Leopoldia comosa prevents metabolic disorders in rats with high-fat diet-induced obesity.

PURPOSE: Obesity is the main feature of a complex illness known as metabolic syndrome. Anti-obesogenic therapies are often associated with side effects and represent a high cost in conventional pharmacological approaches. New strategies based on natural remedies are under continuous investigation. Leopoldia comosa (L.) Parl. (L. comosa) is a spontaneous plant with diuretic, anti-inflammatory and antioxidant properties. Recently, a hypoglycemic activity mediated by inhibition of carbohydrate digestion has been identified. The aim of this study was to evaluate the effects of a diet supplemented with L. comosa extracts on a rat model of diet-induced obesity. METHODS: Leopoldia comosa bulb extracts were obtained using a dynamic extractor. Phytochemical properties and in vitro determination of the antioxidant activity and of the inhibitory effects on lipase and pancreatic amylase were performed. Rats were fed (12 weeks) a standard diet, or a high-fat diet (HFD), or an HFD plus L. comosa (20 or 60 mg/die) extracts. The metabolic and anthropometric parameters were recorded. RESULTS: Results indicated that L. comosa inhibited lipase and pancreatic amylase activities. In vivo data showed that the supplementation with both doses of L. comosa extracts counteracted the HFD-dependent effects. It reduced body weight, abdominal obesity and dyslipidemia, and improved glucose tolerance with a reduction of lipidic tissue hypertrophy and liver steatosis, as compared to HFD-fed rat. In liver, L. comosa reduced protein expression levels of PEPCK and G6Pase. CONCLUSION: We suggest that L. comosa extracts prevent obesity-dependent metabolic disorders. This paves the way for their therapeutic application as a natural anti-obesity drug.

Exome sequencing in multiplex autism families suggests a major role for heterozygous truncating mutations.

Autism is a severe neurodevelopmental disorder, the aetiology of which remains mainly unknown. Family and twin studies provide strong evidence that genetic factors have a major role in the aetiology of this disease. Recently, whole exome sequencing (WES) efforts have focused mainly on rare de novo variants in singleton families. Although these studies have provided pioneering insights, de novo variants probably explain only a small proportion of the autism risk variance. In this study, we performed exome sequencing of 10 autism multiplex families with the aim of investigating the role of rare variants that are coinherited in the affected sibs. The pool of variants selected in our study is enriched with genes involved in neuronal functions or previously reported in psychiatric disorders, as shown by Gene Ontology analysis and by browsing the Neurocarta database. Our data suggest that rare truncating heterozygous variants have a predominant role in the aetiology of autism. Using a multiple linear regression model, we found that the burden of truncating mutations correlates with a lower non-verbal intelligence quotient (NVIQ). Also, the number of truncating mutations that were transmitted to the affected sibs was significantly higher (twofold) than those not transmitted. Protein-protein interaction analysis performed with our list of mutated genes revealed that the postsynaptic YWHAZ is the most interconnected node of the network. Among the genes found disrupted in our study, there is evidence suggesting that YWHAZ and also the X-linked DRP2 may be considered as novel autism candidate genes.

First reported long-term two- and three-dimensional echocardiographic follow-up with histopathological analysis of a transcatheter pulmonary valve implantation in a pet dog.

Transcatheter pulmonary valve implantation (TPVI) is indicated for use in the management of failing pulmonary valves in humans. We report here the long-term follow-up of the first documented transcatheter pulmonary valve implanted in a client-owned dog. A one-year-old Beagle dog with severe congenital type A valvular pulmonic stenosis first underwent percutaneous balloon pulmonary valvuloplasty, leading two years later to severe pulmonary regurgitation. A TPVI using a Melody™ bioprosthetic valve was then successfully performed, with normalization of the right heart cavities. Repeated two- and three-dimensional transthoracic echocardiographic examinations combined with Doppler modes confirmed the appropriate position and function of the valve for four years. Mitral myxomatous valvular degeneration led to refractory left-sided congestive heart failure, and the dog was humanely euthanized. After postmortem examination, X-ray imaging and histopathological evaluation of the stent and the valve were performed. Ex-vivo imaging of the implanted valve using a Faxitron® Path radiography system and microscopic evaluation of the implanted stent and bioprosthetic leaflets did not show any relevant leaflet or stent alterations. This case provides a proof of concept in interventional veterinary cardiology, showing that TPVI can be performed in dogs with subsequent long-term maintaining normal pulmonary valve function.

