Regaining chondrocyte phenotype in thermosensitive gel culture

Chondrocyte tissue engineering continues to be a challenging problem. When chondrocytes are duplicated in vitro, it is imperative to obtain an adequate number of cells of optimal phenotype. A temperature-sensitive polymer gel, a copolymer of poly(N-isopropylacrylamide) and acrylic acid (PNiPAAm-co-Aac), has the ability of gelling at 37 degrees C (the lower critical solution temperature, LCST) or above and liquefying below that temperature (Vernon and Gutowska, Macromol. Symp. 1996;109:155-167). The hypothesis of this study was that chondrocytes could (1) duplicate in the copolymer gel; (2) regain their chondrocyte phenotype; and (3) be easily recovered from the gel by simply lowering the temperature below 37 degrees C. Chondrocytes from adult rabbit scapular cartilage were harvested and cultured in a monolayer culture until confluency (approximately 2 weeks). Next, the cells were harvested and seeded into the copolymer gel and cultured for 2-4 weeks. The phenotype of the cultured cells was then characterized. Two groups of control cultures, monolayer and agarose gel, were used to compare their ability to maintain chondrocyte phenotype. The results showed that chondrocytes isolated from rabbit scapula can re-express chondrocyte phenotype in agarose culture and polymer gel culture but not in monolayer culture. Also, cultured chondrocytes can be easily recovered from polymer gel culture by simply lowering the temperature. This new in vitro method of chondrocyte culture is recommended for chondrocyte propagation and regaining chondrocyte phenotype before cell seeding or transplantation.     

The immediate early genes of human cytomegalovirus require only proximal promoter elements to upregulate expression of interleukin-1 beta.

Human cytomegalovirus (HCMV) can infect monocytes and macrophages. The immediate early one (IE1) gene product of HCMV positively regulates its own expression, as well as the expression of the interleukin-1 beta (IL-1) gene. This study describes the IL-1 promoter proximal region required for upregulation of IL-1 gene expression by the HCMV IE1 or IE1 plus IE2 gene products. An IL-1 chloramphenicol acetyltransferase (CAT) construct containing the IL-1 genomic upstream sequence from position -1097 to +14 and four additional IL-1CAT plasmids containing progressive deletions of the -1097 to -131 sequence were used to evaluate the effect of the HCMV IE gene products on IL-1 gene expression. IL-1CAT plasmids were transfected into a monocytic cell line, THP-1, with plasmids containing either the IE promoter-regulatory region upstream of the bona fide IE1 (pIE1), IE2 (pIE2), or IE1+2 genes (pIE1+2) or a control plasmid containing the IE promoter-regulatory region alone (pLink760). In the presence of pIE1+2, there was an approximate 15-fold increase in CAT activity compared with the control, pLink760, in cells with CAT plasmids containing the -1097 to +14 IL-1 sequence. Plasmids with progressive deletions of this sequence, including the plasmid containing the shortest upstream segment (-131 to +14) also had an approximate 15-fold increase in CAT activity. The upregulation of IL-1 expression was mediated, primarily, by IE1 and not by IE2. This effect was promoter specific because an IL-1CAT plasmid with a complete deletion of the proximal promoter elements (-234 to +146) did not respond to the HCMV IE gene products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lassa fever. Effective therapy with ribavirin

