RACK1 cooperates with NRASQ61K to promote melanoma in vivo.

Melanoma is the deadliest skin cancer. RACK1 (Receptor for activated protein kinase C) protein was proposed as a biological marker of melanoma in human and domestic animal species harboring spontaneous melanomas. As a scaffold protein, RACK1 is able to coordinate the interaction of key signaling molecules implicated in both physiological cellular functions and tumorigenesis. A role for RACK1 in rewiring ERK and JNK signaling pathways in melanoma cell lines had been proposed. Here, we used a genetic approach to test this hypothesis in vivo in the mouse. We show that Rack1 knock-down in the mouse melanoma cell line B16 reduces invasiveness and induces cell differentiation. We have developed the first mouse model for RACK1 gain of function, Tyr::Rack1-HA transgenic mice, targeting RACK1 to melanocytes in vivo. RACK1 overexpression was not sufficient to initiate melanomas despite activated ERK and AKT. However, in a context of melanoma predisposition, RACK1 overexpression reduced latency and increased incidence and metastatic rate. In primary melanoma cells from Tyr::Rack1-HA, Tyr::NRasQ61K mice, activated JNK (c-Jun N-terminal kinase) and activated STAT3 (signal transducer and activator of transcription 3) acted as RACK1 oncogenic partners in tumoral progression. A sequential and coordinated activation of ERK, JNK and STAT3 with RACK1 is shown to accelerate aggressive melanoma development in vivo.

Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy.

AXL receptor tyrosine kinase (RTK) is implicated in proliferation and invasion of many cancers, particularly in pancreatic ductal adenocarcinoma (PDAC), for which new therapeutic options are urgently required. We investigated whether inhibition of AXL activity by specific monoclonal antibodies (mAbs) is efficient in limiting proliferation and migration of pancreatic cancer cells. Expression of AXL was evaluated by immunohistochemistry in 42 PDAC. The AXL role in oncogenesis was studied using the short hairpin RNA approach in a pancreatic carcinoma cell line. We further generated antihuman AXL mAbs and evaluated their inhibitory effects and the AXL downstream signaling pathways first in vitro, in a panel of pancreatic cancer cell lines and then in vivo, using subcutaneous or orthotopic pancreatic tumor xenografts. AXL receptor was found expressed in 76% (32/42) of PDAC and was predominantly present in invasive cells. The AXL-knockdown Panc-1 cells decreased in vitro cell migration, survival and proliferation, and reduced in vivo tumor growth. Two selected anti-AXL mAbs (D9 and E8), which inhibited phosphorylation of AXL and of its downstream target AKT without affecting growth arrest-specific factor 6 (GAS6) binding, induced downexpression of AXL by internalization, leading to an inhibition of proliferation and migration in the four pancreatic cancer cell lines studied. In vivo, treatment by anti-AXL mAbs significantly reduced growth of both subcutaneous and orthotopic pancreatic tumor xenografts independently of their KRAS mutation status. Our in vitro and preclinical in vivo data demonstrate that anti-human AXL mAbs could represent a new approach to the pancreatic cancer immunotherapy.

Canine melanoma diagnosis: RACK1 as a potential biological marker.

Melanoma diagnosis in dogs can be challenging due to the variety of histological appearances of canine melanocytic neoplasms. Markers of malignancy are needed. Receptor for activated C-kinase 1 (RACK1) was found to characterize melanomas in other mammals. We investigated the value of RACK1 detection in the classification of 19 cutaneous and 5 mucosal melanocytic neoplasms in dogs. These tumors were categorized as melanocytomas or benign and melanomas or malignant after evaluation of their morphology, mitotic index, and Ki-67 growth fraction. Using immunofluorescence, we confirmed microphthalmia-associated transcription factor (MITF) as a marker of normal and transformed melanocytic cells in dog tissues. All control (n = 10) and tumoral (n = 24) samples stained positively for MITF (34/34, 100%). Whereas RACK1 was not detected in healthy skin melanocytes, melanocytic lesions were all positive for RACK1 signal (24/24, 100%). RACK1 cytoplasmic staining appeared with 2 distinct distribution patterns: strong, diffuse, and homogeneous or granular and heterogeneous. All melanoma samples (13/13, 100%) stained homogeneously for RACK1. All melanocytomas (11/11, 100%) stained heterogeneously for RACK1. Immunohistochemistry was less consistent than immunofluorescence for all labelings in melanocytic lesions, which were often very pigmented. Thus, the fluorescent RACK1-MITF labeling pattern helped to distinguish melanomas from melanocytomas. Furthermore, RACK1 labeling correlated with 2 of 11 morphological features linked to malignancy: cell and nuclear size. These results suggest that RACK1 may be used as a marker in dog melanomas.

