Changes in serum levels of TNF-alpha, IL-6, OPG, RANKL and their correlation with radiographic and clinical assessment in fragility fractures and high energy fractures.

Stages of bone turnover during fracture repair can be assessed employing serum markers of osteoblastic and osteoclastic activity, inflammatory cytokines, clinical evaluation and imaging instruments. Our study compare the fracture healing process in fragility fractures and high energy fractures by evaluating serum changes of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), osteoprotegerin (OPG) and receptor activator of the nuclear factor-kB ligand (RANKL) in combination with radiographic (Radiographic Union Scale for Tibial fractures, RUST) and clinical (Lower extremity measure, LEM) assessments. We enrolled 56 patients divided into four corresponding groups: group A with high energy trauma fracture (tibial/femoral shaft); group B with low energy trauma fracture (femoral fractures); healthy (control A) and osteoporotic subjects (control B). Blood samples were collected before surgery (T0) and after 10 weeks (T10). Serum concentrations of IL-6, TNF-alpha, RANKL and OPG were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. Our results show that RANKL values are significantly higher at T10 than at T0 in low energy trauma fractures (group B). OPG is significantly lower in each control group than that of the respective fractured group and its concentration at T0 and at T10 is significantly lower in high than in low energy fractures. RANKL/OPG ratio is significantly higher in both controls than in fractured groups, and significantly increases after 10 weeks. IL-6 and TNF-alpha concentrations significantly decrease during fracture healing and are higher in high (group A) than in low energy fractures (group B). Significant differences were also found in both RUST score and LEM between groups A and B. Changes in TNF-alpha and IL-6 levels correlate with RUST and LEM in fragility and high energy fractures, while RANKL/OPG ratio is associated with these clinical parameters only in fragility fractures. These findings suggest that serum levels of IL-6, TNF-alpha, RANKL and OPG might be used to monitor the stages of fracture repair. Further studies will be needed to confirm the role of these cytokines in fracture repair.

A pilot study on the transcriptional response of androgen- and insulin-related genes in peripheral blood mononuclear cells induced by testosterone administration in hypogonadal men.

The aim of the present study is to determine whether testosterone (T) administration changes the expression profile of androgen- and insulin-related genes in peripheral blood mononuclear cells (PBMC). To this end, we evaluated the gene expression profile of 19 genes (AKT2, CCND1, GSK3ALPHA, IGF1, GSK3BETA, FOXO3, IL6, IGFBP2, UGT2B17, ARA55, CREBBP, CYP11A, HSD17B1, HSD17B7, UGT2B7, SELADIN 1, CLU, PGC1, AKR1C1) selected according their function in the androgen pathways, in a series of 11 hypogonadal men pharmacologically treated with T. We noted that 7 genes were differentially expressed, five of them were up-regulated (AKT2 FC=2.39, CREBBP FC=11.2, GSK3beta FC=5.6, UGT2B7 FC=4.49, UGT2B17 FC=2.88) and two were down-regulated (ARA55 FC= -2.0, CYP11A FC= -2.47). This experience suggests that androgen- and insulin-related genes can be considered useful blood genomic biomarkers for specific steroid drugs.

Androgen- and insulin-related gene signature using a specific low density oligoarray AndroChip 2 in peripheral blood mononuclear cells in agonists, recreational athletes and sedentary subjects.

The early detection of genomic biomarkers (e.g. RNAs) through analysis of circulating blood cells could have a substantial impact on biomedicine, particularly in monitoring clinical trials, drug toxicity and doping in athletes. To achieve this goal, it is essential to develop methods that are sufficiently sensitive to detect biomarker alterations during normal biologic processes, pathogenic processes, and or in response to therapeutic or other intervention. Using a low density microarray (AndroChip 2) we detected a transcriptional profiling signature of 190 genes related to androgen and insulin metabolism pathway, in peripheral blood mononuclear cell (PBMC) in subjects with different intensities of sports activities. We demonstrated that androgen and insulin gene transcriptional levels are independent to sports activity and therefore potentially suitable for drug monitoring and/or drug doping (such as anabolic androgen steroid AAS abuse) and or gene doping.

Degenerate PCR method for identification of an antiapoptotic gene in BHV-1.

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type-1) BHV-1 DNA. An alignment of carboxyl-terminal domains belonging to three proteins encoded by gamma34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type-1 (BHV-1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that UL14 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti-apoptotic function may derive from its heat shock protein-like properties.

The immunosuppressive cytokines influence the fetal survival in patients with pregnancy-induced hypertension.

