Segregation of structural collagen genes in adolescent idiopathic scoliosis.

The etiology of idiopathic scoliosis remains unknown. The condition results in a characteristic deformity of the spine and surrounding tissues. Both Types I and II collagen are important constituents of the affected tissues, and thus defective collagens are reasonable candidates for the primary abnormality in adolescent idiopathic scoliosis (AIS). Direct analyses of the amount and solubility of collagen have revealed differences between normal individuals and those with AIS. However, these changes may be secondary to the mechanical effects of the spinal deformity. Segregation analysis was done of genetic markers linked to the structural genes encoding Types I and II collagen to test these candidate loci in four pedigrees with dominantly inherited AIS. In one pedigree, markers linked to both of the Type I collagen loci (COL1A1 and COL1A2) were found to be inherited independently of the abnormal phenotype. Two pedigrees were discordant at one of the Type I loci. The condition also segregated independently of the locus for Type II collagen (COL2A1) in three pedigrees. This is evidence against idiopathic scoliosis generally being caused by mutations in the Types I and II collagen genes.     

Benzo(a)pyrene inhibits epidermal growth factor binding and receptor autophosphorylation in human placental cell cultures

Studies investigated the effects of benzo(a)pyrene (BP) treatment on epidermal growth factor (EGF) receptor binding and kinase activity in human placental cell cultures. Specific binding of 125I-EGF to cells from early gestation placentae was significantly decreased by 37 and 60% following exposure to 1 and 10 microM BP, respectively, for 24 hr. In contrast, cells cultured from term placentae showed no inhibitory effect of either concentration of BP. Specific binding of 125I-labeled insulin and insulin-like growth factors-I and -II to early gestation cells was decreased only 15-18% at 10 microM BP, which indicates that loss of membrane receptors appears to be selective for EGF. Scatchard analysis of early gestation cells revealed that BP was associated with a dose-dependent loss in the number of high affinity EGF binding sites. Evidence from cross-linking and autophosphorylation experiments confirmed that the Mr 170,000 binding protein was decreased in a dose-dependent manner following BP treatment. In comparison, term placental cells exhibit a 26% loss of EGF receptor autophosphorylation without alteration in binding following exposure to 10 microM BP. Thus, early gestation cells exhibit a BP-related down-regulation of EGF receptors, whereas term placental cells show receptor desensitization. No adverse effect of BP treatment was observed on the incorporation of [35S] methionine into proteins secreted by early gestation cells. Further experiments compared the effects of BP with the related poly-cyclic compounds beta-naphthoflavone, alpha-naphthoflavone, and 3-methylcholanthrene. In early gestation cells, EGF binding and receptor autophosphorylation were measurably decreased at 10 microM concentrations of these polycyclic compounds, but to a lesser extent than observed with BP. In term placental cells, however, EGF binding was unchanged or increased, whereas receptor autophosphorylation was decreased 10-26%. Thus, exposure of term placental cells to these polycyclic compounds leads to a dissociation between EGF binding and receptor protein kinase activity. Finally, aryl hydrocarbon hydroxylase activity was induced 20- to 200-fold in early placental cells exposed to BP, beta-naphthoflavone, and 3-methylcholanthrene. In summary, the direct effects of BP and related compounds observed on placental EGF receptors may indicate altered function of EGF in the regulation of cell growth and differentiation in the human placenta.     

Midazolam: kinetics and effects on memory, sensorium, and haemodynamics.

This study aimed not only to compare the pharmacokinetics of oral and intravenous doses of the new water-soluble benzodiazepine, midazolam, but also to study the effects on haemodynamics, sensorium, and memory performance. Eight normal human volunteers each received a single 15 mg dose of midazolam base orally and intravenously in randomized sequence 2 weeks apart. Serial venous samples were obtained for 12 h after dosing. Vital signs, sensorium testing and memory testing using word lists were also performed. Computerized non-linear least squares curve-fitting of the two-compartment open model to the oral and intravenous data simultaneously yielded the following estimates: V1, 0.33 1 kg-1, VdSS, 1.08 1 kg-1, t1/2,lambda, 0.10 h, t1/2,Z, 1.89 h, ka 1.17 h-1 and bioavailability, 49%. The intravenous dose decreased the systolic pressure 22 mm Hg during the first half-hour and the oral dose had 50% less effect. Most subjects became drowsy halfway through the infusion and were only rousable to voice by its end. The sensorium was clear by 2-3 h. After oral dosing the peak sensorium effects of ataxia-dysarthria were seen at 30 min and had cleared by 2 h. Memory testing showed that memory acquisition was markedly impaired for at least 90 min after the intravenous dose and slight recovery was apparent at this time after the oral dose. Memory performance was proportionately more impaired than the sensorium score. We conclude that: midazolam kinetics are characterized by rapid absorption, but incomplete bioavailability and rapid elimination, midazolam intravenously may lower blood pressure significantly, and the level of consciousness correlates poorly with the degree of memory impairment.     

