Therapy with nonglycosaminoglycan-binding mutant CCL7: a novel strategy to limit allograft inflammation.

Chemokines are immobilized by binding to glycosaminoglycans (GAGs). A non-GAG-binding mutant CCL7 (mtCCL7) was developed that retained its affinity for chemokine receptors. This mtCCL7 induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration (p<0.01). Unlike wild-type CCL7, mtCCL7 persisted in the circulation of BALB/c mice for more than 6 h and prevented leukocyte infiltration of skin isografts (p<0.05). Treatment with mtCCL7 marginally increased the survival of C57BL/6 to BALB/c skin allografts and reduced graft infiltration by CD3+ cells (p<0.05). Importantly, mtCCL7 promoted long-term (>40 day) graft survival following minor histocompatibility (HY) antigen mismatched C57BL/6 skin transplantation; control grafts were rejected by day 24. Treatment with mtCCL7 produced a significant decrease in the frequency of IFN-gamma producing donor-reactive splenic T cells, reduced CCR2 expression by circulating leukocytes for 6 h (p<0.01) and blocked the normal increase in affinity of alpha4beta1 integrins for VCAM-1 following transient chemokine stimulation. These data suggest that mtCCL7 persists in the circulation and reduces both specific T-cell priming and the capacity of circulating immune cells to respond to GAG-bound chemokine at sites of developing inflammation.

Chronic allograft nephropathy: intraepithelial signals generated by transforming growth factor-beta and bone morphogenetic protein-7.

It has been suggested that TGFbeta can cause chronic allograft nephropathy by inducing epithelial to mesenchymal transition (EMT); some studies show a reverse transition can be produced by bone morphogenetic protein-7 (BMP7). The current study assessed the relative contribution of signals generated within tubular epithelial cells by TGFbeta and BMP7 in normal kidney and after transplantation. Epithelial cells in normal human kidneys expressed phosphorylated forms of both Smad2/3 and Smad1/5/8 within their nuclei; cell culture experiments showed that these signaling molecules were generated in response to TGFbeta and BMP7, respectively. Phospho(p)-Smad2/3 was expressed at increased levels by tubular epithelial cells during acute renal allograft rejection and chronic allograft nephropathy but pSmad1/5/8 was expressed at very low levels; this staining profile was associated with induction of the EMT marker, S100A4. Further in vitro experiments demonstrated that this pattern of Smad signaling was a consequence of the stimulation of tubular epithelial cells with TGFbeta in the absence of BMP7. Importantly, addition of BMP7 to TGFbeta-stimulated cells enhanced the expression of pSmad1/5/8 and reduced expression of S100A4. These results suggest that exogenous BMP7 could restore the homeostatic balance of pSmad signaling found in normal kidneys, thereby preventing or reversing the development of chronic allograft nephropathy.

Tracking family practice graduates.

Lancaster General Hospital in 1991 surveyed graduates of its 21-year-old Family Practice Residency on a number of medical and non-medical issues. The results, overviewed in this article, may lay the groundwork for future efforts to obtain a national consensus regarding family practice education in its current format.

Production of pregnancy-associated plasma protein-A (PAPP-A) by cultured tumour granulosa cells.

Ten ovarian and 2 cervical tumour cell lines were analysed for the production of pregnancy-associated proteins. Pregnancy-associated plasma protein-A (PAPP-A) was detected by radioimmunoassay in culture media of 2 out of 4 (50%) tumour granulosa cell lines (mean = 104 microIU/10(5) cells/24 h) but not in any ovarian (n = 6) or cervical (n = 2) tumour cell lines. By contrast, human chorionic gonadotrophin (hCG), pregnancy specific beta 1-glycoprotein and alpha-fetoprotein (AFP) were not detected in any of the PAPP-A positive media. Only two cell lines produced hCG (58.5 and 25.5 mIU/10(5) cells/24 h). No AFP was produced by any of these 12 cell lines, whereas placental protein 5 was positive in 7. None of these proteins were detected in the culture media of 4 cell lines. In vitro derived PAPP-A was immunologically indistinguishable from either pregnancy or ovarian follicular PAPP-A. All PAPP-A species interacted reversibly with immobilised heparin and were determined by molecular sieve chromatography to have an apparent molecular weight of 820,000 daltons. Cultured tumour granulosa cells specifically synthesised and secreted a large protein which was immunologically and physicochemically indistinguishable from in vivo (pregnancy and ovarian follicular) derived PAPP-A.

The influence of pergonal on in vitro production of placental protein 5 (PP5) by ovarian tumour cells.

A radioimmunoassay for the measurement of placental protein 5 (PP5) has been developed using a second antibody-polyethyleneglycol method to separate free from bound ligand. PP5 immunoreactivity was detected in culture media from a cell line derived from a small cell carcinoma of the ovary (SCCWm2). The culture-derived PP5 shares immunological identity with pregnancy and ovarian follicular PP5. Affinity interactions between PP5 and matrices such as heparin or thrombin-Sepharose were similar and independent of the origin of PP5. PP5 was heterogeneous with two forms, a monomer (18k) and dimer (36k) being detected in all biological fluids examined. The dimer was predominant in ovarian follicles and the SCCWm2 cell line, while the monomer predominated in pregnancy serum. The basal rate of PP5 secretion by the SCCWm2 cell line was 0.61AU/24h/10(5) cells. Incubation of cells in the presence of 150mIU Pergonal stimulated PP5 production 20.6 fold and following removal of Pergonal the production rate was maintained at 18.0 times the basal rate. No choriconic gonadotrophin, pregnancy-specific beta 1-glycoprotein, pregnancy associated plasma protein-A or alpha fetoprotein was detected in the culture medium of the SCCWm2 cell line.