Tiziana Punzi

Transdermal Delivery of Heparin Using Pulsed Current Iontophoresis

PurposeIn clinical practice heparin has to be administered by injection with obvious disadvantages; thus, transdermal delivery by electrically assisted methods have been studied. In this study we evaluated the efficacy of a Food and Drug Administration-approved pulsed current iontophoresis system in delivering heparin through living rat skin.MethodsFluorescent and radioactive heparin as well as a commercial heparin preparation were delivered through rat skin via a pulsed current iontophoresis system.ResultsPulsed current iontophoresis allowed fluorescent heparin to cross the stratum corneum localizing in epidermis and dermis. Unfractionated, high-, and low molecular weight fraction pools, obtained by fractionating [35S]-unfractionated heparin on a molecular weight sieve, were then separately tested. Pulsed current iontophoresis elicited the transdermal delivery of low molecular weight heparin, but not that of high molecular weight heparin. Finally, pulsed current iontophoresis of an unfractionated pharmaceutical heparin preparation significantly decreased plasmatic factor Xa activity.ConclusionsWe hypothesize that this technique could be used to administer low molecular weight heparin in a cost-efficient and safe manner without the need for syringes and needles.

Cadmium modulates proliferation and differentiation of human neuroblasts

Cadmium is an environmental pollutant inducing numerous pathological effects, including neurological disorders and brain diseases. However, little is known about the molecular mechanisms of cadmium in affecting neurons and in inducing neurotoxicity in the development of the human brain. We have recently established, cloned, and propagated in vitro a primary long‐term cell culture (FNC‐B4) obtained from the human fetal olfactory neuroepithelium. In the present study, we show that different concentrations of cadmium chloride (CdCl2) induced dose‐dependent biological effects in FNC‐B4 cells. A low concentration (10 μM) of CdCl2 stimulated neuroblast growth, whereas a high concentration (100 μM) inhibited the growth and the viability of neuroblasts inducing morphological and cytoskeletal alterations as well as apoptotic cell death. We also observed that CdCl2 affected, in a dose‐dependent manner, the differentiation of FNC‐B4 neuroblasts, with increased mRNA and protein levels of differentiation markers and decreased expression levels of neuronal stem markers. Furthermore, differentiated cells co‐expressed glial and neuronal markers. We suggest that CdCl2 in FNC‐B4 neuroblasts might represent a selective cue by which, in a heterogeneous primary culture, the more differentiated mature cells die, whereas the undifferentiated cells, at the same time glial and neuronal progenitors, are forced to access a state of differentiation.

Iron Indices and Vitamin D Receptor Polymorphisms in Hemodialysis Patients

Cardiovascular disease caused by accelerated atherosclerosis is the major determinant of morbidity and mortality in chronic kidney disease patients. Vitamin D and its analogs provide survival benefit for hemodialysis (HD) patients. Vitamin D exerts its effects through the vitamin D receptor (VDR) that is coded for by a gene showing several polymorphisms that, in turn, are associated with a variety of diseases and differential responses to vitamin D. In this study, we evaluated the association between 4 VDR polymorphisms (ie, those identified by the restriction enzymes BsmI, ApaI, TaqI, and FokI) and iron indices (serum iron, transferrin, transferrin saturation, and ferritin) in 88 hemodialysis patients routinely treated with vitamin D. The absence or presence of the BsmI, ApaI, TaqI, and FokI restriction sites were denominated B and b, A and a, T and t, F and f, respectively. Our results show that in HD patients with transferrin saturation <20%, the F allele was more frequent than in HD patients with transferrin saturation >20% (P = .03). This relationship may provide a link between VDR alleles and iron and nutritional markers, which are highly predictive variables of cardiovascular morbidity and mortality in hemodialysis patients.

C-reactive protein levels and vitamin d receptor polymorphisms as markers in predicting cachectic syndrome in cancer patients.