ELISA for leptospiral 3-hydroxyacyl-CoA dehydrogenase in urine is a promising screening tool for acute leptospirosis.

Introduction. Leptospirosis is a zoonotic disease that is prevalent worldwide. Leptospiral 3-hydroxyacyl-CoA dehydrogenase (3-HADH) is excreted in the urine of infected individuals. However, the potential use of 3-HADH as a biomarker for the diagnosis of leptospirosis using enzyme-linked immunosorbent assay (ELISA) has not been investigated. A technique that identifies Leptospira in a patient in urine sample will be valuable in regular diagnostics and epidemic scenarios, as opposed to existing serological approaches. This study aimed to develop and evaluate an ELISA that can detect 3-HADH in the urine of patients with confirmed acute leptospirosis and to assess its potential as a screening test for leptospirosis. Methods. Laboratory confirmation of acute leptospirosis was done by flaB-nested polymerase chain reaction (PCR) of plasma samples from suspected patients. ELISA-based determination of the presence of 3-HADH in the urine of PCR-positive patients versus PCR-negative patients matched for fever date was performed by coating ELISA plates with urine supernatants and using rabbit anti-3-HADH as the primary antibody. Receiver operating characteristic curve analysis was used to determine the cutoff values for the ELISA. The diagnostic measures between the PCR-positive and PCR-negative patients were compared using the Mann-Whitney U test. Results. In total, 158 febrile patients were assessed, of whom 121 (76.6 %) were male. Of the 15 flaB-nested PCR-positive patients, 12 were in the acute phase of the febrile illness. The best cutoff was an average optical density (ODav) value of 0.2200 for febrile patients. Sensitivity and specificity were 83.33% [95 % confidence interval (CI), 51.59-97.91 %) and 83.33 % (95 % CI, 76.05-89.13 %), respectively. The ODav values for PCR-positive patients in the acute phase of the disease (≤7 days of fever) were significantly higher than those for PCR-negative patients (P<0.001, U=114.0, z=-4.946). Conclusion. Detection of 3-HADH in urine by ELISA appears to be promising for the screening of acute leptospirosis in suspected patients.

Regulatory T cells expressing CD19-targeted chimeric antigen receptor restore homeostasis in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is a progressive disease leading to immune-mediated tissue damage, associated with an alteration of lymphoid organs. Therapeutic strategies involving regulatory T (Treg) lymphocytes, which physiologically quench autoimmunity and support long-term immune tolerance, are considered, as conventional treatment often fails. We describe here a therapeutic strategy based on Tregs overexpressing FoxP3 and harboring anti-CD19 CAR (Fox19CAR-Tregs). Fox19CAR-Tregs efficiently suppress proliferation and activity of B cells in vitro, which are relevant for SLE pathogenesis. In an humanized mouse model of SLE, a single infusion of Fox19CAR-Tregs restricts autoantibody generation, delay lymphopenia (a key feature of SLE) and restore the human immune system composition in lymphoid organs, without detectable toxicity. Although a short survival, SLE target organs appear to be protected. In summary, Fox19CAR-Tregs can break the vicious cycle leading to autoimmunity and persistent tissue damage, representing an efficacious and safe strategy allowing restoration of homeostasis in SLE.

High-density SNP association study and copy number variation analysis of the AUTS1 and AUTS5 loci implicate the IMMP2L-DOCK4 gene region in autism susceptibility.

Autism spectrum disorders are a group of highly heritable neurodevelopmental disorders with a complex genetic etiology. The International Molecular Genetic Study of Autism Consortium previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we performed a high-density association analysis in AUTS1 and AUTS5, testing more than 3000 single nucleotide polymorphisms (SNPs) in all known genes in each region, as well as SNPs in non-genic highly conserved sequences. SNP genotype data were also used to investigate copy number variation within these regions. The study sample consisted of 127 and 126 families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Further investigation of the strongest association results was conducted in an independent European family sample containing 390 affected individuals. Association and copy number variant analysis highlighted several genes that warrant further investigation, including IMMP2L and DOCK4 on chromosome 7. Evidence for the involvement of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family.