Induction of parturition with aglepristone in various sized bitches of different breeds.

The objective of this study was to confirm in various breeds of dogs the efficacy and safety of a parturition induction treatment described to be successful in Beagle dogs. Parturition was induced in seven various sized pregnant bitches of different breeds, with 15 mg aglepristone per kg at day 59-61 post-estimated ovulation day, followed 24 h later by 0.15 IU oxytocin per kg subcutaneous injections every 2 h. Two bitches were small-sized bitches (<10 kg), three bitches were large-sized bitches (30-40 kg) and two bitches were giant bitches (>40 kg). The results were compared to a control group (n = 6), in which bitches underwent a natural delivery in the same environmental conditions as the induced group. In the induced group, parturition was successfully induced in 7/7 bitches. The first pup in a litter was born on average 25.9 +/- 3.29 h after aglepristone administration (21-30 h). Two of seven bitches from the small-sized group delivered some of their pups before the first administration of oxytocin. The mean duration of parturition was 9.6 +/- 5.4 h vs 8.0 +/- 4.8 h in the control group. The mean interval between two successive pups being delivered was 115.6 +/- 82.8 min (34-265) vs 68.8 +/- 24.5 min in the control group (p < 0.03). The mean weight at parturition did not differ significantly between the two groups. One litter of four Yorkshire Terrier pups in the induced group were premature at the time of birth and died between 19 and 29 h post-delivery. This study, although on a very limited number of dogs, confirms the efficacy of the aglepristone/oxytocin protocol to induce parturition in dogs.

Diagnosis of endometritis in the bitch: a new approach.

There are a few investigations into endometritis in the bitch and its relationship with failure to conceive remains unclear. This may be because of the difficulty in collecting uterine samples for further investigations. Recently, transcervical catheterization by vaginal endoscopy has been introduced allowing the evaluation of the endometrium. In this study, uterine cytology and bacteriology were evaluated in 26 infertile bitches. Endometritis was bacterial in origin in most cases (70% of affected bitches), but these results may be underestimated, as some other pathogens (anaerobic bacteria, mycoplasms and fungi) were not investigated. Endometritis, in our opinion, should be investigated in each case of unexplained infertility in bitches. The method used here seems reliable although defining more accurate classification criteria will improve the efficiency of this non-invasive technique.

Disseminated acute concomitant aspergillosis and mucormycosis in a pony.

A 6-year-old female pony died after 2 days of prostration. Clinical signs included hyperthermia and abnormal pulmonary auscultation sounds. Necropsy revealed diffuse severe necrohaemorrhagic colitis and splenitis, multiple visceral ecchymoses, petechial haemorrhages in the brain and lungs. Microscopical examination showed acute necrohaemorrhagic colitis, encephalitis, pneumonia and splenitis associated with fibrinoid vasculitis, thrombosis and fungal hyphae within and around vessels. Immunohistologically, concomitant aspergillosis (caused by Aspergillus fumigatus) and mucormycosis (causde by Absidia corymbifera) were identified in the colonic and pulmonary lesions, whereas pure mucormycosis was observed in cerebral and splenic lesions. Dual mycotic infections are very rarely described, and the present case emphasizes the need of immunohistochemistry in order to obtain a clear-cut diagnosis of mixed fungal infections.

Sclerosing peritoneal mesothelioma in a dog - a case report.

A case of peritoneal sclerosing mesothelioma in a 3-year-old German shepherd dog is reported. The dog presented a severe abdominal distension. Cytological examination of the peritoneal fluid revealed anaplastic epithelioid cells. Necropsy findings revealed an irregular-shaped mass attached to the pancreas and stomach with numerous nodules covering the intestinal and urinary bladder serosa. The diagnosis was made by histology and immunohistochemistry, with cytokeratin, vimentin and calretinin antibodies. Differential diagnosis with chronic peritonitis and spreading of abdominal primary carcinoma is discussed.

