GIP mediates the incretin effect and glucose tolerance by dual actions on α cells and ß cells.

Glucose-dependent insulinotropic polypeptide (GIP) communicates nutrient intake from the gut to islets, enabling optimal levels of insulin secretion via the GIP receptor (GIPR) on ß cells. The GIPR is also expressed in α cells, and GIP stimulates glucagon secretion; however, the role of this action in the postprandial state is unknown. Here, we demonstrate that GIP potentiates amino acid-stimulated glucagon secretion, documenting a similar nutrient-dependent action to that described in ß cells. Moreover, we demonstrate that GIP activity in α cells contributes to insulin secretion by invoking paracrine α to ß cell communication. Last, specific loss of GIPR activity in α cells prevents glucagon secretion in response to a meal stimulus, limiting insulin secretion and driving glucose intolerance. Together, these data uncover an important axis by which GIPR activity in α cells is necessary to coordinate the optimal level of both glucagon and insulin secretion to maintain postprandial homeostasis.

Sustained release of a GLP-1 and FGF21 dual agonist from an injectable depot protects mice from obesity and hyperglycemia.

There is great interest in identifying a glucagon-like peptide-1 (GLP-1)-based combination therapy that will more effectively promote weight loss in patients with type 2 diabetes. Fibroblast growth factor 21 (FGF21) is a compelling yet previously unexplored drug candidate to combine with GLP-1 due to its thermogenic and insulin-sensitizing effects. Here, we describe the development of a biologic that fuses GLP-1 to FGF21 with an elastin-like polypeptide linker that acts as a sustained release module with zero-order drug release. We show that once-weekly dual-agonist treatment of diabetic mice results in potent weight-reducing effects and enhanced glycemic control that are not observed with either agonist alone. Furthermore, the dual-agonist formulation has superior efficacy compared to a GLP-1/FGF21 mixture, demonstrating the utility of combining two structurally distinct peptides into one multifunctional molecule. We anticipate that these results will spur further investigation into GLP-1/FGF21 multiagonism for the treatment of metabolic disease.

A novel angiopoietin-derived peptide displays anti-angiogenic activity and inhibits tumour-induced and retinal neovascularization.

BACKGROUND AND PURPOSE: Pathological angiogenesis is associated with various human diseases, such as cancer, autoimmune diseases and retinopathy. The angiopoietin (Ang)-Tie2 system plays critical roles in several steps of angiogenic remodelling. Here, we have investigated the anti-angiogenic effect of a novel angiopoietin-derived peptide. EXPERIMENTAL APPROACH: Using computational methods, we identified peptides from helical segments within angiopoietins, which were predicted to inhibit their activity. These peptides were tested using biochemical methods and models of angiogenesis. The peptide with best efficacy, A11, was selected for further characterization as an anti-angiogenic compound. KEY RESULTS: The potent anti-angiogenic activity of A11 was demonstrated in a multicellular assay of angiogenesis and in the chorioallantoic membrane model. A11 bound to angiopoietins and reduced the binding of Ang-2 to Tie2. A11 was also significantly reduced vascular density in a model of tumour-induced angiogenesis. Its ability to inhibit Ang-2 but not Ang-1-induced endothelial cell migration, and to down-regulate Tie2 levels in tumour microvessels, suggests that A11 targets the Ang-Tie2 pathway. In a rat model of oxygen-induced retinopathy, A11 strongly inhibited retinal angiogenesis. Moreover, combination of A11 with an anti-VEGF antibody showed a trend for further inhibition of angiogenesis, suggesting an additive effect. CONCLUSIONS AND IMPLICATIONS: Our results indicate that A11 is a potent anti-angiogenic compound, through modulation of the Ang-Tie2 system, underlining its potential as a therapeutic agent for the treatment of ocular and tumour neovascularization, as well as other pathological conditions that are dependent on angiogenesis.

Fourteen years of hemodialysis with a central venous catheter: mechanical long-term complications.

