Keloid fibroblast responsiveness to epidermal growth factor and activation of downstream intracellular signaling pathways.

Keloids, which overgrow the boundaries of the original injury, represent aberrations in the fundamental process of wound healing that include over-abundant cell in-migration, cell proliferation, and inflammation, as well as increased extracellular matrix synthesis and defective remodeling. To understand the key events that result in the formation of these abnormal scars would open new avenues for better understanding of excessive repair, and might provide new therapeutic options. We examined epidermal growth factor receptor (EGFR)-induced cell motility in keloid fibroblasts, as this receptor initiates cell migration during normal wound repair. We show that keloid fibroblasts respond to EGF-induced cell migration but the response is somewhat diminished compared to normal adult fibroblasts (approximately 30% reduced); the mitogenic response was similarly blunted (approximately 5% reduced). Keloid fibroblasts express near normal levels of EGFR (82%), but show a much more attenuated activation of EGFR itself and the motility-associated phospholipase C-gamma. This was reflected in part by rapid loss of EGFR upon exposure to EGF. Interestingly, while extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) activation was relatively robust in keloid fibroblasts, the downstream triggering of the motility-associated calpain activity was blunted. This was reflected by high cell-substratum adhesiveness in the keloid fibroblasts. Thus, the blunted migratory response to EGF noted in keloid fibroblasts appears due to limited activation of two important biochemical switches for cell motility.     

PET imaging of oncogene overexpression using 64Cu-vasoactive intestinal peptide (VIP) analog: comparison with 99mTc-VIP analog.

The purpose of this study was to assess the feasibility of PET imaging of oncogene VPAC1 receptors overexpressed in human breast cancer cells. METHODS: Vasoactive intestinal peptide (VIP) analog (TP3982) was synthesized to harbor a carboxy-terminus lysine (Lys) residue separated from VIP-asparagine (Asn(28)) by 4-aminobutyric acid (Aba) as a spacer. Lys was derivatized with diaminopropionic acid coupled to a pair of dibenzoylthioglycolic acid residues as protecting groups. The analog was labeled with (64)Cu at pH 9 ((64)Cu-TP3982) and (99m)Tc at pH 12 ((99m)Tc-TP3982). (99m)Tc-TP3982 and VIP derivatized with Aba-GAGG and labeled with (99m)Tc ((99m)Tc-TP3654) were used as reference agents. Smooth muscle relaxivity assays performed with each derivative and compared with unaltered VIP(28) demonstrated functional integrity. In vitro stability of (64)Cu-TP3982 was determined by challenging the complex with 100-mol excess of diethylenetriaminepentaacetic acid (DTPA), human serum albumin (HSA), and cysteine. In vivo stability was determined in urine and serum for up to 24 h. The mass of the Cu-TP3982 complex was determined by mass spectrometry. Human T47D breast tumor xenografts were grown in athymic nude mice. Planar scintigraphic imaging was performed at 4 and 24 h after the intravenous administration of (99m)Tc-TP3982 and (99m)Tc-TP3654 and PET imaging was performed using a small animal MOSAIC PET scanner, also at 4 and 24 h after injection of (64)Cu-TP3982. Tissue-distribution studies were also performed. In a separate experiment, receptors were blocked by intravenous injection of authentic VIP(28) 30 min before the administration of (64)Cu-TP3982 and tissue distribution was examined. RESULTS: (64)Cu-TP3982 labeling yields were 98% +/- 1.2% and those for (99m)Tc-TP3982 and (99m)Tc-TP3654 were 98.2% +/- 1.1% and 97% +/- 1.6%, respectively. The biologic activity of both VIP analogs was uncompromised. When (64)Cu-TP3982 was challenged with 100-mol excess of DTPA, HSA, or cysteine, >98% radioactivity remained as (64)Cu-TP3982. In vivo, >98% of (64)Cu circulating in plasma remained as (64)Cu-TP3982. Of the (64)Cu excreted in urine 4, 20, and 24 h after injection, >98%, 89.9% +/- 0.9%, and 85% +/- 3%, respectively, were bound to TP3982. The mass of Cu-TP3982 as determined by surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) was 4,049.7 Da. Four hours after receptor blocking with VIP(28), there was a significant reduction in uptake of all tissues except in the liver. With (64)Cu-TP3982, the 4-h postinjection tumor uptake was 10.8 +/- 2.1 %ID/g versus 0.5 +/- 0.02 %ID/g and 0.24 +/- 0.08 %ID/g for (99m)Tc-TP3982 and (99m)Tc-TP3654, respectively. Twenty-four hours after injection, the corresponding numbers were 17 +/- 0.7 %ID/g, 0.77 +/- 0.1 %ID/g, and 0.23 +/- 0.1 %ID/g. The severalfold greater uptake (21.2-74) of (64)Cu-TP3982 is attributable to the in vivo stability of the agent. CONCLUSION: The results suggest that the uncompromised biologic activity and the significantly greater tumor uptake of (64)Cu-TP3982, combined with the high sensitivity and enhanced resolution of PET imaging, make (64)Cu-TP3982 highly desirable for further studies in PET imaging of oncogene receptors overexpressed in breast and other types of cancers.     

