Weight Loss Outcomes Among Early High Responders to Exenatide Treatment: A Randomized, Placebo Controlled Study in Overweight and Obese Women.

Objective: As there is significant heterogeneity in the weight loss response to pharmacotherapy, one of the most important clinical questions in obesity medicine is how to predict an individual's response to pharmacotherapy. The present study examines patterns of weight loss among overweight and obese women who demonstrated early robust response to twice daily exenatide treatment compared to those treated with hypocaloric diet and matched placebo injections. Methods: We randomized 182 women (BMI 25-48 kg/m2) to treatment with exenatide alone or matched placebo injections plus hypocaloric diet. In both treatment groups, women who demonstrated ≥ 5% weight loss at 12 weeks were characterized as high responders and those who lost ≥10% of body weight were classified as super responders. Our primary outcome was long-term change in body weight among early high responders to either treatment. An exploratory metabolomic analysis was also performed. Results: We observed individual variability in weight loss with both exenatide and hypocaloric diet plus placebo injections. There was a trend toward a higher percentage of subjects who achieved ≥ 5% weight loss with exenatide compared to diet (56% of those treated with exenatide, 76% of those treated with diet, p = 0.05) but no significant difference in those who achieved ≥ 10% weight loss (23% of individuals treated with exenatide and 36% of those treated with diet, p = 0.55). In both treatment groups, higher weight loss at 3 months of treatment predicted super responder status (diet p=0.0098, exenatide p=0.0080). Both treatment groups also demonstrated similar peak weight loss during the study period. We observed lower cysteine concentrations in the exenatide responder group (0.81 vs 0.48 p < 0.0001) and a trend toward higher levels of serotonin, aminoisobutyric acid, anandamide, and sarcosine in the exenatide super responder group. Conclusion: In a population of early high responders, longer term weight loss with exenatide treatment is similar to that achieved with a hypocaloric diet. Clinical Trial Registration: www.clinicaltrialsgov, identifier NCT01590433.

The Contemporary Prevalence of Diabetic Neuropathy in Type 1 Diabetes: Findings From the T1D Exchange.

OBJECTIVE: To evaluate the contemporary prevalence of diabetic peripheral neuropathy (DPN) in participants with type 1 diabetes in the T1D Exchange Clinic Registry throughout the U.S. RESEARCH DESIGN AND METHODS: DPN was assessed with the Michigan Neuropathy Screening Instrument Questionnaire (MNSIQ) in adults with ≥5 years of type 1 diabetes duration. A score of ≥4 defined DPN. Associations of demographic, clinical, and laboratory factors with DPN were assessed. RESULTS: Among 5,936 T1D Exchange participants (mean ± SD age 39 ± 18 years, median type 1 diabetes duration 18 years [interquartile range 11, 31], 55% female, 88% non-Hispanic white, mean glycated hemoglobin [HbA1c] 8.1 ± 1.6% [65.3 ± 17.5 mmol/mol]), DPN prevalence was 11%. Compared with those without DPN, DPN participants were older, had higher HbA1c, had longer duration of diabetes, were more likely to be female, and were less likely to have a college education and private insurance (all P < 0.001). DPN participants also were more likely to have cardiovascular disease (CVD) (P < 0.001), worse CVD risk factors of smoking (P = 0.008), hypertriglyceridemia (P = 0.002), higher BMI (P = 0.009), retinopathy (P = 0.004), reduced estimated glomerular filtration rate (P = 0.02), and Charcot neuroarthropathy (P = 0.002). There were no differences in insulin pump or continuous glucose monitor use, although DPN participants were more likely to have had severe hypoglycemia (P = 0.04) and/or diabetic ketoacidosis (P < 0.001) in the past 3 months. CONCLUSIONS: The prevalence of DPN in this national cohort with type 1 diabetes is lower than in prior published reports but is reflective of current clinical care practices. These data also highlight that nonglycemic risk factors, such as CVD risk factors, severe hypoglycemia, diabetic ketoacidosis, and lower socioeconomic status, may also play a role in DPN development.

