ZnT8 Arg325Trp polymorphism influences zinc transporter expression and cytokine production in PBMCs from patients with diabetes.

AIMS: ZnT8 Arg325Trp polymorphism has been associated with type 2 diabetes (T2DM) susceptibility. The Arg-325 risk variant shows accelerated zinc (Zn) transport kinetic and reduced glucose-stimulated insulin secretion in pancreatic cells. However, it remains unexplored the role of Znt8 polymorphism in the regulation of Zn homeostasis and inflammatory response in peripheral blood mononuclear cells (PBMCs) from T2DM patients. METHODS AND RESULTS: A total of 556 healthy controls and 413 T2DM patients were genotyped for ZnT8 Arg325Trp polymorphism confirming the association of Arg-325 variant with an increased T2DM risk (OR = 1.35 95% C.I: 1.10-1.66; p = 0.0044). Moreover, PBMCs from Arg/Arg T2DM subjects showed increased intracellular free Zn, higher gene expression of Metallothioneins, Znt1, Znt8, Zip2 genes, and reduced Znt4 and Znt7. Higher release of IL-1α, IL-1ß, IFN-γ, IL-12p70 and TNF-α and a reduced IL-10 secretion after lipopolysaccharide (LPS) stimulation were observed in PBMCs from Arg/Arg T2DM carriers as compared to subjects with the Trp variant. CONCLUSIONS: Our data provide evidence of a substantial different Zn homeostasis regulation between Znt8 Arg-325 and Trp-325 carriers in PBMCs from T2DM patients. Moreover, Znt8 Arg-325 risk variant shows an enhanced inflammatory response upon LPS stimulation that might aggravate insulin resistance and the progression of diabetes cardiovascular complications.

Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E(2)-dependent plaque instability.

BACKGROUND: Studies have implicated a role for prostaglandin (PG) E(2)-dependent matrix metalloproteinase (MMP) biosynthesis in the rupture of atherosclerotic plaque. Cyclooxygenase-2 (COX-2) and PGE synthase (PGES) are coregulated in nucleated cells by inflammatory stimuli. The aim of this study was to characterize the expression of COX-2 and PGES in carotid plaques and to correlate it with the extent of inflammatory infiltration and MMP activity and with clinical features of patients' presentation. METHODS AND RESULTS: Plaques were obtained from 50 patients undergoing carotid endarterectomy and divided into 2 groups (symptomatic and asymptomatic) according to clinical evidence of recent transient ischemic attack or stroke. Plaques were analyzed for COX-2, PGES, MMP-2, and MMP-9 by immunocytochemistry and Western blot, whereas zymography was used to detect MMP activity. Immunocytochemistry was used to identify CD68+ macrophages, CD3+ T lymphocytes, and HLA-DR+ cells. The percentage of macrophage-rich areas was larger (P<0.0001) in symptomatic plaques. COX-2, PGES, and MMPs were detected in all specimens; enzyme concentration, however, was significantly higher in symptomatic plaques. COX-2, PGES, and MMPs were especially noted in shoulders of symptomatic plaques, colocalizing with HLA-DR+ macrophages. All symptomatic plaques contained activated forms of MMPs. Finally, inhibition of COX-2 by NS-398 was accompanied by decreased production of MMPs that was reversed by PGE(2). CONCLUSIONS: This study demonstrates the colocalization of COX-2 and PGES in symptomatic lesions and provides evidence that synthesis of COX-2 and PGES by activated macrophages is associated with acute ischemic syndromes, possibly through metalloproteinase-induced plaque rupture.

Aneurysm of a jejunal branch of the superior mesenteric artery in a patient with Marfan's syndrome.

A case of post-traumatic aneurysm of a jejunal branch of the superior mesenteric artery in a patient with Marfan's syndrome is reported. Ascending aortic involvement is well known in Marfan's syndrome but no association with visceral artery aneurysms has been previously described. The blunt abdominal trauma preceding the detection of the aneurysm may have been the precipitating cause in a predisposed patient. Because of the high risk of rupture, aneurysms of the superior mesenteric artery branches should be treated. Excision or ligation without restoring continuity are the most common surgical procedures; endovascular embolization is an alternative option especially in high risk patients.

Efficacy of rifampin-levofloxacin as a prophylactic agent in preventing Staphylococcus epidermidis graft infection.

