion of Na⁺ from the host cytoplasm. Confocal and electron microscopy immunolocalization analyses of Na⁺/K⁺ exchangers in the root nodule showed the plasma membrane (MtNHX7) and endosome/tonoplast (MtNHX6) signal in non-infected cells; however, in mature infected cells the proteins were depleted from their target membranes and expelled to vacuoles. This mistargeting suggests partial loss of the exchanger’s functionality in these cells. In the mature part of the nodule 7 of the 20 genes encoding ion transporters, channels, and Na⁺/K⁺ exchangers were either not expressed or substantially downregulated. In nodules from plants subjected to salt treatments, low temperature-scanning electron microscopy and X-ray microanalysis revealed the accumulation of 5–6 times more Na⁺ per infected cell versus non-infected one. Hence, the infected cells’ inability to withstand the salt may be the integral result of preexisting defects in the localization of proteins involved in Na⁺ exclusion and the reduced expression of key genes of ion homeostasis, resulting in premature senescence and termination of symbiosis.     

CD4+ T-Cell Plasticity in Non-Infectious Retinal Inflammatory Disease

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4⁺ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4⁺FoxP3⁺CD25⁺ regulatory T cells (Tregs) were known to suppress function of effector CD4⁺ T cells and contribute to resolution of disease. It has been recently reported that some CD4⁺ T-cell subsets demonstrate shared phenotypes with another CD4⁺ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4⁺ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.     

Peripheral Blood Lymphocyte Subsets (CD4+, CD8+ T Cells,NK Cells) in Patients with Cardiovascular and Neurological Complications after Carotid Endarterectomy

Background: The aim of the study was to evaluate the differences in the circulating immune cells’ subgroups after the atherosclerotic plaque removal in patients presenting with postoperative complications as compared to the patients without complications after carotid endarterectomy (CEA). Methods: Patients with significant carotid atherosclerosis (n = 124, age range: 44 to 87 years) who underwent CEA were enrolled in a prospective study. The immunology study using flow cytometry was performed to determine the percentages of peripheral blood T cells (CD4⁺, CD8⁺, Treg—CD4⁺/CD25⁺) and NK (natural killer) cells before and after the procedure. The data were expressed as the percentage of total lymphocytes ± the standard error of mean. Results: The mean percentage of lymphocytes (61.54% ± 17.50% vs. 71.82% ± 9.68%, p = 0.030) and CD4 T lymphocytes (T helper, 38.13% ± 13.78% vs. 48.39% ± 10.24%, p = 0.027) was significantly lower six hours after CEA in patients with postoperative 30-day cardiovascular and neurological complications as compared to the group without complications. On the other hand the mean NK level in the group with complications was significantly higher (21.61% ± 9.00% vs. 15.80% ± 9.31%, p = 0.048). Conclusions: The results of this study suggest that after carotid endarterectomy the percentages of circulating immune cells subsets differ in patients with and without postoperative complications.     

Characterization in Effective Stimulation on the Magnitude,Gating, Frequency Dependence,and Hysteresis of INa Exerted by Picaridin (or Icaridin), a Known Insect Repellent