AbstractBackground and Objective: In patients with advanced cancer, cachexia correlates with low performance status and poor quality of life. In addition, cachexia may be associated with reduced response to chemo-radiotherapy and a poor prognosis in cancer patients. Nearly all forms of cachexia are closely associated with chronic inflammation and elevated levels of inflammatory and pro-inflammatory circulating factors, including C-reactive protein (CRP), which is considered a valid laboratory and clinical marker. Among the different pathways involved in the production of inflammatory cytokines and chemokines, the vitamin D-vitamin D receptor (VDR) axis plays a fundamental role.In this study, we explore the possible association between CRP and key factors pertaining to the vitamin D axis — in particular, VDR gene polymorphisms — in cancer patients with cachexia. Although certain tumor types are more commonly associated with cachexia, even within the same tumor type there are significant differences in the extent and duration of cachexia. Such variations may be due to polymorphisms of the VDR gene that could lead to cachexia-prone genotypes or to cachexia-resistant genotypes. Identification of such genotypes could be very helpful in the management of cancer patients. Methods: Forty-three cancer patients were recruited by the Nutritional Unit of the Prato Hospital. Data on age, gender, type of cancer, stage of cancer, and nutritional assessment, as well as transferrin, ferritin, albumin, and CRP levels, were collected. Genomic DNA was extracted from peripheral blood leukocytes and amplified by polymerase chain reaction. BsmI, ApaI, TaqI, and FokI polymorphisms of the VDR gene were investigated using the respective restriction enzymes. For the different VDR polymorphisms, the absence or presence of the restriction sites were designated with capital or small letters, respectively. For example, for the BsmI polymorphism, the presence of the undigested fragment identified the B allele, whereas the presence of the digested fragment identified the b allele. Results: Cancer patients with cachexia have higher CRP levels compared with non-cachectic cancer patients, independently from the genotype. In cachectic patients, the presence of specific VDR BsmI and TaqI alleles was associated with higher CRP levels. In particular, the VDR b and T alleles were more frequent in cachectic cancer patients with elevated CRP levels than in cachectic patients with normal CRP levels. Conclusion: From these results, we hypothesize that there is an association between BsmI and TaqI VDR gene polymorphisms and the cachectic syndrome. In particular, we propose that in cancer patients, the concomitance of b and T alleles with elevated CRP levels may represent an early clinical predictor for the development of a more aggressive form of cachexia.

Angiotensin-converting enzyme/vitamin D receptor gene polymorphisms and bioelectrical impedance analysis in predicting athletic performances of Italian young soccer players.

Micheli, ML, Gulisano, M, Morucci, G, Punzi, T, Ruggiero, M, Ceroti, M, Marella, M, Castellini, E, and Pacini, S. Angiotensin converting enzyme/vitamin D receptor gene polymorphisms and bioelectrical impedance analysis in predicting athletic performances of Italian young soccer players. J Strength Cond Res 25(8): 2084-2091, 2011—We evaluated the association between 2 genetic polymorphisms known to be involved in fitness and performance, and anthropometric features, body composition, and athletic performances in young male soccer players with the goal of identifying genetic profiles that can be used to achieve maximal results from training. One hundred twenty-five medium-high-level male soccer players were genotyped for angiotensin-converting enzyme (ACE) I/D, and vitamin D receptor (VDR) FokI gene polymorphisms and scored for anthropometric measurements, body composition, and athletic performance. Body mass index, fat mass, fat-free mass, resistance, reactance, impedance, phase angle (PA), and body cell mass were measured. Athletic performance was evaluated by squat jump, countermovement jump (CMJ), 2-kg medicine ball throw, 10- and 20-m sprint time. We observed that the homozygous ff genotype of the VDR gene was significantly more represented in young soccer players than in a matched sedentary population. Values of reactance and PA were differently distributed in ACE and VDR genotypes with high mean values in subjects with DD (ACE) and FF (VDR) genotypes. No correlation was observed between ACE or VDR genotypes and 2-kg medicine ball throw, 10- and 20-m sprint times. The ID genotype of ACE was associated with the best performances in squat jump and CMJ. Our results suggest that determination of ACE and VDR genotypes might help select those young athletes harboring the most favorable genetic potential to succeed in soccer.

New Frontiers in Sport Training: Genetics and Artistic Gymnastics

Abstract Morucci, G, Punzi, T, Innocenti, G, Gulisano, M, Ceroti, M, and Pacini, S. New frontiers in sport training: Genetics and artistic gymnastics. J Strength Cond Res 28(2): 459–466, 2014—The increasing understanding of the genetic influences in sport has prompted an association study between the athletic performances and the polymorphisms of the angiotensin-converting enzyme (ACE), the &agr;-actinin-3 (ACTN3), and the vitamin D receptor genes. The details of these gene polymorphisms can provide useful information to improve and plan new modern training programs for elite athletes. Eighty Italian male high level gymnasts were trained and tested for gymnastic-specific exercises and tested in all the mens artistic gymnastic apparatus (floor, pommel horse, rings, vault, parallel bars, and horizontal bar), and then genotyped. The training parameters of volume, intensity, and density of each gymnast were periodically measured during the season in each apparatus from the tests performed, and the seasonal average values were calculated. Gene polymorphisms were determined by polymerase chain reaction restriction fragment length polymorphism assay and studied in association with the performance results. The performances of ACE II gymnasts were significantly lower than that of the ACE ID/DD gymnasts in the apparatus expressing power features, confirming the predisposition of these athletes toward power-oriented sport. Gymnasts with ACTN3 RR/RX genotypes did not show a predisposition to the power-oriented apparatus, having worse performances compared with that of the ACTN3 XX gymnasts. Similarly, gymnasts with ACE II + ACTN3 RR/RX combined genotypes showed lower performances in comparison with that of the other gymnasts. Vitamin D receptor polymorphisms showed no significant association with the athletic performances. Because ACE insertion/deletion (I/D) and ACTN3 R577X polymorphisms heavily affect the physical performance of elite male gymnasts, the Italian Gymnastic Federation trainers have started to customize the current high-level training programs.

