Asymmetrical nitrido tc-99m heterocomplexes as potential imaging agents for benzodiazepine receptors

The design, synthesis, and biological evaluation of nitrido technetium-99m complexes for imaging benzodiazepine receptors are described. The design was performed by selecting the precursor biologically active substrate desmethyldiazepam, and the reactive metal-containing fragment [(99m)Tc(N)(PXP)](2+) (PXP = diphosphine ligand) as molecular building-blocks for assembling the structure of the final radiopharmaceuticals through the application of the so-called 'bifunctional' and 'integrated' approaches. This required the synthesis of the ligands H(2)BZ1, H(2)C1, and H(2)C2 (Figures 1 and 2) derived from desmethyldiazepam. In turn, these ligands were reacted with [(99m)Tc(N)(PXP)](2+) to afford the complexes [(99m)Tc(N)(PXP)(L)] (L = BZ1, C1, C2). The chemical nature of the resulting Tc-99m radiopharmaceuticals was investigated using chromatographic methods, and by comparison with the analogous complexes prepared with the long-lived isotope Tc-99g and characterized by spectroscopic and analytical methods. Results showed that the complexes [(99m)Tc(N)(PXP)(L)] are neutral and possess an asymmetrical five-coordinated structure in which two different bidentate ligands, PXP and L, are coordinated to the same Tc[triple bond]N core. With the ligand H(2)BZ1, two isomers were obtained depending on the syn or anti orientation of the pendant benzodiazepine group relative to the Tc[triple bond]N multiple bond. Biodistribution studies of Tc-99m complexes were carried out in rats, and affinity for benzodiazepine receptors was assessed through in vitro binding experiments on isolated rat's cerebral membranes using the corresponding Tc-99g complexes.     

Retinoic acid stimulates matrix calcification and initiates type I collagen synthesis in primary cultures of avian weight-bearing growth plate chondrocytes

The effect of retinoic acid (RA) on primary cultures of growth plate chondrocytes obtained from weight-bearing joints was examined. Chondrocytes were isolated from the tibial epiphysis of 6- to 8-week-old broiler-strain chickens and cultured in either serum-containing or serum-free media. RA was administered at low levels either transiently or continuously after the cells had become established in culture. Effects of RA on cellular protein levels, alkaline phosphatase (AP) activity, synthesis of proteoglycan (PG), matrix calcification, cellular morphology, synthesis of tissue-specific types of collagen, and level of matrix metalloproteinase (MMP) activity were explored. RA treatment generally increased AP activity, and stimulated mineral deposition, especially if present continuously. RA also caused a shift in cell morphology from spherical/polygonal to spindle-like. This occurred in conjunction with a change in the type of collagen synthesized: type X and II collagens were decreased, while synthesis of type I collagen was increased. There was also a marked increase in the activity of MMP. Contrasting effects of continuous RA treatment on cellular protein levels were seen: they were enhanced in serum-containing media, but decreased in serum-free HL-1 media. Levels of RA as low as 10 nM significantly inhibited PG synthesis and caused depletion in the levels of PG in the medium and cell-matrix layer. Thus, in these appendicular chondrocytes, RA suppressed chondrocytic (PG, cartilage-specific collagens) and enhanced osteoblastic phenotype (cell morphology, type I collagen, alkaline phosphatase, and mineralization). J. Cell. Biochem. 65:209–230. © 1997 Wiley-Liss, Inc.     

Inhibition of terminal differentiation and matrix calcification in cultured avian growth plate chondrocytes by Rous sarcoma virus transformation

Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification. To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization. The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology. The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin. RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells. There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes. RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells. Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix. These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization. They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification. J. Cell. Biochem. 69:453–462, 1998. © 1998 Wiley-Liss, Inc.     

Chondrocytes isolated from tibial dyschondroplasia lesions and articular cartilage revert to a growth plate-like phenotype when cultured in vitro

We report here a comparative study of the development and behavior of chondrocytes isolated from normal growth plate tissue, tibial dyschondroplasic lesions, and from articular cartilage. The objective of these studies was to determine whether the properties exhibited by chondrocytes in dysplasic lesions or in articular cartilage were due to their cellular phenotype, their environment, or both. We had previously analyzed the electrolytes and amino acid levels in the extracellular fluid of avian growth plate chondrocytes. Using these data, we constructed a culture medium (DATP5) in which growth plate cells essentially recapitulate their normal behavior in vivo. Here, we used DATP5 to examine the behavior of chondrocytes isolated from lesions of tibial dyschondroplasia (TD). We found that once isolated from lesion and grown in this supportive medium, dysplasic chondrocytes behaved essentially like normal growth plate cells. These findings suggest that the cause of TD is local factors operating in vivo to prevent these cells from developing normally. With respect to articular chondrocytes, our data indicate that they more closely retain normal protein and proteoglycan synthesis when grown in serum-free media. These cells readily induced mineral formation in vitro, both in the presence and absence of serum. However, in serum-containing media, mineralization was significantly enhanced when the cells were exposed to retinoic acid (RA) or osteogenic protein-1 (OP-1). Our studies support previous work indicating the presence of autocrine factors produced by articular chondrocytes in vivo that prevent mineralization and preserve matrix integrity. The lack of inhibitory factors and the presence of supporting factors are likely reasons for the induction of mineralization by articular chondrocytes in vitro.     

Metabolic Syndrome and Breast Cancer Risk: A Case-Cohort Study Nested in a Multicentre Italian Cohort

Background

Metabolic syndrome (defined as at least three among abdominal obesity, high blood triglycerides, low high-density lipoprotein cholesterol, high blood glucose, and high blood pressure) is emerging as a risk factor for breast cancer; however few studies – most confined to postmenopausal women – have investigated associations between breast cancer risk and metabolic syndrome. The purpose of this study was to examine the association between metabolic syndrome and its components, and risk of breast cancer in postmenopausal and premenopausal women.

