Platelet number is positively and independently associated with glycated hemoglobin in non-diabetic overweight and obese subjects.

BACKGROUND AND AIMS: A significant increase in platelet count may be a risk factor for atherosclerotic cardiovascular disease. This study investigates the association between platelet number and glucose metabolism, evaluated by glycated hemoglobin (HbA1c) levels, in a apparently healthy population represented by overweight and obese subjects with normal glucose and HbA1c levels. METHODS AND RESULTS: As many as 240 subjects, 177 women and 63 men, aged 18-70 years, were enrolled. Body mass index (BMI), waist circumference (WC), systolic and diastolic blood pressure levels, platelet count and fasting blood glucose, insulin, insulin resistance, HbA1c, uric acid, triglyceride, total cholesterol, high and low density lipoprotein cholesterol concentrations were evaluated. Concerning the univariate correlation analyses between platelets number and all other variables, platelet count was significantly (and positively) correlated only with HbA1c (P < 0.05) and female sex (P < 0.01). HbA1c (P < 0.05), female sex (P < 0.001), and diastolic blood pressure (P < 0.01), positively, and age (P < 0.05) and systolic blood pressure (P < 0.05), negatively, were significantly and independently associated to platelet count in a final multiple regression analysis. CONCLUSION: This is the first study showing a strong positive and independent relationship between HbA1c and platelet number in non-diabetic overweight and obese subjects.

Platelet number is negatively and independently associated with carotid intima-media thickness in apparently healthy overweight/obese subjects.

BACKGROUND AND AIM: A significant change of platelet number may be a risk factor for atherosclerotic cardiovascular disease. The aim of this study was to investigate the association between platelet number and early signs of atherosclerosis, evaluated by carotid intima-media thickness (c-IMT), in a apparently healthy population mainly represented by obese subjects. METHODS AND RESULTS: As many as 961 subjects, 686 women and 275 men, aged between 18 and 74 years, were enrolled in the study. Of these, 54 individuals (5.6% of all subjects) were normal weight, 259 individuals (27.0% of all subjects) were overweight, and 648 individuals (67.4% of all subjects) were obese. Waist circumference (WC) and blood glucose, insulin, total cholesterol, high and low density lipoprotein cholesterol, triglycerides and platelet count were also detected in all subjects, who underwent carotid echo color doppler ultrasound to measure c-IMT. c-IMT was significantly and positively associated to age (r = 0.204, P < 0.0001), fasting glucose (r = 0.073, P < 0.0240), total cholesterol (r = 0.096, P = 0.0031), and systolic and diastolic blood pressure (r = 0.140, P < 0.0001 and r = 0.119, P < 0.0003 respectively); c-IMT was significantly and negatively correlated with platelet count (r = -0.165, P < 0.0001). Only age (P < 0.0001) and systolic blood pressure (P = 0.0393), positively, and platelet number (P < 0.0001), negatively, were significantly and independently associated to c-IMT in a final multiple regression analysis. CONCLUSION: Lower platelet number represented an independent determinant of c-IMT in a population, mainly represented by obese patients. These results suggest that a decrease of platelet number may well be an early defensive mechanism in subjects developing the thickening of carotid artery.

Extracorporeal shock waves induce osteogenic differentiation of human bone-marrow stromal cells.

The effects of treatment with shock waves (SW) on osteoblastic cells have already been described. Furthermore, the effects of treatment with SW are also determined by the contextual stimulation of other cell lines, in particular of mesenchymal cells. This is the first experimental study of stimulation of a human mesenchymal stem cell line, taken from bone marrow, using SW (electromagnetic device), with two energy levels. The results showed a significant increase in expression of the main osteoblastic differentiation genes: BMP2, alkaline phosphatase, osteocalcin, COL1A1, RUNX2. The monitoring within 96 hours demonstrated a progressive increase of cell adhesion and an intense cell proliferation at 48 h. The differentiation response and proliferation of stem cells after treatment with SW shows that this therapy is an effective method of regenerative medicine.

Pharmacogenetics and pharmacogenomics: role of mutational analysis in anti-cancer targeted therapy.