Picaridin (icaridin), a member of the piperidine chemical family, is a broad-spectrum arthropod repellent. Its actions have been largely thought to be due to its interaction with odorant receptor proteins. However, to our knowledge, to what extent the presence of picaridin can modify the magnitude, gating, and/or the strength of voltage-dependent hysteresis (Hys_(V)) of plasmalemmal ionic currents, such as, voltage-gated Na⁺ current [I_Na], has not been entirely explored. In GH₃ pituitary tumor cells, we demonstrated that with exposure to picaridin the transient (I_Na(T)) and late (I_Na(L)) components of voltage-gated Na⁺ current (I_Na) were differentially stimulated with effective EC₅₀’s of 32.7 and 2.8 μM, respectively. Upon cell exposure to it, the steady-state current versus voltage relationship I_Na(T) was shifted to more hyperpolarized potentials. Moreover, its presence caused a rightward shift in the midpoint for the steady-state inactivate curve of the current. The cumulative inhibition of I_Na(T) induced during repetitive stimuli became retarded during its exposure. The recovery time course from the I_Na block elicited, following the conditioning pulse stimulation, was satisfactorily fitted by two exponential processes. Moreover, the fast and slow time constants of recovery from the I_Na block by the same conditioning protocol were noticeably increased in the presence of picaridin. However, the fraction in fast or slow component of recovery time course was, respectively, increased or decreased with an increase in picaridin concentrations. The Hys_(V)’s strength of persistent I_Na (I_Na(P)), responding to triangular ramp voltage, was also enhanced during cell exposure to picaridin. The magnitude of resurgent I_Na (I_Na(R)) was raised in its presence. Picaritin-induced increases of I_Na(P) or I_Na(R) intrinsically in GH₃ cells could be attenuated by further addition of ranolazine. The predictions of molecular docking also disclosed that there are possible interactions of the picaridin molecule with the hNa_V1.7 channel. Taken literally, the stimulation of I_Na exerted by the exposure to picaridin is expected to exert impacts on the functional activities residing in electrically excitable cells.     

Unique Regulation of Intestinal Villus Epithelial Cl−/HCO3− Exchange by Cyclooxygenase Pathway Metabolites of Arachidonic Acid in a Mouse Model of Spontaneous Ileitis

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na⁺/H⁺ and Cl⁻/HCO₃⁻ exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl⁻/HCO₃⁻ exchange, measured as DIDS-sensitive ³⁶Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered K_m with no effects on V_max. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl⁻/HCO₃⁻ exchangers, were unaltered. Thus, inhibition of villus cell Cl⁻/HCO₃⁻ exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl⁻, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.     

Metalloendopeptidase ADAM-like Decysin 1 (ADAMDEC1) in Colonic Subepithelial PDGFRα+ Cells Is a New Marker for Inflammatory Bowel Disease

Metalloendopeptidase ADAM-Like Decysin 1 (ADAMDEC1) is an anti-inflammatory peptidase that is almost exclusively expressed in the gastrointestinal (GI) tract. We have recently found abundant and selective expression of Adamdec1 in colonic mucosal PDGFRα⁺ cells. However, the cellular origin for this gene expression is controversial as it is also known to be expressed in intestinal macrophages. We found that Adamdec1 mRNAs were selectively expressed in colonic mucosal subepithelial PDGFRα⁺ cells. ADAMDEC1 protein was mainly released from PDGFRα⁺ cells and accumulated in the mucosal layer lamina propria space near the epithelial basement membrane. PDGFRα⁺ cells significantly overexpressed Adamdec1 mRNAs and protein in DSS-induced colitis mice. Adamdec1 was predominantly expressed in CD45⁻ PDGFRα⁺ cells in DSS-induced colitis mice, with only minimal expression in CD45⁺ CD64⁺ macrophages. Additionally, overexpression of both ADAMDEC1 mRNA and protein was consistently observed in PDGFRα⁺ cells, but not in CD64⁺ macrophages found in human colonic mucosal tissue affected by Crohn’s disease. In summary, PDGFRα⁺ cells selectively express ADAMDEC1, which is localized to the colon mucosa layer. ADAMDEC1 expression significantly increases in DSS-induced colitis affected mice and Crohn’s disease affected human tissue, suggesting that this gene can serve as a diagnostic and/or therapeutic target for intestinal inflammation and Crohn’s disease.     

