Radiographic and CT features of metallosis in a lame dog after total hip replacement: the cloud sign.

A 2-year-old female American Akita was referred for CT of the pelvis and hindlimbs due to a left hindlimb lameness after a left total hip replacement. Referral radiographs and CT images demonstrated amorphous soft-tissue and mineral opacities surrounding the proximal femur and the prosthetic stem, consistent with the "cloud sign" reported as a characteristic of metallosis in humans. Dorsomedial displacement of the prosthetic head, multiple foci of geographic osteolysis alongside the "cloud sign", presumed pseudotumor lesions, and medial iliac lymphadenopathy were also identified with CT. Metallosis was confirmed based on ultrasound-guided cytology, revision surgery, and histopathology.

Osteochondrodysplasia and the c.1024G>T variant of TRPV4 gene in Scottish Fold cats: genetic and radiographic evaluation.

OBJECTIVES: The objectives of this study were to investigate the c.1024G>T SNP in the TRPV4 gene in Scottish Straight and Fold cats, and to evaluate the pattern of skeletal phenotype and the evolution of radiological signs of Scottish Fold osteochondrodysplasia (SFOCD) over time in heterozygous subjects. METHODS: DNA was obtained from blood samples of 17 cats (Scottish Fold: n = 12; Scottish Straight: n = 5) and subsequently genotyped by sequencing in a 249 bp region of the TRPV4 gene (exon 6), including the known c.1024G>T causative mutation for osteochondrodysplasia. Orthopaedic and radiographic analyses were performed on animals carrying the mutant allele. RESULTS: Genotyping by sequencing confirmed that all and only the Scottish Fold cats carried the mutant allele in a heterozygous asset. Furthermore, two other exon variants, already described in the literature as silent variants, were found in some of the sampled cats. Comparative orthogonal radiographic views of the shoulder, elbow, carpus, hip, stifle and tarsus were obtained. A mediolateral projection of the thoracic and lumbar column was also performed. Three out of four cats were clinically and radiographically examined again 1.5 years later. CONCLUSIONS AND RELEVANCE: Although the presence of the mutant allele in all the tested Scottish Fold cats was confirmed, only 1/12 showed clinical signs of SFOCD. Furthermore, no cats in the 1.5-year follow-up showed skeletal changes. Although significant, the c.1024G>T mutation in the TRPV4 gene, supposedly, is not the only cause or risk of developing SFOCD.

Fall time may be a reliable discriminator between neoplastic and non-neoplastic urinary bladder lesions in dogs undergoing contrast-enhanced ultrasound: a pilot study.

Contrast-enhanced ultrasound (CEUS) can provide quantitative information on enhancement patterns and perfusion of lesions, based on time-intensity curves (TICs). No published studies have compared CEUS parameters in neoplastic and non-neoplastic urinary bladder lesions in dogs. The aim of the current prospective, pilot study was to quantitatively characterize the CEUS pattern of neoplastic and non-neoplastic urinary bladder lesions in dogs, assessing the influence of contrast arrival time (CAT) on the final appearance of the curves. Fourteen dogs with cyto-histopathological diagnoses were included (seven malignant and seven inflammatory lesions). B-mode ultrasound was performed followed by CEUS examination after an intravenous bolus injection of 0.04 mL/kg of contrast medium, and TICs were elaborated by dedicated software. Receiver operating characteristic curves (ROC) for each TIC parameter were obtained. Neoplastic lesions had subjectively shorter rise time (RT), time to peak (TTP) and fall time (FT) than inflammatory lesions. Based on ROC curve analyses, fall time ≥ 10.49 s was the most reliable parameter for diagnosing non-neoplastic disease in this small sample of dogs (area under the curve [AUC] 0.75, sensitivity 83.33%, specificity 66.67%). No difference was found between ROCs calculated for each parameter of TICs by adding or removing CAT. Results of the current study provide background for future, larger scale studies evaluating use of a CEUS FT threshold of 10.49 s as a possible discriminator for urinary bladder neoplastic lesions in dogs.

Preoperative planar lymphoscintigraphy allows for sentinel lymph node detection in 51 dogs improving staging accuracy: Feasibility and pitfalls.