Obstructive right ventricular outflow tract myxosarcoma in an adult dog.

An 8-year-old female spayed German Shepherd cross was presented for acute onset of respiratory distress. Four days before presentation, the owner noticed a reduced appetite and reluctance to move. Clinical examination identified muffled lung sounds and a left base, diamond-shaped systolic murmur graded 4/6. Echocardiography identified pleural and pericardial effusion, ascites and a myxoid mass (39 mm/18.9 mm) obstructing the right ventricular outflow tract and interfering with the pulmonary valve function. Given the poor prognosis, the dog was euthanatised, and a postmortem examination was performed. Grossly, a mass with a heterogeneous appearance was identified below the pulmonary valve leaflets. Based on histopathological and immunohistochemical findings, a diagnosis of intracardiac myxosarcoma affecting the subvalvular region of the pulmonary artery was made. To the author's knowledge, this is the first report of right ventricle out flow tract myxosarcoma in the canine species.

Beneficial effect of oral administration of zinc sulfate on 5-fluorouracil-induced gastrointestinal mucositis in rats.

OBJECTIVE: This experimental study explored the potential of oral zinc sulfate to protect the gut mucosa from 5-fluorouracil (5-FU)-induced degenerative lesions in Wistar rats. MATERIALS AND METHODS: Female Wistar rats were used and divided into 2 interventional groups (Z with 6 animals and F with 5 animals) and one control group (M with 5 rats). After 2 hours of fasting, group Z received via oral gavage 1.5 ml of solution, corresponding to 15 mg zinc sulfate for 9 consecutive days. Groups F and M received only the vehicles. On day 3, 400 mg/kg of 5-FU was administered intraperitoneally to groups Z and F. Tissue samples were collected from the duodenum, jejunum, colon and liver. Histological assessment for each gastrointestinal tract segment was determined semi-quantitatively by rating 11 histological features from normal (0) to severe (3). The independent groups were analyzed using the Kruskal-Wallis test and the Mann-Whitney U-test, with a Bonferroni correction for alpha (p ≤ 0.016). RESULTS: In group F the jejunum was the most affected area with a mean histological score of  27 (25-32). In the Z group, significantly lower histological scores were obtained compared with group F (duodenum Z vs. F: U = 0, p = 0.004; jejunum Z vs. F: U = 0, p = 0.006 and colon: Z vs. F: U = 0, p = 0.005). Graded liver necro-inflammatory lesions were significantly lower in group Z compared with group F (U = 0, p = 0.004), suggesting fewer bacterial intestinal translocation processes. CONCLUSIONS: Zinc sulfate has a beneficial role, decreasing the severity of gut mucosal injuries induced by 5-FU in Wistar rats.

QSAR models for biocides: The example of the prediction of Daphnia magna acute toxicity.

Biocides are multi-component products used to control undesired and harmful organisms able to affect human or animal health or to damage natural and manufactured products. Because of their widespread use, aquatic and terrestrial ecosystems could be contaminated by biocides. The environmental impact of biocides is evaluated through eco-toxicological studies with model organisms of terrestrial and aquatic ecosystems. We focused on the development of in silico models for the evaluation of the acute toxicity (EC50) of a set of biocides collected from different sources on the freshwater crustacean Daphnia magna, one of the most widely used model organisms in aquatic toxicology. Toxicological data specific for biocides are limited, so we developed three models for daphnid toxicity using different strategies (linear regression, random forest, Monte Carlo (CORAL)) to overcome this limitation. All models gave satisfactory results in our datasets: the random forest model showed the best results with a determination coefficient r2 = 0.97 and 0.89, respectively, for the training (TS) and the validation sets (VS) while linear regression model and the CORAL model had similar but lower performance (r2 = 0.83 and 0.75, respectively, for TS and VS in the linear regression model and r2 = 0.74 and 0.75 for the CORAL model).