Macrophages of the mucosa-associated lymphoid tissue (MALT) as key elements of the immune response to vitamin D binding protein-macrophage activating factor

Macrophages are key elements of the immune response and vitamin D binding protein-macrophage activating factor (DBP-MAF, also known as GcMAF) has been successfully used in treatment of immunodeficiency (J Med Virol 81:16-26, 2009). Here we report the effects of DBP-MAF on the immune system of HIV/AIDS patients as well as the effects of an original probiotic preparation, putatively containing DBP-MAF. Eight HIV/AIDS patients were treated with 100 ng/week DBP-MAF (from www.gcmaf.eu) i.v. for 15 weeks. During treatment, patients did not assume antiretroviral drugs. Blood monocyte count rose in six patients, indicating a response to DBP-MAF consistent with the effects of DBP-MAF described in Immunol Cell Biol 76:237-44, 1998. Individual response appeared to be associated with vitamin D receptor (VDR) gene polymorphisms ( BsmI and FokI ). Within the time frame of administration, however, no significant increase in CD4 cell count or decrease in viral load was observed. Therefore, searching for an alternative approach, we tested an original milk-derivative (MAF 3 14®) that contains microorganisms introduced in order to maximize natural DBP-MAF production. We hypothesized that natural DBP-MAF, once ingested, activated the Mucosa-Associated Lymphoid Tissue (MALT) widely diffused in the walls of the entire gastrointestinal tract. In fact, enzymes of certain strains of microorganisms contained in yogurt and kefir are able to convert milk Gc-protein into active DBP-MAF and it is known that kefir modulates the immune response in mice, increasing the phagocytic activity (i.e. activating) of peritoneal and pulmonary macrophages (Immunobiology 211:149-56, 2006). It is also known that probiotic yogurt consumption is associated with an increase of CD4 count among people living with HIV/AIDS (J Clin Gastroenterol 44:e201-5, 2010). Thus, members of the research team consumed 125 ml/day of MAF 3 14® for three weeks. Participants did not assume any drug or supplement and did not modify their usual diet and lifestyle. Blood analyses were performed two weeks before beginning consumption, and after three week consumption. After three week consumption, CD4 count dramatically increased in those of us who started with low CD4 count (subject # 1, before consumption CD4: 372; CD8: 206. After consumption: CD4: 609; CD8: 448), or abnormal CD4/CD8 ratio (subject # 2, before consumption: CD4: 857; CD8: 794. After consumption: CD4: 1279; CD8: 640). Also these effects appeared to be associated with VDR gene polymorphisms.

Tensegrity and plasma for skin regeneration.

Mechanical stresses induce variations in tissue tensegrity leading to cell proliferation and differentiation thus contributing to tissue remodelling. Besides mechanical forces, skin remodelling may be induced by the application of plasma, a new type of energy delivery resulting in controlled heat damage. Here we demonstrate that mechanical stress induced by the application of vacuum increases the efficacy of plasma in skin regeneration treatment.

Friend erythroleukemia cells induce angiogenesis in chick embryo chorioallantoic membrane and in human umbilical vein endothelial cells

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias.

Effects of Cadmium and vitamin D binding protein-derived macrophage activating factor (DBP-MAF) in human breast cancer cells

We previously demonstrated that chronic exposure of the human breast cancer cell line MCF-7 to non-cytotoxic concentrations of Cadmium reduced viability and angiogenic potential of this cell line. In order to better understand these effects, cells, after Cadmium exposure, were treated with vitamin D binding protein-derived macrophage activating factor (DBP-MAF). DBP-MAF is a potent macrophage-activating factor derived from vitamin D binding protein, a polymorphic serum glycoprotein with multiple functions also known as a group specific component or Gc protein. Besides stimulating macrophages, DBP-MAF has anti-tumour properties. Our data demonstrate that the decrease of MCF-7 cell viability following Cadmium treatment was completely reversed when DBP-MAF was present in the cell medium. Following this observation, we further investigated the role of DBP-MAF in modulating angiogenesis, morphology and cytoskeleton structure of MCF-7 cell line. As shown by chorioallantoic membrane assay, DBP-MAF inhibited MCF-7 cancer cell-stimulated angiogenesis. Concerning cell morphology (studied by contrast phase light microscopy and after Papanicolaou staining), following DBP-MAF treatment, cell shape and growth pattern were significantly modified. Vimentin expression (studied by immunohistochemistry and Western blot analysis), considered a hallmark of human breast cancer progression, after DBP-MAF treatment, significantly varied. Intermediate filament status changes, consisting in a shift from a keratin-rich to a vimentin-rich network (epithelial-mesenchymal transition), were observed. In conclusion, we demonstrate that the anti-cancer effects of DBP-MAF can be attributed to multiple actions independent of macrophage stimulation such as reversal of Cadmium effects on cell viability, reversal of morphological malignant phenotype and inhibition of cancer cell-stimulated angiogenesis. For these reasons, DBP-MAF might represent an useful tool to control progression and differentiation of human breast cancer.