A critical review of the effectiveness of rodent pharmaceutical carcinogenesis testing in predicting for human risk.

The pharmaceutical industry and regulatory agency toxicology testing paradigms in the United States currently appear successful, in part because of the continuously increasing life expectancy and the declining age-adjusted cancer rates in the United States. Although drugs likely have a minimal impact on the population statistics for cancer rates, pharmaceutical pathologists and toxicologists must focus on the individual risk for pharmaceutical carcinogenesis. As our understanding of carcinogenesis increases exponentially, and after hundreds if not thousands of rodent cancer tests, significant improvement in the precision of human pharmaceutical carcinogenesis hazard identification should now be possible and would enable a reduction in the substantial false-negative and false positive-rates reported herein. The appropriate use of acute, subchronic, chronic, and special toxicology tests to identify the major associated cancer risk factors, specifically, hormonal modulation, immunosuppression, genetic toxicity, and chronic toxicity, can be recognized through this review of pharmaceutical carcinogens. Significant opportunities exist for improving the effectiveness and efficiency of the current cancer risk assessment paradigm.

Role of hospital stay and antibiotic use on Pseudomonas aeruginosa gastrointestinal colonization in hospitalized patients.

The prospective cohort study presented here assessed the risk factors associated with Pseudomonas aeruginosa gastrointestinal colonization (PAGIC) in 933 patients hospitalized in five different wards in a French university hospital. A total of 195 patients were colonized. By logistic regression, hospitalization in an intensive care unit and length of hospital stay were independent risk factors. A significant association was observed between fluoroquinolone use and PAGIC caused by an ofloxacin-resistant strain (p < 0.0001), imipenem use and PAGIC caused by an imipenem-resistant strain (p < 0.0002) and ceftazidime use and PAGIC caused by a ceftazidime-resistant strain (p < 0.02). The ecological impact of antibiotic use is of great clinical relevance and clinicians should consider antimicrobial resistance in order to limit the development and dissemination of resistant microorganisms.

Risk-factors for gastrointestinal colonisation with resistant Enterobacteriaceae among hospitalised patients: a prospective study.

This study assessed the incidence of gastrointestinal colonisation by resistant Enterobacteriaceae among hospitalised patients, and identified risk-factors for ceftazidime and ofloxacin resistance. A prospective cohort study was performed in five wards in a French teaching hospital during a 2-year period. Patients hospitalised for > 48 h were enrolled between 17 April 2000 and 30 April 2002. A rectal swab was taken at admission, then once-weekly and/or on the day of discharge. In total, 933 patients were investigated and 585 amoxycillin-resistant isolates were obtained. Resistance rates for ceftazidime and ofloxacin were 9.4% and 4.8%, respectively. Multivariate analysis indicated that previous hospitalisation (p < 0.004) and exposure to amoxycillin-clavulanate (p < 0.003) and ceftriaxone (p < 0.002) were associated significantly with ceftazidime resistance. Hospitalisation in the urology ward (p < 0.02) and previous exposure to fluoroquinolones (p < 0.01) were the two independent risk-factors associated with ofloxacin resistance. The results of the study confirmed that antibiotic use selected resistant Enterobacteriaceae from the gut flora. Resistance was observed mostly in patients with previous antibiotic exposure and previous hospitalisation in wards with a high antibiotic selection pressure.

Umbilical vein and placental vessels from newborns with hereditary haemorrhagic telangiectasia type 1 genotype are normal despite reduced expression of endoglin.

