The bovine foot skin microbiota is associated with host genotype and the development of infectious digital dermatitis lesions.

BACKGROUND: Bovine Digital Dermatitis (BDD) is a prevalent infectious disease, causing painful foot skin lesions and lameness in cattle. We describe herein the bovine foot skin microbiota and its associations with BDD using 16S rRNA gene amplicon and shotgun metagenomic sequencing on samples from 259 dairy cows from three UK dairy farms. RESULTS: We show evidence of dysbiosis, and differences in taxonomy and functional profiles in the bovine foot skin microbiome of clinically healthy animals that subsequently develop BDD lesions, compared to those that do not. Our results suggest that taxonomical and functional differences together with alterations in ecological interactions between bacteria in the normal foot skin microbiome may predispose an animal to develop BDD lesions. Using genome-wide association and regional heritability mapping approaches, we provide first evidence for interactions between host genotype and certain members of the foot skin microbiota. We show the existence of significant genetic variation in the relative abundance of Treponema spp. and Peptoclostridium spp. and identify regions in the bovine genome that explain a significant proportion of this variation. CONCLUSIONS: Collectively this work shows early changes in taxonomic and functional profiles of the bovine foot-skin microbiota in clinically healthy animals which are associated with subsequent development of BDD and could be relevant to prevention of disease. The description of host genetic control of members of the foot skin microbiota, combined with the association of the latter with BDD development offer new insights into a complex relationship that can be exploited in selective breeding programmes. Video Abstract.

The dysbiosis of ovine foot microbiome during the development and treatment of contagious ovine digital dermatitis.

BACKGROUND: Contagious Ovine Digital Dermatitis (CODD) is an emerging and common infectious foot disease of sheep which causes severe welfare and economic problems for the sheep industry. The aetiology of the disease is not fully understood and control of the disease is problematic. The aim of this study was to investigate the polybacterial aetiopathogenesis of CODD and the effects of antibiotic treatment, in a longitudinal study of an experimentally induced disease outbreak using a 16S rRNA gene amplicon sequencing approach. RESULTS: CODD was induced in 15/30 experimental sheep. During the development of CODD three distinct phenotypic lesion stages were observed. These were an initial interdigital dermatitis (ID) lesion, followed by a footrot (FR) lesion, then finally a CODD lesion. Distinct microbiota were observed for each lesion in terms of microbial diversity, clustering and composition. Porphyromonadaceae, Family XI, Veillonellaceae and Fusobacteriaceae were significantly associated with the diseased feet. Veillonellaceae and Fusobacteriaceae were most associated with the earlier stages of ID and footrot rather than CODD. Following antibiotic treatment of the sheep, the foot microbiota showed a strong tendency to return to the composition of the healthy state. The microbiota composition of CODD lesions collected by swab and biopsy methods were different. In particular, the Spirochaetaceae family were more abundant in samples collected by the biopsy method, suggesting that these bacteria are present in deeper tissues of the diseased foot. CONCLUSION: In this study, CODD presented as part of a spectrum of poly-bacterial foot disease strongly associated with bacterial families Porphyromonadaceae, Family XI (a family in Clostridiales also known as Clostridium cluster XI), Veillonellaceae and Fusobacteriaceae which are predominately Gram-negative anaerobes. Following antibiotic treatment, the microbiome showed a strong tendency to return to the composition of the healthy state. The composition of the healthy foot microbiome does not influence susceptibility to CODD. Based on the data presented here and that CODD appears to be the severest end stage of sheep infectious foot disease lesions, better control of the initial ID and FR lesions would enable better control of CODD and enable better animal welfare.

16S rRNA amplicon sequencing reveals a polymicrobial nature of complicated claw horn disruption lesions and interdigital phlegmon in dairy cattle.