1,25(OH)2D3 Inhibited Ferroptosis in Zebrafish Liver Cells (ZFL) by Regulating Keap1-Nrf2-GPx4 and NF-κB-hepcidin Axis

Ferroptosis is a kind of iron-dependent programed cell death. Vitamin D has been shown to be an antioxidant and a regulator of iron metabolism, but the relationship between vitamin D and ferroptosis is poorly studied in fish. This study used zebrafish liver cells (ZFL) to establish a ferroptosis model to explore the effect of 1,25(OH)₂D₃ on cell ferroptosis and its mechanism of action. The results showed that different incubation patterns of 1,25(OH)₂D₃ improved the survival rate of ZFL, mitigated mitochondrial damage, enhanced total glutathione peroxidase (GPx) activity, and reduced intracellular reactive oxygen species (ROS), lipid peroxidation (LPO), and malondialdehyde (MDA), as well as iron ion levels, with the best effect at 200 pM 1,25(OH)₂D₃ preincubation for 72 h. Preincubation of ZFL at 200 pM 1,25(OH)₂D₃ for 72 h downgraded keap1 and ptgs2 gene expression, increased nrf2, ho-1, fth1, gpx4a,b expression, and lowered the expression of the nf-κb p65,il-6,il-1β gene, thus reducing the expression of hamp1. The above results indicate that different incubation patterns of 1,25(OH)₂D₃ have protective effects on ferroptosis of ZFL induced by ferroptosis activator RSL3 and 1,25(OH)₂D₃ can inhibit ferroptosis of ZFL by regulating Keap1–Nrf2–GPx4 and NF-κB–hepcidin axis.     

The β2-Subunit (AMOG) of Human Na+, K+-ATPase Is a Homophilic Adhesion Molecule

The β₂ subunit of Na⁺, K⁺-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While β₁ isoform have been shown to trans-interact in a species-specific mode with the β₁ subunit on the epithelial neighboring cell, the β₂ subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of β₂ or described its adhesion mechanism. Until now, the interactions pronounced for β₂/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether β₂ is a cell–cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell–cell aggregation, and protein–protein interaction assays. We observed that the glycosylated extracellular domain of β₂/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human β₂ subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein–protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant β₂ subunit show that the β₂ subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human β₂ subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).     

Interleukin-21 in Viral Infections

Interleukin (IL)-21 is a cytokine that affects the differentiation and function of lymphoid and myeloid cells and regulates both innate and adaptive immune responses. In addition to regulating the immune response to tumor and viral infections, IL-21 also has a profound effect on the development of autoimmune and inflammatory diseases. IL-21 is produced mainly from CD4⁺ T cells—in particular, follicular helper T (Tfh) cells—which have a great influence on the regulation of antibody production. It is also an important cytokine for the activation of CD8⁺ T cells, and its role in recovering the function of CD8⁺ T cells exhausted by chronic microbial infections and cancer has been clarified. Thus, IL-21 plays an extremely important role in viral infections, especially chronic viral infections. In this review, I will introduce the findings to date on how IL-21 is involved in some typical viral infections and the potential of treating viral diseases with IL-21.     

25(OH)D Is Effective to Repress Human Cholangiocarcinoma Cell Growth through the Conversion of 25(OH)D to 1α,25(OH)2D3

Cholangiocarcinoma (CCA) is a devastating disease without effective treatments. 1α,25(OH)₂D₃, the active form of Vitamin D, has emerged as a new anti-cancer regimen. However, the side effect of hypercalcemia impedes its systemic administration. 25(OH)D is biologically inert and needs hydroxylation by CYP27B1 to form 1α,25(OH)₂D₃, which is originally believed to only take place in kidneys. Recently, the extra-renal expression of CYP27B1 has been identified and in vitro conversion of 25(OH)D to 1α,25(OH)₂D₃ has been found in some cancer cells with CYP27B1 expression. In this study, CYP27B1 expression was demonstrated in CCA cells and human CCA specimens. 25(OH)D effectively represses SNU308 cells growth, which was strengthened or attenuated as CYP27B1 overexpression or knockdown. Lipocalcin-2 (LCN2) was also found to be repressed by 25(OH)D. After treatment with 800 ng/mL 25(OH)D, the intracellular 1α,25(OH)₂D₃ concentration was higher in SNU308 cells with CYP27B1 overexpression than wild type SNU308 cells. In a xenograft animal experiment, 25(OH)D, at a dose of 6 μg/kg or 20 μg/kg, significantly inhibited SNU308 cells’ growth without inducing obvious side effects. Collectively, our results indicated that SNU308 cells were able to convert 25(OH)D to 1α,25(OH)₂D₃ and 25(OH)D CYP27B1 gene therapy could be deemed as a promising therapeutic direction for CCA.     