Hereditary haemorrhagic telangiectasia, HHT, is an autosomal dominant disorder that affects approximately 1 in 8000 people. HHT1 is associated with mutations in the ENG (Endoglin) gene and with haploinsufficiency. The disorder is characterized by focally dilated vessels, which can lead to arteriovenous malformations and serious complications even in young children. In the current study, umbilical cord and placenta samples from newborns with ENG mutations were analyzed to estimate the level of corresponding protein and look for potential vascular dysplasia. We confirmed, using metabolic labelling and flow cytometry, that endoglin levels were significantly reduced to median values of 47 per cent (range 32-56 per cent) and 58 per cent (46-90 per cent), respectively, in human umbilical vein endothelial cells derived from newborns with ENG mutations (HHT1 group; n=18) relative to samples from newborns shown not to have the familial mutation (non-HHT group). We also quantified the relative expression of endoglin by estimating the endoglin/PECAM-1 staining ratio in tissue sections. We observed significantly lower values in the HHT1 group, compared to the non-HHT group for the umbilical vein (n=9; median 0.6 vs 0.9; ranges 0.2-1.0 and 0.5-1.5) and for placental stem villus vessels (n=9 and 10; median 0.42 vs 0.93; ranges 0.24-0.58 and 0.56-1.18). No differences in the estimated umbilical vein cross-sectional area and in the proportion of vessels present in placental villi were observed in sections from the HHT1 group relative to the non-HHT group. Thus, blood vessels from HHT1 individuals are maintained intact in the umbilical vein and placenta during pregnancy and delivery, despite a significant reduction in endoglin expression.

Expression and function of CD105 during the onset of hematopoiesis from Flk1(+) precursors.

During ontogeny, the hematopoietic system is established from mesoderm-derived precursors; however, molecular events regulating the onset of hematopoiesis are not well characterized. Several members of the transforming growth factor beta (TGF-beta) superfamily have been implicated as playing a role during mesoderm specification and hematopoiesis. CD105 (endoglin) is an accessory receptor for members of the TGF-beta superfamily. Here it is reported that during the differentiation of murine embryonic stem (ES) cells in vitro, hematopoietic commitment within Flk1(+) mesodermal precursor populations is characterized by CD105 expression. In particular, CD105 is expressed during the progression from the Flk1(+)CD45(-) to Flk1(-)CD45(+) stage. The developmentally regulated expression of CD105 suggests that it may play a role during early hematopoiesis from Flk1(+) precursors. To determine whether CD105 plays a functional role during early hematopoietic development, the potential of CD105-deficient ES cells to differentiate into various hematopoietic lineages in vitro was assessed. In the absence of CD105, myelopoiesis and definitive erythropoiesis were severely impaired. In contrast, lymphopoiesis appeared to be only mildly affected. Thus, these findings suggest that the regulated expression of CD105 functions to support lineage-specific hematopoietic development from Flk1(+) precursors.

Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphate.

It remains uncertain how calcium, phosphate and calcitriol regulate parathyroid cell growth. The present study was aimed at examining possible direct effects of these modulators and of the calcimimetic NPS R-467 on parathyroid cell growth in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell cycle antigen Ki 67 expression in a parathyroid cell culture model derived from uraemic patients. The effect of NPS R-467 on parathyroid hormone (PTH) secretion and intracellular [Ca2+]i response was also examined. Increasing the [Ca2+] in the medium from 0.5 to 1.7 mM increased DNA synthesis (P < 0.005) and the number of Ki 67-positive cells (P < 0.005). However, NPS R-467 (0.01-1 microM) inhibited 3[H]thymidine incorporation by 35% in the presence of 0.5 mM [Ca2+]e. Exposure of cells to Ca2+ or NPS R-467 led to a rapid increase of intracellular Ca2+, although the pattern of increase differed. Addition of calcitriol (10-10-10-7 M) to the culture medium suppressed [3H]thymidine incorporation dose-dependently. Finally, high levels of phosphate (3.5 mM) in the medium led to a significant (P < 0.05) increase in [3H]thymidine incorporation. The observed stimulatory effect of Ca2+ in the medium in vitro appears to be at variance with the inhibitory effect of calcimimetic NPS R-467 in vitro. In an attempt to solve these apparent discrepancies, and based on the notion of a reduced calcium-sensing receptor (CaR) expression in parathyroid tissues of uraemic patients, we hypothesize that Ca2+ may regulate parathyroid cell proliferation via two different pathways, with predominant growth inhibition in cases of high CaR expression or activation, but prevailing stimulation of proliferation in cases of low CaR expression.