Sentinel lymph node (SLN) mapping is the current gold standard for the oncological staging of solid malignancies in humans. This prospective observational study describes the feasibility and the limits of preoperative lymphoscintigraphy for SLN detection in dogs with spontaneous malignancies and the improvements in staging accuracy. Client-owned dogs with confirmed malignant neoplasia and absence of distant metastasis were prospectively enrolled. Lymphoscintigraphy was performed after the peritumoral injection of Technetium-99m labeled nanocolloids. Regional dynamic and static images were acquired, with and without masking of the injection site with a lead shield. The dogs were then subjected to surgery for tumor excision and SLN extirpation. Intraoperative SLN detection was performed by combining methylene blue dye and a dedicated gamma probe. Overall, 51 dogs with a total of 60 solid malignant tumors were enrolled. Lymphoscintigraphy identified at least one SLN in 57 of 60 cases (95%). The SLN did not always correspond to the regional lymph node (35/57, 61.4%). The use of a lead shield, masking the injection site, markedly improved the SLN visibility. The median time of SLN appearance was 11.4 ± 9.3 min. No side effects were observed. Preoperative lymphoscintigraphy allows for SLN detection in dogs and can improve staging accuracy by either identifying the SLN in a different lymphosome than clinically expected or discriminating the draining node in uncertain cases. The combined use of preoperative and intraoperative techniques is recommended to increase the SLN detection rate.

Prostatic leiomyosarcoma in a dog treated using a multimodal approach.

Prostatic leiomyosarcoma is an uncommon tumor encountered in male dogs, with only 2 cases reported in the veterinary literature with no follow-up described. A 12-year-old male intact German Wirehaired Pointer presented for evaluation of straining to defecate and urinate. Whole body computed tomography (CT) examination identified a spherical multicavitary expansile mass arising from the prostate gland and severely obliterating the pelvic canal. Partial subcapsular prostatectomy was performed, and histological and immunohistochemical results were consistent with prostatic leiomyosarcoma. Metronomic cyclophosphamide and nonsteroidal anti-inflammatory drugs were administered as adjuvant chemotherapy. Follow-up CT 10 months later indicated no signs of recurrence or metastasis. To the best of our knowledge, this patient represents the first report of successful multidisciplinary treatment consisting of partial subcapsular prostatectomy and adjuvant chemotherapy for prostatic leiomyosarcoma in a dog. After 15 months of follow-up, the patient remained recurrence-free without metastasis.

CT features of feline lipiduria and renal cortical lipid deposition.

OBJECTIVES: The aims of this study were to document the presence and prevalence of feline lipiduria and renal lipid deposition on CT, and to search for associations between the presence of lipiduria and sex, urinary tract abnormalities and urolithiasis. METHODS: The CT examinations of 252 cats were reviewed for the presence of an antigravitational hypodense bubble in the urinary bladder with density values between -180 Hounsfield units (HU) and -20 HU. To identify associations between lipiduria and sex, urinary tract abnormalities and urolithiasis, Fisher's exact test was used. Renal cortical density measurement was performed in all cats. The Mann-Whitney test was performed to compare renal cortical density between lipiduric and unaffected cats. RESULTS: A total of 27 domestic cats (10.7%) had CT evidence of lipiduria. Lipiduric cats had a significantly lower renal cortical density than unaffected cats (P <0.01). Male neutered cats had a significantly higher frequency of lipiduria and lower renal cortical density compared with female neutered cats (P <0.01). There was no significant difference between the groups regarding renal, ureteral or urethral abnormalities. CONCLUSIONS AND RELEVANCE: Lipiduria is a common physiological phenomenon in cats that can be detected on routine CT examinations. Decreased renal cortical density is associated with lipiduria. This may aid in the diagnosis of feline lipiduria and help to differentiate its presence from other pathological depositions and excretions.

Contrast-enhanced ultrasound for sentinel lymph node mapping in the routine staging of canine mast cell tumours: A feasibility study.