OBJECTIVES: To investigate the efficacy of levofloxacin in the prevention of vascular prosthetic graft infection in a rat model. METHODS: Graft infections were established in the subcutaneous tissue of 225 male Wistar rats by implantation of Dacron prostheses followed by topical inoculation with methicillin-susceptible and methicillin-resistant S. epidermidis. The study included a group without contamination, two contaminated groups without prophylaxis, two contaminated groups with intraperitoneal levofloxacin prophylaxis, two contaminated groups with intraperitoneal cefazolin prophylaxis, two contaminated groups with intraperitoneal teicoplanin prophylaxis and six contaminated groups with rifampin-soaked graft and intraperitoneal levofloxacin, cefazolin or teicoplanin prophylaxis. The grafts were removed after 7 days and evaluated by quantitative culture. RESULTS: The efficacy of levofloxacin against the methicillin-susceptible strain was not different to that of cefazolin or teicoplanin. Levofloxacin showed slight less efficacy than teicoplanin against the methicillin-resistant strain. The combination levofloxacin-rifampin demonstrated to be similarly effective to the combination rifampin-teicoplanin and more effective than the combination rifampin-cefazolin against both strains. CONCLUSIONS: Rifampin-levofloxacin combination seems useful for the prevention of late-appearing vascular graft infections caused by S. epidermidis.

alpha-MSH modulates local and circulating tumor necrosis factor-alpha in experimental brain inflammation.

Tumor necrosis factor (TNF-alpha) underlies pathological processes and functional disturbances in acute and chronic neurological disease and injury. The neuroimmunomodulatory peptide alpha-MSH modulates actions and production of proinflammatory cytokines including TNF-alpha, but there is no prior evidence that it alters TNF-alpha induced within the brain. To test for this potential influence of the peptide, TNF-alpha was induced centrally by local injection of bacterial lipopolysaccharide (LPS). alpha-MSH given once i.c.v. with LPS challenge, twice daily intraperitoneally (i.p.) for 5 d between central LPS injections, or both i.p. and centrally, inhibited production of TNF-alpha within brain tissue. Inhibition of TNF-alpha protein formation by alpha-MSH was confirmed by inhibition of TNF-alpha mRNA. Plasma TNF-alpha concentration was elevated markedly after central LPS, indicative of an augmented peripheral host response induced by the CNS signal. The increase was inhibited by alpha-MSH treatments, in relation to inhibition of central TNF-alpha. Presence within normal mouse brain of mRNA for the alpha-MSH receptor MC-1 suggests that the inhibitory effects of alpha-MSH on brain and plasma TNF-alpha might be mediated by this receptor subtype. The inhibitory effect of alpha-MSH on brain TNF-alpha did not depend on circulating factors because the effect also occurred in brain tissue in vitro. This indicates that alpha-MSH can act directly on brain cells to inhibit their production of TNF-alpha. If central TNF-alpha contributes to pathology in CNS disease and injury, and promotes inflammation in the periphery, agents that act on brain alpha-MSH receptors should decrease the pathological TNF-alpha reaction and promote tissue survival.

A potential mechanism of local anti-inflammatory action of alpha-melanocyte-stimulating hormone within the brain: modulation of tumor necrosis factor-alpha production by human astrocytic cells.

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) occurs in CNS tissue in neurological disorders, infection, and injury. Its excessive production is believed to contribute to local pathology, in which case modulation of TNF-alpha production should promote survival of neural tissue. The neuropeptide alpha-melanocyte stimulating hormone [alpha-MSH (1-13)] inhibits TNF-alpha production in vivo and in vitro, and in this research we tested the capacity of the peptide, and of an anti-inflammatory COOH-terminal tripeptide fragment of it, to inhibit TNF-alpha production induced by bacterial endotoxin in cells of a human glioma line (A-172, anaplastic astrocytoma cells). Both peptides were effective, although the alpha-MSH (1-13) sequence was more potent. Preincubation of the cells with alpha-MSH (1-13) markedly increased its effectiveness. The anticytokine effect of alpha-MSH in glioma cells may be mediated by human melanocortin-1 receptors; mRNA for this receptor subtype was isolated from resting A-172 cells. These results, combined with prior evidence of effectiveness of alpha-MSH molecules in modulating inflammatory processes and of their low toxicity, suggest that the molecules may be useful in the treatment of CNS disorders that have an inflammatory component.

alpha-MSH modulates experimental inflammatory bowel disease.

