Smac127 Has Proapoptotic and Anti-Inflammatory Effects on Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Rheumatoid arthritis (RA) is characterized by synovial inflammation and hyperplasia. Fibroblast-like synoviocytes (FLSs) are apoptosis-resistant and contribute to the pathogenesis of RA by producing cytokines and proteolytic enzymes, which degrade the extracellular matrix. We evaluated the proapoptotic and anti-inflammatory activity of the small molecule Smac127 on RA-FLSs cultured in synovial fluid (SF), in order to reproduce the physiopathological environmental characteristic of RA joints. In this context, Smac127 induces apoptosis by inhibiting apoptosis proteins (IAPs). This inhibition activates caspase 3 and restores the apoptotic pathway. In addition, Smac127 induces a significant inhibition of the secretion of IL-15 and IL-6, stimulation of pannus formation, and damage of bone and cartilage in RA. Also the secretion of the anti-inflammatory cytokine IL-10 is dramatically increased in the presence of Smac127. The cartilage destruction in RA patients is partly mediated by metalloproteinases; here we show that the MMP-1 production by fibroblasts cultured in SF is significantly antagonized by Smac127. Conversely, this molecule has no significant effects on RANKL and OPG production. Our observations demonstrate that Smac127 has beneficial regulatory effects on inflammatory state of RA-FLSs and suggest a potential use of Smac127 for the control of inflammation and disease progression in RA.

Electromagnetic character of the competitive γγ/γ-decay from 137mBa.

Second-order processes in physics is a research topic focusing attention from several fields worldwide including, for example, non-linear quantum electrodynamics with high-power lasers, neutrinoless double-ß decay, and stimulated atomic two-photon transitions. For the electromagnetic nuclear interaction, the observation of the competitive double-γ decay from 137mBa has opened up the nuclear structure field for detailed investigation of second-order processes through the manifestation of off-diagonal nuclear polarisability. Here, we confirm this observation with an 8.7σ significance, and an improved value on the double-photon versus single-photon branching ratio as 2.62 × 10-6(30). Our results, however, contradict the conclusions from the original experiment, where the decay was interpreted to be dominated by a quadrupole-quadrupole component. Here, we find a substantial enhancement in the energy distribution consistent with a dominating octupole-dipole character and a rather small quadrupole-quadrupole component in the decay, hindered due to an evolution of the internal nuclear structure. The implied strongly hindered double-photon branching in 137mBa opens up the possibility of the double-photon branching as a feasible tool for nuclear-structure studies on off-diagonal polarisability in nuclei where this hindrance is not present.

Cyclic nucleotide-dependent relaxation in human umbilical vessels.

Umbilical vessels have a low sensitivity to dilate, and this property is speculated to have physiological implications. We aimed to investigate the different relaxing responses of human umbilical arteries (HUAs) and veins (HUVs) to agonists acting through the cAMP and cGMP pathways. Vascular rings were suspended in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U44069, concentration-response curves to the nitric oxide (NO) donor sodium nitroprusside (SNP), the soluble guanylate cyclase (sGC) stimulator BAY 41-2272, the adenylate cyclase (AC) activator forskolin, the ß-adrenergic receptor agonists isoproterenol (ADRB1), salmeterol (ADRB2), and BRL37344 (ADRB3), and the phosphodiesterase (PDE) inhibitors milrinone (PDE3), rolipram (PDE4), and sildenafil (PDE5) were performed. None of the tested drugs induced a relaxation higher than 30% of the U44069-induced tone. Rings from HUAs and HUVs showed a similar relaxation to forskolin, SNP, PDE inhibitors, and ADRB agonists. BAY 41-2272 was significantly more efficient in relaxing veins than arteries. ADRB agonists evoked weak relaxations (< 20%), which were impaired in endothelium-removed vessels or in the presence of the NO synthase inhibitor L-NAME, sGC inhibitor ODQ. PKA and PKG inhibitors impaired ADBR1-mediated relaxation but did not affect ADRB2-mediated relaxation. ADRB3-mediated relaxation was impaired by PKG inhibition in HUAs and by PKA inhibition in HUVs. Although HUA and HUV rings were relaxed by BRL37344, immunohistochemistry and RT-qPCR analysis showed that, compared to ADRB1 and ADRB2, ADRB3 receptors are weakly or not expressed in umbilical vessels. In conclusion, our study confirmed the low relaxing capacity of HUAs and HUVs from term infants. ADRB-induced relaxation is partially mediated by endothelium-derived NO pathway in human umbilical vessels.