Lameness represents an intractable problem for the dairy industry. Complicated claw horn disruption lesions, interdigital hyperplasia, and interdigital phlegmon are important lameness causing foot lesions. Their aetiology is multifactorial, but infectious processes are likely implicated in disease pathogenesis. Our aim was to investigate the bacterial profiles of these lesions using 16S rRNA gene sequencing of samples obtained from 51 cattle across ten farms in the UK. In this study, interdigital hyperplasia, interdigital hyperplasia with signs of interdigital dermatitis, interdigital phlegmon, complicated sole ulcers, complicated toe ulcers lesions, and complicated white line lesions were investigated; corresponding healthy skin control samples were also analysed. All diseased tissues displayed reduced microbial richness and diversity (as described by Chao1, Shannon, and Simpson alpha-diversity indices) compared to their healthy skin control samples. Our results confirm the association of Treponema spp with some of these disorders. Other anaerobic bacteria including Fusobacterium spp., Fastidiosipila spp. and Porphyromonas spp. were implicated in the aetiology of all these lesions with the exception of interdigital hyperplasia. Complicated claw horn disruption lesions, and interdigital phlegmon were found to have similar bacterial profiles. Such sharing of bacterial genera suggests many of the infectious agents detected in these foot lesions are acting opportunistically; this finding could contribute towards future treatment and control strategies.

Arsenic uptake and translocation by plants in pot and field experiments.

A work undertaken by pot and field experiments to assess the suitability of poplars and ferns for the in-situ, phytoextraction, of a dumping site with residues from the roasting process of arseno-pyrite is reported. The main characteristic of this site is the high content of both the As metalloid and heavy metals (e.g., Al, Fe, Cu, Co, Cr, Pb). Two poplar clones (Populus deltoides 'Dvina' and Populus x canadensis 'Orion') and Pteris vittata (Chinese brake fern) were planted in the contaminated soil both ex situ in pots and in situ. Plant survival, As accumulation in plant tissues, leaf content of pigments, soluble proteins, activity of catalase and SH-groups in both roots and leaves were evaluated during a 24-month study period. Both poplar and fern plants exhibited an increase in the activity of catalase and SH group contents when grown in the presence of pyrite ashes. The results showed that the co-planting system (arsenic-hyperaccumulator fern Pteris vittata and Populus clones) was suitable for phytoextraction of multi-contaminated dumping sites. Agronomic measures such as irrigation, soil tillage and amendments also seem to be necessary for the successful establishment of poplar trees and ferns in contaminated soils in order to enhance plant growth through the improvement of soil conditions.

The role of basophil activation test in special populations with mastocytosis and reactions to hymenoptera sting.

BACKGROUND: Systemic mastocytosis (SM) may be associated with hymenoptera allergy. In such cases, immunotherapy is a life-saving treatment, but a circumstantiated diagnosis is needed for its prescription. Patients with SM and previous reactions to stings, but with negative tests represent a diagnostic dilemma. The basophil activation test (BAT) may be helpful in refining the diagnosis. OBJECTIVE: We assessed the usefulness of BAT in subpopulations of mastocytosis patients, including those with negative tests for insect allergy. METHODS: Within a population of patients with mastocytosis and previous stings, we studied by BAT and augmented intradermal test (IDT) (10 µg/ml) two groups: (1) with reactions to stings and negative tests; (2) without reactions and negative tests. Basophil activation test was performed with different venoms, assessing at flow cytometry basophils' activation. RESULTS: Sixty-three patients had mastocytosis and 52 had reactions to previous hymenoptera stings. Of them, seven proved negative to diagnostic tests. In six of seven of those patients, BAT was negative with all venoms, and in one, basophils resulted activated also with the negative control. In six patients without previous reactions and negative tests, BAT was totally negative in five of six patients and weakly positive to Hornet in one. Finally, the IDT at 10 µg/ml venom produced nonspecific positive results in most cases. CONCLUSION: In patients with mastocytosis, the negative results of standard tests are reliable, because BAT and IDT at higher concentration do not add useful information.

Type 1 diabetes onset and pandemic influenza A (H1N1).