The Janus Face of Tumor Microenvironment Targeted by Immunotherapy.

The tumor microenvironment (TME) is a complex entity where host immune and non-immune cells establish a dynamic crosstalk with cancer cells. Through cell-cell interactions, which are mediated by key signals, such as the PD-1/PD-L1 axis, as well as the release of soluble mediators, this articulated process defines the nature of TME determining tumor development, prognosis, and response to therapy. Specifically, tumors are characterized by cellular plasticity that allows for the microenvironment to polarize towards inflammation or immunosuppression. Thus, the dynamic crosstalk among cancer, stromal, and immune components crucially favors the dominance of one of the Janus-faced contexture of TME crucial to the outcome of tumor development and therapeutic response. However, mostly, TME is dominated by an immunosuppressive landscape that blocks antitumor immunity and sustain tumor progression. Hence, in most cases, the immunosuppressive components of TME are highly competent in suppressing tumor-specific CD8+ T lymphocytes, the effectors of cancer destruction. In this complex context, immunotherapy aims to arm the hidden Janus face of TME disclosing and potentiating antitumor immune signals. Herein, we discuss recent knowledge on the immunosuppressive crosstalk within TME, and share perspectives on how immunotherapeutic approaches may exploit tumor immune signals to generate antitumor immunity.

Antitumor Effects of Epidrug/IFNα Combination Driven by Modulated Gene Signatures in Both Colorectal Cancer and Dendritic Cells.

Colorectal cancer results from the progressive accumulation of genetic and epigenetic alterations. IFN signaling defects play an important role in the carcinogenesis process, in which the inability of IFN transcription regulatory factors (IRF) to access regulatory sequences in IFN-stimulated genes (ISG) in tumors and in immune cells may be pivotal. We reported that low-dose combination of two FDA-approved epidrugs, azacytidine (A) and romidepsin (R), with IFNα2 (ARI) hampers the aggressiveness of both colorectal cancer metastatic and stem cells in vivo and triggers immunogenic cell death signals that stimulate dendritic cell (DC) function. Here, we investigated the molecular signals induced by ARI treatment and found that this drug combination increased the accessibility to regulatory sequences of ISGs and IRFs that were epigenetically silenced in both colorectal cancer cells and DCs. Likewise, specific ARI-induced histone methylation and acetylation changes marked epigenetically affected ISG promoters in both metastatic cancer cells and DCs. Analysis by ChIP-seq confirmed such ARI-induced epigenetically regulated IFN signature. The activation of this signal endowed DCs with a marked migratory capability. Our results establish a direct correlation between reexpression of silenced ISGs by epigenetic control and ARI anticancer activity and provide new knowledge for the development of innovative combined therapeutic strategies for colorectal cancer. Cancer Immunol Res; 5(7); 604-16. ©2017 AACR.

Inhibition of MMP-9 expression by ritonavir or saquinavir is associated with inactivation of the AKT/Fra-1 pathway in cervical intraepithelial neoplasia cells.

A reduced incidence and decreased clinical progression of uterine cervical intraepithelial neoplasia (CIN) has been observed in women infected with human immunodeficiency virus (HIV) treated with HIV-protease inhibitors (PIs). The HIV-PIs saquinavir (SQV) and ritonavir (RTV) have been demonstrated to efficiently inhibit invasion of human primary CIN cells by downregulating the expression of matrix metalloproteinase (MMP)-9. The present study further investigated the molecular mechanisms underlying the activity of SQV and RTV in CIN. The results of the present study indicate that the treatment of human primary CIN cells with SQV or RTV directly impairs events leading to MMP-9 expression, including the phosphorylation of AKT and the nuclear localisation of the Fos-related antigen transcription factor. In addition, neither SQV nor RTV affected the expression of human papilloma virus proteins, such as E6 or E7. In view of the important role that the AKT/Fra-1/MMP-9 signalling pathway serves in CIN progression to invasive cervical carcinoma, these data further support the use of HIV-PIs in the treatment of CIN in women infected with HIV and women who are not infected with HIV. Furthermore, the present study identified a molecular mechanism underlying the anti-invasive effects of SQV/RTV, providing useful information for the development of SQV/RTV derivatives, which may be employed as novel anticancer drugs.