Potential role of modifier genes influencing transforming growth factor-beta1 levels in the development of vascular defects in endoglin heterozygous mice with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder because of mutations in the genes coding for endoglin (HHT1) or ALK-1 (HHT2). The disease is associated with haploinsufficiency and a murine model was obtained by engineering mice that express a single Endoglin allele. Of a total of 171 mice that were observed for 1 year, 50 developed clinical signs of HHT. Disease prevalence was high in 129/Ola strain (72%), intermediate in the intercrosses (36%), and low in C57BL/6 backcrosses (7%). Most mice first presented with an ear telangiectasia and/or recurrent external hemorrhage. One-third of mice with HHT showed severe vascular abnormalities such as dilated vessels, hemorrhages, liver and lung congestion, and/or brain and heart ischemia. Disease sequelae included stroke, hydrocephalus, fatal hemorrhage, and congestive heart failure. Thus the murine model reproduces the multiorgan manifestations of the human disease. Levels of circulating latent transforming growth factor (TGF)-beta1 were significantly lower in the 129/Ola than in the C57BL/6 strain. Intercrosses and 129/Ola mice expressing reduced endoglin also showed lower plasma TGF-beta1 levels than control. These data suggest that modifier genes involved in the regulation of TGF-beta1 expression act in combination with a single functional copy of endoglin in the development of HHT.

Analysis of endoglin expression in normal brain tissue and in cerebral arteriovenous malformations.

BACKGROUND AND PURPOSE: A high incidence of arteriovenous malformations (AVMs) is associated with hereditary hemorrhagic telangiectasia type 1. Endoglin, the gene mutated in this disorder, is expressed at reduced levels on blood vessels of these patients. Since endoglin is a component of the transforming growth factor-beta receptor complex critical for vascular development and homeostasis, we determined its expression in sporadic cerebral AVMs and in normal brain vessels. METHODS: Twenty cerebral AVMs and 10 normal brain samples were analyzed for endoglin, platelet endothelial cell adhesion molecule 1 (PECAM-1), alpha-smooth muscle cell actin, vimentin, and desmin by immunohistochemistry. RESULTS: In normal brain, endoglin was found not only on the endothelium of all vessels but also on the adventitial layer of arteries and arterioles. In cerebral AVMs, the numerous vessels present expressed endoglin on both endothelium and adventitia. Arterialized veins, identified by lack of elastin and uneven thickness of smooth muscle cells, revealed endoglin-positive mesenchymal cells in the adventitia and perivascular connective tissue. These cells were fibroblasts since they expressed vimentin but not actin and/or desmin. CONCLUSIONS: This is the first report of endoglin expression on adventitia of normal brain arteries and on arterialized veins in cerebral AVMs. Increasing numbers of endoglin-positive endothelial and adventitial cells were seen in sporadic cerebral AVMs, but endoglin density was normal. Thus, it is not involved in the generation of these lesions. However, the presence of endoglin on fibroblasts in the perivascular stroma suggests an active role for this protein in vascular remodeling in response to increased blood flow and shear stress.

Absence of response to human parathyroid hormone in athymic mice grafted with human parathyroid adenoma, hyperplasia or parathyroid cells maintained in culture.