Therapeutic strategies utilization and resource consumption in patients treated for psoriatic arthritis: findings from a real-world analysis in an Italian setting.

PURPOSE: The purpose of this study was to analyze the therapeutic strategies and estimate the health care resource consumption in patients with psoriatic arthritis (PsA). PATIENTS AND METHODS: An observational retrospective cohort analysis of administrative databases of six Italian Local Health Units was performed. Patients ≥18 years with a hospitalization discharge diagnosis of PsA (International Classification of Diseases, Ninth Revision code: 696.0) or exemption code (045.696.0) for PsA from January 1, 2010 to December 31, 2015 (inclusion period), with at least one prescription of any therapy used for PsA were included. The index date (ID) was the first date matching with at least one of the inclusion criteria during the inclusion period. All patients were followed up after the ID until the end of data availability. Baseline C-reactive protein (CRP) levels (±6 months in relation to the ID) were also analyzed. RESULTS: A total of 2,408 (prevalence 0.83 per 1,000) patients with PsA (male 52%; median age 54 years) were included in the study; patients were already treated for PsA in 42.4% of cases. At 1 year of follow-up, 73% of the patients received one systemic drug, while 22% of patients received two systemic drugs; in addition, our results show an increase in the number of add-on or switches in a longer follow-up period. The utilization of biologic agents was higher among patients with previous PsA treatment, showing a progression of the pathology. Overall, a medium/high level of CRP at baseline was observed among more than half of the overall sample, with slight changes across subgroups in analysis. The average health care costs were €1,966.4 and €13,914 per year for patients treated with conventional systemic therapy and biological agents, respectively. CONCLUSION: A better knowledge of prescription therapeutic scheme and economic burden of PsA could stimulate the rational development of health programs aimed at potentiating services for its management.

Fusion of the wrist in rheumatoid arthritis: a clinical and functional evaluation of two surgical techniques.

We retrospectively compared wrist arthrodesis using the Mannerfelt technique in 19 or an AO-plate in 23 patients with long-standing rheumatoid arthritis. The mean follow-up was for 76 months. Compared with the Mannerfelt fusion group, patients in the AO-plate group reported greater satisfaction with their wrist function (74% vs 37%, p = 0.015). Complications were reported in six wrists in the AO-plate group and two wrists in the Mannerfelt fusion group (p = 0.258). At final follow-up, 95% of patients (41) reported either no pain or only mild pain. There was improvement in flexion of the finger joints in both groups but no significant improvement in the extension lag in either group. Both methods relieve pain and improve function. Overall, the activities of daily living scores and the patients' subjective assessment of outcome tended to be higher in the AO-plate group than in the Mannerfelt fusion group, although the difference was not statistically significant. Similarly, although more postoperative complications occurred in the AO-plate group, the difference between the two groups was not statistically significant.

Anticancer effects of zoledronic acid against human osteosarcoma cells.

Based on neoadjuvant chemotherapy, the prognosis of osteosarcoma patients has improved dramatically. However, due to therapy resistance in patient subgroups, the development of new treatment strategies is still of utmost importance. The aim of our study was to test the effects of the nitrogen-containing bisphosphonate zoledronic acid (ZOL) on osteosarcoma cell lines (N = 9). Exposure to ZOL at low micromolar concentrations induced a dose- and time-dependent block of DNA synthesis and cell cycle progression followed by microfilament breakdown and apoptosis induction. The ZOL-induced cell cycle accumulation in S phase was accompanied by significant changes in the expression of cyclins and cyclin-dependent kinase inhibitors with a prominent loss of cyclin E and D1. ZOL not only inhibited growth but also migration of osteosarcoma cells. The mevalonate pathway intermediary geranyl-geraniol (GGOH) but not farnesol (FOH) significantly inhibited the anticancer effects of ZOL against osteosarcoma cells. Correspondingly, ZOL sensitivity correlated with the blockade of protein geranylgeranylation indicated by unprenylated Rap1. Overexpression of even high levels of P-glycoprotein, as frequently present in therapy-resistant osteosarcomas, did not impair the anticancer activity of ZOL. Summarizing, our data suggest that ZOL, which selectively accumulates in the bone, represents a promising agent to improve osteosarcoma therapy.

