[Prognostic factors for substantial clinical benefit from musculoskeletal pathologies of the shoulder]. / Factores pronósticos de beneficio clínico sustancial en patologías musculoesqueléticas de hombro.

INTRODUCTION: shoulder plain is a common cause of complain, however a precise diagnosis is hard to achieve. This is why finding factors associated to a good prognosis could help to improve our clinical practice. OBJECTIVE: to identify demographic and clinical characteristics from the initial assessment associated with substantial clinical benefit (SCB) in patients with shoulder pain one month after treatment or at patient's discharge. MATERIAL AND METHODS: this was a secondary analysis of a prospective cohort of patients with shoulder pain. Demographic and clinical (self-reported scales) factors associated with SCB at discharge or four weeks after the initial assessment, were analyzed. SCB was defined as a score +5 on a Global Rating of Change. A logistic regression model was made in order to identify predictors of SCB. The area under the curve ROC was used to assess the performance of the model with its independent variables. RESULTS: 101 patients of 138 were analyzed. The median age was 55 (RIQ 37-61) years old, there were 55 (54.5%) women in the sample. The variables independently associated to SCB were fracture as a reason for admission [adjusted OR 11.8 (95% CI 1.4-101.8); p = 0.024], and durations of shoulder symptoms shorter than seven months [adjusted OR 4.63 (95% CI 1.9-11.1); p = 0.001]. CONCLUSION: the diagnosis of fracture and durations of shoulder symptoms shorter than seven months were independently associated with a SCB after one month of treatment or at the patient's discharge.

INTRODUCCIÓN: las patologías de hombro representan una condición clínica frecuente, pero suele ser complejo realizar un diagnóstico preciso. Es por esto, que conocer qué variables permiten realizar un pronóstico del resultado del tratamiento puede ser útil para la práctica clínica. OBJETIVO: el objetivo del presente estudio fue identificar características clínicas y demográficas asociadas a beneficio clínico sustancial (BCS) en pacientes con afecciones musculoesqueléticas de hombro al mes o al alta del inicio de tratamiento fisioterápico. MATERIAL Y MÉTODOS: se realizó un análisis secundario de una cohorte prospectiva de pacientes con dolor de hombro. Se evaluó qué factores demográficos y clínicos se asociaban a BCS. Se consideró BCS un puntaje +5 en la Global Rating of Change. Se realizó un modelo de regresión logística para identificar predictores de BCS. A su vez, se utilizó el área bajo de la curva ROC para determinar el desempeño del modelo con sus respectivas variables independientes. RESULTADOS: de 138 sujetos se analizaron 101 pacientes. La mediana de edad fue de 55 (RIQ 37-61) años, hubo 55 (54.5%) mujeres dentro de la muestra. Las variables que se asociaron independientemente a BCS fueron fractura como motivo de ingreso [OR ajustado 11.8 (IC95% 1.4-101.8); p = 0.024] y tiempo de evolución menor a siete meses [OR ajustado 4.63 (IC95% 1.9-11.1); p = 0.001]. CONCLUSIÓN: el diagnóstico de fractura y el tiempo de evolución menor a siete meses se asociaron de manera independiente a BCS al cumplir un mes de tratamiento kinésico o al alta.

Rickettsia rickettsii growth and temperature-inducible protein expression in embryonic tick cell lines.

Rickettsia rickettsii has limited adverse effects on its arthropod vector, but causes severe disease in man. To model differences in host-parasite interaction, R. rickettsii growth and protein expression were examined at temperatures reflective of host environment in the tick cell lines DALBE3 and IDE2, the human endothelial cell line ECV304, and the African green monkey kidney cell line Vero76. At low multiplicities of infection, rickettsial titres increased 10(2)-10(3)-fold in all cell lines after incubation for 3 days at 34 degrees C. At higher multiplicities and with extended incubation, R. rickettsii showed enhanced survival in tick versus mammalian cells. No difference in rickettsial ultrastructure or protein profiles was detected between different host cell types. Rickettsial proteins of 42, 43, 48, 75 and 100 kDa are induced in tick cells shifted from 28 degrees to 34 degrees C, but not in cells maintained at 28 degrees C. This temperature response may be associated with expression of rickettsial determinants that are pathogenic to mammalian hosts.

Improved plaque assays for Rickettsia prowazekii in Vero 76 cells.

Typhus group rickettsiae, including Rickettsia prowazekii and R. typhi, produce visible plaques on primary chick embryo fibroblasts and low-passage mouse embryo fibroblasts but do not form reproducible plaques on continuous cell culture lines. We tested medium overlay modifications for plaque formation of typhus group rickettsiae on the continuous fibroblast cell line Vero76. A procedure involving primary overlay with medium at pH 6.8, which was followed 2 to 3 days later with secondary overlay at neutral pH containing 1 microgram of emetine per ml and 20 micrograms of NaF per ml, resulted in visible plaques at 7 to 10 days postinfection. A single-step procedure involving overlay with medium containing 50 ng of dextran sulfate per ml also resulted in plaque formation within 8 days postinfection. These assays represent reproducible and inexpensive methods for evaluating the infectious titers of typhus group rickettsiae, cloning single plaque isolates, and testing the susceptibilities of rickettsiae to antibiotics.