The mechanisms underlying inflammatory bowel disease (IBD) remain obscure but the importance of inflammatory processes is clear and most pharmacological therapies inhibit inflammation. The search for more effective agents with low toxicity continues. To test the possibility that the antiinflammatory/anticytokine peptide alpha-MSH can be used to control IBD, the peptide was administered to a murine colitis model. The peptide treatment had marked salutary effects: it reduced the appearance of fecal blood by over 80%, inhibited weight loss, and prevented disintegration of the general condition of the animals. Mice given alpha-MSH showed markedly lower production of TNF alpha by tissues of the lower colon stimulated with concanavalin A; the inhibitory effect of alpha-MSH on production of inflammatory nitric oxide by lower bowel tissue was even greater. The combined results indicate that alpha-MSH modulates experimental IBD, perhaps by inhibiting production within the gut of the local proinflammatory agents TNF alpha and nitric oxide, or by inhibiting inflammatory processes closely linked to these mediators.

[Video-thoracoscopic pulmonary lobectomy. Our experience]. / Lobectomie polmonari videotoracoscopiche. Nostra esperienza.

Video-endoscopic surgery developed from laparoscopic removal of the gallbladder and can be used for treatment of certain disease processes of the chest. The major application of this technique is the performance of pulmonary resections (lobectomies and pneumonectomies). Video-thoracoscopic lobectomy requires general anesthesia with a double lumen intubation. After collapse of the involved lung, two intercostal incisions are performed (the first one in seventh space, mid axillary and the second one in fifth space, below the angle of the scapula) and an additional mini-thoracotomy (submammillary in the fifth space) is made. The lobar arteries, veins and bronchus are occluded with an automatic endoscopic stapling device. Seven cases of pulmonary neoplasms (stage I) have been treated by video pulmonary lobectomy (3 right lower lobectomies, 2 right upper lobectomies, 2 left lower lobectomies). Two procedures have been converted for an advanced neoplastic stage that required a radical pneumonectomy and an uncontrolled bleeding. This new procedure has the advantage of greatly reducing the pain and ventilatory disability associated with conventional open thoracic surgery.

[Immediate reconstruction after radical mastectomy for breast carcinoma with a Becker-type expander prosthesis]. / Ricostruzione immediata dopo mastectomia radicale per carcinoma mammario con protesi-espansore tipo Becker.

In recent years, the breast has won importance from the female psychology point of view. After ablative surgery for cancer, the loss of the breast gives rise in the woman to a feeling of refusal of her new body shape. Reconstruction produces positive feelings in the patient. The best-used surgical technique for operable breast cancer is modified radical mastectomy. An immediate reconstruction of the missing breast is considered when the following conditions are fulfilled: desire for reconstruction, age of the patient, clinical and pathologic evaluation (tumor size-tumor grade-lymph-node status). After modified radical mastectomy with "en bloc" removal of axillary lymph nodes, an immediate reconstruction is performed by lining the surfaces with human fibrin glue (Tissucol) followed by the insertion of a prosthesis of appropriate size (Becker Expander Mammary Prosthesis-Type 1) in the subserratus-subpectoral position. Human fibrin lining reduces the incidence of capsular contracture and effusion production. A suction drain is placed in the axillary area and is removed after 5 days. The use of expanding-type prostheses means that the patient must return for further expanding. When the pathologic features are negative, the delayed nipple areola reconstruction is possible with the full-thickness skin grafts in the groin or in pre-existent appendectomy incision. By this procedure, adjuvant chemotherapy or radiation therapy can be performed if required. From 1987 to 1991 the procedure has been performed in 35 cases of mammary neoplasm (mean age 40 years) at INRCA Department of Surgery of Ancona). No evidence of adverse effect on the natural course of the breast disease for have been seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 bolus therapy induces immediate and selective disappearance from peripheral blood of all lymphocyte subpopulations displaying natural killer activity: role of cell adhesion to endothelium.

