Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage.

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.

An I2 imidazoline ligand, RS 45041, potentiates hyperalgesia in acute arthritis.

Descending inhibition is increased after the induction of inflammation of the knee. The present study investigated whether this effect is mediated through alpha 2-adrenoceptors and/or I2 imidazoline receptors in the rat. An alpha 2-adrenoceptor antagonist, RX 821002, a selective I2 imidazoline ligand, RS 45041, and idazoxan, which has affinity for both these receptor types, were administered. After the induction of acute arthritis by intra-articular injection of kaolin and carrageenan, the agents binding to I2 imidazoline receptors further reduced the paw withdrawal latency to radiant heat beyond that induced by acute arthritis, i.e. these drugs were pronociceptive, potentiating hyperalgesia. These results suggest that I2 imidazoline receptors have an important role to play in modulation of hyperalgesia during acute inflammation. Development of I2 imidazoline drugs may prove useful in the treatment of hyperalgesia.

Effect of subcutaneous recombinant human erythropoietin in cancer patients receiving radiotherapy: preliminary results of a randomized, open-labeled, phase II trial.

PURPOSE: To determine the efficacy and safety of subcutaneous administration of recombinant human erythropoietin (r-HuEPO) at a dose of 200 units/kg/day to cancer patients undergoing radiotherapy. METHODS AND MATERIALS: This is a randomized, open-labeled, Phase II study. Only patients receiving radiotherapy +/- chemotherapy are included. Eligibility is restricted to patients with lung cancer, carcinoma of the uterine cervix, prostatic adenocarcinoma, or adenocarcinoma of the breast. Patients in the control and treatment arms receive radiotherapy with similar policies, and their doses of radiotherapy and treatment volumes are determined by the site and stage of the disease. Patients in the "treatment arm" receive 200 units/kg/day of r-HuEPO, subcutaneously, five times a week with iron (Fe SO4, 325 mg. p.o., t.i.d.) supplements. Complete blood counts are obtained weekly. Quality of life is assessed weekly by the patients themselves by a few simple entries on an analog scale. RESULTS: Twenty-six patients have been entered in the study so far. Twelve patients were placed in the control arm and 14 in the treatment arm. Pre-randomization demographic and laboratory mean values in both arms were comparable, with none of 16 parameters compared reaching statistical significance. Our results can be summarized as follows: (a) Mean hemoglobin, hematocrit, and red blood cell values increased gradually in the treatment arm patients. Week-by-week comparison showed that mean values for these three parameters were significantly higher in the treatment arm than in the control arm. For example, the p values for the differences in hemoglobin mean values for weeks 1-6 were 0.015, 0.002, 0.003, 0.0002, 0.0006, and 0.007, respectively. Similar trends were observed for red blood cells and the hematocrit values. (b) No significant toxicity has been encountered. (c) No significant differences in the mean values of white blood cells and platelet counts were seen between the two arms. The values of these two parameters declined over the course of radiotherapy. (d) The mean weekly increase in hemoglobin levels in the treatment arm was 0.43 gm/dl. CONCLUSION: (a) The safety and efficacy of r-HuEPO, with 200 units/kg/day of subcutaneous administration, have been confirmed in our study group. (b) However, the rate of increase in hemoglobin levels is not very rapid with the doses used. (c) Dose escalation studies are needed for determination of the feasibility of improving hemoglobin levels by about 1 gm/dl/week. (d) The question whether improvement in hemoglobin with r-HuEPO therapy can improve outcome by improving tumor oxygenation needs to be studied in carcinoma of the uterine cervix and squamous cell carcinoma of the head and neck.

Specific control of follicle stimulating hormone in the male: postulated site of action of inhibin.

Serum luteinizing and follicle stimulating hormone concentrations were related to gonadal function, as reflected by sperm count and serum testosterone concentrations, in a group of men studied over 10 years after surgical correction of bilateral cryptorchidism in childhood. The results indicated that while all degrees of gonadal function occurred in these patients, the main adverse effect of cryptorchidism was on spermatogenesis. In some of the patients with oligospermia and normal Leydig cell function there was an isolated increase of basal serum FSH concentrations, suggesting a specific impairment of the testicular production of 'inhibin'. Gonadotrophin releasing hormone (LHRH) tests in these patients were compared to those in patients with normal basal gonadotrophins and to those with elevated basal LH and FSH concentrations. A selective exaggeration of the FSH response to exogenous LHRH in the group of patients with a monotropic elevation of FSH concentrations suggests that 'inhibin' modulates the secretion of FSH by an action on the pituitary rather than by modifying endogenous LHRH production by the hypothalamus.