The goal of cancer pharmacogenomics is to obtain benefit from personalized approaches of cancer treatment and prevention. Recent advances in genomic research have shed light on the crucial role of genetic variants, mainly involving genes encoding drug-metabolizing enzymes, drug transporters and targets, in driving different treatment responses among individuals, in terms of therapeutic efficacy and safety. Although a considerable amount of new targeted agents have been designed based on a finely understanding of molecular alterations in cancer, a wide gap between pharmacogenomic knowledge and clinical application still persists. This review focuses on the relevance of mutational analyses in predicting individual response to antitumor therapy, in order to improve the translational impact of genetic information on clinical practice.

Effect of low-level laser irradiation on osteoblast proliferation and bone formation.

Applications of laser therapy in biostimulation and healing injured tissues are widely described in medical literature. The present study focuses on the effects of laser irradiation on the growth rate and differentiation of human osteoblast-like cells seeded on titanium or zirconia surfaces. Cells were laser irradiated with low therapeutical doses at different intervals and the effects of irradiation were evaluated at each time-point. After 3 hours lasered cells showed an enhanced mitogen activity compared to non-lasered control cells and a higher alkaline phosphatase activity, marker of bone formation. At the same time, the mRNA of RUNX2 and OSTERIX, two genes involved in osteoblast differentiation, showed a clear decrease in lasered cells. This reached the lowest value 6 to 12 hours after irradiation, after which the transcripts started to increase, indicating that the laser treatment did promote the osteogenic potential of growth-induced cells. These results indicate that Low Level Laser Treatment (LLLT) stimulates osteogenic cell proliferation.

Cross-linking of Fas by antibodies to a peculiar domain of gp120 V3 loop can enhance T cell apoptosis in HIV-1-infected patients.

Previous studies have demonstrated that T cell-reactive antibodies in HIV-1 infection contribute to lymphocyte depletion by cytotoxicity that involves differential membrane targets, such as the 43.5-kD receptor on CEM cells. Here, we show that these antibodies bind Fas as result of a molecular mimicry of the gp120. Both flow cytometry and immunoblotting using the human Fas-transfected mouse WC8 lymphoma revealed positive binding of immunoglobulin G from several patients to a 43.8-kD membrane receptor that also reacts with the CH11 anti-Fas monoclonal antibody. Specificity to Fas was further confirmed to chimeric recombinant human Fas-Fc by ELISA, whereas overlapping peptide mapping of a Fas domain (VEINCTR-N) shared by gp120 V3 loop demonstrated a predominant affinity to the full-length 10-mer peptide. Four anti-Fas affinity preparations greatly increased the subdiploid DNA peak of CEM cells similar to agonist ligands of Fas. In addition, anti-Fas immunoglobulin G strongly inhibited the [3H]thymidine uptake of CEM cells in proliferative assays, inducing a suppression as high as provoked by both CH11 mAb and recombinant human Fas ligand. Since anti-Fas were reactive to gp120, it is conceivable that antibodies binding that domain within the V3 region are effective cross-linkers of Fas and increase apoptosis in peripheral T cells. These results suggest that autologous stimulation of the Fas pathway, rather than of lymphocytotoxic antibodies, may aggravate lymphopenia in a number of HIV-1+ subjects.

Defective levels of both circulating dendritic cells and T-regulatory cells correlate with risk of recurrence in cutaneous melanoma.

BACKGROUND: Immune markers in the peripheral blood of melanoma patients provide useful information for clinical management although there is poor consensus on circulating cells which could putatively reflect the disease activity and play a prognostic role. Here, we investigated both dendritic cells (DCs) and T-regulatory cells (Tregs). METHODS: The number of DC subsets as myeloid (m) and plasmacytoid was measured by flowcytometry in 113 melanoma patients in different clinical stages and correlated with the disease activity to evaluate the recurrence free survival (RFS) calculated as difference between baseline and post-surgical values in relation to the criteria for the melanoma staging, as primary tumor removal, sentinel lymph node biopsy and completion of lymph node dissection. RESULTS: Circulating mDC levels were significantly lower in metastatic melanoma than in other stages and inversely correlated to Treg values while both populations were similarly expressed in inactive disease at stage I-III. Furthermore, the levels of these cells after melanoma removal were apparently related to the disease activity since their persistent defect reflected high risk of recurrence and reduced the RFS. CONCLUSIONS: This work highlighted the role of immune cell measurement for the management of melanoma activity and the identification of patients at potential risk of recurrence based on the mDC ratio.