Type 1 diabetes (T1D) is a heterogeneous disorder characterized by destruction of pancreatic beta cells, culminating in loss of insulin secretion. Data from large epidemiologic studies worldwide indicate that during the last decades the incidence of T1D has increased significantly, reaching percentages of 2-5% annually. This increase suggests that there is a significant environmental contribution impacting the development of the disease, since genetic factors alone can hardly explain the rapid increase. Studies regarding T1D epidemiology in diverse populations aim to identify the disease causal factors and new targets for intervention. Viruses are one of the environmental factors implicated in the development of T1D in susceptible individuals. Recent studies suggest an association of T1D with H1N1 influenza. We would like to comment on this association and report our experience. Prospective studies are necessary to assess whether H1N1 infection is involved in T1D pathogenesis and provide directions on how to deal with viral infections in diabetes-susceptible individuals.

Ultrasonography in early assessment of elderly patients with polymyalgic symptoms: a role in predicting diagnostic outcome?

OBJECTIVE: To study the usefulness of ultrasonography (US) in predicting the diagnostic outcome in patients with polymyalgic symptoms. METHODS: Sixty-one elderly patients with polymyalgic syndrome were recruited in a secondary care setting and followed up in a prospective way. Clinical, laboratory, and US data obtained at onset were re-evaluated after 1 year when diagnostic outcome was defined. RESULTS: A diagnostic shift was observed in 32 polymyalgic patients (52%). Calcium pyrophosphate deposition disease (CPDD) was diagnosed in nine patients, elderly-onset rheumatoid arthritis (EORA) in 18, and elderly-onset spondyloarthritis (EOSpA) in five. In polymyalgia rheumatica (PMR) patients US demonstrated synovitis in 90% of cases, in both proximal (90%) and peripheral joints (41%). The best predictive US model for the definitive diagnosis of PMR comprised: the presence of subacromial-subdeltoid bursitis [odds ratio (OR) 5.603, p = 0.003], low frequency of wrist (OR 0.074, p < 0.001), metacarpophalangeal (OR 0.052, p < 0.001), and metatarsophalangeal effusion/synovitis (OR 0.107, p < 0.027), low frequency of knee menisci chondrocalcinosis (OR 0.091, p = 0.013), tendinous calcaneal calcifications (OR 0.078, p = 0.006), and Achilles enthesitis (OR 0.107, p = 0.027), and low power Doppler US (PDUS) scores at wrist (OR 0.052, p < 0.001). CONCLUSIONS: US and PDUS can be useful in distinguishing, at onset of disease, pure PMR from other diseases mimicking this condition.

Bedside ultrasonography of musculoskeletal complications in brain injured patients.

INTRODUCTION: The aim of this study was to evaluate the role of bedside ultrasonography (US) in early diagnosis of musculoskeletal complications (MSC) of acquired brain injuries, to describe its incidence and US features in a neurorehabilitation setting. MATERIALS AND METHODS: All 163 patients admitted in tertiary-level neurorehabilitation unit with diagnosis of stroke or severe brain injury (SBI), with symptoms or signs of musculoskeletal pathology, underwent bedside US. RESULTS: MSC were diagnosed in 51.5%. In 86.9% US clarified diagnosis and/or modified therapeutic approach. Shoulder pain was observed in 27.6%. US showed a shoulder subluxation in 73.3% and a frozen shoulder in 8.8% of painful shoulders. In all the cases rotator cuff abnormalities were noted. Wrist-hand syndrome was observed in 29.4%. US showed mild effusion in wrist joints and tendon sheaths and subcutaneous edema without significant vascularity. Neurogenic heterotopic ossification was observed in 1.8%. US demonstrated the "zone phenomenon" or heterogeneously hypoechoic mass with low resistance vessels within the lesions. Contractures and spasticity were observed in 18.4%. US allowed reliable guidance for Botulinum toxin A injection. Relapsing osteoarthritis and acute arthritis were diagnosed in 15.3% and 7.3% respectively. Patients with MSC had lower Functional Independence Measurement (FIM) and Katz index scores in discharge (p < 0.04 and p < 0.0294 respectively) and more length of hospital stay (p = 0.0024). DISCUSSION: Musculoskeletal pathology frequently complicates the course of acquired brain injuries and it delays functional recovery. Bedside US is a cheap and sensitive diagnostic tool and it can aid clinicians to define diagnosis and to choose therapeutic approach.