Canine mast cell tumours (MCTs) typically spread to lymph nodes (LNs) before reaching distant sites, and LN assessment is an important part of MCT staging. Sentinel LN (SLN) mapping techniques to identify draining LNs are being developed and could improve the accuracy of MCT staging. The primary objective of this feasibility study was to determine the safety and effectiveness of contrast-enhanced ultrasound (CEUS) to identify SLNs. Secondary objectives were to determine if the SLNs identified by CEUS coincided with the regional LN predicted by the anatomical lymphosomes, if previous MCT excision altered CEUS SLN findings, and if CEUS could identify MCT nodal metastases. Between June 2017 and March 2019, 59 dogs with 62 MCTs were enrolled. No adverse events related to CEUS were reported. CEUS detected at least 1 SLN in 59/62 MCTs (95.2%, 95% CI: 86.5-99.0%). In only 32/59 (54.2%) MCTs, clinicians would have correctly predicted the SLN(s) identified by CEUS. Among the 35 MCTs that had histological examination of the SLN(s), the prevalence of metastasis was 60% (95% CI: 42.1-76.1%). Additional staging procedures did not reveal any metastases in dogs with histologically non-metastatic SLNs. Integration of CEUS SLN mapping into the routine staging of MCTs is promising, but future studies are required to refine this procedure and to investigate if it would translate into a clinical benefit.

Effect of Stifle Flexion Angle on the Repeatability of Real-Time Elastosonography of the Patellar Ligament in Medium- to Large-Breed Dogs.

OBJECTIVE: The aims of this study were to describe the elastosonographic findings of the patellar ligament in healthy dogs >15 kg in different positions and determine the most appropriate stifle angle to perform elastosonography of the patellar ligament. STUDY DESIGN: Eighteen clinically healthy dogs, weighing >15 kg, were prospectively recruited. B-mode ultrasound and real-time elastosonography of both patellar ligaments were performed on days 0 and 14. Elastosonography examinations were performed with the stifle in four positions: standing position, lateral recumbency with the stifle positioned at 135 degrees extension and in full passive flexion and extension. RESULTS: The percentage hardness of the patellar ligament was significantly lower when the elastosonographic examination was performed with the dog standing, compared with the other positions (p < 0.005). The variability in the elastosonography readings for each stifle position was lowest when the dog was standing (89.32%). CONCLUSION: Elastosonography is a feasible technique for evaluating the elasticity of the normal canine patellar ligament in dogs >15 kg and would be a useful technique for investigating the mechanical changes within the patellar ligament following stifle surgery. Elastosonography of the patellar ligament should be performed with the dog standing to provide the most accurate elastosonograms with lowest variability between readings.

Use of contrast-enhanced ultrasound for the diagnosis of idiopathic renal hematuria in a dog.

Background: Idiopathic renal hematuria (IRH) generally occurs in healthy large-breed dogs that are <2-years-old. It is characterized by recurrent bleeding from the kidneys of unidentified cause. The final diagnosis is reached through the exclusion of primary urinary system and systemic causes of hematuria along with the direct visualization by cystoscopy of hematic urine jets at the ureteral orifice. Case Description: An 8-year-old female neutered Whippet was presented for investigation of a 4-week history of chronic intermittent macroscopic hematuria. Physical examination, systolic blood pressure, extensive laboratory workup (including coagulation profile and platelet count), urine culture, thoracic and abdominal radiographs, pneumocystogram, and double-contrast cystography were all unremarkable. B-mode ultrasound showed no abnormalities apart from a moderate amount of suspended echogenic amorphous material visible within the urinary bladder lumen. In the contrast-enhanced ultrasound (CEUS) study, a large amount of echogenic ill-defined material was noted projecting into the urinary bladder lumen from the right ureterovesical junction in the fundamental mode. This material was more conspicuous and markedly contrast-enhancing in the harmonic mode. Ultrasound contrast medium has the unique property to strictly remain within the vessels without interstitial trapping or elimination by the kidneys. Indeed, the presence of the micro-bubbles into the urinary tract lumen in the CEUS study was interpreted as a direct sign of active urinary tract bleeding. A diagnosis of IRH was reached through CEUS and B-mode ultrasound along with an extensive laboratory workup and periodic follow-up of the patient. No therapy was administered and at a 1-year follow-up, the patient was alive with no current episodes of macroscopic hematuria reported. Conclusion: To the authors' knowledge, this is the first report describing the use of CEUS for the characterization of IRH. CEUS could represent a safe, non-invasive, affordable novel alternative technique to cystoscopy or cystotomy for the real-time diagnosis of IRH.

Ultrasound and computed tomography of sacculitis and appendicitis in a rabbit.