Development of Activity Assays for High-Volume Evaluation of Human Immunodeficiency Virus (HIV) Protease Inhibitors in Rat Serum: Results with Ditekiren

We showed previously that a commercially available synthetic tetradecapeptide, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, produces authentic angiotensin I (Ang I) upon incubation with the HIV-1 protease (S. K. Sharma et al., Anal. Biochem. 198:363, 1991). Therefore, we developed an Ang-I based activity assay for HIV protease inhibitors based on the technology developed earlier (M. J. Ruwart et al., Pharm. Res. 7:407, 1990; S. K. Sharma et al., Anal. Biochem. 186:24, 1990) for tracking renin inhibitors in rat sera. Ditekiren was either extracted from sera with ethyl acetate or assayed after the interfering substances in sera were precipitated with acetonitrile. Purified recombinant HIV-1 protease was added to extracted rat serum and the enzymatic reaction was initiated in the presence of the tetradecapeptide substrate. The inhibition of Ang I production was measured by a commercially available RIA kit. The cleanup methodology also enabled a commercially available Proteinase Scintillation Proximity Assay (SPA, Amersham) to quantify ditekiren in rat serum through the addition of recombinant HIV-1 protease and cleavage of substrate from SPA beads. Results were confirmed by HPLC or by the renin assay for ditekiren, which inhibits both aspartyl proteases. These technologies should prove useful for assessing serum levels of HIV protease inhibitors in rat.     

Lymph node infarction – a rare complication associated with disseminated intra vascular coagulation in a case of dengue fever

Background  

Lymph node infarction is known to occur in association with many non-neoplastic and neoplastic conditions however its occurrence in association with DIC is not reported hitherto in the literature.     

Use of multiple antigenic peptides related to antigenic determinants of infectious bursal disease virus (IBDV) for detection of anti-IBDV-specific antibody in ELISA--quantitative comparison with native antigen for their use in serodiagnosis

Multiple antigenic peptides (MAPs) prepared for the predicted antigenic determinants on the VP2 protein of infectious bursal disease virus (IBDV) were used as antigens in enzyme-linked immunosorbent assay (ELISA)--an alternative to whole viral antigen to detect anti-IBDV antibodies in the chicken sera. Two MAPs were synthesized, which could specifically detect the anti-IBDV antibodies in serum samples by ELISA. The optimum quantity of MAP1 and MAP2 required to coat the wells of the ELISA plate was 5 ng/ml, whereas the amount of purified IBDV whole viral antigen was 500 ng/ml, indicating the high efficiency of MAPs. In this study, we mainly focused on the antigenicity of two eight-branched MAPs to detect anti-IBDV antibodies in ELISA, which would serve as safe, chemically defined, noninfectious alternative antigens to whole virus in serodiagnosis. The specificity and sensitivity of both MAP1 and MAP2 were found to be relatively better than the whole viral antigen.     

Increased venous proinflammatory gene expression and intimal hyperplasia in an aorto-caval fistula model in the rat

We hypothesized that the venous limb of an arteriovenous (AV) fistula would evince up-regulation of genes relevant to vascular remodeling along with neointimal hyperplasia and relevant histological changes. Using the aorto-caval model of an AV fistula model in the rat, we demonstrate marked up-regulation in such proinflammatory genes as monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, and endothelin-1, 2 weeks after the creation of the fistula. Neointimal hyperplasia occurred in variable degrees by 5 weeks after establishing the fistula, and by 16 weeks, such neointimal hyperplasia was progressive and pronounced; at this time point, abundant extracellular matrix was also observed. Smooth muscle cells were present in the hyperplastic neointima as evidenced by staining for alpha-smooth muscle actin; ultrastructurally, smooth muscle cells with a synthetic as well as a contractile phenotype were readily observed. Accumulation of extracellular matrix in the model at 16 weeks was accompanied by increased expression of transforming growth factor-beta1 mRNA, the latter finding contrasting with the suppression of transforming growth factor-beta1 mRNA observed in this model at 2 weeks. In summary, we describe marked up-regulation in proinflammatory genes and progressive neointimal formation in the venous vasculature in an AV fistula model in the rat. We suggest that such alteration in gene expression and histological injury, in conjunction with the relative simplicity of this model, offer a new approach in the study of such timely biological and clinically relevant phenomena as differential gene expression in response to hemodynamic forces, processes involved in vascular remodeling, mechanisms of injury in venous bypass grafts, and mechanisms of dysfunction of AV fistulae used in hemodialysis.     

Murine Brca1: sequence and significance for human missense mutations

We have cloned and sequenced a mouse homologue of the humanbreast and ovarian cancer susceptibility gene, BRCA1. The predictedmouse Brca1 protein is composed of 1812 amino acids. The murineprotein is 60% identical and 72% similar to the human BRCA1protein. Two regions of high homology have been identified betweenthe two proteins. First is the Cys3—His—Cys4 typezinc-finger domain that is identical between the two proteins.The second region is defined by 115 amino acids near the carboxyend of the Brca 1 protein that is 83% identical to human BRCA1sequence. Seven of eight amino acids involved in human missensemutations that are associated with the disease were found tobe conserved between the two species. In contrast, most of theamino acids that are involved in polymorphic variations werenot conserved. We therefore propose that the interspecies conservationof predicted amino acid sequences can be used as an additionalcriterion to determine the significance of human missense mutations.