Tumor suppressor proteins are differentially affected in human ependymoblastoma and medulloblastoma cells exposed to nerve growth factor.

The aim of our study was to investigate the role of nerve growth factor (NGF) on the expression of the p73 protein in human ependymoblastoma (EP) and medulloblastoma (MB) cells. It was found that NGF exposure on MB cells blocks proliferation, as well as on EP cells and induces overexpression of p73. NGF reduces the number of cells and promotes the expression of TrkA of these neoplastic cells. Moreover, NGF plus cisplatin treatment reduces the cytotoxic effect of cisplatin. These observations indicate that NGF by interfering with mechanisms associated with cells proliferation and survival might induce the differentiation event through TrkA pathways.

Changes of thyroid function during long-term hGH therapy in GHD children. A possible relationship with catch-up growth?

BACKGROUND: Growth hormone (GH) treatment in patients with GH deficiency (GHD) can determine changes in the thyroid function. The clinical significance of these changes remains controversial, and all studies have so far covered rather a short period--usually no longer than one year. OBJECTIVE: To determine the effect of long-term recombinant hGH treatment in children with idiopathic GHD on the thyroid function. PATIENTS AND METHODS: Nineteen prepubertal children (12 boys and 7 girls, mean age 9.2 +/- 3.1 years) with idiopathic GHD were studied and followed for twenty-four months. None of the patients showed multiple pituitary hormone deficiencies. Nineteen healthy children matched for age and sex acted as controls. RESULTS: Patients with GHD showed a significant increase in TT (3) at twelve months and in FT (3) at six and twelve months after starting GH treatment, with a significant decrease at eighteen and twenty-four months. TT (4) level decreased significantly at twelve months and increased significantly at eighteen and twenty-four months. FT (4) also decreased, but only slightly, after twelve months of hGH treatment, and then increased significantly at twenty-four months. TSH levels did not vary significantly during the course of therapy. TT (3)/TT (4) and FT (3)/FT (4) ratios increased significantly after six and twelve months of therapy and significantly decreased later, approaching pre-therapy values. The SDS of Growth Velocity (SDS-GV) increased remarkably during the first year of therapy and then decreased significantly during the second year, although it remained significantly higher than the pre-therapy values. TT (3) and TT (3)/TT (4) ratio displayed a significant correlation with SDS-GV at twelve months of therapy. In a multiple regression analysis with age, bone age, parental height, GH dose, TT (3,) TT (3)/TT (4), and the SDS of IGF-I, only the TT (3)/TT (4) ratio at twelve months of therapy (p < 0.001) was identified as a significant predictor of SDS-GV. CONCLUSION: Our data confirm that changes in thyroid function are present in GHD children during long-term hGH therapy. These changes probably resulted from the effect of hGH on the peripheral metabolism of thyroid hormones and appear to be transitory, disappearing during the second year of hGH treatment. We speculate on the functional significance of these changes, and in particular, on their role in catch-up growth after hGH therapy.

Gene expression profile analysis in human T lymphocytes from patients with Down Syndrome.

Down Syndrome (DS) is caused by the presence of three copies of the whole human chromosome 21 (HC21) or of a HC21 restricted region; the phenotype is likely to have originated from the altered expression of genes in the HC21. We apply the cDNA microarray method to the study of gene expression in human T lymphocytes with trisomy 21 in comparison to normal cells. Two patients with DS were investigated, along with two normal subjects as a control, all being tested in independent, duplicated cell culture experiments. The most consistent finding was the overexpression of the superoxide dismutase gene (SOD1), located on 21q, and of MHC DR beta 3 (HLA-DRB3), GABA receptor A gamma 2 (GABRG2), acetyltransferase Coenzyme, A 2 (ACAT2) and ras suppressor protein 1 (RSU1) genes. When the data were clustered according to chromosome localization, the HC21 gene set showed, on average, the highest expression in DS cells in all the experiments. Moreover, separate clustering of patients and controls was obtained when analysis was restricted to HC21 gene expression values. These findings reinforce the specific gene dosage theory for the pathogenesis of the DS phenotype, and show a consistent overexpression of the SOD1 gene on 21q.