Higher mitochondrial DNA content in human IUGR placenta.

IUGR has been associated to a specific placental phenotype with reduced uptake of specific nutrients. Recently, it has been hypothesized that IUGR may be determined during early gestation. This period is characterized by decidual trophoblast invasion and by intense cellular growth, replication and differentiation. Since a huge energetic availability is required during gestation, we hypothesize that mitochondria may play a crucial role in this process being the main energetic producer in the cell. The aim of this study was to investigate the role of mitochondria in IUGR pathogenesis, evaluating the number of mitochondrial DNA copies (mtDNA) in IUGR placentae compared to controls. Placental samples were collected from 50 singleton pregnancies at the time of elective caesarean section. Twenty-six pregnancies were controls with normal intrauterine growth (AGA) and 24 were studied after the in utero diagnosis of IUGR. All samples were analyzed by real-time quantitative PCR and statistical analysis was performed by non-parametric tests. The median value of mitochondrial DNA content (IQR) in AGA and IUGR placentae was significantly different (455 and 698, respectively, p=0.004). The cell types responsible for the difference observed is unknown and it is possible that changes observed in the proportion of cell types may influence this measurement. Moreover, a significant negative relationship was observed between mtDNA and umbilical venous pO(2), with the highest levels detected in the most severe IUGR cases according to Doppler findings and to the presence of preeclampsia. These data suggest a relationship between the pathogenesis of IUGR and increased placental mtDNA copies. From our results we can speculate that increased mtDNA represents an adaptation of the metabolic placental mechanism to the calorie restriction of the fetus. Furthermore, we found that this rise was inversely related to oxygen tension in the umbilical vein. Although no specific pathogenetic role can be implied, mtDNA increases with hypoxia in placentas of IUGR.

Mitochondrial DNA content: A new biomarker for sudden intrauterine unexplained death syndrome (SIUDS).

In literature there are no data related to mitochondrial DNA (mtDNA) content in sudden intrauterine unexplained death syndrome (SIUDS). To test the hypothesis that a quantitative excess of mtDNA could play a role in the pathogenesis of SIUDS, mtDNA content was measured in cerebral cortex of 9 SIUDS and in 7 controls. The median (interquartile range) mtDNA in SIUDS and controls was 14,000 (8600-33,500), 3400 (0-8500) copies per nuclear DNA, respectively (p=0.007). If mitochondria are involved in SIUDS, then higher mitochondrial DNA content may be a biomarker of this syndrome.

Inhibitory effect of pasireotide and octreotide on lymphocyte activation.

Somatostatin (SST) regulates the function of the central and peripheral nervous system, the endocrine and exocrine organs, as well as the vascular and immune system. These actions are mediated by five specific membrane somatostatin receptors. This study compares the effects on human lymphocytes of two long-acting somatostatin analogues that have different receptor affinity: octreotide and pasireotide. Both analogues have an antiproliferative effect on human lymphocyte proliferation, but they act at different concentration and, while octreotide enhances IL10 and inhibits gamma IFN pasireotide inhibits IL2 and gamma IFN. In both sets of experiment the different behaviour of the two analogues could be due to their different affinity to the SSTR subtypes. Finally this study suggest that the growth inhibitory action of somatostatin analogues is an apoptotic phenomenon and it can be mediated by SSTR2a, in the case of octreotide, and by SSTR3 when pasireotide is used or it can be mediated by the heterodimerization of the two receptor.