Overexpression of interleukin-12 and T helper 1 predominance in lupus nephritis.

Imbalance of cytokine homeostasis is a prominent feature of both experimental and human systemic lupus erythematosus (SLE). Because interleukin (IL)-12 promotes interferon (IFN)-gamma production leading to polarization of peripheral cells toward a T helper (Th) 1 phenotype, we investigated its role in lupus nephritis (LN). Soluble Th1 and Th2 cytokines were measured by enzyme-linked immunosorbent assay (ELISA) in sera and urines of SLE patients and controls. Th1/Th2 peripheral lymphocyte polarization was determined by flow cytometry. Glomerular accumulation of IL-12 was evaluated by immunohistochemistry, whereas urinary IL-12 was evaluated by ELISA. Higher serum IL-12 levels in SLE were associated with LN, whereas IL-4 was unrelated to the renal damage. Peripheral cells from LN patients showed a Th1 phenotype with a high IFN-gamma expression that paralleled the severity of renal damage. IL-12 was present within glomerular mononuclear cells in classes IV and V LN, and its accumulation was correlated strongly with urinary levels. IL-12 overexpression in SLE may contribute to the development of LN. Both serum and urinary IL-12 elevation reflect its glomerular production and parallel Th1 polarization of peripheral T cells and high IFN-gamma production. In SLE patients, IL-12 measurement may thus be predictive of the development of LN.

Primary intimal sarcoma of the thoracic aorta.

Primary aortic tumors are well known for both their rarity and variability in clinical presentation and usually are diagnosized post-operatively or by post-mortem examination. Intimal sarcoma is a recurrent histological variant and the involvement of the thoracic aorta is an unusual presentation. Angiography and computed tomography are accurate methods to evaluate aortic tumors though transesophageal echocardiography is actually used for the differential diagnosis. Here, we describe an unusual intimal sarcoma of the thoracic aorta whose clinical feature strongly mimicked a diffuse thrombotic disease.

Sjögren's syndrome: an autoimmune disorder with otolaryngological involvement.

Sjorgen's syndrome (SS) is an autoimmune exocrinopathy characterized by lymphocyte infiltration of salivary and lacrimal glands that leads to progressive xerostomia and xerophtalmia. One-third of patients suffer of systemic manifestations including arthritis, fever, fatigue and mucosal dryness whereas those with major salivary involvement show an increased risk to develop low-grade non-Hpdgkin lymphomas. In addition, a minority of patients show symptoms related to progressive hearing loss whose pathogenesis remains undefined. Both deposition of autoantibodies to antigens of the inner-ear structures and infiltration by autoreactive T-cells have been implicated in its pathogenesis. In this context, high levels of autoantibodies to both cardiolipin and M3 muscarinic receprtors as well as to ciliar epitopes of the cochlear cells have been recently described. Here we review recent advances on the pathodgenesis of SS with a particular focus to otolaryngological manifestations.

Cancer treatment-induced bone loss (CTIBL): pathogenesis and clinical implications.

Osteopenia and osteoporosis are often long-term complications of anti-neoplastic treatments, defined as "cancer treatment-induced bone loss" (CTIBL). This pathological condition in oncologic patients results in a higher fracture risk than in the general population, and so has a significant negative impact on their quality of life. Hormone treatment is the main actor in this scenario, but not the only one. In fact, chemotherapies, radiotherapy and tyrosine kinase inhibitors may contribute to deregulate bone remodeling via different mechanisms. Thus, the identification of cancer patients at risk for CTIBL is essential for early diagnosis and appropriate intervention, that includes both lifestyle modifications and pharmacological approaches to prevent bone metabolism failure during anti-tumor treatments.

Overexpression of Fas antigen on T cells in advanced HIV-1 infection: differential ligation constantly induces apoptosis.