In athymic mice we have developed a model of long-term human PTH hypersecretion, using xenotransplantation of respectively parathyroid gland fragments obtained from patients with primary (primary) or secondary (secondary) uremic hyperparathyroidism (HPT), and parathyroid cells maintained in culture from patients with secondary uremic HPT. Both grafted parathyroid tissue fragments and cultured cells induced prolonged and marked secretion of human intact PTH (iPTH) in nude mice. Despite extremely high plasma iPTH levels, hypercalcemia or hypophosphatemia was not observed. Moreover, PTH secretion was not significantly modified by low-calcium, high-phosphate diet for 3 weeks. Four mice which had a mean plasma human iPTH level of 237+/-152 pg/ml for more than 9 months and 4 age-matched, sham-grafted control mice with undetectable human iPTH levels underwent bone histomorphometry examination. No difference was found between the two groups with respect to active bone resorption surface or number of osteoclasts/mm2. We hypothesize that the characteristic deficit of T cell function and of cytokine and growth factor production may protect nude mice with chronic hypersecretion of human PTH from hypercalcemia and bone lesions. We suggest that this strain of mice could be used for better understanding the relationship between cytokines and bone turnover.

Endoglin expression is reduced in normal vessels but still detectable in arteriovenous malformations of patients with hereditary hemorrhagic telangiectasia type 1.

Endoglin is predominantly expressed on endothelium and is mutated in hereditary hemorrhagic telangiectasia (HHT) type 1 (HHT1). We report the analysis of endoglin in tissues of a newborn (family 2), who died of a cerebral arteriovenous malformation (CAVM), and in a lung specimen surgically resected from a 78-year-old patient (family 5), with a pulmonary AVM (PAVM). The clinically affected father of the newborn revealed a novel mutation that was absent in his parents and was identified as a duplication of exons 3 to 8, by quantitative multiplex polymerase chain reaction. The corresponding mutant protein (116-kd monomer) and the missense mutant protein (80-kd monomer) present in family 5 were detected only as transient intracellular species and were unreactive by Western blot analysis and immunostaining. Normal endoglin (90-kd monomer) was reduced by 50% on peripheral blood-activated monocytes of the HHT1 patients. When analyzed by immunostaining and densitometry, presumed normal blood vessels of the newborn lung and brain and vessels adjacent to the adult PAVM showed a 50% reduction in the endoglin/PECAM-1 ratio. A similar ratio was observed in the CAVM and PAVM, suggesting that all blood vessels of HHT1 patients express reduced endoglin in situ and that AVMs are not attributed to a focal loss of endoglin.

Identification of hereditary hemorrhagic telangiectasia type 1 in newborns by protein expression and mutation analysis of endoglin.

Hereditary hemorrhagic telangiectasia (HHT) is a dominantly inherited vascular disorder that is heterogeneous in terms of age of onset and clinical manifestations. Endoglin is the gene mutated in HHT1, which is associated with a higher prevalence of pulmonary arteriovenous malformations than HHT2, where ALK-1 is the mutated gene. Endoglin is constitutively expressed on endothelial cells and inducible on peripheral blood activated monocytes so that protein levels can be measured by metabolic labeling and immunoprecipitation. We report the analysis of umbilical vein endothelial cells in 28 newborns from 24 families with a clinical diagnosis of HHT. Reduced levels of endoglin were observed in umbilical vein endothelial cells in 15/28 subjects and in activated monocytes of all clinically affected relatives tested, suggesting that these individuals had HHT1. No mutant protein was expressed at the cell surface in any of these cases, and a transient intracellular species was seen in samples of only two families, supporting a haploinsufficiency model. Quantitative multiplex PCR fragment analysis was established for the endoglin gene and revealed six mutations that were confirmed by automated DNA sequencing. An additional 10 mutations were identified in newborns by sequencing all exons. Of the 16 mutations, 10 were novel, three had been independently identified in related families, and three were previously known. Our data confirm that endoglin levels correlate with the presence or absence of mutation in HHT1 families, allowing the early identification of affected newborns that should be screened clinically to avoid serious complications of this disorder, such as cerebral arteriovenous malformations.

Apoptosis in parathyroid hyperplasia of patients with primary or secondary uremic hyperparathyroidism.