As early as 10-15 min after the start of a 30 min interleukin-2 (IL-2) infusion, a rapid, virtually complete disappearance of all natural killer (NK) lymphocyte subpopulations (including both CD3- CD56+ and CD3+ CD56+ cells with either alpha/beta or gamma/delta T-cell receptor) was observed from peripheral blood. In contrast, the number of T lymphocytes (CD3+ CD56-) was unmodified for at least 2 h after IL-2 injection. The IL-2-induced, rapid disappearance from peripheral blood of NK and NK-like lymphocytes may be related to their massive adherence to the activated endothelium. In this regard, IL-2 infusion caused a very rapid rise of tumour necrosis factor-alpha (TNF-alpha) plasma concentration, whereas other cytokines, such as interferon-gamma (IFN-gamma), were induced only at later times. In vitro experiments indicated that IL-2, either alone or better combined with TNF-alpha, exerts a rapid and selective stimulatory effect on NK adhesion to endothelial cells. On the basis of these findings, we suggest that the activation of NK lymphocytes induced by IL-2, alone or combined with TNF-alpha, plays a key role in mediating the massive and selective adherence of NK and NK-like cells following IL-2 bolus infusion.

Two-step differentiation of AML-193 leukemic line: terminal maturation is induced by positive interaction of retinoic acid with granulocyte colony-stimulating factor (CSF) and vitamin D3 with monocyte CSF.

The human AML-193 cell line requires exogenous granulocyte-monocyte colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) for growth in liquid or semisolid medium. However, these CSFs do not stimulate the differentiation of the cell line. We show that addition of all-trans retinoic acid (RA) or 1,25 dihydroxyvitamin D3 (D3) induces AML-193 cells to differentiate into the granulocytic or monocytic lineage, respectively. On the other hand, addition of either G- or M-CSF alone exerts virtually no differentiative effect. Terminal granulocytic or monocytic differentiation was observed when AML-193 cells were treated with RA and G-CSF, or D3 and M-CSF, respectively, as evaluated by cell morphology, analysis of surface antigens, and phagocytic functions. These positive interactions indicate that the differentiating activity of G- and M-CSF on leukemic cells may be unmasked by preliminary treatment with RA and D3, respectively, ie, the physiologic inducers override the leukemic differentiation blockade and CFSs exert their differentiative activity on the unblocked leukemic cells. These preliminary observations on a single cell line may pave the way for the designing of clinical protocols combining physiologic inducer(s) and hematopoietic growth factor(s) in the treatment of acute leukemia.

Adoptive immunotherapy with high-dose interleukin-2: kinetics of circulating progenitors correlate with interleukin-6, granulocyte colony-stimulating factor level.

Immunotherapy with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells results in significant tumor regression in patients with advanced cancer. We have investigated the kinetics of circulating erythroid (BFU-E) and granulocytic-macrophage (CFU-GM) progenitors after IL-2 therapy in 11 cancer patients, mainly affected by metastatic melanoma and renal cell carcinoma. Administration of IL-2 from day 1 through day 5 constantly induced a dramatic decrease of the number of circulating BFU-E and CFU-GM, which then showed a striking rebound (up to values fourfold and sevenfold higher, respectively, than the pretherapy levels) on discontinuation of IL-2, ie, from day 5 through day 10. A similar kinetic pattern was observed during and after the second cycle of IL-2 administration. 3[H]-thymidine killing experiments showed that the cycling activity of the progenitors was virtually unmodified in the rebound phases. To explore the mechanism(s) underlying this kinetic pattern, we have analyzed the plasma concentration of several hematopoietic growth factors, including IL-1 beta, IL-3, IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and erythropoietin (Ep). No modifications in the levels of IL-3, GM-CSF, or IL-1 beta were observed, whereas a pronounced increase of IL-6 and G-CSF concentration was monitored, starting at day 3 and peaking at day 5 of treatment (a parallel, but modest, increase of Ep level was also observed). The elevation of IL-6 and G-CSF concentration is directly correlated with and may, at least in part, underlie the subsequent rebound of circulating hematopoietic progenitors. Furthermore, the increase in IL-4 level observed at day 10 of therapy may mediate the eosinophilia gradually starting at this stage of treatment.

IL-6/BSF-2 selectively stimulates the GO----S progression of CD8+ lymphocytes.

