Miniaturized titanium implants placed in the diaphysis of mouse femurs were used as a model to examine concurrent effects on bone marrow lymphocytes, while attempting to accelerate the osseointegration of titanium implants by applying human recombinant Transforming Growth Factor Beta1 (TGF-beta 1) in carrier medium to the implant site, prior to implant placement. While the response of lymphocytes to titanium implants has been addressed, at present, the response of these cells in vivo to TGF-beta 1 remains unknown. The use of double immunofluorescence to bind molecular markers has allowed the detection of the various phenotypic stages during B cell differentiation, including both precursor B cells and early progenitor cells, as well as CD4-, and CD8- T cells, in femurs receiving 1mug, 10mug, and 100mug TGF-beta 1/100mul carrier medium, and carrier medium alone prior to implant placement. (Abstract shortened by UMI.)

Capacitation of spermatozoa is essential for fertilization. This process is associated with a change of motility pattern of the spermatozoon called hyperactivation. The possibility that reactive oxygen species play a role in mouse sperm hyperactivation and capacitation was investigated. In this work, we demonstrate that the generation of reactive oxygen species by the reaction xanthine + xanthine oxidase stimulates hyperactivation and capacitation. Reactive oxygen species scavengers such as N-tert-butyl-$ alpha$-phenylnitrone, superoxide dismutase, catalase, dimethysulfoxide, benzoic acid and salicylic acid inhibit basal as well as (X + XO)-induced sperm hyperactivation and capacitation. These results suggest that ${ rm cdot}O sb{2-}$ and H$ sb2$O$ sb2$ react together through the iron catalysed Haber-Weiss reaction to form OH. Investigation of possible $ cdot$OH targets suggest that this radical promotes sperm hyperactivation and capacitation via modification of levels of membrane lipid peroxides and of cellular free -SH groups on small molecules, proteins or both.

Apoptosis, a mechanism for modulating tissue growth, is triggered by signals (Fas ligand (FasL), tumor necrosis factor-alpha (TNF-alpha)) derived from other cells as well as external factors such as ultraviolet radiation (UV). We have investigated the possibility that FasL, Fas, M-CSF and flt3/flk2 ligand are released on shed vesicles from HuT 78 cells, tumor cell lines (MIP-101 and CX-1), and Chinese hamster ovary (CHO) cells transfected with cDNA encoding for M-CSF and flt3/flk2 ligand, respectively. Further, we examined the effects of UV-B irradiation on the capacity of CHO cells to shed vesicles per se, and on the capacity of CHO cells transfected with cDNA encoding flt3/flk2 ligand to express flt3/flk2 ligand on their cell surface. Vesicles and plasma membranes from CHO cells solubilized in N-octyl-beta-D-glycopyranoside, electrophoresed in polyacrylamide and reacted with anti-human M-CSF or anti-flt3/flk2 ligand antibody revealed bands of 90 and 45 kDal or 32 kDal bands, respectively. Following irradiation with UV-B (600 Joules/m2), CHO cells transfected with flt3/flk2 ligand cDNA failed to express the protein at their cell surface. When plasma membranes and shed vesicles collected from HuT 78 cells, solubilized, electrophoresed in polyacrylamide and reacted with anti-FasL IgG, immunoreactive FasL protein was detected in membranes and vesicles from HuT 78 cells. When exposed to FasL-bearing SVs 67.2 +/- 0.59% of Fas-expressing tumor cells survived as compared to cells treated with SVs derived from CX-1 cells (control), results which were comparable to the level of cell death observed following treatment with anti-Fas antibody (69.1 +/- 1.25%). Tumor cells treated with FasL- and Fas-bearing SVs simultaneously, exhibited 96.4 +/- 1.04% viability, indicating that FasL and Fas interact when expressed on SVs, in vitro. Our results suggest that diminished release of shed vesicles represents a potential early event in radiation-induced apoptosis occurring even pri