3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells.

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches.

Importance of primary indication and liver function between stages: results of a multicenter Italian audit of ALPPS 2012-2014.

BACKGROUND: Posthepatectomy liver failure is one of the most feared complications in extended hepatic resections. In 2012, a novel two-stage liver resection was developed, able to induce rapid and extensive hypertrophy by portal vein ligation and in situ liver splitting - Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS). The technique became more widely employed but its use remained controversial due to reporting of high complication and mortality rates. METHOD: A national audit was performed to gather information about the safety of the procedure and to better understand the complications. The audit was offered to all high-volume hepatobiliary centers in Italy. RESULTS: Of all Italian centers approached in January 2012, 12 centers with experience in ALPPS enrolled and participated in collection of data. Fifty patients underwent ALPPS between 2012 and 2014. In 48/50 patients completion of hepatectomy was performed successfully. Major morbidity occurred in 54% with a 20% 90-day mortality. Uni- and multivariate analysis showed that ALPPS for cholangiocarcinoma and a peak of bilirubin over 5 mg/dl between stages was associated with increase of 90-day mortality and worse survival. DISCUSSION: It is proposed that a moratorium be introduced for classic ALPPS in cholangiocarcinoma and to abort ALPPS in patients who develop an interstage increase in bilirubin, due to the high risk of liver failure and mortality.

IFN-α potentiates the direct and immune-mediated antitumor effects of epigenetic drugs on both metastatic and stem cells of colorectal cancer.

Epigenetic alterations, including dysregulated DNA methylation and histone modifications, govern the progression of colorectal cancer (CRC). Cancer cells exploit epigenetic regulation to control cellular pathways, including apoptotic and metastatic signals. Since aberrations in epigenome can be pharmacologically reversed by DNA methyltransferase and histone deacetylase inhibitors, epigenetics in combination with standard agents are currently envisaged as a new therapeutic frontier in cancer, expected to overcome drug resistance associated with current treatments. In this study, we challenged this idea and demonstrated that the combination of azacitidine and romidepsin with IFN-α owns a high therapeutic potential, targeting the most aggressive cellular components of CRC, such as metastatic cells and cancer stem cells (CSCs), via tight control of key survival and death pathways. Moreover, the antitumor efficacy of this novel pharmacological approach is associated with induction of signals of immunogenic cell death. Of note, a previously undisclosed key role of IFN-α in inducing both antiproliferative and pro-apoptotic effects on CSCs of CRC was also found. Overall, these findings open a new frontier on the suitability of IFN-α in association with epigenetics as a novel and promising therapeutic approach for CRC management.

Sociodemographic, clinical and organisational factors associated with delayed hospital discharges: a cross-sectional study.