OBJECTIVES: To investigate Fas in peripheral lymphocytes from HIV-1-positive patients at different disease stages with respect to the extent of apoptosis. DESIGN: The study included analysis of Fas involvement in T-cell apoptosis observed during HIV-1 infection. Because ligation of Fas can result in costimulation of proliferation or the induction of apoptosis in uninfected cells, we evaluated the effect on T cells of Fas activation by monoclonal antibodies (MAb) of different specificity from both UB2 and CH11 clones and activation by the Fas ligand (Fas-L). METHODS: Fas was measured by FACS in peripheral blood and in phytohaemagglutinin (PHA)-driven cultures derived from 59 HIV-1-positive individuals with different Centers for Disease Control and Prevention stages. The percentage of apoptotic cells was detected by propidium iodide cell staining. The effect of Fas ligation was assessed in peripheral T cells from patients and healthy controls by a proliferative test measuring the 3H-thymidine uptake. RESULTS: FACS analysis revealed that Fas was predominantly expressed in advanced disease, although it was promptly exposed in PHA cultures from asymptomatic individuals. In several instances, Fas overexpression was associated with substantial subdiploid DNA content in cells from severely lymphopenic patients. The proliferative assay showed a significant inhibition of 3H-thymidine uptake in T cells from all patients following Fas ligation by the immunoglobulin (Ig) G1 MAb from the UB2 clone. This was in contrast to the apparent cell activation detected in controls and the weak suppression observed in Fas-positive cell lines. In addition, the IgM anti-Fas and recombinant Fas-L concentrations inducing a moderate inhibition of fresh T cells from controls strongly depressed the proliferative rate of cells from patients. CONCLUSIONS: Our data suggest that Fas overexpression parallels the progression of the disease and that the increased susceptibility of T cells from HIV-1-infected individuals to undergo apoptosis may include a Fas pathway. Functionally exhausted T cells in advanced HIV-1 infection are primed to apoptosis because of their high sensitivity to Fas stimulation even using the IgG1 MAb, which is unreactive to the death domain of Fas. This suggests that the increased sensitivity of Fas is apparently unrelated to its trimeric ligation and supports the hypothesis that Fas pathway plays a role in increasing the lymphocyte apoptosis during the disease.

Anti-Fas (CD95/Apo-I) autoantibodies and soluble Fas levels concur in T cell depletion in HIV type 1 infection.

Deregulation of the Fas/FasL pathway in activated T cells is suspected to contribute to the abnormal apoptosis that drives their progressive depletion during HIV-1 infection. However, the role of serum soluble Fas (sFas) is unclear. Here we investigated both sFas and anti-Fas IgG levels in a cohort of 227 HIV-1-infected patients with respect to their T cell apoptosis. By using optimized ELISAs, we found that serum titers of sFas and anti-Fas were linearly correlated in 17 severely lymphopenic subjects as compared with other patients grouped in relation to their single expression of anti-Fas and sFas, or with double-negative control patients. Cytofluorimetric measurement of the subdiploid DNA-containing cell population by both PI and TUNEL revealed an increased occurrence of cell death in vitro, in particular in patients with elevations of sFas. We also found that fresh CD4(+) cells from these patients showed high levels of both caspase 3 (CPP32) and its molecular targets, namely PARP and CK18. In addition, their in vitro proliferative rate was inhibited by sFas, in particular in patients with undetectable levels of the soluble receptor in vivo as well as in normal donors. In these subjects the Fas-related caspase 8 (FLICE) was significantly increased in cells treated with the recombinant Fas. These results support the contention that functionally exhausted T cells may undergo apoptosis in response to the persistent in vivo stimulation by sFas. This may elucidate the described occurrence of enhanced cell death in advanced HIV-1 infection in association with serum elevations of the soluble receptor.

LFA-1 expression on CD4(+)CD45RO(+) peripheral blood T-lymphocytes in RR MS: effects induced by rIFNbeta-1a.