Radiation therapy is currently under investigation as a therapeutic option for the prevention and the treatment of restenosis following percutaneous coronary interventions. Restenosis after vessel injury is associated with a transient state of cell proliferation and bears many similarities with wound healing. It is known that ionizing radiation can delay or impair wound healing. One proposed approach uses the deployment of a radioactive stent and continuous low dose-rate treatment. Our experimental studies have been conducted to better evaluate the radiobiology of vascular cells and as a preamble to the design of an original radioactive stent based on a 45Ca-DTPA polymer-coated stent. / We performed morphometric analysis of human coronary segments with restenosis. Most of vessel area was occupied by atherosclerotic plaque and neointima accounted for a limited part. Adventitial and medial thicknesses were significantly thinner than the atherosclerotic plaque and neointima. The medial layer was the most cellular. Our results indicated that target volumes for endovascular catheter-based brachytherapy or radioactive stents remain small. After delivery of doses such as proposed in the current experience, only a few thousand cells would remain clonogenic. Depending of the residual growth stimulus, this would create a permanent impairment or a significant delay in the healing and the restenosis process. / We compared in vitro responses of human and pig fibroblasts, smooth muscle cells and endothelial cells exposed to high dose-rate gamma irradiation. Using clonogenic assays, growth inhibition and filter elution techniques, we found that human and pig cells have similar radiation responses. Our results suggest that vascular cell lines cover a broad range of radiosensitivities without specific differences between cell types. / We developed an experimental set-up to evaluate the in vitro effects of low energy beta-emitters on vascular cells. The complex is the 45Ca-diethylenetriaminepentaacetate (45Ca-DTPA) with a maximal energy of 255 KeV. There was a good correlation between calculated doses and doses measured by dosimeters placed at the bottom of petri dishes. However, it seems that doses administered to cell monolayers would be better evaluated by the dose calculated by the Medical Internal Radiation Dose (MIRD) instead of half the calculated dose, as previously suggested. / We evaluated the dose-rate effect of vascular smooth muscle cells exposed to low dose-rate irradiation with the 45Ca-DTPA complex. (Abstract shortened by UMI.)

Oxytocin (OT) and the related hormone vasopressin (AVP) are both expressed in the hypothalamus but are never co-expressed in the same neurons. The OT and AVP genes are believed to derive from one common gene by gene duplication and both genes are adjacent to each other on the chromosome in all species studied. Because of the lack of suitable cell lines expressing the OT gene, scientists have taken advantage of the availability of promoter sequences of numerous species to overcome this deficit. Alignment of the OT promoters available revealed stretches of high homology and further analysis of these stretches revealed that they are binding sites for the nuclear receptor superfamily of transcription factors such as the estrogen receptor (ER), retinoic acid receptor (RAR) and the thyroid hormone receptor (TR). Here, I identify that the orphan receptors, chicken ovalbumin up-stream promoter transcription factor II (COUP-TFII), ErbA-related factor 2 (Ear-2) and retinoid orphan receptor alpha1 (RORalphal) as potential regulators of the OT gene promoter. / In contrast to the positive effect of the ER, RAR and TR on the OT promoter, COUP-TFII and Ear-2 have a negative effect on the human OT promoter. The effects of COUP-TFs on the OT promoter are two-fold; they are able to silence or actively repress basal promoter activity and secondly, inhibit the activation by other nuclear receptors via a mechanism of competitive binding. Two additive DNA elements were identified to mediate the effect of COUP-TFII and Ear-2. The first one is a direct repeat of the AGGTCA motif spaced by zero nucleotide (DR0) that is embedded in the OT/estrogen response element (OT/ERE). The second element, located downstream of the OT/ERE, consists of three AGGTCA motifs each spaced by four nucleotides. Inhibition of the estrogenic induction only requires the DR0 whereas both elements are required for full silencing of basal activity. DNaseI footprinting and gel shift assays show that recombinant COUP-TFII and Ear-2 are able to bind to the DR0 and the downstream repeats. Mutations that affect the ability of COUP-TFII and Ear-2 to bind to these elements also affect their activity in transient transfection studies. The importance of COUP-TFII and Ear-2 in the regulation of OT gene expression is supported by their expression in the epithelial layer of the rat parturient uterus. The uterus is a known site of OT expression where a dynamic regulation of OT is observed. (Abstract shortened by UMI.)

To better define the differences between laparoscopic (LC) and mini cholecystectomy (MC) in treating cholelithiasis, we conducted a randomized controlled trial with 70 patients (LC:38, MC:32). / Both groups were comparable at baseline. The median length of post-operative hospital stay and time to full diet were significantly shorter in LC than MC (p $<$ 0.005 for both). Mean duration of convalescence was 11.9 ($ pm$9.1) days for LC and 20.2 ($ pm$16.5) days for MC (p = 0.04). Kaplan-Meier survival analysis confirmed these results. Using Cox's proportional hazards model, duration of convalescence was only found to be associated with the type of cholecystectomy performed. Three quality of life scores showed that LC patients improved more quickly than MC patients after cholecystectomy. / Surgeons underestimated convalescence on average by 25% (p $<$ 0.01) when compared to nurses' measurements. / In conclusion, even though recovery after MC was shorter than generally anticipated, time to recovery from LC was still shorter and more predictable than MC.