Clinical, tomographic, and postmortem aspects of a rare congenital Dicephalus Monauchenos Iniodymus in a Nelore calf produced in vitro from Brazil: case report / Aspectos clínicos, tomográficos e pós-morte de um raro Dicephalus Monauchenos Iniodymus congênito em um bezerro Nelore produzido in vitro no Brasil: relato de caso

The aim of this work was to report the occurrence of dicephalus iniodymus monauchenos in a Nellore newborn. A three-days old calf, from in vitro production, with duplication of the head and a history of cesarean birth was attended. On physical examination, the dicephalus, iniodymus and monauchenos, which were almost the same size and shape, had four eyes and four ears. Computed tomography showed the presence of two skulls fused with a common occipital foramen, two nasopharynxes, oropharynxes with the presence of a cleft lip and a cleft palate in the right head, which continued in a single esophagus and a single trachea. At necropsy, the presence of duplication of the cerebrum and cerebellum was observed, with union of the parts in the region of the trapezoid body of the brainstem and continued as a single spinal cord. This study characterizes the clinical, tomographic, and necropsy findings of a dicephalus Nelore neonate.(AU)

O objetivo deste trabalho foi relatar a ocorrência de Dicephalus Iniodymus Monauchenos em um neonato da raça Nelore de produção in vitro. Foi atendida uma fêmea bovina, de três dias de idade, com duplicação das cabeças e histórico de nascimento por meio de cesariana. No exame físico, observou-se a dicefalia, Iniodymus e Monauchenos, apresentando quatro olhos e quatro orelhas. Na tomografia computadorizada, constatou-se a presença de dois crânios fundidos com um forame occipital comum, duas nasofaringes, orofaringes com presença de lábio leporino e fenda palatina na cabeça direita, que continuavam em um único esôfago e em uma única traqueia. Na necropsia, observou-se a presença de duplicação do encéfalo e cerebelo, com união das partes na região do corpo trapezoide do tronco encefálico, que continuavam como uma única medula espinhal. Este estudo caracteriza os achados clínicos, tomográficos e de necropsia de um neonato Nelore dicefálico.(AU)

First occurrence of Arnold Chiari type II malformation and associated abnormalities in a Gir calf produced in vitro from Brazil - case report / [Primeira ocorrência de malformação de Arnold Chiari tipo II e anormalidades associadas em uma bezerra Gir de produção in vitro no Brasil - relato de caso]

This study characterized the clinical, radiological, ultrasound, and necroscopic findings of a case of Arnold-Chiari type II malformation in a Gir breed calf from Brazil. The animal was hospitalized at sixty days of age, in permanent sternal recumbency, cutaneous appendix at the 4th lumbar vertebra and kyphoscoliosis of the caudal and lumbosacral thoracic spine. Radiographic examination of the spine and skull revealed spina bifida and suspected occipital hypoplasia. Upon examination of myelography with an injection of lumbar and atlantooccipital contrast, it was possible to visualize the meningocele at the 4th lumbar vertebra region and findings at the rhombencephalon level of increased regional pressure with failure to fill the contrast in the posterior fossa, in the presence of clear demarcation of the circumvolutions of the cerebral cortex and the subarachnoid space of the cervical spinal cord. Ultrasonographic examination of the cerebellum showed an insinuation of the cerebellar worm through the foramen magnum. The animal did not show changes in complete blood count, biochemical series, and cerebrospinal fluid and was negative for Pestivirus. There was a worsening of the clinical conditions and the animal died. This malformation of unknown etiology must be studied as a differential diagnosis of the nervous system disorders.(AU)