We investigate the in vivo and in vitro effects of short-term treatment with recombinant Interferon beta-1a (rIFNbeta-1a) on CD4(+)CD45RO(+) activated/memory peripheral blood T-lymphocytes (PBTLs) expressing Leukocyte Function Antigen-1 (LFA-1; CD11a/CD18) in relapsing-remitting (RR) Multiple Sclerosis (MS) patients. Blood samples were obtained from 10 RR MS patients before and after 2, 4 and 6 months of rIFNbeta-1a (Avonex) treatment. For each sample, the percentage of CD4(+)CD45RO(+)CD11a(+) (CD11a(dim) and CD11a(bright)) T-cells was evaluated in in vivo PBTLs and in untreated or rIFNbeta-1a (1000 U/ml) or recombinant soluble Intercellular Adhesion Molecule-1 (ICAM-1, the ligand for LFA-1) (400 ng/ml) treated cultured PBTLs by triple fluorescence flow-cytometry (FACS analysis). Soluble ICAM-1 (sICAM-1) serum levels were evaluated by ELISA. In vivo, the percentage of CD4(+)CD45RO(+), CD4(+)CD45RO(+)CD11a(+), CD4(+)CD45RO(+)CD11a(dim) PBTLs increased after 4 and 6 months of rIFNbeta-1a treatment compared to pretreatment and 2 months of treatment (p<0.05). The CD11a expression per se did not change during the time course. Soluble ICAM-1 (sICAM-1) serum levels also increased (p<0.05) after 4 and 6 months of treatment. When T-cells, obtained from the blood of the same patients before and during in vivo treatment, were cultured either untreated or treated with rIFNbeta-1a, they showed an increase in the percentage of CD4(+)CD45RO(+) T-cells expressing CD11a(bright) (p<0.05). The addition of recombinant sICAM-1 to untreated cultures decreased the percentage of CD4(+)CD45RO(+) T-cells expressing CD11a. This last finding seems to support an indirect effect in vivo of rIFNbeta-1a via sICAM-1 on this T-cell subset, since the ICAM-1 soluble form, induced in vivo in serum by rIFNbeta-1a but lacking in in vitro conditions, keeps the percentage of CD11a(+) unchanged within CD4(+)CD45RO(+) T-cells and induces their expression of CD11a(dim), probably preventing T-cells from transmigrating.

Benign monoclonal gammopathy with IgG anti-DNA, anti-Sm and anti-F(ab')2 activity.

OBJECTIVE: To study anti-DNA idiotypic markers and anti-DNA activity in human monoclonal immunoglobulin proteins. METHODS: Seventy human IgG M-components intentionally selected for cationic electrophoretic characteristics were studied for F4 and 31 anti-DNA idiotypic markers and anti-DNA as well as anti-F(ab')2 antibody activity. RESULTS: Eight of 70 M-components showed significant anti-DNA activity. In two both anti-DNA and anti-F(ab')2 activity occurred together. One IgG-2 kappa M-component showed extremely high anti-ds DNA, anti-Sm, anti-F(ab')2 and anti-Sm/ RNP ELISA activity. Cross inhibition studies showed that each reactive antigen inhibited the other. N-terminal V-region sequencing showed the VH3, VK3 subgroup. Anti-idiotypic rabbit antibody produced against this M-component showed strong reactivity with affinity purified IgG anti-DNA and anti-F(ab')2 from most SLE patients and normal subjects. CONCLUSION: Monoclonal human immunoglobulins may contain multiple autoantibody specificities including anti-DNA; anti-Sm, anti-Sm/RNP, and anti-F(ab')2. Many antibodies with these specificities share common V-region antigens. Such relationships could contribute to idiotypic immune regulation and control.

Serum elevations of soluble Fas (CD95/apo-I) concur in deregulating T cell apoptosis during active lupus disease.

Apoptosis is deregulated in active systemic lupus erythematosus and Fas is overexpressed by T cells, although the role of its soluble form (sFas) is unclear. We have explored both the biological significance and structure of sFas in relation to the disease activity. Serum levels of both sFas and sFas-L were correlated with T cell apoptosis in 26 systemic lupus eythematosus patients along with measurement of poly (ADP) ribose polymerase and CK18. In addition, both proliferative rate and change of ploidy were measured in CD3+ cells after treatment with sFas. Both sFas and sFas-L correlated with apoptosis in patients with active systemic lupus eythematosus. Incubation with sFas greatly suppressed proliferation of CD3+ cells from inactive patients and healthy donors, whereas immunoprecipitation revealed both the 48-kDa full-length Fas and the 26-kDa splicing variant in sera from active patients. We postulate that sFas is released to exert a pro-apoptogen effect. It seems possible that the apoptosis program itself includes the shedding/secretion of different forms of Fas to spread a death signal.