Vasomotor effects of hydrogen sulfide in human umbilical vessels.

Hydrogen sulfide (H2S) has recently emerged as a biologically active gas with multiple effects on the cardiovascular system. We aimed to investigate the vasomotor actions of sodium sulfide (Na2S), which forms H2S and HS- in solution, in human umbilical artery (HUA) and vein (HUV) rings. In addition, we examined by immunocytochemistry the expression and localization of cystathionine ß-synthase (CBS), cystathionine lyase (CSE), and 3-mercaptopyruvate sulphurtransferase (MPST), the enzymes responsible for endogenous H2S production. Human umbilical vessels were compared with chicken embryo umbilical vessels. HUA and HUV expressed a robust signal for CSE, CBS, and 3-MPST in both endothelial and smooth muscle cells. However, HUA rings did not respond to Na2S (10-6M-10-3M) either at resting tone or during contraction evoked by serotonin or KCl. Similarly, the extraembryonic part of chicken allantoic artery did not respond to Na2S. In contrast, Na2S induced a concentration-dependent contraction in HUV rings under resting tone and a concentration-dependent relaxation when the H2UV rings were contracted with serotonin (42 ± 5% relaxation) or KCl (12 ± 5% relaxation). Na2S-induced contraction of HUV was impaired following removal of extracellular Ca2+, endothelial denudation, NO synthase inhibition (L-NAME), or soluble guanylate cyclase (sGC) inhibition (ODQ). Na2S-induced relaxation of HUV was impaired by the KATP channel inhibitor glibenclamide. In conclusion, H2S does not have vasomotor effects on HUA but induced contraction (mediated through inactivation of the NO/sGC axis) and relaxation (mediated through KATP channels) in HUV. Our data suggest a role for H2S in the venous side of human umbilical circulation.

Cycling and early pregnant endometrium as a site of regulated expression of the vitamin D system.

In addition to its calciotropic function, the secosteroid 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has potent anti-proliferative/immunomodulatory effects on various tissues. Consistently, the enzyme that catalyzes the synthesis of 1,25(OH)(2)D(3), 1alpha-hydroxylase (1alpha-OHase) and the vitamin D receptor have a widespread tissue distribution. Among site-specific functions, the hormone has been suggested to be involved in uterine physiology. However, molecular analysis of the vitamin D system in normal endometrium throughout the menstrual cycle as well as its regulation in the context of endometrial physiological and pathological events have received very limited attention. Thus, we have studied expression, localization and regulation of 1alpha-OHase in human cycling and early pregnant endometrium. The capacity for 1alpha-hydroxylation and the presence of vitamin D receptor in endometrial cells have also been evaluated. The functional significance of these findings has been tested by evaluating gene expression of the catabolic enzyme, vitamin D 24-hydroxylase, and of the adhesion protein, osteopontin. Finally, to verify any potential dysfunction of the vitamin D system in endometriosis, a reproductive disease characterized by immune-mediated anomalies, we have analyzed expression of 1alpha-OHase in both eutopic and ectopic endometrium of affected patients. Results obtained showed that the active form of the 1alpha-OHase gene was expressed in human endometrial stromal cells independent of the cycle phase but with a significant increase in early pregnant decidua. A similar profile was observed for the protein, which was abundantly expressed in the cytoplasm of both endometrial stroma and epithelial glands. Both cycling and early pregnant endometrial cells also expressed the vitamin D receptor. In the same cells, 1alpha-OHase mRNA levels were significantly stimulated by the pro-inflammatory cytokine interleukin (IL)-1beta (50 and 500 pg/ml) while addition of the active form of the hormone could modulate both CYP24 and osteopontin gene expression. The 1alpha-OHase gene was also expressed in ectopic endometrium and its levels were increased in proliferative phase cultures derived from patients with endometriosis. Human cycling endometrium may be included among the extrarenal sites able to synthesize vitamin D. The IL-1beta-mediated induction of 1alpha-OHase gene and the hormonal modulation of osteopontin support a role for the hormone in the immunological mechanisms underlying uterine function. Abnormalities of this system are present in endometriosis.