The ideal dialysis access ensures adequate blood flow for dialysis, has a long life, and is associated with a low complication rate. Although no current type of access fulfills all these criteria, the native arteriovenous fistula (AVF) is close to doing so. Unfortunately, various kinds of vascular access (VA) are becoming more and more necessary to enable hemodialysis (HD). The central venous catheter (CVC), which is associated with higher morbidity and mortality, could be the only viable option to maintain permanent VA. We report an unusual complication in a patient, a 74-year-old female, who had been undergoing HD via a CVC for 14 yrs. A polyurethane CVC with a double lumen was inserted into the right internal jugular vein because an AVF was not feasible, and a polytetrafluoroethylene (PTFE) prosthesis was obstructed. In 2003, the CVC was removed due to stenosis and occlusion of the superior vena cava. A new CVC, also made of polyurethane and with a double lumen, was inserted into the left femoral vein. In January 2005, the patient reported a small rupture of about 3-4 mm located under the cuff of the CVC. For this reason, the left femoral vein had to be used, replacing the Optiflow one with a 40-cm long Tesio CVC, and the second catheter was inserted into the right femoral artery by conventional surgery. After 10 months, the patient returned once more, after the CVC in the left femoral vein had been removed because of malfunction and that the at-tempts to cannulate the same vein again had failed. Currently, two 70-cm long Tesio catheters implanted in the right femoral vein (whose tips almost reach the diaphragm) are used for dialysis sessions. The number of CVC implants has progressively increased amongst HD patients who are elderly, diabetic or who have been on long-term HD. The patient described in this case report is currently using a 70-cm long double Tesio catheter (single Tesio CVC in SPI silicon) placed in the right femoral vein. She has resumed therapy with dicumarol anticoagulants, maintaining INR within the 2.5-3.5 range. In conclusion, both the increase in the use of venous catheters for HD and in the survival of dialysis patients contribute towards the observation of rare complications associated with CVC use.

Increased hepatitis C virus (HCV)-specific CD4+CD25+ regulatory T lymphocytes and reduced HCV-specific CD4+ T cell response in HCV-infected patients with normal versus abnormal alanine aminotransferase levels.

CD4+CD25+ T regulatory cells may play a role in the different clinical presentations of chronic hepatitis C virus (HCV) infection by suppressing CD4+ T cell responses. Peripheral CD4+CD25+ T cells from chronic HCV carriers with normal and abnormal alanine aminotransferase (ALT) were analysed for specificity and effect on HCV-specific CD4+ T cell reactivity by flow cytometry for intracellular cytokine production and proliferation assay. HCV-specific CD4+CD25(+high) T cells consistently produced transforming growth factor (TGF)-beta but only limited amounts of interleukin (IL)-10 and no IL-2 and interferon (IFN)-gamma. The HCV-specific TGF-beta response by CD4+CD25(+high) T cells was significantly greater in patients with normal ALT compared to patients with elevated ALT. In addition, a significant inverse correlation was found between the HCV-specific TGF-beta response by CD4+CD25(+high) T cells and liver inflammation. In peripheral blood mononuclear cells (PBMC), both HCV antigen-induced IFN-gamma production and proliferation of CD4+ T cells were greater in patients with elevated ALT compared with patients with normal ALT. Depletion of CD4+CD25+ cells from PBMC resulted in an increase of both IFN-gamma production and proliferation of HCV-specific CD4+ T cells that was significantly greater in patients with normal ALT levels compared with patients with elevated ALT. In addition, CD4+CD25+ T cells from patients with normal ALT levels proved to be significantly more potent to suppress CD4+ T cell reactivity with respect to those from patients with elevated ALT. In conclusion, these data support the hypothesis that CD4+CD25+ cells may play a role in controlling chronic inflammatory response and hepatic damage in chronic HCV carriers.

Quinoxalinylethylpyridylthioureas (QXPTs) as potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Further SAR studies and identification of a novel orally bioavailable hydrazine-based antiviral agent.

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

Lack of evidence for the Th2 predominance in patients with chronic hepatitis C.

A T helper (Th)1 to Th2 shift has been proposed to be a critical pathogenic determinant in chronic hepatitis C. Here, we evaluated mitogen-induced and hepatitis C virus (HCV) core antigen-induced cytokine production in 28 patients with biopsy-proven chronic hepatitis C. Flow cytometry demonstrated that after mitogenic stimulation the percentage of Th2 cells (IL-4 + or IL-13 +) and Th0 cells (IFN-gamma/IL-4 + or IL-2/IL-13 +) did not differ between patients and controls. In contrast, the percentage of Th1 cells (IFN-gamma + or IL-2 +) was significantly increased in CD4 +, CD8 +, 'naive'-CD45RA + and 'memory'-CD45RO + T-cell subsets from patients versus controls. Similar results were obtained by ELISA testing supernatants from mitogen-stimulated, unfractionated peripheral blood mononuclear cell (PBMC) cultures. Interferon-alpha treatment was associated with a reduction in the mitogen-induced Th1 cytokine response in those patients who cleared their plasma HCV-RNA. Analysis of cytokine expression by CD4 + T cells after HCV core antigen stimulation in a subgroup of 13 chronic hepatitis C patients demonstrated no cytokine response in 74% of these patients and an IFN-gamma-restricted response in 26%. Finally, no Th2 shift was found in lipopolysaccharide-stimulated monocytes. These data indicate that a Th1 to Th2 shift does not occur in chronic hepatitis C.