BACKGROUND: Evidence from studies conducted in Western countries indicates that a significant proportion of hospital beds are occupied by patients who experience a delayed hospital discharge (DHD). However, evidence about this topic is lacking in Italy, and little is known on the patients' and organisational characteristics that influence DHDs. Therefore, we carried out a survey in all the hospitals of a Northern Italian region to analyse the prevalence and the determinants of DHD. METHODS: A cross-sectional study was carried out during an index period of 15 days in 256 operative units in Emilia-Romagna, a Northern Italian region with 4.4 million inhabitants, to identify patients medically fit for discharge but still hospitalised. The characteristics of these patients (n = 510) were compared with all the other patients (n = 5,815) hospitalised in the same operative units during the index period using multilevel logistic regression models. RESULTS: The one-day prevalence of DHD was 8.1%. More than half of DHD patients (52.7%) waited to access long-term/rehabilitation units or residential care homes, 16.7% experienced a delay for family-related reasons, and 14.5% were waiting to be admitted to other rehabilitation services. Among DHD patients hospitalised in long-term/rehabilitation units, 45.3% were waiting to be transferred to residential care homes. Patients' characteristics associated with a higher likelihood of DHD in multilevel logistic regression were older age, provision of intensive care, a diagnosis of dementia, tumours or femoral/shoulder fractures, and a number of comorbidities. Patients hospitalised in long-term/rehabilitation units, as well as in orthopaedics/traumatology units, were significantly more likely to have a DHD compared with patients hospitalised in general surgery units. Moreover, compared with Local Health Authority Hospitals, being hospitalised in Hospital Trusts was associated with a higher likelihood of DHD. CONCLUSIONS: Although the prevalence of DHD in the present study is markedly lower than that reported in the literature, we submit that the DHD problem should be addressed with major organisational innovations, with a special focus on the ageing of the population and epidemiological trends. Organisational changes imply new ways of managing emerging clusters of patients whose needs are not efficiently or effectively met by traditional organisation models and services.

HIV-1 tat promotes integrin-mediated HIV transmission to dendritic cells by binding Env spikes and competes neutralization by anti-HIV antibodies.

Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.

Apicidin and docetaxel combination treatment drives CTCFL expression and HMGB1 release acting as potential antitumor immune response inducers in metastatic breast cancer cells.

Currently approved combination regimens available for the treatment of metastatic tumors, such as breast cancer, have been shown to increase response rates, often at the cost of a substantial increase in toxicity. An ideal combination strategy may consist of agents with different mechanisms of action leading to complementary antitumor activities and safety profiles. In the present study, we investigated the effects of the epigenetic modulator apicidin in combination with the cytotoxic agent docetaxel in tumor breast cell lines characterized by different grades of invasiveness. We report that combined treatment of apicidin and docetaxel, at low toxicity doses, stimulates in metastatic breast cancer cells the expression of CTCF-like protein and other cancer antigens, thus potentially favoring an antitumor immune response. In addition, apicidin and docetaxel co-treatment specifically stimulates apoptosis, characterized by an increased Bax/Bcl-2 ratio and caspase-8 activation. Importantly, following combined exposure to these agents, metastatic cells were also found to induce signals of immunogenic apoptosis such as cell surface expression of calreticulin and release of considerable amounts of high-mobility group box 1 protein, thus potentially promoting the translation of induced cell death into antitumor immune response. Altogether, our results indicate that the combined use of apicidin and docetaxel, at a low toxicity profile, may represent a potential innovative strategy able to activate complementary antitumor pathways in metastatic breast cancer cells, associated with a potential control of metastatic growth and possible induction of antitumor immunity.

Fibroblast Growth Factor-2 and the HIV-1 Tat Protein Synergize in Promoting Bcl-2 Expression and Preventing Endothelial Cell Apoptosis: Implications for the Pathogenesis of AIDS-Associated Kaposi's Sarcoma.

Kaposi's sarcoma (KS) is a vascular tumor frequently occurring in Human Immunodeficiency Virus- (HIV-) 1-infected individuals. Our previous work indicated that the angiogenic fibroblast growth factor (FGF)-2 and the Tat protein of HIV-1, both expressed in KS lesions of HIV-infected patients, synergize at inducing angioproliferative, KS-like lesions in mice. Here we show that the development of angioproliferative lesions promoted in mice by combined Tat and FGF-2 associates with an increase in the levels of expression of the antiapoptotic Bcl-2 protein. Upregulation of Bcl-2 expression by combined FGF-2 and Tat occurs also in vitro, and this protects human primary endothelial cells from programmed cell death. As Bcl-2 is expressed in human KS lesions in a fashion paralleling the progression of the disease, these findings suggest a molecular mechanism by which Tat and FGF-2 cooperate in KS maintenance and progression in HIV-infected individuals.