GeneRecords: a relational database for GenBank flat file parsing and data manipulation in personal computers.

UNLABELLED: Extracting the desired data from a database entry for later analysis is a constant need in the biological sequence analysis community; GeneRecords 1.0 is a solution for GenBank biological flat file parsing, as it implements a structured representation of each feature and feature qualifier in GenBank following import in a common database managing system usable in a personal computer (Macintosh and Windows environments). This collection of related databases enables the local management of GenBank records, allowing indexing, retrieval and analysis of both information and sequences on a personal computer. AVAILABILITY: The current release, including the FileMaker Pro runtime application (built for Windows and Macintosh environments), is freely available at http://apollo11.isto.unibo.it/software/

The murine DSCR1-like (Down syndrome candidate region 1) gene family: conserved synteny with the human orthologous genes.

A recently recognized gene family, conserved from yeast to humans, includes Down syndrome candidate region 1 gene (DSCR1), Adapt78 (recognized as the hamster ortholog of the DSCR1 isoform 4), ZAKI-4 (renamed DSCR1-like 1, DSCR1L1) and DSCR1L2 (a novel gene on human chromosome 1), along with yeast and C. elegans single members (Strippoli P., Lenzi L., Petrini M., Carinci P., Zannotti M., 2000. A new gene family including DSCR1 (Down Syndrome Candidate Region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member. Genomics 64, 252-263). The proposed family labels were a putative single-strand nucleic acid binding domain similar to the RNA recognition motif, and a unique, highly-conserved serine-proline motif. We have used a bioinformatics-driven molecular biology approach to characterize the murine members of DSCR1-like gene family. Systematic expressed-sequence-tags (EST) database search and reverse-transcription polymerase chain rection (RT-PCR) product sequencing allowed identification of the murine DSCR1, DSCR1L1 and DSCR1L2. The sequences of the respective protein products are of 198, 197 and 241 amino acids, respectively, and are very similar to the corresponding human proteins. The very broad expression pattern of the murine DSCR1 genes is similar to that of the human genes. Using a radiation hybrid panel, we mapped the murine DSCR1-like family members. The murine DSCR1 ortholog is located on the chromosome 16, in a region corresponding to that on human chromosome 21 just upstream of the Down syndrome candidate region. DSCR1L1 and DSCR1L2 murine genes are also located in chromosomal segments of chromosome 17 and 4, respectively, exactly corresponding to those containing the respective human homologs on chromosomes 6 and 1. Description of the mouse orthologs for DSCR1-like genes will allow knockout mice to be obtained for specific family members.

A new gene family including DSCR1 (Down Syndrome Candidate Region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member (DSCR1L2).

A new gene family has been identified on the basis of in-depth bioinformatics analysis of the Down syndrome candidate region 1 (DSCR1) gene, located on 21q22.1. We have determined the complete coding sequences of similar genes in Saccharomyces cerevisiae and Caenorhabditis elegans, as well as that of a novel human gene, named DSCR1L2 (DSCR1-like 2). Peripheral blood leukocyte cDNA sequencing predicts as its product a 241-amino-acid protein highly similar to products of the human genes DSCR1 and ZAKI-4 (HGMW-approved symbol DSCR1L1). The highest level of expression of DSCR1L2 mRNA was found by Northern blot analysis in heart and skeletal muscles, liver, kidney, and peripheral blood leukocytes (three transcripts of 3.2, 5. 2, and 7.5 kb). The gene consists of four exons and spans about 22 kb on chromosome 1 (1p33-p35.3) (Human Chromosome 1, Sanger Centre). Exon/intron organization is highly conserved between DSCR1 and DSCR1L2. Two alternative DSCR1L2 mRNA splicing forms have been recognized, with one lacking 10 amino acids in the middle of the protein. Analysis of expressed sequence tags (ESTs) shows DSCR1L2 expression in fetal tissues (heart, liver, and spleen) and in adenocarcinomas. ESTs related to the murine DSCR1L2 orthologue are found in the 2-cell stage mouse embryo, in developing brain stem and spinal cord, and in thymus and T cells. The most prominent feature identified in the protein family is a central short, unique serine-proline motif (including an ISPPXSPP box), which is strongly conserved from yeast to human but is absent in bacteria. Moreover, homology with the RNA-binding domain was weakly but consistently detected in a stretch of 80 amino acids at the amino-terminus by fine sequence analysis based on tools utilizing both hidden Markov models and BLAST. The identification of this new gene family should allow a better understanding of the functions of the genes belonging to it.