Effects of chimeric somatostatin-dopamine molecules on human peripheral blood lymphocytes activation.

BIM 23A761, selective for somatostatin receptors subtypes 2, 5 and the dopamine receptor subtype 2, and BIM 23A757 with affinity for SSTR2 and DAR2 were studied on human PBL proliferation and activation. BIM 23A761 was significantly more potent than specific SSTR and DAR2 agonists in suppressing lymphocyte proliferation induced by mitogen or alloantigen, while BIM 23A757 was more potent than specific SSTR2 and DAR2 agonists in suppressing antigen induced proliferation only. Both molecules displayed enhanced potency in suppressing IFNgamma and IL-6 secretion compared with the SSTR and DAR2 analogs, while only BIM 23A761 was able to inhibit IL-2 secretion and its effect is more potent than the control analogs. Furthermore BIM 23A761 inhibit cell progression into the S phase and then into the G2/M, while BIM 23A757 inhibited bromodeoxyuridine incorporation only during the S phase. Both chimeric molecules resulted significantly more effective than the respective controls.

Proapoptotic activity of a monomeric smac mimetic on human fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Inhibitors of apoptosis proteins (IAPs) block cell death in response to diverse stimuli. The mitochondrial protein, second mitochondria-derived activator of caspase (Smac), negatively regulates IAP inhibition of caspase activity. We investigated the proapoptotic activity of a synthetic Smac (Smac 066) on fibroblast-like synoviocytes (FLS) derived from patients with active rheumatoid arthritis (RA). We found that Smac 066 induced significant apoptosis in all RA-FLS samples. Furthermore, IAPs, which are upregulated in RA-FLS, were downregulated by Smac 066. This suggested that IAPs upregulation was responsible for RA-FLS sensitivity to Smac 066. Next, we analysed caspase activation and found that Smac 066 was associated with caspase 8 and caspase 3 activities. We then investigated the mechanism underlying Smac 066 downregulation of IAPs in RA-FLS with an apoptotic pathway array. Interestingly, Smac 066 significantly upregulated IGFBP-5, a protein involved in differentiation, apoptosis, and osteoblastic activation. Smac 066 may represent a new therapeutic approach to RA treatment.

The apoptotic effect of somatostatin analogue SMS 201-995 on human lymphocytes.

The antiproliferative effect of a synthetic octapeptide, somatostatin analogue SMS 201-995 (SMS), and its capacity to bind were evaluated on human peripheral blood lymphocytes (PBL) activated by phytohemoagglutinin (PHA). We then addressed our work to investigate if SMS inhibits PHA activation of PBL by a cytostatic rather than a cytotoxic mechanism. Consequently, we studied the cell cycle distribution and the activation of caspase-3, measuring the presence of the cleavage product of poly(ADP-ribose) polymerases (PARP), and we evaluated the presence of apoptotic DNA by using a monoclonal antibody specific for the single-stranded regions of DNA. All our results indicate that SMS induces apoptosis in activated lymphocytes.

Efficacy of photodynamic therapy against doxorubicin-resistant murine tumors.

Photodynamic therapy (PDT) is a relatively new cancer treatment modality that employs light excitation of a photosensitizer to yield cytotoxic oxygen-related species. In the present study we explored whether PDT would have therapeutic effect against doxorubicin-resistant murine tumors. We compared the efficacy of PDT with aluminium disulphonated phthalocyanine (A1S2Pc) and laser light on the doxorubicin-sensitive murine tumors, B16 melanoma (B16), L1210 leukemia (L1210), P388 lymphoma (P388) and the corresponding doxorubicin-resistant lines (B16/Dx, L1210/Dx and P388/Dx). Mice bearing L1210-L1210/Dx, P388-P388/Dx and B16-B16/Dx, were treated with 5 mg/kg of A1S2Pc and laser light (100 mW/cm2 x 10 min of exposure) or with doxorubicin (10 or 12 mg/kg i.v.). The results show that PDT is active versus all tumors while doxorubicin is effective only against the three sensitive tumor lines (L1210, P388 and B16). These observations suggest that PDT might be a beneficial alternative treatment for drug-resistant tumors.