A 9-month-old neutered male rabbit was referred for lethargy, anorexia, and gastrointestinal stasis. Routine hematology, serum biochemistry, and diagnostic imaging were performed. Computed tomography revealed a wall thickening of the sacculus rotundus and appendix, which was further confirmed on abdominal ultrasound. Full thickness biopsies were collected with histopathology diagnosing a chronic multifocal heterophilic granulomatous sacculitis and appendicitis. The patient was treated medically and at 6 weeks follow-up, clinical signs and intestinal changes had completely regressed. Inflammation of the sacculus rotundus and appendix should be considered as a cause of gastrointestinal stasis in rabbits.

Computed tomographic appearance of canine tonsillar neoplasia: 14 cases.

The palatine tonsil is an uncommon site of oral canine neoplasia. For affected tonsils, squamous cell carcinoma is the most frequent type of neoplasia, followed by melanoma and lymphoma. Computed tomography (CT) is increasingly used for investigation of canine oropharyngeal pathology; however, limited information is available on the CT appearance of tonsillar neoplasms. Objectives of this retrospective descriptive case series were to characterize the CT features of canine tonsillar neoplasia and determine whether specific CT features differentiate nonneoplastic from neoplastic tonsils. Computed tomographic studies of 14 dogs diagnosed with tonsillar neoplasia were retrieved from two referral hospitals and reviewed by two observers. Diagnosis was based on histology or cytology. Carcinoma was diagnosed in 11 dogs, melanoma in two and lymphoma in one dog. Specific CT features of the tonsil and regional lymph nodes did not differentiate neoplastic from nonneoplastic tonsillar diseases, but regional lymph node CT features were useful for diagnosis in some cases. Marked enlargement (width ≥ 18 mm, 12/18), heterogeneity (16/18), and loss of the hypoattenuating hilus (18/18) of the medial retropharyngeal lymph node were common concomitant features of tonsillar neoplasia. The medial retropharyngeal and mandibular lymphadenomegaly was ipsilateral to the neoplastic tonsil in 8/12 and 6/9 dogs, respectively. Five dogs demonstrated little or no enlargement of the tonsil despite the associated metastatic lymphadenomegaly. Tonsillar neoplasia should therefore be considered as a differential diagnosis for dogs with CT evidence of isolated medial retropharyngeal lymphadenomegaly (regardless of normally sized tonsils), or of any enlarged tonsil with no associated lymphadenomegaly.

Real-time elastosonography of lipomatous vs. malignant subcutaneous neoplasms in dogs: Preliminary results.

Real-time elastography is a recently introduced ultrasound technique allowing the investigation of elastic properties of tissues. A diagnostic accuracy study was conducted to test the performance of this technique in the assessment of subcutaneous lesions in dogs. Fifty-two dogs were prospectively included in the preliminary study (34 malignant and 18 benign lesions). B-mode ultrasound was performed assessing the shape, margins, heterogeneity, and echotexture of the lesions. On elastosonography, assessment of the percentage of softness/hardness was recorded. A qualitative assessment was performed according to the Tsukuba elasticity score with a 1-5 score, representing the increased percentage of high stiffness areas. Results were compared with cytology/histopathology of the lesions. Receiver Operating Curves of the overall diagnostic sensitivity and specificity were obtained. Fisher's exact test and Pearsons's Chi-squared test estimated the relationship between the B-mode appearance of the lesions and final diagnosis. A hardness cutoff of 50.25% was identified between lesions, with malignant neoplasms having higher percentages. A 100% specificity and 89% sensitivity for correctly detecting the nature of the lesion on elastosonography was established. Qualitative assessment of the Tsukuba elasticity score established 1.5 as the cutoff between elastograms of lipomatous and malignant lesions, with 100% sensitivity and 61% specificity in differentiating them. Real-time elastosonography is a novel, noninvasive, and accurate technique for differentiating malignant from benign lipomatous lesions in dogs. This method could be considered as a complementary tool with additional diagnostic value for routine invasive procedures, such as fine needle aspirates.

Dynamic tomographic studies of interscapular feline injection-site sarcoma: essential or useless practice?