Interstitial cells of Cajal in the striated musculature of the mouse esophagus.

Interstitial cells of Cajal (ICC) are important regulatory cells in the smooth muscle coats of the digestive tract. Expression of the Kit receptor tyrosine kinase was used in this study as a marker to study their distribution and development in the striated musculature of the mouse esophagus. Sections and whole-mounts were studied by immunohistochemistry. KitW-lacZ transgenic mice, which carry the lacZ reporter gene inserted in place of the first exon of the Kit gene, were processed for Xgal histochemistry, for quantitative analysis and for ultrastructural studies. Spindle-shaped ICC were scarce in both muscle layers of the thoracic esophagus, while their number increased steeply toward the cardia in the striated portion of the intraabdominal esophagus. They did not form networks and had no relationship with intrinsic myenteric ganglia and motor end-plates. They were often close to nerve fibers immunoreactive for neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) or neuropeptide Y (NPY), but not to fibers immunoreactive for substance P (SP), calcitonin gene related peptide (CGRP), enkephalin, or the capsaicin receptor VRI. They were present in the fetus but absent in adult ICC-deficient KitW-lacZ/KitWv mice. Interstitial cells of Cajal were identified by electron microscopy by their ultrastructure in the striated muscle of the esophagus and exhibited Xgal labeling, while fibroblasts and muscle cells were unlabeled. Interstitial cells of Cajal are scattered between striated muscle cells in the mouse esophagus. They are close to nerves with defined neurochemical coding and could possibly represent specialized esophageal spindle proprioceptors.

Kinetics of Bartonella birtlesii infection in experimentally infected mice and pathogenic effect on reproductive functions.

The kinetics of infection and the pathogenic effects on the reproductive function of laboratory mice infected with Bartonella birtlesii recovered from an Apodemus species are described. B. birtlesii infection, as determined by bacteremia, occurred in BALB/c mice inoculated intravenously. Inoculation with a low-dose inoculum (1.5 x 10(3) CFU) induced bacteremia in only 75% of the mice compared to all of the mice inoculated with higher doses (> or =1.5 x 10(4)). Mice became bacteremic for at least 5 weeks (range, 5 to 8 weeks) with a peak ranging from 2 x 10(3) to 10(5) CFU/ml of blood. The bacteremia level was significantly higher in virgin females than in males but the duration of bacteremia was similar. In mice infected before pregnancy (n = 20), fetal loss was evaluated by enumerating resorption and fetal death on day 18 of gestation. The fetal death and resorption percentage of infected mice was 36.3% versus 14.5% for controls (P < 0.0001). Fetal suffering was evaluated by weighing viable fetuses. The weight of viable fetuses was significantly lower for infected mice than for uninfected mice (P < 0.0002). Transplacental transmission of Bartonella was demonstrated since 76% of the fetal resorptions tested was culture positive for B. birtlesii. The histopathological analysis of the placentas of infected mice showed vascular lesions in the maternal placenta, which could explain the reproductive disorders observed. BALB/c mice appeared to be a useful model for studying Bartonella infection. This study provides the first evidence of reproductive disorders in mice experimentally infected with a Bartonella strain originating from a wild rodent.

Expression of the receptor tyrosine kinase KIT in mature beta-cells and in the pancreas in development.

In the pancreas, ligands of receptor tyrosine kinases (RTKs) are thought to be implicated in the development and function of the islets of Langerhans, which represent the endocrine part of the pancreas. In a previous study, we randomly screened by reverse transcriptase-polymerase chain reaction for RTKs expressed in the embryonic pancreas. One cDNA fragment that was cloned during this screen corresponded to the KIT receptor. The objective of the present study was to analyze the pattern of Kit expression in the pancreas. We demonstrated that Kit is expressed and functional in terms of signal transduction in the insulin-producing cell line INS-1. Indeed, upon treatment with the KIT ligand (KITL), the extracellular signal-regulated protein kinase was phosphorylated, and the expression of early responsive genes was induced. We also demonstrated that Kit mRNAs are present in fetal and adult rat islets. We next used mice that had integrated the lacZ reporter gene into the Kit locus. In these mice, beta-galactosidase (beta-gal) served as a convenient marker for expression of the endogenous Kit gene. Kit was found to be specifically transcribed in beta-cells (insulin-expressing cells), whereas no expression was found in other endocrine cell types or in the exocrine tissue. Interestingly, not all mature beta-cells expressed Kit, indicating that Kit is a marker of a subpopulation of beta-cells. Finally, by following beta-gal expression in the pancreas during fetal life, we found that at E14.5, Kit is expressed in both insulin- and glucagon-expressing cells present at that stage, and also in a specific cell population present in the epithelium that stained negative for endocrine markers. These data suggest that these Kit-positive/endocrine-negative cells could represent a subpopulation of endocrine cell precursors.