Down regulation of Qa gene expression on drug-modified tumor cells.

BACKGROUND: Mouse leukemia, L1210, strongly enhances its immunogenicity following in vivo treatment with 5-(3-3'-dimethyl-1-triazeno) imidazole-4-carboxamide (DTIC). Previous experiments have shown that transformed cells elicit a cell-mediated response accountable for rejection and resistance to a subsequent injection of parental tumor into a syngeneic host. L1210 expresses classical H-2 class I molecules, and since it has been shown that DTIC treatment does not modify the expression of these molecules, this is a suitable model to study nonclassical class I antigens, such as Qa2 glycoproteins, and their potential role in tumorigenicity. METHODS: Cloned cells from L1210 were treated with DTIC and then H-2D, and Qa antigen expression was studied on four clones, before and after xenogenization with DTIC. RESULTS AND CONCLUSIONS: a strong decrease of Qa2 molecule expression was demonstrated by radioimmunoassay and immunofluorescent staining and was confirmed by FACS and 2D-gel analysis. The presence or the absence of Qa antigens on tumor cells could thus be involved in tolerance or rejection of tumor cells in syngeneic animals.

Monoclonal antibodies against recombinant human growth hormone as probes to study immune function.

Monoclonal antibodies were raised against human recombinant growth hormone (rhGH) and those that did not cross-react with other human recombinant proteins like prolactin (PRL), interleukin 2 (IL-2), insulin, or bovine pituitary growth hormone were selected. The selected hybridoma supernatants were studied for their ability to influence T lymphocyte proliferation when induced either by a mitogen, such as phytohemagglutinin (PHA), or by alloantigen. All supernatants inhibited proliferation. Three MAbs were then purified by several passages on antimouse IgG (or IgM)-agarose columns, and characterized. These MAbs recognized three different epitopes, as revealed by competition study, although their inhibitory effect on PHA-induced T cell proliferation was quite similar. The data demonstrate that the MAbs were not cytolytic, that they did not interfere with the PHA binding to T cell membranes, and, as revealed by FACS analysis, did not bind to the membrane. Finally, these MAbs immunoprecipitated a 44-kDa molecule from PHA-activated T cell-concentrated supernatants. These data indicate that the MAbs recognized a soluble factor that plays a central role in T cell proliferation and that is probably the immune growth hormone.

Monoclonal antibody against human growth hormone receptor.

GHR shows a high degree of homology with the prolactin receptor and with the other receptors that belong to the hemopoietic receptor superfamily. This paper describes a monoclonal antibody (MAb) (2B4B6) specific for both the extracellular domain of human GHR and human growth hormone (GH) binding protein. Mice were immunized against a seven-aminoacid peptide sequence screened by FASTA (sequence similarity search served by Genome-Net) from the European Bioinformatics Institute to exclude the existence of human membrane proteins with significant sequence homology. MAbs were screened against the peptide sequence and 2B4B6 was selected for its capability to recognize the full-length hGHBP. As evaluated by both enzyme-linked immunoadsorbent assay (ELISA) and FACS analysis, this MAb seems to recognize and bind to a hGHR positive cell line, IM-9, as well as a murine cell line, BaF3 (8/6), transfected with a chimeric construct, hGHR/hG-CSFR and expressing hGHR on the cell membrane. Studies investigating the biological effects of this MAb showed that anti-hGHR mediated inhibition of cell proliferation was not due to competition with GH binding but rather to prevention of receptor dimerization. Because of its specificity, this MAb may be usefully applied in situations in which GHR and receptors with a high degree of homology, such as PRLR (prolactin receptor), are expressed simultaneously, as occurs in the immune system.