Effect of different activation stimuli on the cytokine response of human macrophages to CD40L.

Here we show that CD40L (ligand for CD40) failed to induce the production of tumour necrosis factor alpha (TNF-alpha), interleukin (IL-)-1 beta, IL-10 and IL-12 in macrophages matured in vitro in the absence of growth factors or in the presence of macrophage colony-stimulating factor (M-CSF). In contrast, enzyme-linked immunoabsorbent assay (ELISA) testing and cytofluorimetric (FACS) analysis demonstrated significant production of TNF-alpha and IL-1 beta, but not of IL-10 and IL-12 in macrophages maturated in the presence of CD40L and re-stimulated with CD40L. The priming effect of CD40L on TNF-alpha and IL-1 beta production was related to induction of CD40 expression. Finally, CD40L priming did not modify the cytokine response of macrophages to lipopolysaccharide. In conclusion, our results suggest that CD40/CD40L interactions are important for the activation of macrophages as effector cells that mediate inflammation and tissue damage in T cell-mediated inflammatory processes.

Granulocyte-macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanisms.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has multiple effects on the antigen phenotype and function of macrophages. In this study we investigated the effect of GM-CSF on cytokine production by macrophages. We found that GM-CSF may modify the tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) response to lipopolysaccharide (LPS) through two different mechanisms. Relatively early in culture, GM-CSF increases the amount of cytokines synthesized by responding cells; this effect appears to be unrelated to modulation of CD14 expression and LPS-binding capacity. After prolonged incubation, GM-CSF up-regulates both CD14 expression and LPS-binding capacity, and the frequency of cytokine-producing cells. Release of CD14 in the culture supernatant was decreased in the presence of GM-CSF, suggesting that a reduced shedding was responsible for the effect of GM-CSF on CD14 expression. Enhancement of cytokine production was also observed in GM-CSF-treated macrophages after stimulation by phorbol 12-myristate 13-acetate (PMA), thus indicating that GM-CSF affects both CD14-dependent and -independent cytokine production. Finally, GM-CSF did not modulate the LPS- and PMA-induced production of IL-10 and IL-12. We conclude that GM-CSF may play a role in manipulating the activation-induced expression of pro-inflammatory cytokines by macrophages. Enhanced production of these cytokines could play an important role in the pathogenesis of Gram-negative septic shock syndrome and in defence against infectious agents.

Human immunodeficiency virus type 1 infection modulates the interleukin (IL)-1beta and IL-6 responses of human macrophages to CD40 ligand stimulation.

Better understanding of the mechanisms of proinflammatory cytokine production during human immunodeficiency virus (HIV) type 1 infection is of pivotal importance. The effect of HIV-1 infection on recombinant CD40 ligand (CD40L)-induced interleukin (IL)-1beta and IL-6 production by human macrophages was analyzed. ELISA and cytofluorometric analysis demonstrated that CD40L stimulation of HIV-1-infected macrophages resulted in substantial production of IL-1beta and IL-6. In contrast, no cytokine response was observed in uninfected cells. No modulation of the receptor for CD40 was found to account for the enhanced response to CD40L. The CD40L effect was not due to lipopolysaccharide contamination and was completely abrogated by preincubation with a monoclonal anti-CD40L antibody. mRNA studies indicated that the priming effect of HIV-1 on the macrophage response to CD40L was regulated at the transcriptional level. Finally, the effect of HIV-1 on the cytokine response could not be abolished by the HIV-1 protease inhibitor U75875 at concentrations that completely suppressed HIV-1 replication.

[Enterohemorrhagic Escherichia coli O157:H7 infection]. / L'infezione da Escherichia coli entero-emorragico sierotipo O157:H7.