Differential activity of Rickettsia rickettsii opmA and ompB promoter regions in a heterologous reporter gene system.

The outer membrane of the Gram-negative obligate intracellular parasite Rickettsia rickettsii contains two large surface protein antigens with approximate molecular masses of 200 and 135 kDa termed rOmpA and rOmpB, respectively. rOmpB is the most abundant protein in the outer membrane, while rOmpA is a relatively minor constituent. Densitometry of intrinsically radiolabelled protein profiles from R. rickettsii-infected Vero cells indicated a molar ratio of approximately 1:9 between rOmpA and rOmpB. The putative promoter-5' untranslated regions (5' UTR) from their recently characterized genes (rompA and rompB) were placed in the promoter assay vector pKK232-8 to test whether these elements conserve aspects of differential expression in a heterologous host-reporter system. Primer extension analysis of RNA from Escherichia coli clones containing the constructs indicated that E. coli RNA polymerase faithfully utilizes rompA and rompB transcription start sites identified previously in R. rickettsii. The rompB insert directs 28-fold higher levels of chloramphenicol acetyl transferase activity than the rompA insert.

The dispersion of defective endogenous murine retroviral elements suggests retrotransposition-mediated amplification.

The dispersion of four replication-defective endogenous proviruses, originally detected in 129 strain mice and shown to have extensive deletions of gag, pol, and env gene regions, was investigated in 13 inbred strains and substrains of mice. Using probes to sequences flanking the integration sites in 129 mice, unique genomic Eco RI fragments were assigned to each of the four endogenous proviral elements. Analyses revealed that certain of these proviral elements are present both in strains closely related to strain 129 (i.e., strains 101 and LP/J) and in more distantly related strains (i.e., strains BALB/cJ, A/J, and C3H/HeJ). In mouse strains lacking proviral integration at a particular locus, the size of the corresponding Eco RI genomic fragment and absence of a characteristic Kpn I site indicated the lack of a residual solitary long terminal repeat. Hybridization of oligonucleotide probes that distinguish the specific deletions present within these elements identified additional analogous proviral integrations at many different sites in all strains investigated. These data indicate that the diversification of these proviral elements found in inbred strains is generated by integration of new copies, rather than excision through homologous recombination. Moreover, the results are consistent with other endogenous retroviruses providing the trans-acting proteins necessary to package the defective viral RNA.

The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor.

A Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub-species specific antigenic determinants. The 5' portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5' terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3' portion of this large open reading frame. This suggests a role for post-translational processing of rOmp B from a large precursor molecule.

Truncated gag products encoded by Gv-1-responsive endogenous retrovirus loci.

The conversion of endogenous or exogenous murine retroviruses to a leukemogenic phenotype involves recombination with retroviral sequences present in host genomic DNA. In the 129 Gix+ inbred strain, these endogenous sequences are replication defective but still express retroviral proteins under the apparent transcriptional control of the Gv-1 regulatory locus. To study the protein-coding potential of Gv-1-regulated endogenous retroviral loci, we used oligonucleotide probes directed to env deletion breakpoints identified in previously characterized cDNA clones. Four endogenous retroviral loci were isolated from a library of 129 Gix+ genomic DNA with these probes. Three loci cloned with the env deletion probe del env-1 had virtually identical proviral inserts by restriction analysis. A unique locus was identified and cloned with the del env-2 probe, which must therefore represent a Gv-1-responsive element. Restriction enzyme and nucleotide sequence analyses indicated that the del env-1 and del env-2 loci represented members of the polytropic and modified polytropic classes of endogenous retrovirus, respectively. Despite this divergence, members of both classes contained identical deletions of 19 nucleotides within p30gag and of 1,474 nucleotides from p10gag into the reverse transcriptase-coding region of pol, suggesting that a recombination event had occurred between these proviral sequences prior to insertion within the genome. The del env-1 and del env-2 loci retained coding capacity for truncated gag polyproteins, confirmed by in vitro translation and immunoprecipitation of the protein products. Nucleotide sequence comparison of the untranslated leader (L) regions of the del env-1 and del env-2 loci to a replication-competent ecotropic virus indicated regions that might be important to dispersion of these endogenous retroviral elements throughout the host genome.

A map of the hCG beta-LH beta gene cluster.