Objectives Feline injection-site sarcomas (FISSs) are soft tissue tumours typically characterised by an interscapular location and highly infiltrative behaviour. CT is considered the modality of choice for FISS staging and double positioning (dynamic approach) was reported to successfully detect the exact extent of infiltration into the muscles. The aim of the present study was to investigate the utility of the dynamic approach in feline patients referred for preoperative staging of interscapular FISS. Methods Tumour volume estimates were compared between the ellipsoid and the semi-automated segmentation methods. Two radiologists blinded to the patient coding used images from each position to assess the extent of muscular infiltration. The distance between the neoplasm and the adjacent skeletal structures (scapulae, spinous processes) was recorded in both positions by a single radiologist. Results Fifty-nine of 84 neoplasms invaded the adjacent muscular structures, with up to 15 muscles infiltrated. Between the extended and flexed position the average estimated numbers of muscles infiltrated were 1.9 (extended) and 1.84 (flexed) for observer A and 1.89 (extended) and 1.85 (flexed) for observer B. Good agreement between observers was established, with higher tumour volumes detected via the ellipsoid method. Moreover, tumours with smaller volumes showed slightly decreased muscular infiltration. Marked difference in the recorded distance between the skeletal structures and the neoplasm in the two different positions was established (mean ± SD difference spinous processes: 9.74 ± 9.57 mm; mean ± SD difference scapulae: 15.15 ± 11.76 mm). Conclusions and relevance A dynamic approach should be used for a complete evaluation of the invasiveness of FISS along with appropriate methodology for tumour volume measurement, which could potentially alter the tomographic estimation of the real dimension of the neoplasms.

Advances in the anatomic study of the interscapular region of the cat.

BACKGROUND: New clinical oriented approaches are supported by the integration of advanced imaging techniques, e.g. computed tomography and magnetic resonance, with gross anatomy imaging. The interscapular region of the cat is a typical site of a highly invasive infiltrative pathology, i.e. Feline Injection-Site Sarcoma. Even if chemotherapy and radiotherapy have been considered as pre-surgical therapies, extensive surgery is still the recommended treatment. Evidence suggested that the relationships between muscles, infiltrative mass and adjacent musculoskeletal structures could change according to the forelimb positions: a fact to be duly considered while planning the surgical approach. Anatomic and tomographic atlases provide only images of the interscapular region from cats positioned with their forelimbs extended cranially, which means that, they do not record musculoskeletal modifications due to the forelimb movements. Aim of this study was to provide detailed images of the changes occurring in the musculoskeletal structures of the interscapular region of cats according to their forelimb position by comparing cross-sectional gross anatomy, computed tomography and magnetic resonance imaging. RESULTS: We provide an atlas of normal cross-sectional anatomy, computed tomography and magnetic resonance imaging of the interscapular region of the cat, from the fifth cervical vertebra to the fifth thoracic vertebra. We compare and couple the slices obtained both in flexed and extended forelimb positioning with the animal maintained in sternal recumbency. CONCLUSION: This study shows a new and dynamic way to investigate the interscapular region of the cat and provides anatomical references for in vivo computed tomography and magnetic resonance imaging, considering changes in the muscular form according to the forelimb positioning. We believe that an in-depth anatomical knowledge of the interscapular region may be helpful to approach the study of any pathology located there and, in particular, to set up an appropriate therapy for the feline injection-site sarcoma.

Modulation of human estrogen receptor alpha F promoter by a protein kinase C/c-Src-dependent mechanism in osteoblast-like cells.

The human estrogen receptor alpha (ERalpha) gene is driven by multiple promoters, of which the F promoter alone is found to be active in primary osteoblasts. The study was aimed at identifying new regulatory pathways affecting transcription of the receptor in this cell lineage. We generated human osteoblast-like cells, Saos-2, stably transfected with a luciferase-reporter gene downstream of the human ERalpha F promoter (Saos F-Luc), and assayed the reporter response to differentiation-related signals. Over-confluence, shown to stimulate osteoblast differentiation, caused a time-dependent increase of F-promoter activity and correlated with an inactivation of protein kinase C alpha (PKCalpha ). PKC downregulation, obtained by long-term treatment with phorbol 12-myristate 13-acetate (PMA), resulted in promoter stimulation at similar levels in sub-confluent cells. The F promoter contains a putative PMA-responsive AP-1 site, but AP-1 activation was unremarkable in over-confluent cells. Treatment with PP1, a specific inhibitor of the non-receptor tyrosine-kinase c-Src, which is a negative regulator of osteoblast differentiation, showed that the activity of this kinase inhibits the F promoter. In PP1-treated cells, F-promoter activity was not further increased by PMA. Treatment with the generic kinase inhibitor 4-dimethylaminopyridine (DMAP) resulted in a dose-dependent induction of the promoter, which matched a parallel decrease of active c-Src. The effect was c-Src dependent, as DMAP caused no further promoter induction in PP1-treated cells. Overexpression of exogenous human ERalpha resulted in modest promoter stimulation, which required the ligand-independent activator function 1 of the receptor. In murine primary osteoblasts, additional ERalpha signal was observed upon induction of F promoter. In conclusion, we demonstrated a robust PKC/c-Src-dependent and estrogen-independent mechanism modulating transcription of ERalpha in osteoblasts, probably affecting estrogen responsiveness during cell differentiation.