Methods

We performed a case-cohort study on 22,494 women recruited in 1993-1998 to four Italian centres (Turin, Varese, Naples, Ragusa) of the European Prospective Investigation into Cancer and Nutrition (EPIC) and followed-up for up to 15 years. A random subcohort of 565 women was obtained and 593 breast cancer cases were diagnosed. Hazard ratios (HR) with 95% confidence intervals (CI), adjusted for potential confounders, were estimated by Prentice-weighted Cox proportional hazards models.

Results

Presence of metabolic syndrome was associated with significantly increased breast cancer risk in all women (HR 1.52, 95%CI 1.14-2.02). When the analyses were repeated separately for menopausal status, the association was limited to postmenopausal women (HR 1.80, 95%CI 1.22-2.65) and absent in premenopausal women (HR 0.71, 95%CI 0.43-1.16); P for interaction between metabolic syndrome and menopausal status was 0.001. Of metabolic syndrome components, only high blood glucose was significantly associated with increased breast cancer risk in all women (HR 1.47, 95%CI 1.13-1.91) and postmenopausal women (HR 1.89, 95%CI 1.29-2.77), but not premenopausal women (HR 0.80, 95%CI 0.52-1.22; P interaction=0.004).

Conclusions

These findings support previous data indicating that metabolic syndrome is an important risk factor for breast cancer in postmenopausal women, but not in premenopausal women, and suggest that prevention of metabolic syndrome through lifestyle changes could confer protection against breast cancer.     

IFN-Alpha Receptor-1 Upregulation in PBMC from HCV Na?ve Patients Carrying CC Genotype. Possible Role of IFN-Lambda

Background and Aims

IL-28B gene polymorphisms predict better therapeutic response and spontaneous clearance of HCV. Moreover, higher expression of IFN-lambda has been reported in patients with the rs12979860 CC favourable genotype. The study aim was to establish possible relationships between IL-28B rs12979860 genotypes and expression of IFN-alpha receptor-1 (IFNAR-1) in naïve HCV patients, and to explore the possible role of IFN-lambda.

Methods

IFNAR-1 mRNA levels were measured in PBMC from naïve patients with chronic hepatitis C with different IL-28 genotypes. The ability of IFN-lambda to up-regulate the expression of IFNAR-1 was established in PBMC from healthy donors carrying different IL-28B genotypes.

Results

Lower IFNAR-1 mRNA levels were observed in PBMC from HCV-infected naïve patients as compared to healthy donors. In healthy donors, IFNAR-1 mRNA levels were independent from IL-28B genotype, while in HCV patients, an increasing gradient was observed in TT vs CT vs CC carriers. In the latter group, a direct correlation between IFNAR-1 and endogenous IL-28B expression was observed. Moreover, IFN-lambda up-regulated IFNAR-1 expression in normal PBMC in a time-and dose-dependent manner, with a more effective response in CC vs TT carriers.

Conclusion

Endogenous levels of IFN-lambda may be responsible for partial restoration of IFNAR-1 expression in HCV patients with favourable IL-28 genotype. This, in turn, may confer to CC carriers a response advantage to either endogenous or exogenous IFN-alpha, representing the biological basis for the observed association between CC genotype and favourable outcome of either natural infection (clearance vs chronicization) or IFN therapy.     

Diagnostic performance of anti-Zika virus IgM,IgAM and IgG ELISAs during co-circulation of Zika,dengue, and chikungunya viruses in Brazil and Venezuela

BackgroundSerological diagnosis of Zika virus (ZIKV) infection is challenging because of the antibody cross-reactivity among flaviviruses. At the same time, the role of Nucleic Acid Testing (NAT) is limited by the low proportion of symptomatic infections and the low average viral load. Here, we compared the diagnostic performance of commercially available IgM, IgAM, and IgG ELISAs in sequential samples during the ZIKV and chikungunya (CHIKV) epidemics and co-circulation of dengue virus (DENV) in Brazil and Venezuela.Methodology/Principal findingsAcute (day of illness 1–5) and follow-up (day of illness ≥ 6) blood samples were collected from nine hundred and seven symptomatic patients enrolled in a prospective multicenter study between June 2012 and August 2016. Acute samples were tested by RT-PCR for ZIKV, DENV, and CHIKV. Acute and follow-up samples were tested for IgM, IgAM, and IgG antibodies to ZIKV using commercially available ELISAs. Among follow-up samples with a RT-PCR confirmed ZIKV infection, anti-ZIKV IgAM sensitivity was 93.5% (43/46), while IgM and IgG exhibited sensitivities of 30.3% (10/33) and 72% (18/25), respectively. An additional 24% (26/109) of ZIKV infections were detected via IgAM seroconversion in ZIKV/DENV/CHIKV RT-PCR negative patients. The specificity of anti-ZIKV IgM was estimated at 93% and that of IgAM at 85%.Conclusions/SignificanceOur findings exemplify the challenges of the assessment of test performance for ZIKV serological tests in the real-world setting, during co-circulation of DENV, ZIKV, and CHIKV. However, we can also demonstrate that the IgAM immunoassay exhibits superior sensitivity to detect ZIKV RT-PCR confirmed infections compared to IgG and IgM immunoassays. The IgAM assay also proves to be promising for detection of anti-ZIKV seroconversions in sequential samples, both in ZIKV PCR-positive as well as PCR-negative patients, making this a candidate assay for serological monitoring of pregnant women in future ZIKV outbreaks.