IL-6 preferentially promotes the DNA synthesis of human peripheral blood CD8+, rather than CD4+, lymphocytes in presence of PHA: this effect is observed in serum-free cultures of greater than 99% purified CD8+ lymphocytes. However, IL-6 is able to stimulate DNA synthesis of CD8+ lymphocytes triggered by a mitogenic anti-CD2 mAb, but not by anti-CD3 mAb: these results suggest that IL-6 selectively induces activation of CD8+ lymphocytes through the CD2 rather than the CD3 pathway. Limiting dilution analysis indicates that accessory cells are not required to mediate the action of IL-6 on CD8+ cells. Furthermore, this action is not blocked by addition of mAb neutralizing either IL-2 or IL2R, thus suggesting that IL-6 does not act via IL-2. CD8+ lymphocytes grown in the presence of PHA + IL-6 incorporate (3H)-thymidine to the same extent as those stimulated with PHA + IL-2, but do not increase in number until day 6 of culture. It is hence apparent that the stimulating activity of IL-6 on CD8+ lymphocytes is restricted to the GO----S phase progression, but does not lead to mitosis. IL-6 receptors are expressed on resting CD4+ and CD8+ lymphocytes: their expression is significantly enhanced on both activated CD4+ and CD8+ cells. Scatchard analysis of (125I)-IL-6 binding data showed the presence of high (Kd, 3 x 10(-10) M) and low (Kd, 6 x 10(-8) M) affinity IL6R on both lymphocyte populations. Similarly, mRNA encoding IL6R was detected in both CD4+ and CD8+ lymphocytes. Thus, our studies indicate that IL-6 directly and selectively stimulates the GO----S progression of CD8+ lymphocytes in the presence of mitogen and absence of IL-2: this phenomenon may be of interest for the elucidation of mechanisms activating cytotoxic T lymphocytes.

Increased human immunodeficiency virus (HIV) expression in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3: roles of interferon and tumor necrosis factor in regulation of HIV production.

We have investigated the roles of cytokines in the modulation of human immunodeficiency virus (HIV) production in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3. HIV-infected U937 cells exhibited markedly lower levels of CD4 and HLA-DR antigens than uninfected cells did. Vitamin D3 induced a time-dependent macrophagelike differentiation, as determined by monitoring the expression of some surface antigens by means of the monoclonal antibodies OKM1, OKM5, OKM13, OKM14, OKT4, anti-HLA-DR, TecMG2, TecMG3, LeuM3, LeuM1, anti-HLA-DP, and anti-HLA-DQ. Treatment with hydroxyvitamin D3 resulted in a marked increase in HIV production compared with control cultures. Interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) were detected in the culture media, whereas interferon (IFN) was not generally found. Using the polymerase chain reaction technique, we found HIV-infected U937 cells to express detectable levels of mRNAs for alpha interferon (IFN-alpha), IFN-beta, TNF-alpha, and IL-1 beta. The addition of TNF resulted in a marked increase of HIV production, whereas IL-1 beta was ineffective. In contrast, both IFN-alpha and IFN-beta exerted some inhibitory effect on HIV production, which was more marked in vitamin D3-treated cultures than in untreated cultures. HIV production was significantly increased by antibodies to IFN-alpha in both untreated and vitamin D3-treated cultures. Anti-IFN-beta antibody increased HIV production only in vitamin D3-treated cells. In contrast, anti-TNF-alpha antibodies markedly decreased HIV production in both control and differentiating U937 cells. Vitamin D3 treatment resulted in a higher expression of TNF receptors in differentiating cells than in control HIV-infected cells. These data demonstrate a strong correlation between HIV production and macrophagelike differentiation in chronically infected U937 cells and suggest that endogenous IFN and TNF exert opposite effects in the regulation of virus production in both undifferentiated and vitamin D3-treated cell cultures.

Adoptive immunotherapy of human cancer: the cytokine cascade and monocyte activation following high-dose interleukin 2 bolus treatment.

Serum concentration kinetics of gamma-interferon (IFN-gamma), neopterin, 2'-5' A synthetase and tumor necrosis factor alpha were determined in five cancer patients undergoing adoptive immunotherapy with high-dose interleukin 2 (IL-2) bolus infusion and lymphokine-activated killer cells according to the National Cancer Institute, NIH protocol. In all cases a significant increase of these markers was observed after IL-2 treatment. This suggests that the antitumor effect of high-dose IL-2 bolus administration may be in part mediated by activation of a cascade of endogenous cytokines including IFN-gamma and tumor necrosis factor alpha. After IL-2 bolus injection, the kinetics of neopterin was similar but delayed when compared to that of IFN-gamma: this suggests that macrophages, the specific source of neopterin, become activated by IFN-gamma following IL-2-mediated lymphocyte induction, thus implying a possible role for macrophages in the antitumor effects mediated by IL-2 and lymphokine-activated killer cells.