Tumor osteolysis in osteopetrotic mice

Details of the cellular and biochemical mechanisms involved in focal destruction of bone at sites of tumor osteolysis are unknown. It has been shown that tumors from sarcoma (2472) cell lines induce focal osteolysis in mice by stimulating formation and activation of osteoclasts. In this report, the influence of 2472 tumors on the skeletons of osteoclast-deficient animals (op/op) was studied. After op/op femora had been inoculated with 2472 cells, tumors developed and focal osteolysis occurred. There were more osteoclasts per histologic section in sham-injected femora (19 ± 5) than in tumor-bearing femora (412 ± 129) (p < 0.05). The size of the osteoclasts also increased from 304 ± 81 μm² in sham-injected limbs to 407 ± 62 μm² in tumor-bearing limbs (p < 0.001). Conditioned media from 2472 op/op tumor explants contained macrophage colony-stimulating factor. A deficiency of osteoclasts in op/op mice is the result of the absence of this factor; therefore, these data introduce the possibility that macrophage colony-stimulating factor derived from 2472 tumor may be responsible for directing osteoclast-mediated osteolysis at sites of the tumor.     

Hepatic artery involvement in polymyalgia arteritica.

Disturbances of liver function tests are common in polymyalgia arteritica, but the underlying liver lesion has not been defined. We report a patient who was demonstrated to have a giant cell arteritis involving both the hepatic and temporal arteries, and we discuss the possibility that such an arteritis involving the hepatic arteries is responsible for the abnormalities of liver function seen in this condition.     

Functional depletion of T- and B-memory cells and other lymphoid cell subpopulations-during trypanosomiasis.

T. brucei infection in mice causes generalized immunosuppression with multiple changes in the cells of the lymphoid tissue. Loss of B cell responsiveness to antigens and mitogens, and the induction of suppressive T-cells and macrophages, have been previously reported (Hudson, Byner, Freeman & Terry, 1976; Corsini, Clayton, Askonas & Ogilvie, 1977; Jayawardena & Waksman, 1977). In this study, purified B- or T-cell populations from infected mice have been tested functionally in vitro or in vivo by transfer into syngeneic irradiated hosts to separate the cells from trypanosomes or their products. B-memory cells for thymus dependent (DNP-KLH) and thymus independent (DNP-Ficoll) antigens are depleted or lose their potential to respond to the antigen during T. brucei infection. Similarly, purified T-helper cells, and T-cells reactive to allogeneic target cells in mixed lymphocyte reactions are functionally defective. By 16 days of infection all these responses are less than 10% of the normal level. The loss of B-cell function follows the peak parasitaemia and is accompanied by increases in the serum levels of both IgM and IgG. Enhanced Ig production and decline in B-cell potential also occur in T-deprived mice and in CBA/N mice which lack a subset of T-independent B-cells. Cells affecting delayed hypersensitivity reactions retain their activity throughout trypanosome infection and so far provide the only exception to the general decline in immune potential.     

Delineation of an estimated 6.7 MB candidate interval for an anophthalmia gene at 3q26.33-q28 and description of the syndrome associated with visible chromosome deletions of this region

Anophthalmia or microphthalmia occur in approximately one in 10 children who have severe visual impairment. These eye malformations are often of unknown aetiology, but can be inherited in autosomal dominant, recessive or X-linked forms, and can also occur in association with specific chromosome abnormalities. Four children are described in the medical literature with microphthalmia or anophthalmia in association with chromosome rearrangements involving distal 3q, suggesting the presence of a micro/anophthalmia gene in this region. We have identified two further patients with micro/anophthalmia and chromosome rearrangements involving 3q26-->3q27 and identified a 6.7 MB common deleted region. Patient 1 had multiple abnormalities including bilateral anophthalmia, abnormalities of the first and second cranial nerves and partial absence of the corpus callosum. His karyotype was 46,XY,del(3)(q26.33q28). Patient 2 had right anophthalmia and left extreme microphthalmia. Her karyotype was 46,XX,del(3)(q26.33q28)t(3;7)(q28;q21.1). Both patients had intrauterine growth retardation (IUGR) and strikingly similar dysmorphic facies consisting of bossed forehead, downward-slanting palpebral fissures, grooved bridge of the nose, prominent low-set ears, small down-turned mouth and small mandible. We identified BAC clones mapping to distal 3q from the ENSEMBL and NCBI Entrez databases. These BAC clones were used as fluorescence in situ hybridisation (FISH) probes to identify the minimum deleted region common to both patients. This interval, between clones RPC11-134F2 and RPC11-132N15, was estimated to be 6.7 MB. We conclude that there is an anophthalmia locus within this interval. Candidate genes mapping to this region include Chordin and DVL3, a homologue of the Drosophila Dishevelled gene.     

An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.