Vitamin D protects human endothelial cells from oxidative stress through the autophagic and survival pathways.

CONTEXT: Recently, vitamin D (VitD) has been recognized as increasingly importance in many cellular functions of several tissues and organs other than bone. In particular, VitD showed important beneficial effects in the cardiovascular system. Although the relationship among VitD, endothelium, and cardiovascular disease is well established, little is known about the antioxidant effect of VitD. OBJECTIVE: Our objective was to study the intracellular pathways activated by VitD in cultured human umbilical vein endothelial cells undergoing oxidative stress. DESIGN: Nitric oxide production, cell viability, reactive oxygen species, the mitochondrial permeability transition pore, membrane potential, and caspase-3 activity were measured during oxidative stress induced by administration of 200 µM hydrogen peroxide for 20 minutes. Experiments were repeated in the presence of specific vitamin D receptor ligand ZK191784. RESULTS: Pretreatment with VitD alone or in combination with ZK191784 is able to reduce the apoptosis-related gene expression, involving both intrinsic and extrinsic pathways. At the same time, it has been shown the activation of pro-autophagic beclin 1 and the phosphorylation of ERK1/2 and Akt, indicating a modulation between apoptosis and autophagy. Moreover, VitD alone or in combination with ZK191784 is able to prevent the loss of mitochondrial potential and the consequent cytochrome C release and caspase activation. CONCLUSIONS: The present study shows that VitD may prevent endothelial cell death through modulation of the interplay between apoptosis and autophagy. This effect is obtained by inhibiting superoxide anion generation, maintaining mitochondria function and cell viability, activating survival kinases, and inducing NO production.

Triazene compounds in the treatment of acute myeloid leukemia: a short review and a case report.

Acute myeloid leukemia (AML) is a highly lethal disease, especially in old patients. Chemoresistance and the absence of host immune responses against autochthonous malignancy play a major role in the poor prognosis of AML. The triazene compounds Dacarbazine and Temozolomide are monofunctional alkylators that donate methyl groups to many sites in DNA, including the O(6)-position of guanine producing O(6)-methylguanine (O(6)-MeG). If not repaired, O(6)-MeG frequently mispairs with thymine during DNA duplication. O(6)-MeG:T mismatches can be recognized by the mismatch repair (MMR) system which activates a cascade of molecular events leading to cell cycle arrest and cell death. If MMR is defective, cells continue to divide and GC → AT transition mutations occur. In preclinical models, such mutations can lead to the appearance of abnormal proteins containing non-self peptides ("chemical xenogenization" CX) that can be recognized by host cell-mediated immunity. Repair of O(6)-MeG is achieved by the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT), which removes the methyl adduct in an autoinactivating stoichiometric reaction. High MGMT levels attenuate the pharmacodynamic effects of triazenes. In the last few years, triazenes, alone or with MGMT inhibitors, have been tested in AML. In view of their potential activity as CX inducers, triazenes could offer the additional advantage of host anti-leukemia immune responses. The present paper describes several studies of leukemia treatment with triazenes and a case of acute refractory leukemia with massive skin infiltration by malignant cells. Treatment with Temozolomide and Lomeguatrib, a potent MGMT inhibitor, produced a huge, although transient, blastolysis and complete disappearance of all skin lesions.

Hypertrophic donavanosis in a young pregnant woman.

BACKGROUND: Donovanosis is a chronic bacterial illness, progressive and indolent, which normally attacks the skin and mucous membranes in the genital and perigenital regions. CASE: An 18-year-old pregnant female presented with large, hypertrophic lesions in the ano-genital region. HIV serology was negative. Pap smear revealed a CIN 1 associated with HPV infection. Biopsy yielded macrophages laden with Gram-negative Donovan bodies. SUMMARY AND CONCLUSION: A diagnosis of vulvar and perianal donovanosis was reached; the patient decided to terminate the pregnancy and was treated with azithromycin, which led to clinical resolution.