Established human lung cancer exhibits a complex pattern of genetic changes as well as several distinct autocrine growth factor loops for regulatory peptides. The best studied example is that of gastrin-releasing peptide or GRP, the mammalian homolog of the amphibian bombesin. Three human bombesin receptor subtypes have been cloned. They are the gastrin-releasing peptide receptor (GRP-R), the neuromedin-B receptor (NMB-R), and the bombesin-receptor subtype-3 (BRS-3). Low level expression of the GRP-R and NMB-R mRNA was detected in cultures of human bronchial epithelium. BRS-3 mRNA was undetected in these cells. An elevation of intracellular calcium concentration was observed in response to bradykinin, but not to bombesin, vasopressin or cholecystokinin. Such responses are frequently noted in lung cancer cell lines. This suggests that broad responsiveness to multiple neuropeptides is acquired during bronchial carcinogenesis. However, homologous desensitization of intracellular calcium responses to bombesin is present in lung cancer cells and can be mimicked by protein kinase C-activating phorbol esters. However, upon inhibition of protein kinase C, homologous desensitization to bombesin persists. The data indicate that protein kinase C is not a dominant component in the homologous desensitization to bombesin in small cell lung cancer cells.

The composition and heterogeneity of human intervertebral disc proteoglycans from different aged individuals were investigated. (1) The glycosaminoglycan content in the nucleus pulposus was greater than that in the annulus fibrosus at all ages. Glycosaminoglycan content increased from the infant to the young adult, and then decreased from the young to the mature adult. (2) Disc contained a higher content of hyaluronate than articular cartilage at all ages. (3) Proteolytically modified LP3 was predominant in the disc. (4) Very low link protein concentrations were observed in adult disc proteoglycan preparations and adult disc extracts analysed by SDS/PAGE and immunoblotting suggesting a deficiency in link protein content. In contrast, radioimmunoassay of disc extracts suggests no deficiency in link protein content. (5) The existence of extremely fragmented link protein in adult disc is postulated to reconcile the results of the two analytic methods. (6) Non-proteoglycan forms of biglycan and decorin are present in the disc. The difference between the two non-proteoglycan forms of biglycan appears to be due to different N-linked oligosaccharide substitution rather than proteolysis.

The Duplan strain of Radiation Leukemia Virus (RadLV) induces an AIDS-like syndrome when injected intraperitoneally into adult C57BL/6 mice. The mice develop T cell depletion, polyclonal B cell proliferation (lymphadenopathy, splenomegaly), hypergammaglobulinemia, and immune deficiency. This syndrome has recently been designated MAIDS (murine acquired immunodeficiency syndrome). This virus preparation, passaged in vivo, is crude and known to contain different strains of retroviruses. To identify the etiologic agent of this disease, we first infected fibroblasts in vitro with this crude virus mixture. Supernatants of these cultures induced disease. Unintegrated viral DNA from fibroblasts newly-infected with this virus preparation was extracted by the Hirt supernatant method and analysed with various viral probes: 8.8 and 4.8 kb DNA species were detected and molecularly cloned in pUC 18 (clones Du9S and Du5H, respectively). An unique DNA sequence derived from the gag region of the defective Du5H genome did not hybridize to various other retroviral DNA's (Moloney MuLV, N-Cl-35 or Du9S). The restriction of Du9S is similar to the map of other RadLV's. The defective Du5H DNA was transfected into fibroblasts in the presence of the neomycin resistance gene and rescued with the non-leukemogenic ecotropic RadLV (G6T2). This virus complex, as well as the ecotropic virus alone, were inoculated into C57BL/6 mice to test their pathogenic potential. The rescued cloned defective virus caused MAIDS, whereas the helper virus (G6T2) alone did not cause disease. Sequence data shows that the defective virus has large deletions in pol and env, and that the gag region is conserved and harbors a novel p12 sequence. This mouse model may help in understanding some important mechanisms underlying the biology of retrovirus-induced immunodeficiency syndromes, such as AIDS.

Gamma-glutamyl carboxylase (GGCX) is an enzyme essential for the post-translational modification of glutamic acid residues found within the gamma-carboxyglutamic acid (GLA) domain of vitamin K-dependent blood coagulation factors as well as other proteins principally involved in bone development such as Osteocalcin and Matrix Gla Protein. We propose that Marrow Stromal Cells (MSC) may serve as a useful Factor IX (FIX) delivery vehicle in vivo. As part of the validation of this cellular delivery platform for gene therapy, we determined whether MSCs endogenously express the GGCX gene. We demonstrated that GGCX was present in MSCs and that it was upregulated as MSCs differentiate into osteoblasts. These results will be of use in the rational development of Marrow Stromal Cells as a delivery vehicle of synthetic gamma-carboxylated therapeutic proteins, including FIX for therapy of Hemophilia B. Furthermore, we wanted to improve upon factor IX gamma-glutamyl carboxylation by generating a fusion FIX protein with enhance carboxylation capabilities. We interchanged the propeptide responsible of the efficiency of this post-translational process of factor IX by the one of the most efficiently gamma-carboxylated proteins, prothrombin.