Este estudo caracterizou os achados clínicos, radiológicos, ultrassonográficos e necroscópicos de um caso de malformação de Arnold-Chiari tipo II em uma bezerra Gir no Brasil. O animal foi hospilatizado aos 60 dias de idade, apresentando decúbito esternal permanente, apêndice cutâneo na altura da quarta vértebra lombar e cifoescoliose da coluna vertebral torácica caudal e lombossacra. Ao exame radiográfico da coluna e do crânio, foram observadas espinha bífida e suspeita de hipoplasia occipital. Ao exame de mielografia com injeção de contraste lombar e atlanto-occipital, foi possivel visualizar a meningocele na altura da quarta vértebra lombar e achados em nível rombencefálico de aumento da pressão regional com falha de preenchimento do contraste na fossa posterior, na presença de nítida demarcação das circunvoluções do córtex cerebral e do espaço subaracnoide da medula espinhal cervical. Ao exame ultrassonográfico do cerebelo, foi observada insinuação do verme cerebelar através do forame magno. O animal não apresentou alterações em hemograma completo, série bioquímica e fluido cérebro-espinhal e foi negativo para Pestivirus. Houve uma piora do quadro clínico e o animal morreu. Essa malformação de etiologia desconhecida deve ser estudada como um diagnóstico diferencial.(AU)

Molecular analysis of a filamentous phage (fsl) of Vibrio cholerae O139.

A filamentous bacteriophage from Vibrio cholerae O139 strain A1-4450 was isolated (fsl). The phage fsl had a ssDNA genome and dsDNA as a replicative form (RF) in lysogenic host cell. The DNA sequence of fsl RF was determined. It consisted of 6340 bp and had a G + C content of 43.5%. Fifteen possible ORFs were found in fsl. One of them, ORF384, was estimated to encode 384 amino acid residues (44.6 kDa) and had homologous regions with the zot gene of V. cholerae and gene I of the coliphage group. ORF104, located upstream of ORF384, was homologous to gene 93 protein of Pf3 (filamentous phage of Pseudomonas sp.) corresponding to gene VI of coliphage. Other than ORF384 and ORF104, the ORF81, ORF44, ORF29, and ORF193 were speculated to correspond to gene V, gene VII, gene IX, and gene III, respectively, in the order as reported on f1 phage.

Repair of articular cartilage defects treated by microfracture and a three-dimensional collagen matrix.

The objective of our study was to evaluate the behavior of ovine chondrocytes and bone marrow stromal cells (BMSC) on a matrix comprising type-I, -II, and -III collagen in vitro, and the healing of chondral defects in an ovine model treated with the matrix, either unseeded or seeded with autologous chondrocytes, combined with microfracture treatment. For in vitro investigation, ovine chondrocytes and BMSC were seeded on the matrix and cultured at different time points. Histological analysis, immunohistochemistry, biochemical assays for glycosaminoglycans, and real-time quantitative PCR for collagens were performed. The animal study described here included 22 chondral defects in 11 sheep, divided into four treatment groups. Group A: microfracture and collagen matrix seeded with chondrocytes; B: microfracture and unseeded matrices; C: microfracture; D: untreated defects. All animals were sacrificed 16 weeks after implantation, and a histomorphometrical and qualitative evaluation of the defects was performed. The in vitro investigation revealed viable cells up to 3 weeks; chondrocytes had a predominantly round morphology, produced glycosaminoglycans, and expressed both collagen markers, whereas BMSC stained positive for antibodies against type-II collagen; however, no mRNA for type-II collagen was amplified. All treatment groups of the animal model showed better defect filling compared to untreated knees. The cell-seeded group had the greatest quantity of repair tissue and the largest quantity of hyaline-like tissue. Although the collagen matrix is an adequate environment for BMSC in vitro, the additionally implanted unseeded collagen matrix did not increase the repair response after microfracture in chondral defects. Only the matrices seeded with autologous cells in combination with microfracture were able to facilitate the regeneration of hyaline-like cartilage.

Biological and clinical significance of concurrent p53 gene alterations, MDR1 gene expression, and S-phase fraction analyses in breast cancer patients treated with primary chemotherapy or radiotherapy.