IgM, IgA and IgG rheumatoid factors in patients with rheumatoid arthritis and normal donors.

IgM, IgA, and IgG Rheumatoid Factors (RF) were measured by ELISA assay in serum from 26 patients with definite rheumatoid arthritis (RA) and 11 normal controls. IgM-RF was assayed by ELISA, radioimmunoassay, and also by the standard latex fixation test in all sera from RA patients. In patients with RA quantitative amounts of IgM, IgA, and IgG-RF as estimated by ELISA were highly correlated. Significant correlations were found between a physician's rating of disease activity and IgG-RF (r = 0.44; p less than .02) and IgA-RF (r = 0.38; p = .06 but not for IgM-RF as measured in any of the three assays.

Familial systemic sclerosis following exposure to organic solvents and the possible implication of genetic factors.

Both genetic and environmental factors are suspected to play a role in the pathogenesis of systemic sclerosis. We compare its occurrence in 3 sisters working in a dry cleaner's shop and exposed to occupational inhalation of organic solvents. Two sisters showing the human leukocyte antigens (HLA)-DR11/DQ7 haplotype were affected. The third has maintained the same job as the others for over 10 years and has no signs of the disease. The fact that she has a different HLA haplotype points to the significance of genetic factors in increasing the risk of systemic sclerosis. It is suggested that the DR11/DQ7 haplotype enhances the development of a clinical subset of systemic sclerosis associated with production of anti-topoisomerase-I antibodies, and that environmental triggers prime the disease in subjects with this genetic background.

[Apoptosis or programmed cell death: regulatory and pathophysiological mechanisms]. / Apoptosi, o morte cellulare programmata: meccanismi regolatori e fisiopatologia.

Apoptosis is an active death process genetically encoded to eliminate abnormal or unwanted cells. The phenomenon is induced by a cascade of molecular events leading to nucleolysis by endonucleases and involves a number of membrane receptors and cytoplasmic proteins. These structures (including Fas, müllerian inhibiting substance, p53 and the c-myc oncogene) contribute, by interactive regulatory mechanisms, to the promotion or inhibition of apoptosis, on the basis of both external stimulus and cell activation state. Since apoptosis is a selective process to suppress defective cells, deregulation of genes encoding for such apoptosis-related proteins could be relevant in the growth of several tumors. Remarkably, overexpression of the bcl-2 gene in a few experimental lymphomas has been associated with neoplastic proliferation because of its inhibitory effect on apoptosis. Conversely, early activation of Fas, an apoptosis-inducing gene on HIV-infected CD4+ lymphocytes, is thought to aggravate T cell lymphopenia in HIV infection by increasing the level of normal apoptosis. Genetic deregulation of apoptosis has also been postulated in the pathogenesis of several diseases. Indeed, while preliminary studies suggest that apoptosis plays a role in autoimmune disorders including systemic lupus erythematosus, the pathogenesis of a few degenerative neuropathies, such as Alzheimer's disease, could depend on a similar altered mechanism in apoptosis of neuronal cells. However, no studies are presently available to suggest that exploitation of molecular events of apoptosis would imply therapeutic progress.

[Common variable immunodeficiency. The authors' personal cases from the last 5 years]. / Immunodeficienza comune variabile. Casistica personale degli ultimi cinque anni.

Common variable immunodeficiency (CVI) is a heterogeneous syndrome characterized by defective antibody formation, resulting in abnormally low serum immunoglobulin levels. Clinical presentation usually includes recurrent infections of the respiratory tract, mostly induced by capsular bacteria. Patients are also highly prone to Giardia lamblia infections and related gastrointestinal disorders, as well as to a variety of autoimmune diseases which appear in approximately 20% of them. In addition, CVI can be frequently associated with a non-Hodgkin lymphoma or gastric carcinoma. In spite of its relatively frequent occurrence, the pathogenesis of CVI still remains poorly defined. An overview of the syndrome is published in this issue (pages 616-622). The authors experience (18 cases during last five years) is here reported.