Molecular and biological features of two new human squamous and adenocarcinoma of the lung cell lines.

Two human cancer cell lines were established from metastatic lesions of an adenocarcinoma (RAL) and a squamous cell (CAEP) carcinoma of the lung. The clinical histories of the patients from whom the cell lines were derived are reported. The lines were maintained in continuous culture with doubling times of 65 (RAL) and 50 (CAEP) hours. The RAL and CAEP cell lines, whose morphology and ultrastructural features are presented, showed extensively rearranged karyotypes with modal number of 85 (RAL) and 98 (CAEP). In particular, chromosome 2 pentasomy and several clonal markers were evident in the RAL cells, whereas a telomeric deletion of chromosome 1, del (1)(q32), was observed in the CAEP cells. The morphologic data were confirmed by high expression of specific antigens for each histotype. A marked positivity of the neuron-specific enolase (NSE) levels was evident by immunoenzymatic assays in the cell lines cytosol with respect to those present in the respective patient's sera. No amplification or rearrangements were evident in the CMYC, LMYC, NMYC, INT-2, ERBB2, HRAS, KRAS, MOS, HST-1 genes by Southern blotting analysis in each cell line. Point mutations in exon 1 of KRAS and in exon 7 of TP53 were evident by polymerase chain reaction (PCR)-DNA sequencing in the RAL cell line, whereas no alterations were present in the HRAS and RB genes. The four genes studied did not show point mutations in the CAEP cell line. The RAL cell line was resistant to all the drugs tested, whereas the CAEP cells were sensitive to vinblastine. These cell lines may represent useful experimental models to investigate lung cancer biology and anticancer drug response.

Molecular genetics and in vitro sensitivity of a new human cell line, KKP, from a gastric adenocarcinoma.

A new cancer cell line (KKP) was established from an ascitic effusion of an advanced gastric adenocarcinoma, intestinal type. The line has been maintained in continuous monolayer culture with a doubling time of 48 hours for more than 2 years. KKP cells, whose ultrastructural features are presented, showed an aneuploid DNA content, a modal number of 53 chromosomes, and the presence of one double minute chromosome. The karyotype showed trisomies of chromosomes 7, 12, 13, and 14, tetrasomy of chromosome 18, a reciprocal translocation [t(1;20)(q21;p11.2)], and a [t(4;?)] rearrangement. No amplification or rearrangements were evident in the c-MYC, c-ERB B2, H-RAS, INT-2, HST-1, c-MOS, and K-RAS genes, whereas somatic rearrangements were present in the sequences corresponding to c-MET and cyclin E genes by Southern blotting analysis. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of P53 and RB genes did not reveal alterations or point mutations in the SSCP pattern of conformers. The chemosensitivity pattern assay of the KKP cell line indicated that it was sensitive to cisplatin, etoposide, and doxorubicin and resistant to 4'-hydroperoxycyclophosphamide. The clinical history of the patient from whom the cell line was derived is reported and compared with the results observed in the cell line in vitro. High levels of the tumor-associated antigens CEA (carcinoembryonic antigen) and CA19-9 were evident in the KKP cytosol, whereas the KKP spent culture medium maintained the same low levels of CEA and CA 19-9 found in the patient's serum. This new cell line may represent a useful tool for studying the biology of gastric cancer and for planning new therapeutic approaches.

Chromosomal alterations, biological features and in vitro chemosensitivity of SCLC-R1, a new cell line from human metastatic small cell lung carcinoma.