In a study of Lassa fever in Sierra Leone, West Africa, we identified two variables associated with a high risk of death, and we evaluated the efficacy of ribavirin and Lassa virus-convalescent plasma for the treatment of Lassa fever. A serum aspartate aminotransferase level greater than or equal to 150 IU per liter at the time of hospital admission was associated with a case-fatality rate of 55 percent (33 of 60). Patients with the same risk factor who were treated for 10 days with intravenous ribavirin, begun within the first 6 days after the onset of fever, had a case-fatality rate of 5 percent (1 of 20) (P = 0.0002 by Fisher's exact test). Patients whose treatment began seven or more days after the onset of fever had a case-fatality rate of 26 percent (11 of 43) (P = 0.01). Viremia with levels greater than or equal to 10(3.6) TCID50 per milliliter on admission was associated with a case-fatality rate of 76 percent (35 of 46). Patients with this risk factor who were treated with intravenous ribavirin within the first six days after onset of fever had a case-fatality rate of 9 percent (1 of 11) (P = 0.006), whereas those treated after seven days or more of illness had a fatality rate of 47 percent (9 of 19) (P = 0.035). Oral ribavirin was also effective in patients at high risk of death. Lassa-convalescent plasma did not significantly reduce mortality in any of the high-risk groups. We conclude that ribavirin is effective in the treatment of Lassa fever and that it should be used at any point in the illness, as well as for postexposure prophylaxis.     

Serum estradiol concentration required to maintain body weight, attractivity, proceptivity, and receptivity in the ovariectomized female rat

Female hooded rats (230 to 260 g) were ovariectomized and given a subcutaneous implant of an estradiol-filled Silastic tube. The length of the tube was varied in order to produce a variety of serum estradiol levels. In the first experiment, animals were weighed over a 6-week period following surgery and then tested for sexual responsiveness to a male. The results demonstrated that ovariectomized females with an implant maintaining a serum estradiol concentration at about 15 pg/ml maintained body weight at the same level as that of intact females. A smaller implant gave rise to a higher weight gain and a larger implant to a lower weight gain. All implants resulted in a continuous state of receptivity. In a second experiment, ovariectomized females were implanted with smaller estradiol-filled implants in order to determine the threshold for maintaining proceptivity and receptivity. The results indicated that with a serum estradiol concentration below 15 pg/ml, the frequency of lordosis and of ear wiggling and darting decreased. Progesterone injections facilitated both proceptive and receptive behavior. In addition, following progesterone injections, the time required for a male to mount a female 10 times was decreased in females with low or no estradiol replacement. These results indicate that a constant concentration of estradiol at about the mean level present throughout the estrous cycle will result in normal body weight regulation and will maintain sexual behaviors that normally occur only during estrus. These results emphasize that Silastic implants of estradiol do not mimic normal endocrine function since, even at low levels, estradiol implants produce continuous receptivity.     

Radioimmunoassay of detirelix ([ N-Ac-D-Nal(2)1,D-p-Cl-Phe2,D-Trp3, D-hArg(Et)6(2),D-Ala10]-luteinizing hormone-releasing hormone) in plasma or serum

A procedure for the radioimmunoassay (RIA) of detirelix in plasma or serum at concentrations as low as 0.15 ng/ml is described. Antiserum was produced by deacetylation of the N-terminus amino groups of detirelix and coupling this analog to bovine serum albumin with a carbodiimide and immunizing rabbits with the resultant conjugate. For RIA, 125I-labeled detirelix was used as the tracer and a double antibody procedure was used to separate the free and bound fractions. No purification of samples was required prior to RIA. Accuracy of the method was assessed by adding known quantities of detirelix to detirelix-free plasma and determining the ratio of measured to added analyte. Linear regression analysis for the concentration range 0.15-150.0 ng/ml yielded a regression equation of y = 0.88 X +1.46 and a correlation coefficient of 0.996. Additional validation was obtained from an in vivo study in which [14C]detirelix was administered to monkeys and plasma clearance profiles were determined by RIA and an HPLC-radiochemical method. The RIA results were in good agreement with those obtained by the HPLC method.     