Ouabain-Induced Changes in the Expression of Voltage-Gated Potassium Channels in Epithelial Cells Depend on Cell–Cell Contacts

Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown that in epithelial cells, it induces changes in properties and components related to apical–basolateral polarity and cell–cell contacts. In this work, we used a whole-cell patch clamp to test whether ouabain affects the properties of the voltage-gated potassium currents (Ik) of epithelial cells (MDCK). We found that: (1) in cells arranged as mature monolayers, ouabain induced changes in the properties of Ik; (2) it also accelerated the recovery of Ik in cells previously trypsinized and re-seeded at confluence; (3) in cell–cell contact-lacking cells, ouabain did not produce a significant change; (4) Na⁺/K⁺ ATPase might be the receptor that mediates the effect of ouabain on Ik; (5) the ouabain-induced changes in Ik required the synthesis of new nucleotides and proteins, as well as Golgi processing and exocytosis, as evidenced by treatment with drugs inhibiting those processes; and (5) the signaling cascade included the participation of cSrC, PI3K, Erk1/2, NF-κB and β-catenin. These results reveal a new role for ouabain as a modulator of the expression of voltage-gated potassium channels, which require cells to be in contact with themselves.     

Characterization in Inhibitory Effectiveness of Carbamazepine in Voltage-Gated Na+ and Erg-Mediated K+ Currents in a Mouse Neural Crest-Derived (Neuro-2a) Cell Line

Carbamazepine (CBZ, Tegretol^®) is an anticonvulsant used in the treatment of epilepsy and neuropathic pain; however, several unwanted effects of this drug have been noticed. Therefore, the regulatory actions of CBZ on ionic currents in electrically excitable cells need to be reappraised, although its efficacy in suppressing voltage-gated Na⁺ current (I_Na) has been disclosed. This study was undertaken to explore the modifications produced by CBZ on ionic currents (e.g., I_Na and erg-mediated K⁺ current [I_K(erg)]) measured from Neuro-2a (N2a) cells. In these cells, we found that this drug differentially suppressed the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of I_Na in a concentration-dependent manner with effective IC₅₀ of 56 and 18 μM, respectively. The overall current–voltage relationship of I_Na(T) with or without the addition of CBZ remained unchanged; however, the strength (i.e., ∆area) in the window component of I_Na (I_Na(W)) evoked by the short ascending ramp pulse (V_ramp) was overly lessened in the CBZ presence. Tefluthrin (Tef), a synthetic pyrethroid, known to stimulate I_Na, augmented the strength of the voltage-dependent hysteresis (Hys_(V)) of persistent I_Na (I_Na(P)) in response to the isosceles-triangular V_ramp; moreover, further application of CBZ attenuated Tef-mediated accentuation of I_Na(P)’s Hys_(V). With a two-step voltage protocol, the recovery of I_Na(T) inactivation seen in Neuro-2a cells became progressively slowed by adding CBZ; however, the cumulative inhibition of I_Na(T) evoked by pulse train stimulation was enhanced during exposure to this drug. Neuro-2a-cell exposure to CBZ (100 μM), the magnitude of erg-mediated K⁺ current measured throughout the entire voltage-clamp steps applied was mildly inhibited. The docking results regarding the interaction of CBZ and voltage-gate Na⁺ (Na_V) channel predicted the ability of CBZ to bind to some amino-acid residues in Na_V due to the existence of a hydrogen bond or hydrophobic contact. It is conceivable from the current investigations that the I_Na (I_Na(T), I_Na(L), I_Na(W), and I_Na(P)) residing in Neuro-2a cells are susceptible to being suppressed by CBZ, and that its block on I_Na(L) is larger than that on I_Na(T). Collectively, the magnitude and gating of Na_V channels produced by the CBZ presence might have an impact on its anticonvulsant and analgesic effects occurring in vivo.     