A new human cancer cell line was established from a metastatic lesion of a small cell lung carcinoma (SCLC-R1) and maintained in continuous culture with a doubling time of 62 h. The SCLC-R1 line, whose ultrastructural features are presented, showed a diploid DNA content, a translocation involving chromosome 16 [t(16;?)(q24;?)] and noticeable deletions in the FHIT (fragile histidine triad) region in the short arm of chromosome 3 [del(3)(p14)] and in the telomeric region of the short arm of chromosome 12 [del(12)(p13)]. The involvement of 12p in metastatic small cell lung cancer is reported here for the first time. No amplification or rearrangements were evident in the c-myc, L-myc, N-myc, int-2, c-erbB-2, H-ras, K-ras, c-mos, and hst-1 genes by Southern blot analysis. Wild-type p53, RB, K-ras and H-ras genes were evident by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. The neuron specific enolase (NSE) level was much higher in the cell line's cytosol than in the patient's serum and the cell line also had high expression of chromogranin A and cytokeratin 19. SCLC-R1 cells were sensitive to cisplatin, carboplatin and doxorubicin. The clinical history of the patient from whom the cell line was derived is reported. The characteristics of this new cell line indicate it to be a useful experimental model to investigate lung cancer biology and anticancer drug response.

Establishment and characterization of two new cell lines derived from human metastatic breast carcinomas.

Two human cancer cell lines (MA 2 and MA 3) were established from pleural effusions of infiltrating ductal carcinomas of the breast. The lines were maintained in continuous monolayer culture with doubling times of 70 (MA 2) and 78 (MA 3) hr for more than two years and possessed extensively rearranged abnormal karyo-types with modal chromosome number of 83 (MA 2) and 81 (MA 3) and DNA index values of 1.65 and 1.77, respectively. No amplifications or rearrangements were evident in the c-myc, int-2, c-erb B2, c-Ha-ras, or hst 1 genes in MA 2 and MA 3 cell lines. The clinical histories of the patients from whom the cell lines were derived are reported and compared with the results observed in the cell lines in vitro. The presence of CEA, CA 15-3, and MCA tumor markers observed in the primary tumor tissues was retained by the established cell lines. While the primary tumor tissues were ER+/PgR borderline+ (MA 2) and ER-/PgR+ (MA 3), the MA 2 line was ER+/PgR- and the MA 3 line remained ER-/PgR+. The MDR P-glycoprotein was not expressed either in primary tumor tissues or in the respective cell lines. High expression of cytokeratins 7, 18, and 19 was evident by immunohistochemical analysis in each cell line. whereas cytokeratins 8 and 17 were poorly or not at all expressed. The treatment history of the patients from whom the cell lines were derived involved CMF followed six months later by novantrone and cisplatin plus VP 16 (MA 2) and FEC followed four years later by CMF (MA 3). The chemosensitivity pattern assay of the cell lines indicated that the MA 2 line was sensitive to doxorubicin, cisplatin, and vinblastine, whereas the MA 3 line was sensitive to doxorubicin and cisplatin. The characteristics of these cell lines indicate them to be a good experimental model to investigate breast cancer biology and anticancer drug response.

[Reference values of assayable pyridinolines as markers of bone turn-over]. / Valori di riferimento delle piridinoline dosabili come marcatori del turn-over osseo.

The authors aimed to identify reference values for pyridine cross-links (HP and LP) in clinically normal populations. These cross-links are thought to be markers of bone resorption and are released by osteoclasts and eliminated in the urine. The normal values reported in the literature vary considerably. For this purpose the authors analysed groups of male and female patients, divided into age brackets, with no apparent bone metabolism disorders. Hydroxylysylpyridinoline (HP) values revealed no statistically significant differences in relation to either sex or age. Lysylpyridinoline (LP) values in subjects aged under 50 showed virtually comparable reference intervals in male and female subjects, whereas patients over 50 showed a different pattern of behaviour. In conclusion it can be said that, once reliable reference values have been identified using a urinary cross-link assay (in particular for LP), it is possible to evaluate bone resorption in postmenopausal women.