Alterations in ovarian function of mice with reduced amounts of KIT receptor.

The KIT receptor, present on oocyte and theca cells in ovarian follicles, and its ligand, KIT LIGAND, produced by granulosa cells, are encoded at the Kit gene and the Mgf gene, respectively. Both Kit and Mgf mutations affect oogenesis and folliculogenesis. In this study, the ovarian function of heterozygous mice with a mutation Kit(W-lacZ) was examined. Firstly, the amounts of KIT and KIT LIGAND proteins in the ovaries of mice at different ages were determined. Secondly, in vivo and in vitro folliculogenesis of wild type and heterozygous mice were compared. Western blotting showed that the amounts of both KIT and KIT LIGAND proteins were decreased in mutant mice. Ovarian follicle populations were counted and more type 5a follicles and fewer type 5b (preantral follicles) were present in ovaries from Kit(W-lacZ/+) ovaries. Furthermore, the relationships between oocyte size and follicle size differed between wild type and heterozygous mice. This finding may be a consequence of altered proliferation of granulosa cells or of altered oocyte growth in mutant mice. Other features of folliculogenesis, such as initiation of follicular growth, total follicle population and follicular atresia, were not affected by the mutation. Analysis of in vitro folliculogenesis did not reveal other differences between wild type and mutant mice. It is concluded that the Kit(W-lacZ) mutation affects the expression of KIT and KIT LIGAND proteins, resulting in alterations in granulosa cell proliferation and/or oocyte growth in preantral follicles.

Distribution and ultrastructure of interstitial cells of Cajal in the mouse colon, using antibodies to Kit and Kit(W-lacZ) mice.

The roles of the interstitial cells of Cajal in the stomach and intestine are becoming increasingly clear. Interstitial cells of Cajal in the colon are less well known, however. We studied the development and distribution of the interstitial cells of Cajal in the mouse colon, using the tyrosine kinase receptor Kit as a marker. Sections and whole mounts were studied by confocal microscopy after double immunofluorescence with specific antibodies. The ultrastructure of Kit-expressing cells was examined by electron microcopy in KitW-lacz/+ transgenic mice, which carry the lacz gene inserted in place of the first exon of the Kit gene. In the subserosa, the interstitial cells of Cajal formed a two-dimensional plexus. In the myenteric area, the interstitial cells of Cajal formed a dense plexus that gradually merged with the interstitial cells of Cajal in the outer half of the circular muscle. The inner half of the circular layer was devoid of interstitial cells of Cajal whereas in the submuscular region the interstitial cells of Cajal formed a two-dimensional plexus. Tertiary nerves with various chemical codings closely followed interstitial cell of Cajal processes. By electron microscopy, Kit-expressing cells in the outer parts of the musculature had scattered caveolae, inconspicuous basal lamina and numerous mitochondria, whereas in the submuscular region they had more pronounced myoid features. Kit-expressing cells in the mouse colon are identifiable as interstitial cells of Cajal by their ultrastructure. The interstitial cells of Cajal in the mouse colon mature postnatally. They are organized into a characteristic plexus, close to the nerves with various chemical codings.

A novel model to study the dorsolateral migration of melanoblasts.

Melanocytes derived from pluripotent neural crest cells migrate initially in the dorsolateral pathway between the ectoderm and dermomyotome. To understand the role of specific proteins involved in this cell migration, we looked for a cellular model that mimics the in vivo behavior of melanoblasts, and that allows functional studies of their migration. We report here that wild-type embryonic stem (ES) cells are able to follow the ventral and dorsolateral neural crest pathways after being grafted into chicken embryos. By contrast, a mutant ES cell line deficient for beta1 integrin subunits, proteins involved in cell-extracellular interactions, had a severely impaired migratory behavior. Interestingly, ES cells deficient for Kit, the tyrosine kinase receptor for the stem cell factor (SCF), behaved similarly to wild-type ES cells. Thus, grafting mouse ES cells into chicken embryos provides a new cellular system that allows both in vitro and in vivo studies of the molecular mechanisms controlling dorsolateral migration.