BACKGROUND: Chronic oversecretion of parathyroid hormone (PTH) is associated with parathyroid hyperplasia, reflecting a disturbed balance between cell proliferation and apoptosis. This study addressed the unsolved issue of apoptosis in hyperparathyroidism. METHODS: Parathyroid glands from 19 patients with primary (1 degrees ) and 11 patients with secondary (2 degrees ) uremic hyperparathyroidism, as well as 13 normal parathyroid glands, were examined. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end-labeling assay (TUNEL). Because the apoptotic process is regulated by several oncoproteins, the expression of Bcl-2 and Bax was analyzed by immunohistochemistry. RESULTS: The numbers of apoptotic cells in 1 degrees parathyroid adenoma (0.99 +/- 0.03 per 1000 cells, mean +/- SE, P < 0.009) and 2 degrees parathyroid hyperplasia (1.20 +/- 0.54 per 1000 cells, P < 0.005) were significantly higher than in normal parathyroid tissue (0.13 +/- 0. 06 per 1000 cells). Light microscopy examination of hyperplastic parathyroid tissue from a uremic patient showed the presence of nuclei with dense chromatin characteristic of apoptosis. Bcl-2 staining was strong in normal tissues but weak or negative in several sections of 1 degrees and 2 degrees hyperparathyroid tissues, mostly in nodular areas. Bax staining was homogeneous in normal tissue but patchy in several hyperplastic tissues. CONCLUSION: These results suggest that hyperparathyroidism is associated with a compensatory increase in apoptosis, possibly favored by a diminished Bcl-2/Bax ratio. This renders highly improbable the hypothesis that parathyroid hyperplasia is due to a decreased rate of apoptosis.

Endoglin-deficient mice, a unique model to study hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterized by dilated vessels and arteriovenous malformations. Phenotypic heterogeneity, such as age of onset, severity of disease and organ involvement, is explained in part by two genes being mutated, endoglin (HHT1) and ALK-1 (HHT2). Haploinsufficiency is the mechanism responsible for HHT. This implies that position and type of mutations cannot explain heterogeneity, because mutant proteins are not expressed at the cell surface and consequently cannot interfere with normal function. Based on this model, we generated mice expressing only one allele of endoglin, but in two different inbred strains, 129/Ola and C57BL/6. Phenotypic heterogeneity was also observed among the HHT mice and was very dependent on the genetic background. Our data strongly suggest that additional genes, contributed by the 129/Ola strain, are responsible for the vascular anomalies associated with HHT. The murine model is faithful to the human disease and should allow us to identify the modifier genes of HHT as well as to test potential therapeutic interventions.

A murine model of hereditary hemorrhagic telangiectasia.

Endoglin (CD105), an accessory protein of the TGF-beta receptor superfamily, is highly expressed on endothelial cells. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is associated with mutations in the Endoglin gene, leading to haploinsufficiency. To generate a disease model and ascertain the role of endoglin in development, we generated mice lacking 1 or both copies of the gene. Endoglin null embryos die at gestational day 10.0-10.5 due to defects in vessel and heart development. Vessel formation appears normal until hemorrhage occurs in yolk sacs and embryos. The primitive vascular plexus of the yolk sac fails to mature into defined vessels, and vascular channels dilate and rupture. Internal bleeding is seen in the peritoneal cavity, implying fragile vessels. Heart development is arrested at day 9.0, and the atrioventricular canal endocardium fails to undergo mesenchymal transformation and cushion-tissue formation. These data suggest that endoglin is critical for both angiogenesis and heart valve formation. Some heterozygotes, either with an inbred 129/Ola or mixed C57BL/6-129/Ola background, show signs of HHT, such as telangiectases or recurrent nosebleeds. In this murine model of HHT, it appears that epigenetic factors and modifier genes, some of which are present in 129/Ola, contribute to disease heterogeneity.

Biologic response to peripheral and central administration of recombinant human leptin in dogs.