Human immunodeficiency virus protease inhibitors reduce the growth of human tumors via a proteasome-independent block of angiogenesis and matrix metalloproteinases.

Human immunodeficiency virus protease inhibitors (HIV-PIs), such as indinavir and saquinavir, have been shown to block angiogenesis and tumor cell invasion and to induce tumor cell apoptosis and growth arrest, respectively, both in vitro and in vivo. These findings have suggested that HIV-PIs or their analogues can be used as antitumor drugs. To this regard, indinavir and saquinavir were assessed for their ability to inhibit in vivo the growth of highly prevalent human tumors, such as lung, breast, colon and hepatic adenocarcinomas. We show here that both HIV-PIs significantly inhibited the growth of all adenocarcinomas tested in the mice model. This was not mediated by effects on proteasome-dependent cell growth arrest or on apoptosis but by the block of angiogenesis and matrix metalloproteinase activity. Accordingly, therapeutic steadystate concentrations of indinavir or saquinavir were highly effective in inhibiting invasion of tumor cells in vitro. In contrast, growth arrest was induced only by high concentrations of saquinavir that are not reached or are only transiently present in plasma of treated patients, likely through a proteasome-mediated mechanism. These data suggest that HIV-PIs or their analogues, characterized by a better biodistribution and lower toxicity, may represent a new class of antitumor drugs capable of targeting both matrix metalloproteinases and the proteasome for a most effective antitumor therapy.

Clinical course of classic Kaposi's sarcoma in HIV-negative patients treated with the HIV protease inhibitor indinavir.

HIV protease inhibitors have been shown to exert antiangiogenic and antitumor actions independently from their antiretroviral effect. Based on these studies, HIV-seronegative patients with classic Kaposi's sarcoma were treated with indinavir and followed for clinical evolution, drug pharmacokinetics and Kaposi's sarcoma biomarkers. A favorable clinical course was associated with high drug plasma levels, reduced production of basic fibroblast growth factor, lower numbers of circulating endothelial cells, and a decrease in antibody titers against human herpesvirus 8.

Macrophages transmit human immunodeficiency virus type 1 products to CD4-negative cells: involvement of matrix metalloproteinase 9.

It was previously reported that human immunodeficiency virus type 1 (HIV-1) spreads in CD4 lymphocytes through cell-to-cell transmission. Here we report that HIV-1-infected macrophages, but not lymphocytes, transmit HIV-1 products to CD4-negative cells of either epithelial, neuronal, or endothelial origin in the absence of overt HIV-1 infection. This phenomenon was detectable as early as 1 h after the start of cocultivation and depended on cell-to-cell contact but not on the release of viral particles from donor cells. Transfer of HIV-1 products occurred upon their polarization and colocalization within zones of cell-to-cell contact similar to virological synapses. Neither HIV-1 Env nor Nef expression was required but, interestingly, we found that an HIV-1-dependent increase in matrix metalloproteinase 9 production from donor cells significantly contributed to the cell-to-cell transmission of the viral products. The macrophage-driven transfer of HIV-1 products to diverse CD4-negative cell types may have a significant role in AIDS pathogenesis.

Control of human herpes virus type 8-associated diseases by NK cells.