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V{alpha}1 gene

Previous studies of the TCR chain gene have located promoterelements 5' to the start of the various V genes. The only fullycharacterized enhancer for the entire chain gene (V, J andC genes) has been located {small tilde}3 kb from the 3' endof C. We now report the existence of additional regulatory elementslocated in the introns of several murine V genes (V1, V3 andVB6.2.16). In the case of V1, this element appears to be a promoterwith bidirectional activity that is not T cell specific. Interestingly,upstream of the promoter in the antisense strand, an open readingframe has been found that codes for a small molecular weightprotein ({small tilde}60 amino acids) that contains a prollne-richregion and a tyrosine-isoleucine motif that has homology toIgß (the B29 gene product). A rabbit antiserum madeagainst this sequence has confirmed its existence by Westernblot and immunoprecipitation. Thus this V1 intronic promoterhas the potential not only to induce the formation of a truncatedV1 gene product, but also regulates the expression of a smallmolecular weight protein that may be involved in lymphocyteantigen receptor signaling. The activity of this promoter isregulated by changes in intracellular calcium. In the presenceof ionomycin the promoter is down-regulated in the sense directionand its activity is enhanced in the antisense direction. Thisresult suggests that this promoter can act differentially toproduce two very different gene products. The bidirectionalV1 promoter appears to be the first in the Ig superfamily toinduce potentially functional proteins in both directions.     

Relationship of glucocorticoid suppression of arachidonic acid metabolism to alteration of neutrophil function

The role of arachidonic acid metabolism in glucocorticoid-induced suppression of neutrophil function was investigated. In vivo treatment of cattle with dexamethasone decreased the production of lipoxygenase products of arachidonic acid metabolism (leukotriene B4 [LTB4] and H-5-hydroxy-6,8,11,14-eicosatetraenoic acid [5-HETE]) in neutrophils. We previously reported that in vivo dexamethasone treatment resulted in alteration of in vitro neutrophil oxidative metabolism, iodination, antibody-dependent cell-mediated cytotoxicity (ADCC), and random migration. To determine if the decrease in production of LTB4 and 5-HETE were responsible for the changes in neutrophil function, neutrophils were treated in vitro with inhibitors of the lipoxygenase enzyme (BW755c and nordihydroguaiaretic acid), and their function was evaluated. A decrease in neutrophil oxidative metabolism and iodination was observed, but there was no effect on neutrophil random migration or ADCC. The results suggest that in vivo treatment with glucocorticoids inhibit neutrophil oxidative metabolism and iodination by decreasing the formation of lipoxygenase products of arachidonic acid metabolism but alter neutrophil random migration and ADCC by a mechanism independent of arachidonic acid metabolism.     

Frequency and replication status of the fragile X, fra(X)(q27-28), in a pair of monozygotic twins of markedly differing intelligence.

Chromosome analysis using conventional staining, G banding, and, after BUdR incorporation, two R banding methods, one using Hoechst and one acridine orange, were performed on lymphocytes from a pair of female monozygotic twins. The culture conditions were designed to show the presence of the fragile X (q27-28) which had previously been found to be segregating in the family. One twin was of higher than normal intelligence and the other had been diagnosed as mentally retarded. The frequency of the occurrence of the early/active fragile X compared to the overall total of informative fragile X was determined using both methods described above and was also compared with previous published data in the form of a graph showing percentage of early/active fragile X against intelligence.     

Surgical management of synchronous coronary artery disease and multiple aortic stenosis: a case report with brief review of the literature

One-stage surgery was successfully performed in a 44-year-old hypertensive man with uncontrolled angina, multiple coarctations of the thoracic and abdominal aorta, and a previous subtotal gastrectomy. There was a gradient of 120 mm Hg between the thoracic and abdominal aorta. A graft was placed retroperitoneally from the infrarenal aorta to the ascending aorta and was followed by a coronary artery bypass graft. Twenty-four months postoperatively, the patient was free of angina, and his hypertension was easily controlled.     

Paralysis and unilateral arthritis: is the association established ?

Summary A patient with long-standing upper limb lower motor neurone paresis more recently developed homolateral upper motor neurone hemiplegia. Subsequent primary generalised osteoarthritis spared only the paralysed upper limb. Detailed review of reported cases suggest that the protective effect of paralysis against subsequent development of arthritis is not as clearly established as generally believed.