We investigated the interrelationship between p53 gene alterations, MDR1 gene expression, and S-phase fraction (SPF) in breast carcinomas treated primarily with chemotherapy or radiotherapy and correlated the results with patient outcome to determine the potential clinical significance of these factors. In a consecutive series of 64 fine-needle samplings of breast cancer patients who underwent either neoadjuvant chemotherapy (n = 53) or radiotherapy (n = 11), p53 (exons 5-9) gene alterations by denaturating gradient gel electrophoresis and subsequent direct sequencing, MDR1 gene expression by semiquantitative reverse transcription-PCR, and SPF by DNA flow cytometry were determined. Our results show that p53 mutations (n = 20) were significantly associated (P = 0.01) with high SPF but not with de novo MDR1 gene expression. Most patients with wild-type p53 tumors were found to be resistant to neoadjuvant chemotherapy. No correlation was observed between p53 mutations and the induction of MDR1 gene expression during treatment. Although a significant correlation between shorter distant disease-free survival and high (>/=5%) SPF (P = 0.016) was found, no correlation between distant disease-free survival and p53 status or intrinsic MDR1 gene expression was found. Poor overall survival was observed in patients with tumors with high SPF (P < 0.0004) or lacking MDR1 gene expression (P = 0.03) before treatment, but not with p53 alterations. These data suggest that SPF remains the most relevant biological factor for breast cancer patients treated by primary chemotherapy or radiotherapy and that p53 and MDR1 status may identify a small subset of patients that may resist therapy or pursue an aggressive course.

Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts.

A well-defined chicken osteoblast culture system(18) has been used to examine fibronectin (FN) mRNA levels, synthesis, and accumulation during in vitro differentiation and matrix mineralization. Immunofluorescent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignment with cytoskeletal elements while at the latter time most FN was associated with the extracellular matrix as a ubiquitous fibrillar network. Western blot analysis of total cell-associated proteins also detected FN at all culture times. However, when results were normalized to cellular DNA, FN levels increased until 12-16 and remained relatively constant thereafter. Similarly, FN synthesis as measured by [35S]-methionine labeling, and immunoprecipitation was greatest in early cultures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during their in vitro maturation. In contrast to other osteoblast markers such as alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proportional to cell number. Results with FN differ from those obtained with collagen which continues to accumulate in the extracellular matrix during osteoblast maturation. These results are consistent with FN being important for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix.

Signal transduction of mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as a mechanically induced gene in osteoblasts.

Mechanical perturbation has been shown to modulate a wide variety of changes in second message signals and patterns of gene expression in osteoblasts. Embryonic chick osteoblasts were subjected to a dynamic spatially uniform biaxial strain (1.3% applied strain) at 0.25 Hz for a single 2-h period, and osteopontin (OPN), an Arg-Gly-Asp (RGD)-containing protein, was shown to be a mechanoresponsive gene. Expression of opn mRNA reached a maximal 4-fold increase 9 h after the end of the mechanical perturbation that was not inhibited by cycloheximide, thus demonstrating that mechanoinduction of opn expression is a primary response through the activation of pre-existing transcriptional factors. The signal transduction pathways, which mediated the increased expression of opn in response to mechanical stimuli, were shown to be dependent on the activation of a tyrosine kinase(s) and protein kinase A (PKA) or a PKA-like kinase. Selective inhibition of protein kinase C (PKC) had no effect on the mechanoinduction of osteopontin even though opn has been demonstrated to be an early response gene to phorbol 12-myristate 13-acetate (PMA) stimulation. Mechanotransduction was dependent on microfilament integrity since cytochalasin-D blocked the up-regulation of the opn expression; however, microfilament disruption had no effect on the PMA induction of the gene. The microtubule component of the cytoskeleton was not related to the mechanism of signal transduction involved in controlling opn expression in response to mechanical stimulation since colchicine did not block opn expression. Mechanical stimulus was shown to activate focal adhesion kinase (FAK), which specifically became associated with the cytoskeleton after mechanical perturbation, and its association with the cytoskeleton was dependent on tyrosine kinase activity. In conclusion, these results demonstrate that the signal transduction pathway for mechanical activation of opn is uniquely dependent on the structural integrity of the microfilament component of the cytoskeleton. In contrast, the PKC pathway, which also activates this gene in osteoblasts, acts independently of the cytoskeleton in the transduction of its activity.