Impaired skeletal development in interleukin-6-transgenic mice: a model for the impact of chronic inflammation on the growing skeletal system.

OBJECTIVE: To identify the mediator responsible for the impact of chronic inflammation on skeletal development in children (bone loss, defective peak bone mass accrual, stunted growth), we evaluated the effects of chronic interleukin-6 (IL-6) overexpression on the skeletons of growing prepubertal mice. METHODS: We studied IL-6-transgenic mice that had high circulating IL-6 levels since birth. Trabecular and cortical bone structure were analyzed by microcomputed tomography. Epiphyseal ossification, growth plates, and calvariae were studied by histology/histomorphometry. Osteoclastogenesis, osteoblast function/differentiation, and the effects of IL-6 on bone cells were studied in vitro. Osteoblast gene expression was evaluated by reverse transcriptase-polymerase chain reaction. The mineral apposition rate was evaluated dynamically in cortical bone by in vivo double fluorescence labeling. RESULTS: In prepubertal IL-6-transgenic mice, we observed osteopenia, with severe alterations in cortical and trabecular bone microarchitecture, as well as uncoupling of bone formation from resorption, with decreased osteoblast and increased osteoclast number and activity. Increased osteoclastogenesis and reduced osteoblast activity, secondary to decreased precursor proliferation and osteoblast function, were present. IL-6-transgenic mice also showed impaired development of growth plates and epiphyseal ossification centers. Intramembranous and endochondral ossification and the mineral apposition rate were markedly affected, showing the presence of defective ossification. CONCLUSION: Chronic overexpression of IL-6 alone induces a skeletal phenotype closely resembling growth and skeletal abnormalities observed in children with chronic inflammatory diseases, pointing to IL-6 as a pivotal mediator of the impact of chronic inflammation on postnatal skeletal development. We hypothesize that IL-6-modifying drugs may reduce skeletal defects and prevent the growth retardation associated with these diseases.

Imbalance of osteoclastogenesis-regulating factors in patients with celiac disease.

UNLABELLED: Celiac disease is an autoimmune disorder characterized by atrophy of the intestine villi triggered by ingestion of gluten in genetically susceptible individuals. The association between celiac disease and low BMD has been recognized, but the mechanisms of disturbance are poorly understood. We show imbalance of cytokines relevant to bone metabolism in celiac patients' sera and the direct effect of these sera on in vitro bone cell activity. INTRODUCTION: Celiac disease is associated with mineral metabolism derangement and low BMD. We investigated whether imbalance of serum factors in celiac patients could affect human bone cell activity in vitro. MATERIALS AND METHODS: We studied two groups of celiac patients--one on a gluten-free diet and another before the diet--both with decreased bone mass. Patients were investigated for bone turnover markers, and their sera were used for culturing bone cells from healthy donors and evaluate changes in cell activity. RESULTS: The N-terminal telopeptide of procollagen type I and interleukin (IL)-6 were higher than normal in patients not on the gluten-free diet. IL-1beta and TNF-alpha/beta were normal in all patients. IL-12 was reduced in all patients, whereas IL-18 was reduced only in patients on the diet. The RANKL/osteoprotegerin (OPG) ratio was increased in patients not on the gluten-free diet. Persistently increased osteoclast numbers were obtained from peripheral blood mononuclear cells of healthy donors on incubation with sera of patients not on the gluten-free diet versus control sera and sera from patients on the diet. In human osteoblasts from healthy individuals, IL-18 was reduced on incubation with sera from all patients, whereas OPG expression was lower when sera from patients not on the diet were used. Proliferation, alkaline phosphatase, and nodule mineralization were increased in osteoblast cultures containing sera from all celiac patients, either on or not on the gluten-free diet. CONCLUSIONS: We conclude that bone loss in celiac disease might also be caused by a cytokine imbalance directly affecting osteoclastogenesis and osteoblast activity.