The "natural killer" (NK) cells preferentially kill targets lacking surface major histocompatibility complex class I (MHC-I) molecule expression. NK cells recognize these targets through membrane receptors, which can trigger activating or inhibitory signals for killing. Several tumors or virus-infected cells downregulate MHC-I expression as a mechanism to evade recognition and killing by cytotoxic T lymphocytes (CTL). They, however, become targets for NK cells cytotoxic activity. NK cell activity is reduced during disease progression in human immunodeficiency virus (HIV) infection, and in individuals with AIDS-associated tumors linked with infection by the oncogenic human herpes virus type-8 (HHV8), including Kaposi's sarcoma (KS) and primary effusion lymphomas (PEL). We have demonstrated that AIDS-related KS (AIDS-KS) is characterized by an increased expression of inhibitory receptors by T lymphocytes, and that HIV-non-infected patients with KS (classic KS, C-KS) have a substantial number of NK cells bearing these same receptors. NK cells from patients with C-KS are normally equipped with cytolytic molecules including granzyme A and perforin. However, the cytotoxic activity of NK cells is reduced in patients with C-KS, AIDS-KS, or PEL patients, who are all infected by the HHV8, and this correlates with disease severity. Moreover, we have found that HHV8-infected cell lines established from PELs have a reduced surface expression of MHC-I molecules and are sensitive to the lysis mediated by NK cells. Since PEL cells express the same HHV8 latency program as KS cells, these data point to MHC-I downregulation by HHV8 as a primary immune evasion mechanism against CTL responses, further reinforced by upregulation of inhibitory receptors on T and NK cells in the setting of HIV and/or HHV8 infection. Thus, studies on killing receptor regulation and signaling in T and NK cells may shed light on the pathogenesis of HHV8-associated tumors both in HIV-infected or -noninfected patients.

Clustering of insulin resistance with vascular dysfunction and low-grade inflammation in type 2 diabetes.

Vascular dysfunction, low-grade inflammation, insulin resistance, and impaired fibrinolysis have each been reported to be present in type 2 diabetes, but their relationships, and the role of obesity, have not been investigated. We measured insulin sensitivity (euglycemic clamp), forearm blood flow responses to graded local acetylcholine (Ach) and sodium nitroprusside (SNP) infusions, plasma concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, von Willebrand factor (vWF), plasminogen activator inhibitor (PAI)-1, tissue plasminogen activator (tPA), and high-sensitivity C-reactive protein (hs-CRP) in 81 diabetic patients. When patients were stratified by insulin resistance, more severe insulin resistance was associated (P < 0.05) with overweight, central fat distribution, hypertension, and dyslipidemia (with similar sex distribution, age, fasting plasma glucose, and HbA1c). With regard to vascular function, both endothelium-dependent (Ach) (-22, -40, and -52%; P < 0.0001) and -independent (SNP) (-3, -7, and -27%; P < 0.02) vasodilatation were progressively reduced across insulin resistance tertiles. In multivariate analysis, inflammatory markers (IL-6, hs-CRP, and TNF-alpha) were independently associated with insulin resistance and fasting glycemia, fibrinolytic markers PAI-1 and tPA with insulin resistance and central fat distribution, and vascular indexes (vWF, Ach, and SNP vasodilation) with insulin resistance and obesity or cytokines (TNF-alpha or IL-6). In type 2 diabetes, insulin resistance is associated with vascular dysfunction/damage, impaired fibrinolysis, and low-grade inflammation independently of obesity and poor glycemic control.

HIV-1 Tat regulates endothelial cell cycle progression via activation of the Ras/ERK MAPK signaling pathway.

Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the alpha5beta1 and alphavbeta3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.

Haematocrit, type 2 diabetes, and endothelium-dependent vasodilatation of resistance vessels.

AIMS: In conditions such as type 2 diabetes, hypertension, and smoking, in which haematocrit (Hct) tends to be higher, endothelial function is impaired. In vitro, haemoglobin neutralizes nitric oxide very effectively. Whether red blood cells participate in the regulation of endothelial function in vivo has not been established. METHODS AND RESULTS: Clinical and haematological parameters and forearm blood flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were measured in 84 type 2 diabetic patients and 19 control subjects. Diabetics showed blunted dose-response curves to both SNP and ACh. In diabetics, across quartiles of Hct, ACh blood flow responses were progressively lower (881+/-96, 652+/-81, 513+/-54, 307+/-46%, P