Infections in the digestive tract are due to multiple organism, which cause different syndromes. Escherichia coli O157:H7, already identified as a human pathogen in 1982, has been recognised as a major public health issue, being responsible for sporadic and epidemic cases of haemorrhagic colitis, often associated, in children and elderly, with the haemolytic uraemic syndrome. E. coli O157:H7 infection may occur everywhere, but is more frequent in North Europe, Canada, USA, Argentina and Japan, with annual incidence rates of 8 per 100,000 population. In Italy until 1997 the Italian National Institute of Health has identified 196 cases of haemolytic uraemic syndrome, in addition, an outbreak caused by E. coli O157:H7 occurred in 1993. In Italy the incidence of the haemolytic uraemic syndrome is 4-5 times lower than in Great Britain, Germany and other European countries. E. coli infection is more frequently associated with the ingestion of food from bovine and sheep origin and with infected water. The clinical spectrum includes an asymptomatic infection, non bloody diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome. When the E. coli infection is suspected, it is necessary to isolate the bacterium in a specialised laboratory. Treatment is essentially supportive in order to control anaemia and to maintain an adequate fluid and electrolyte balance, if necessary with the use of dialysis. The use of antimicrobial agents is currently under debate as there are controversial data on the risk of developing haemolytic uraemic syndrome.

Enhanced production of tumor necrosis factor-alpha and interleukin-6 due to prolonged response to lipopolysaccharide in human macrophages infected in vitro with human immunodeficiency virus type 1.

Elevated levels of circulating tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 have been detected in human immunodeficiency virus (HIV) type 1 infection. The overproduction of these cytokines could contribute to AIDS pathogenesis. Thus, the expression of TNF-alpha and IL-6 in human macrophages infected with HIV-1 was investigated. HIV-1 infection, per se, did not induce any TNF-alpha or IL-6 production or cytokine-specific mRNA expression. In contrast, HIV-1 primed macrophages to a prolonged TNF-alpha and IL-6 response to lipopolysaccharide (LPS) stimulation with respect to uninfected cells. Time-course analysis and flow cytometry demonstrated that cytokine production stopped at 6 h in uninfected macrophages but continued up to 24 h in HIV-1-infected cells. RNA studies suggested that HIV-1 interfered with late steps of cytokine synthesis. No modulation of membrane CD14 was found to account for the enhanced response to LPS. Finally, the effect of HIV-1 on cytokine response could not be abolished by the antiviral compound U75875.

Bile acids with differing hydrophilic-hydrophobic properties do not influence cytokine production by human monocytes and murine Kupffer cells.

Bile acids have been proposed to exert immunological effects of potential pathogenic or therapeutic relevance, yet the experimental evidence remains preliminary. We reexamined the effects of a variety of bile salts with differing hydrophilic-hydrophobic properties on the production of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) from monocytes and Kupffer cells. Monocytes from healthy human donors and Kupffer cells from 5-week-old mice were incubated for up to 18 hours with or without varying concentrations of bile salts and lipopolysaccharide (LPS). Monocyte viability was > or = 95% with up to 250 mumol/L sodium ursodeoxycholate and < or = 90% with 200 mumol/L chenodeoxycholate, decreasing sharply at higher concentrations. Kupffer cells were more vulnerable, particularly to chenodeoxycholate (viabilities of 25% and 0% at concentrations of 100 mumol/L and 200 mumol/L, respectively). In monocytes incubated in the presence of 20% fetal calf serum, neither ursodeoxycholate and chenodeoxycholate, nor a variety of other unconjugated and conjugated bile acids, tested up to their maximal noncytotoxic concentrations, influenced the IL-6 and TNF alpha production, at any level of LPS stimulation. Similar to monocytes, incubation of murine Kupffer cells with ursodeoxycholate and chenodeoxycholate did not influence cytokine release. In contrast, the addition of 10 nmol/L dexamethasone to monocytes significantly decreased TNF-alpha and IL-6 release (69 +/- 11% and 48 +/- 15%, respectively). When monocytes were incubated with 200 mumol/L chenodeoxycholate in the presence of lower concentrations of fetal calf serum (10% and 5%, respectively) a significant inhibition of cytokine release was observed, whereas incubation with ursodeoxycholate did not cause any effect. Flow cytometry using fluoresceinated LPS showed that chenodeoxycholate does not interact with the CD14 receptor, thus excluding the possibility of an interference with the LPS uptake by monocytes. Incubation with [14C]-chenodeoxycholate showed that the intracellular bile acid uptake was inversely related to the concentration of fetal calf serum, being negligible (< 3 fmol/cell) at the highest level. In conclusion, bile acids with widely different hydrophobicities are incapable of influencing the release of IL-6 and TNF alpha by monocytes and Kupffer cells, provided they are studied at noncytotoxic concentrations and in the presence of physiological amounts of proteins.