Effectiveness of Columbianadin,a Bioactive Coumarin Derivative,in Perturbing Transient and Persistent INa

Columbianadin (CBN) is a bioactive coumarin-type compound with various biological activities. However, the action of CBN on the ionic mechanism remains largely uncertain, albeit it was reported to inhibit voltage-gated Ca²⁺ current or to modulate TRP-channel activity. In this study, whole-cell patch-clamp current recordings were undertaken to explore the modifications of CBN or other related compounds on ionic currents in excitable cells (e.g., pituitary GH₃ cells and HL-1 atrial cardiomyocytes). GH₃-cell exposure to CBN differentially decreased peak or late component of voltage-gated Na⁺ current (I_Na) with effective IC₅₀ of 14.7 or 2.8 µM, respectively. The inactivation time course of I_Na activated by short depolarization became fastened in the presence of CBN with estimated K_D value of 3.15 µM. The peak I_Na diminished by 10 µM CBN was further suppressed by subsequent addition of either sesamin (10 µM), ranolazine (10 µM), or tetrodotoxin (1 µM), but it was reversed by 10 µM tefluthrin (Tef); however, further application of 10 µM nimodipine failed to alter CBN-mediated inhibition of I_Na. CBN (10 µM) shifted the midpoint of inactivation curve of I_Na to the leftward direction. The CBN-mediated inhibition of peak I_Na exhibited tonic and use-dependent characteristics. Using triangular ramp pulse, the hysteresis of persistent I_Na enhanced by Tef was noticed, and the behavior was attenuated by subsequent addition of CBN. The delayed-rectifier or erg-mediated K⁺ current was mildly inhibited by 10 µM CBN, while it also slightly inhibited the amplitude of hyperpolarization-activated cation current. In HL-1 atrial cardiomyocytes, CBN inhibited peak I_Na and raised the inactivation rate of the current; moreover, further application of 10 µM Tef attenuated CBN-mediated decrease in I_Na. Collectively, this study provides an important yet unidentified finding revealing that CBN modifies I_Na in electrically excitable cells.     

Rufinamide,a Triazole-Derived Antiepileptic Drug,Stimulates Ca2+-Activated K+ Currents While Inhibiting Voltage-Gated Na+ Currents

Rufinamide (RFM) is a clinically utilized antiepileptic drug that, as a triazole derivative, has a unique structure. The extent to which this drug affects membrane ionic currents remains incompletely understood. With the aid of patch clamp technology, we investigated the effects of RFM on the amplitude, gating, and hysteresis of ionic currents from pituitary GH₃ lactotrophs. RFM increased the amplitude of Ca²⁺-activated K⁺ currents (I_K(Ca)) in pituitary GH₃ lactotrophs, and the increase was attenuated by the further addition of iberiotoxin or paxilline. The addition of RFM to the cytosolic surface of the detached patch of membrane resulted in the enhanced activity of large-conductance Ca²⁺-activated K⁺ channels (BK_Ca channels), and paxilline reversed this activity. RFM increased the strength of the hysteresis exhibited by the BK_Ca channels and induced by an inverted isosceles-triangular ramp pulse. The peak and late voltage-gated Na⁺ current (I_Na) evoked by rapid step depolarizations were differentially suppressed by RFM. The molecular docking approach suggested that RFM bound to the intracellular domain of K_Ca1.1 channels with amino acid residues, thereby functionally affecting BK_Ca channels’ activity. This study is the first to present evidence that, in addition to inhibiting the I_Na, RFM effectively modifies the I_K(Ca), which suggests that it has an impact on neuronal function and excitability.     