The beta-subunit of human chorionic gonadotropin (hCG beta) is reportedly encoded by as many as seven non-allelic genes or pseudogenes. Previous studies have identified a cluster of three hCG beta gene copies and the single-copy lutropin-beta subunit (LH beta) gene, but overlap of this cluster with two additional pairs of hCG beta genes has not been demonstrated, despite the isolation of 18 genomic clones. To define the number and organization of non-allelic hCG beta gene copies, genomic Southern blot analyses were performed using hCG beta cDNA and gene-flanking unique-sequence probes. The data show the linear arrangement of six genes (or pseudogenes) and show that the hCG beta-IH beta gene cluster is present in a single 58-kilobase EcoRI fragment. Our map of the cluster indicates which of the cloned hCG beta genes reflect somatic genotypes rather than recombinant artifacts and will thus permit investigation of factors regulating expression during gestation.

The beta subunit of human chorionic gonadotropin is encoded by multiple genes.

Two recombinant phage clones bearing sequences corresponding to the beta subunit of human chorionic gonadotropin (hCG beta) were isolated from a human genomic library. The beta sequences were mapped by blot hybridization of restriction digests of these phage DNAs and the nonoverlapping inserts were subcloned in pBR322 and sequenced. The nucleotide-sequencing data show that the hCG beta subunit is encoded by at least three nonallelic genes. Moreover, based on restriction analyses of human placental DNA, these genes may be linked in a single cluster with four other hCG beta-like genes. The sequenced genes all differ in their 5' flanking regions, and none of them is completely homologous in sequence to either of two hCG beta cDNA clones used here. In the translated region of one of these genes, three base substitutions result in two changes from the reported amino acid sequence. In the family of beta-containing glycoprotein hormones, the hCG beta subunit is unique in that it contains an extension of 29 amino acids at its COOH end. The DNA sequence corresponding to this region in the sequenced genes is part of a larger exon. These data show that the COOH-terminal extension does not result from splicing of the primary RNA transcript.

Evidence for a signal sequence at the N terminus of the common precursor to adrenocorticothrophin and beta-lipotropin in mouse pituitary cells.

The precursor to corticotropin and beta-endorphin was synthesized in a reticulocyte cell-free system under the direction of mRNA from mouse AtT-20 pituitary tumor cells in the presence of [3H]proline, [3H]phenylalanine, [3H]leucine, [3H]valine, [3H]isoleucine or [35S]methionine. Automatic Edman degradation of the radioactive cell-free product showed the following N-terminal sequence: Pro-1, Met-2, Leu-11, Leu-12, Leu-13, Leu-15, Leu-16, Leu-17, Ile-21 and Val-23. The corticotropin-endorphin precursor was also labeled in AtT-20 cells with [3H]valine, [3H]leucine, [3H]tryptophan, [3H]serine, [35S]methionine or [35S]cysteine. Automatic Edman degradation of the radioactive intact cell form gave the following N-terminal sequence: Trp-1, Cys-2, Leu-3, Ser-5, Ser-6, Val-7, Cys-8, Leu-11, Leu-17, Leu-18 and tentatively Met-27. The sequence of the intact cell form from AtT-20 cells matches the sequence of the cell-free form of bovine pituitary precursor beginning at Trp-27, as determined by recombinant DNA technology [Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S. N., and Numa, S. (1979) Nature (Lond.) 278, 423-427]. The sequence of the mouse pituitary mRNA-directed cell-free translation product also matches the bovine precursor beginning at Pro-2. The results suggest that both the mouse and bovine precursors possess a signal sequence of 26 amino acids which is cleaved in intact cells. CNBr cleavage of [35S]cysteine-labelled intact cell precursor gave rise to an N-terminal fragment of a size compatible with the presence of a methionyl residue at or near position 27.

A cellular basis for the differences in regulation of synthesis and secretion of ACTH/endorphin peptides in anterior and intermediate lobes of the pituitary.

The focus of research in our laboratory over the past few years has been the regulation of synthesis, processing and release of the ACTH/LPH family of peptides. These peptides are derived from a common precursor protein that is found in both the anterior and intermediate lobes of the pituitary (Roberts et al. 1978) and in the hypothalamus (Liotta et al. 1979). In the anterior lobe this protein gives rise to alpha (1-39)ACTH, beta-lipotropin and an N-terminal fragment of undefined function. In addition, a variety of intermediate lobe pituitary peptides can be derived from the precursor by further processing of ACTH and beta-LPH. In this paper we compare the structure of the precursor in the anterior and intermediate lobes of mouse and rat pituitary. Processing of the precursor to its constituent hormones is then contrasted in primary cultures of anterior and intermediate lobe cells using pulse label and pulse chase techniques with radioactive amino acids and sugars. Finally, we discuss the difference in behaviour of anterior and intermediate lobe cells in culture with regard to their rates of secretion and intracellular turnover of hormones and regulation of these processes by hypothalamic factors, glucocorticoids and catecholamines.