In vivo bone metastases, osteoclastogenic ability, and phenotypic characterization of human breast cancer cells.

Mouse bone marrow cells cultured with human breast cancer MCF-7 cell-conditioned media showed osteoclastogenesis with an increment of bone resorption, although conditioned media from an adriamycin-selected MCF-7 clone (MCF-7ADR) had no effect. Consistently, MCF-7 cells induced 5-fold more in vivo experimental osteolytic bone metastases, with no soft tissue lesions, compared to MCF-7ADR cells. Paracrine factors stimulating (interleukin (IL)-6, IL-1beta, tumor necrosis factor-alpha (TNF-alpha)) or inhibiting (IL-12, IL-18, granulocyte macrophage-colony stimulating factor (GM-CSF)) osteoclastogenesis were significantly increased in MCF-7ADR relative to MCF-7 cells, suggesting that the inhibitory cytokines could selectively overwhelm the effects of the stimulatory ones. Treatment of osteoblast primary cultures with MCF-7-conditioned medium induced a selective upregulation of IL-6 expression, suggesting an indirect stimulation of osteoclastogenesis via the osteoblasts. MCF-7 and MCF-7ADR showed no difference in proliferation rate. However, a higher ability to migrate and invade gelatin and matrigel was observed in MCF-7ADR. Enhanced invasiveness might result from increased metalloproteinase (MMP) activity and cytoskeleton rearrangement. MCF-7ADR cells expressed higher levels of c-Src, focal adhesion kinase (FAK), and protein tyrosine kinase 2 (PYK2) involved in cell adhesion and motility. MCF-7 and MCF-7ADR expressed high and faint levels of functional estrogen receptor alpha (ERalpha), respectively. MCF-7ADR also showed significantly higher levels of the protein kinase C (PKC) alpha and beta2 and a selective activation of PKC compared to MCF-7, where the most abundant isoforms were beta1 and delta. Heat shock protein 27 (Hsp27) was more abundant in MCF-7 cells, but failed to translocate to the nucleus in response to heat shock. In conclusion, we have demonstrated that despite the fact that MCF-7ADR cells showed a more invasive phenotype relative to MCF-7, they have low potential to induce osteolytic bone lesions and stimulate osteoclastogenesis and osteoclast activity. Therefore, we believe that reduced aggressiveness of breast carcinomas could correlate with a greater osteolytic activity featuring their bone metastases.

Reduction of c-Src activity by substituted 5,7-diphenyl-pyrrolo[2,3-d]-pyrimidines induces osteoclast apoptosis in vivo and in vitro. Involvement of ERK1/2 pathway.

We employed potent and selective c-Src inhibitors to investigate the functional and molecular consequences of inhibited c-Src tyrosine kinase activity in osteoclasts. These pyrrolopyrimidine derivatives reduced osteoclast numbers and induced osteoclast disruption in vivo. In vitro, they inhibited resorption pit formation and osteoclastogenesis, impaired adhesion ability and actin ring organization, and induced programmed cell death in mature osteoclasts. The cell death receptor Fas and p53 were insensitive to c-Src modulation. The expression of the cyclin-dependent kinase (CDK)-inhibitor p21WAF1/CIP1 was markedly reduced, but neither Bcl-2 nor Bcl-xL or Bax were modulated by c-Src inhibition. Caspase-9, and to a lesser extent caspase-3, but not caspase-8, were transiently cleaved (activated) by treatment with the c-Src inhibitors. c-Src inhibition stabilized p38 mitogen-activated protein kinase (MAPK), whereas the c-Jun N-terminal kinase (JNK) pathway did not appear to be modulated by our compounds. Most interestingly, transient extracellular signal regulated kinase (ERK1/2) dephosphorylation followed by sustained remarkable rephosphorylation overwhelming control levels was observed in response to c-Src inhibition. Blockade of ERK1/2 rephosphorylation by PD98059 reduced osteoclast nuclear disruption, suggesting the involvement of this pathway in apoptosis. Collectively, these data demonstrate that small pyrrolopyrimidine derivatives impair osteoclast function and induce cell damage suggestive of apoptosis in vivo and in vitro, with mechanisms presumably involving selective sustained ERK1/2 phosphorylation.