Midazolam’s Effects on Delayed-Rectifier K+ Current and Intermediate-Conductance Ca2+-Activated K+ Channel in Jurkat T-lymphocytes

Midazolam (MDZ) could affect lymphocyte immune functions. However, the influence of MDZ on cell’s K⁺ currents has never been investigated. Thus, in the present study, the effects of MDZ on Jurkat T lymphocytes were studied using the patch-clamp technique. Results showed that MDZ suppressed the amplitude of delayed-rectifier K⁺ current (I_K(DR)) in concentration-, time-, and state-dependent manners. The IC₅₀ for MDZ-mediated reduction of I_K(DR) density was 5.87 μM. Increasing MDZ concentration raised the rate of current-density inactivation and its inhibitory action on I_K(DR) density was estimated with a dissociation constant of 5.14 μM. In addition, the inactivation curve of I_K(DR) associated with MDZ was shifted to a hyperpolarized potential with no change on the slope factor. MDZ-induced inhibition of I_K(DR) was not reversed by flumazenil. In addition, the activity of intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels was suppressed by MDZ. Furthermore, inhibition by MDZ on both I_K(DR) and IK_Ca-channel activity appeared to be independent from GABAA receptors and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes. In conclusion, MDZ suppressed current density of I_K(DR) in concentration-, time-, and state-dependent manners in Jurkat T-lymphocytes and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes.     

Selective Cytotoxicity of Complexes with N,N,N-Donor Dipodal Ligand in Tumor Cells

The present article demonstrates selective cytotoxicity against cancer cells of the complexes [Co(L^D)₂]I₂∙CH₃OH (1), [CoL^D(NCS)₂] (2) and [VOL^D(NCS)₂]∙C₆H₅CH₃ (3) containing the dipodal tridentate ligand LD = N,N-bis(3,5-dimethylpyrazol-1-ylmethyl)amine), formed in situ. All tested complexes expressed greater anticancer activities and were less toxic towards noncancerous cells than cisplatin. Cobalt complexes (1 and 2) combined high cytotoxicity with selectivity towards cancer cells and caused massive tumour cell death. The vanadium complex (3) induced apoptosis specifically in cancer cells and targeted proteins, controlling their invasive and metastatic properties. The presented experimental data and computational prediction of drug ability of coordination compounds may be helpful for designing novel and less toxic metal-based anticancer species with high specificities towards tumour cells.     

1,25-Dihydroxyvitamin D3 and 20-Hydroxyvitamin D3 Upregulate LAIR-1 and Attenuate Collagen Induced Arthritis

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)₂D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)₂D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)₂D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1^−/− deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.     

Increase in Epithelial Permeability and Cell Metabolism by High Mobility Group Box 1, Inflammatory Cytokines and TPEN in Caco-2 Cells as a Novel Model of Inflammatory Bowel Disease

High mobility group box 1 protein (HMGB1) is involved in the pathogenesis of inflammatory bowel disease (IBD). Patients with IBD develop zinc deficiency. However, the detailed roles of HMGB1 and zinc deficiency in the intestinal epithelial barrier and cellular metabolism of IBD remain unknown. In the present study, Caco-2 cells in 2D culture and 2.5D Matrigel culture were pretreated with transforming growth factor-β (TGF-β) type 1 receptor kinase inhibitor EW-7197, epidermal growth factor receptor (EGFR) kinase inhibitor AG-1478 and a TNFα antibody before treatment with HMGB1 and inflammatory cytokines (TNFα and IFNγ). EW-7197, AG-1478 and the TNFα antibody prevented hyperpermeability induced by HMGB1 and inflammatory cytokines in 2.5D culture. HMGB1 affected cilia formation in 2.5D culture. EW-7197, AG-1478 and the TNFα antibody prevented the increase in cell metabolism induced by HMGB1 and inflammatory cytokines in 2D culture. Furthermore, ZnSO₄ prevented the hyperpermeability induced by zinc chelator TPEN in 2.5D culture. ZnSO₄ and TPEN induced cellular metabolism in 2D culture. The disruption of the epithelial barrier induced by HMGB1 and inflammatory cytokines contributed to TGF-β/EGF signaling in Caco-2 cells. The TNFα antibody and ZnSO₄ as well as EW-7197 and AG-1478 may have potential for use in therapy for IBD.