In vitro inhibition of the replication of human immunodeficiency virus type 1 by beta-mercaptoethylamine (cysteamine).

This study investigates the effects of cysteamine alone and in association with zidovudine or didanosine on the replication of human immunodeficiency virus type 1 (HIV-1). More than 90% viral inhibition was obtained by 200 microM cysteamine in lymphocytes and 100 microM cysteamine in macrophages against 4 primary isolates and 2 laboratory strains of HIV-1. Polymerase chain reaction analysis demonstrated that cysteamine interferes with early steps of HIV-1 replication, before proviral DNA formation. The use of cysteamine in conjunction with zidovudine or didanosine brought about an additive antiviral effect without concomitant increases in toxicity. The concentrations of cysteamine that are effective against HIV-1 in vitro have been well tolerated over long periods by patients under treatment for cystinosis, an inherited disorder. These observations suggest that cysteamine alone or in combination with zidovudine or didanosine could be a new potential treatment of HIV-1 infection.

Human immunodeficiency virus-induced cell death in cytokine-treated macrophages can be prevented by compounds that inhibit late stages of viral replication.

The basis of the cytopathic effect induced by a laboratory strain and several clinical isolates of human immunodeficiency virus (HIV) in human macrophages cultured in the presence of macrophage colony-stimulating factor was studied. Infected macrophages die of necrosis, the consequence of the production of mature virions in infected cells. Cell death can be prevented by antiviral compounds that interfere with the assembly and budding of virions. Programmed cell death (apoptosis), a potential mechanism of HIV-mediated cell death in CD4 T lymphocytes, does not occur in infected macrophages as shown by electron microscopy, cytofluorometric and gel electrophoretic DNA analysis, and nuclear fluorescent staining by Hoechst and terminal dUTP-nick-end-labeling (TUNEL) assay. The data suggest that macrophage killing by HIV may occur in vivo. Thus, combination therapies that include compounds that inhibit the cytopathic effect of HIV in macrophages should be considered for AIDS patients.

[Extranodal non-Hodgkin's lymphomas. Review and our experience]. / I linfomi non-Hodgkin extranodali. Review e nostra esperienza.

The authors report their experience concerning 12 cases of extranodal lymphoma (6 gastric, 1 duodenal, 2 ileal, 1 rectal, 1 splenic, 1 mammary). Extranodal lymphomas are increasing because of the high number of patients with AIDS and new extra-European immigration. Surgery is important not only for diagnosis, but above all for therapy. The great majority of extranodal lymphomas is diffuse rather nodular. Diffuse histiocytic type is the most commun pathologic feature. Prognosis of gastroenteric lymphomas is better than adenocarcinomas of gastrointestinal tract. The surgical techniques are the same as those used for the others tumors, and combined-modality therapy appears superior to local therapy alone for patients with extranodal disease characterized by unfavorable histology, site or stage. The classification is that of Ann Arbor, but for many authors the TNM system was an useful predictor of survival in patients treated with surgery.

Macrophage colony-stimulating factor enhances the susceptibility of macrophages to infection by human immunodeficiency virus and reduces the activity of compounds that inhibit virus binding.

The effects of macrophage colony-stimulating factor (M-CSF) on CD4 receptor expression, susceptibility to human immunodeficiency virus type 1 (HIV) infection, and anti-HIV activity of dextran sulfate and soluble-CD4 were studied in cultured, human primary macrophages. M-CSF stimulated macrophage cells to express the CD4 receptor, and this resulted in an increase of both the number of CD4+ cells and the density of the receptor on the cell surface. M-CSF also significantly enhanced the susceptibility of macrophage cells to HIV infection. Interestingly, the anti-HIV activity of dextran sulfate and soluble-CD4 (two compounds that interfere with HIV-CD4 binding with different mechanisms) was reduced 100-fold and fivefold, respectively, in M-CSF-treated macrophages. Human blood concentrations of M-CSF are reported to be similar to those used in this work (1,000 U/mL); thus, it is conceivable that also in vivo this cytokine may modify the susceptibility of macrophages to HIV and the ability of dextran sulfate and soluble CD4 to inhibit HIV replication. These results suggest that the in vitro study in M-CSF-treated macrophages of promising drugs inhibitors of HIV-CD4 binding could provide further insights into the potential efficacy of these compounds in patients.