OBJECTIVE: Because leptin is believed to act within the central nervous system, the objective of this study was to test that presumption by comparing the biologic responses to recombinant human leptin (rHuLeptin) when delivered either subcutaneously or intrathecally in a large animal species, the beagle dog. METHODS AND PROCEDURES: Adult beagle dogs were used for all studies (n=3 to 14). Treatment with rHuLeptin was either as daily subcutaneous or intermittent intrathecal injections. RESULTS: Subcutaneously administered rHuLeptin was absorbed with peak concentrations appearing at 2 to 4 hours. After intrathecal administration, cerebral spinal fluid concentrations declined in a bi-phasic manner with a terminal half-life of -6 to 8 hours. When lean beagles were given leptin subcutaneously, at 0.05 to 5 g/kg/day for up to 6 months, reductions in body weight (up to 30%) and food intake (up to 75%) were observed. Body fat loss was observed in both lean and obese dogs, and confirmed by dual energy X-ray absorptiometry and histology of adipose tissue. When rHuleptin was delivered intrathecally at 4 to 1000 microg/dose for up to 3 months, the primary effects observed were reductions in body weight and food intake. In general all findings reported in the intrathecal studies were consistent with those noted in the subcutaneous studies; however, the required intrathecal dose was substantially lower than that for subcutaneous delivery. DISCUSSION: These studies demonstrate that both subcutaneous and intrathecal treatment of rHuLeptin was associated with effects on body weight, food intake, and body fat in dogs. These results support the concept that the central nervous system is the probable primary site of action for leptin and suggest that rHuLeptin has similar physiologic activities that influence body weight, body fat, and metabolism in large animals to those reported previously in rodents.

The calcium receptor in health and disease.

The recent cloning of a G-protein-coupled, extracellular calcium [(Ca2+)e]-sensing receptor (CaRG) from the parathyroid, kidney and brain of several species has clarified the molecular mechanisms underlying Ca2+-sensing by parathyroid and other cell types. It has long been suspected that such a receptor existed on parathyroid cells, coupled to intracellular second messengers through guanine nucleotide regulatory (G) protein which is able to recognize and respond to (Ca2+)e. Recently, functional screening of a cDNA library constructed from bovine parathyroid mRNA led to the isolation of a 5.3-kb clone expressing maximal Ca2+-stimulated Cl- currents in oocytes. This 5.3-kb cDNA encodes a protein of 1,085 amino acids with three principal predicted structural domains. The CaRG protein is present in chief parathyroid cells, in C cells of the thyroid, in the cortical thick ascending limb (TAL) and collecting duct of the kidney, and in discrete brain areas. CaRG may play several physiological roles. It is a central element in the control of both parathyroid and calcitonin secretion by (Ca2+)e. Moreover, functional evidence for its participation in the regulation of renal Ca2+ reabsorption in TAL and water reabsorption in the collecting duct has been obtained. Mutations of the CaRG gene are responsible for hereditary and familial parathyroid disorders, and a decrease in CaRG expression has been documented in primary and secondary uremic hyperparathyroidism. The expression of CaRG in several additional organs and tissues allows speculation on the potential involvement in other pathologies.

Characterization and mapping to human chromosome 8q24.3 of Ly-6-related gene 9804 encoding an apparent homologue of mouse TSA-1.