Deficiency of Trp53 rescues the male fertility defects of Kit(W-v) mice but has no effect on the survival of melanocytes and mast cells.

Mutations of the receptor tyrosine kinase, Kit, or its ligand, mast growth factor (Mgf), affect three unrelated cell populations: melanocytes, germ cells, and mast cells. Kit signaling is required initially to prevent cell death in these lineages both in vitro and in vivo. Mgf appears to play a role in the survival of some hematopoietic cells in vitro by modulating the activity of p53. Signaling by Mgf inhibits p53-induced apoptosis of erythroleukemia cell lines and suppresses p53-dependent radiation-induced apoptosis of bone marrow cells. We tested the hypothesis that cell survival in Kit mutant mice would be enhanced by p53 deficiency in vivo. Double-mutant mice, which have greatly reduced Kit receptor tyrosine kinase activity and also lack Trp53, were generated and the affected cell lineages examined. Mast cell, melanoblast, and melanocyte survival in the double Kit(W-v/W-v):Trp53(-/-) mutants was not increased compared to the single Kit(W-v/W-v):Trp53(+/+) mutants. However, double-mutant males showed an increase in sperm viability and could father litters, in contrast to their homozygous Kit mutant, wild-type p53 littermates. This germ cell rescue appears to be male specific, as female ovaries were similar in mice homozygous for the Kit mutant allele with or without p53. We conclude that defective Kit signaling in vivo results in apoptosis by a p53-independent pathway in melanocyte and mast cell lineages but that in male germ cells apoptosis in the absence of Kit is p53-dependent.

Survival of patchwork melanoblasts is dependent upon their number in the hair follicle at the end of embryogenesis.

The recessive patchwork (pwk) mutation in mice is associated with a unique hair follicle phenotype. Mice homozygous for patchwork exhibit a variegated coat containing a mixture of white and fully pigmented hairs, but no partially pigmented hairs. We have investigated the etiology of this mutation. We report here that the white hairs result from the lack of melanocytes in the follicle. As indicated by the coat color pattern of patchwork<-->albino chimeras, the target cell for the patchwork mutation is the melanocyte and/or its precursor. Examination of these chimeras also suggested that patchwork does not act in a cell-autonomous manner. The colonization of the skin by melanoblasts in patchwork embryos was studied using a lacZ transgene. Melanoblasts die by apoptosis in hair follicles from homozygous pwk/pwk fetuses starting at embryonic day 18.5, indicating that patchwork acts from this stage. The combination of pwk and KitW-ei, a mutation responsible for a reduced number of melanoblasts in the hair follicle, suggested that pwk gene product is necessary for low numbers of melanoblasts to survive and differentiate in the hair follicle from embryonic day 18.5 onward. We conclude that the pigmented hairs on the coat of pwk/pwk mice may be attributed to a community effect among melanoblasts in the hair follicle at the end of embryogenesis.

Multiple neuroendocrine tumours in transgenic mice induced by c-kit-SV40 T antigen fusion genes.

Transgenic mice carrying either a 1.008 or a 4.225 kb of the mouse c-kit 5'-flanking sequences linked to the oncogenic large T antigen (TAg) region of the simian virus 40 (SV40) genome were generated to test if the c-kit promoter could be used to develop useful mouse models. Both constructs promote tumourigenesis in the pituitary and the thyroid with high efficiency. The cell types from which each of these tumours derives were identified. Tumours of the pituitary derive from alpha-MSH-expressing cells located in the intermediate lobe. Transformed cells of the thyroid were calcitonin-positive, implying that the tumours derive from C cells or their precursors. Chromogranin A and neuron-specific enolase, general neuroendocrine cell markers, were expressed in both tumour types. Furthermore a variety of tumours appeared in the transgenic mice. Several of them stained positively for chromogranin A and/or neuron-specific enolase. This suggests a previously unsuspected tissue-specificity of the c-kit 5' flanking sequences for neuroendocrine cells. The Kit-TAg transgenic mouse lines may represent a valuable model for the study of the development and the biology of neuroendocrine tumours.