PROBLEM: The present study examines the hypothesis that the elevated levels of transforming growth factor (TGF)-beta1 and interleukin (IL)-10 would be protective for the fetus survival during pregnancy-induced hypertension (PIH). Moreover, we evaluate the IL-12 and IL-15 serum concentrations and their relationships with PIH. METHOD OF STUDY: Serum samples were obtained before the onset of labor from control and PIH groups. Cytokine concentrations were determined by Enzyme-Linked Immunoadsorbent Assay. RESULTS: Our data show that PIH women have significantly higher TGF-beta1 and IL-10 concentrations with respect to control groups (P = 0.0001). Similarly, macrophages from the PIH placentas produce in vitro more elevated TGF-beta1 and IL-10 levels compared to normal pregnant ones (P = 0.02), also in the absence of LPS stimulation. IL-12 and IL-15 serum concentrations were not detectable in all pregnant groups. CONCLUSION: We have found that PIH women have elevated concentrations of anti-inflammatory/immunosuppressive cytokines, suggesting their important role in fetal allograft protection during the normal and pathological pregnancy.

Effect of ouabain binding on the fluorescent properties of the Na+/K+-ATPase.

The influence of occupancy by ouabain of its specific binding site on the stability and conformation of the Na+/K+-ATPase has been investigated. When native Na+/K+-ATPase is exposed to guanidinium chloride or diluted acid, tryptophanyl fluorescence falls to 50% of the initial value. If ouabain is bound, higher concentrations of GdmCl or acidity are needed to reach the same decrease in fluorescence. The rotational diffusion coefficient (relaxation time), shows higher values for the Na+/K+-ATPase (ouabain) complex compared to the enzyme alone, suggesting an increase in molecular asymmetry. This observation is confirmed by the Stern-Volmer analysis that shows an increase in the accessibility of the fluorophores in the Na+/K+-ATPase (ouabain) (KSV = 15.6 M-1) with respect to the native enzyme (KSV = 12.5 M-1). Iodine perturbation of the enzyme labelled with FITC, demonstrates a decrease in the accessibility of the fluorescein probe in the Na+/K+-ATPase(ouabain) (KSV = 4 M-1) compared to the Na+/K+-ATPase (KSV = 7 M-1) indicating that after ouabain binding this site of the enzyme is less exposed to the solvent. These data, in agreement with other reports, suggest an allosteric effect of ouabain binding on the Na+/K+-ATPase conformation.

Natural cell-mediated cytotoxicity: a micro assay suitable for clinical tests.

A micro version (microtest, MIT) of the 51Cr release assay for detecting Natural Killer activity (NK) has been developed. The test retains the sensitivity and the efficiency of conventional macroassay (macrotest, MAT) and provides a 5-fold reduction in the number of effector and target cells employed. In experiments performed with peripheral blood mononuclear cells (MNC), untreated or treated with interferon (IFN) or with hydrocortisone (Hy), comparable values of the percentage of specific lysis and of the number of lytic units were obtained using both MAT and MIT methods. Therefore MIT appears to be useful in monitoring the NK function of patients characterized by low MNC counts.

[CAMP mediated cell growth regulation. I. Characterization of protein kinases in CHO cells]. / Regolazione della crescita cellulare da parte del cAMP. I. Caratterizzazione della proteinkinasi in cellule CHO.

Protein kinases in wild-type CHO cells have been characterized. Cells cultured on MEM were collected, homogenized and the extract assayed for protein kinase activity. DEAE cellulose chromatography of 30.000xg extract yields 2 peaks of protein kinases activity, PKI and PKII. The two peaks when analyzed for the binding of 8-N3-(32P)cAMP show two subunits RI and RII and a RI not associated with the enzymatic activity, named RF. This characterization allows us to discuss the meaning of protein kinases in the modulation of the growth regulating effects of cAMP.

[cAMP mediated cell growth regulation. II. Characterization of the biochemical change responsible for resistance to cAMP treatment]. / Regolazione della crescita cellulare da parte del cAMP. II. Caratterizzazione delle alterazioni biochimiche responsabili delle resistenze al trattamento col cAMP.

In this paper we characterize the biochemical defect of a mutant (10248) of CHO cells, resistant to the cAMP treatment. Cells cultured on MEM were collected each three days, homogenized and centrifuged. The cell extract was assayed for protein kinases activity and the binding of 8-N3-(32P)cAMP. The same extract was also applied on to a DEAE cellulose column, eluted with a linear gradient and the fractions tested for the phosphotransferase activity and 8-N3-(32P)cAMP binding. Mutant 10248 shows a different profile of protein kinases activity as compared to 10001 control. Protein kinases II is absent whereas a normal RII binding activity is present. RI shows altered affinity for cAMP.

[EGF modulated Na+/K+ ATPase in cultured fibroblasts]. / Modulazione della Na+/K+ ATPasi da parte dell'EGF in coltura di fibroblasti.

The behaviour of Na+/K+ ATPase during cell growth has been studied. Human cultured fibroblasts were used in the presence or absence of EGF. Sample and control cultures were stopped by gathering and washing the cells with tris buffer. Homogenates were tested for Na+/K+ ATPase activity by the method of incubating and for the -SH groups content (Ellman). Na+/K+ ATPase activity that slightly increases in the controls is strongly reduced by the addition of EGF. The behaviour shows evidence for a double mechanism of action: I) involvement of the cAMP system 2) decrease of the -SH group availability.