The 9804 gene, which encodes a human Ly-6 protein most similar to mouse differentiation Ag TSA-1/Sca-2, has also been called RIG-E. Like mouse TSA-1, it has a broad tissue distribution with varied expression levels in normal human tissues and tumor cell lines. Like some members of the murine Ly-6 family, the 9804 gene is responsive to IFNs, particularly IFN-alpha. Overlapping genomic fragments spanning the 9804 gene (5543 bp) have been isolated and characterized. The gene organization is analogous to that of known mouse Ly-6 genes. The first exon, 2296 bp upstream from exon II, is entirely untranslated. The three coding exons (II, III, and IV) are separated by short introns of 321 and 131 bp, respectively. Primers were developed for specific amplification of 9804 gene fragments. Screening of human-hamster somatic cell hybrids and yeast artificial chromosomes (YACs) indicated that the gene is distal to c-Myc, located in the q arm of human chromosome 8. No positives were detected from the Centre d'Etude du Polymorphisme Humain mega-YAC A or B panels, nor from bacterial artificial chromosome libraries; two positive cosmids (c101F1 and c157F6) were isolated from a human chromosome 8 cosmid library (LA08NC01). Fluorescence in situ hybridization of metaphase spreads of chromosome 8, containing hybrid cell line 706-B6 clone 17 (CL-17) with cosmid c101F1, placed the 9804 gene close to the telomere at 8q24.3. This mapping is significant, since the region shares a homology with a portion of mouse chromosome 15, which extends into band E where Ly-6 genes reside. Moreover, the gene encoding E48, the homologue of mouse Ly-6 molecule ThB, has also been mapped to 8q24.

Persistence of Ca2+-sensing receptor expression in functionally active, long-term human parathyroid cell cultures.

An original human parathyroid cell culture model from uremic patients with IIo hyperparathyroidism has been developed, with its main feature being long-term functionally active viability up to 5 months, as assessed by persistent responsiveness to changes of extracellular Ca2+ concentrations ([Ca2+]e). In addition to the inhibitory effect of increasing [Ca2+]e, increasing extracellular phosphate exerted a biphasic effect on parathyroid hormone (PTH) secretion. The presence of the Ca2+-sensing receptor (CaR), on which depends the response to [Ca2+]e and its persistence, has been demonstrated in our culture system both by direct detection and by inhibition of its activity. CaR protein was detected by Western blot analysis with a specific anti-CaR antibody. CaR gene transcripts have been identified by reverse transcription-polymerase chain reaction analysis. mRNA (by in situ hybridization) and protein (by immunocytochemistry) expression were detected for both CaR and PTH. Adding a specific anti-CaR antibody to the medium induced a marked reduction of low [Ca2+]e-stimulated PTH release, which decreased to levels equivalent to those obtained in high [Ca2+]e medium. The described long-term functionality could be due to several factors, including the clustered cell type of culture yielded by our preparation procedure, the growth characteristics of hyperplastic uremic tissue, and the use of a phosphate-rich medium. The present model, because of its long-term functionality, is a unique tool for the exploration of PTH synthesis and secretion and for studies of parathyroid cell growth in vitro.

Mapping epitopes to distinct regions of the extracellular domain of endoglin using bacterially expressed recombinant fragments.

Endoglin (CD105) is a homodimeric cell surface component of the TGF-beta 1 receptor complex, which is expressed at high levels on vascular endothelium and at lower levels on activated monocytes. It is also the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1. To date, each family has a distinct endoglin mutation, most of which generate premature stop codons. The purpose of the current study was to identify monoclonal antibodies capable of binding to normal and mutated forms of the protein. We generated stable transfectants of full-length human endoglin in murine fibroblasts and engineered and expressed in bacteria several fragments of the extracellular domain. Relatively pure polypeptides were recovered with good yield from inclusion bodies and were tested by ELISA and Western blot; 11 monoclonal antibodies were shown to react specifically with the endoglin transfectants. Ten of these monoclonal antibodies reacted with the bacterial fragments, and their epitopes were assigned to 3 distinct regions of endoglin. Monoclonal antibodies P3D1, TEC4 and GRE reacted with the N-terminal region of 204 amino acids encoded by exons 1 to 5. Monoclonal antibodies P4A4, 44G4, E-9, MAEND3 and PN-E2 all bound to a region of 54 amino acids encoded mostly by exon 7. Monoclonal antibodies CLE4 and RMAC8 reacted with the C-terminal region of the extracellular domain, coded for by exons 8 to 12. Knowing the localization of these epitopes will facilitate the structural and functional analysis of normal and mutated forms of endoglin.