Spatial and temporal patterns of c-kit-expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos.

In the mouse, the Kit receptor and its ligand, the stem cell factor (SCF), are encoded at the W/Kit and Steel loci, respectively. The Kit/SCF transduction pathway is involved in promoting cellular migration, proliferation and/or survival of melanoblasts, hematopoietic progenitors and primordial germ cells. Furthermore, a functional Kit/SCF pathway is required for the development of interstitial cells of Cajal (ICC) in the small intestine. Whereas all c-kit-expressing cells in embryogenesis were not identified, previous studies clearly demonstrated that the c-kit expression pattern extends well beyond cells known to be affected by W mutations. To investigate further Kit function, we specifically marked the c-kit-expressing cells and followed their fate during embryogenesis. A mutation was introduced by gene targeting at the W/Kit locus in mouse embryonic stem cells. The lacZ reporter gene was inserted into the first exon of c-kit, thus creating a null allele, called WlacZ. The lacZ expression reflects normal expression of the c-kit gene in WlacZ/+ embryos. The comparison of the patterns of lacZ-expressing cells between WlacZ/+ and WlacZ/WlacZ embryos allowed us to detect where and when melanoblasts, primordial germ cells and hematopoietic progenitors failed to survive in the absence of Kit. We also observed that ICC express c-kit during embryogenesis. ICC are found identically in WlacZ/+ and WlacZ/WlacZ embryos. Therefore, ICC do not depend on Kit expression during embryogenesis. These results indicate that the function of the c-kit gene is only required for the postnatal development of the ICC. Unexpected sites of c-kit expression were uncovered in embryos, including endothelial, epithelial and endocrine cells. None of these cells are dependent on Kit expression for their migration, proliferation and/or survival during embryogenesis. Nevertheless, we assume that the Kit/SCF pathway could be involved in the growth of transformed endothelial, epithelial and endocrine cells.

A new device for endothelial cell seeding of a small-caliber vascular prosthesis.

A device permitting homogeneous endothelial cell seeding of a small-caliber arterial prosthesis has been developed. The prosthesis is maintained firmly attached to a rotative scaffolding device. This device is activated by an electrical motor at constant and adjustable speed. The whole system is maintained at 37 degrees C in a cell culture incubator. The 4 mm internal diameter polytetrafluoroethylene (PTFE) prosthesis was coated with biological glue and seeded with human saphenous vein endothelial cells obtained by mechanical detachment. Cell seeding density was 2.10(4) cells/cm2 (Group A, n = 6) or 10(5) cells/cm2 (Group B, n = 6). Rotation speed was 8 revolutions per hour (rph) during 90 min. Analysis of the homogeneity of cell seeding was permitted by cell counts on five different segments of the prosthesis. Each longitudinal segment was analyzed at three different subsegments of the circumference. The average adhesion was 43 +/- 4% in Group A and 38% +/- in Group B of seeded cells. No difference could be observed between the different segments and subsegments. In the two groups, cells were spread, and in Group B, a complete endothelial cell layer was obtained on the graft surface. This study permits validation of the device to allow homogeneous cell seeding in an arterial prosthesis.

In vitro endothelialization of carbon-coated Dacron vascular grafts.

Physical vapour deposition is used to coat vascular prostheses with pyrolytic carbon. This coating may facilitate the development of an endothelial monolayer in grafts implanted in laboratory animals. This in vitro study compared the adherence and growth of cultured porcine aortic endothelial cells (EC) seeded in vitro on carbon-coated Dacron, to uncoated prostheses. The cells were incubated at 5 x 10(4) cells/cm2. Progress was monitored at different times (TO + 2 hours, D 1, D 4, D 8) by microscopic observation, when cell counts were made; and by scanning electronic microscopy (SEM) to evaluate morphological EC-graft interactions. Cell adherence was independent of the carbon coating but cellular growth occurred only on carbon-coated Dacron. The SEM observations showed both the shape of the adherent cells, which were rounded on uncoated Dacron and extensively spread on carbon-coated prostheses, and the morphology of the carbon coating.