A new electron microscope positive staining method for viruses in suspension.

A new procedure for the positive staining of viruses in suspension, the Tokuyasu staining procedure (TSP), was evaluated using a non-enveloped virus, rotavirus; an enveloped virus, rubella virus and two glutaraldehyde-treated enveloped viruses, Human T Cell Lymphotropic Virus Type I (HTLV-I) and Human Immunodeficiency Virus Type 1 (HIV-1) as models. The TSP involves an initial staining of the virus with uranyl acetate (UA) followed by thin embedding in a mixture of UA and polyvinyl alcohol (PVA). Using aqueous UA for the TSP, a combination of positively and negatively stained particles was seen for both rotavirus and rubella virus. With glutaraldehyde-fixed HTLV-I and HIV-1, stain penetration did not occur and only negative staining was observed. The substitution of methanolic UA for aqueous UA in the TSP resulted in only positive staining of rotavirus and rubella virus. The change in procedure also resulted in stain penetration of the glutaraldehyde-fixed HTLV-I and HIV-1 to give positively stained particles. Some novel morphological features of rotavirus and rubella virus structure were observed by the TSP.     

Growth hormone therapy in achondroplasia.

A pilot study was carried out to examine the safety and efficacy of recombinant human growth hormone for growth-promoting therapy of achondroplasia. The data suggest that the agent in doses used to treat non-GH-deficient forms of short stature (0.3 mg/kg/wk) modestly increases overall height velocity in some children with achondroplasia. The effect was seen mainly in children with the lowest growth velocities prior to treatment. No untoward effects were noted. Several questions were raised that require further study.     

Rapid detection of polyomavirus BK by a shell vial cell culture assay.

Polyomavirus BK (BKV) causes asymptomatic latent infection in the human host that is reactivated during periods of immune suppression. Detection by conventional tube cell culture is difficult and time consuming because BKV exhibits slow growth with late (14 to 28 days) and subtle cytopathic effects. We developed a shell vial cell culture assay (SVA) using a cross-reactive monoclonal antibody to the T antigen of simian virus 40 to detect BKV rapidly by indirect immunofluorescence. Nuclear fluorescence was seen in BKV-infected cells as early as 16 h postinoculation; 6 to 28 times more foci were present at 36 h postinoculation. Human embryonic kidney cells infected with BKV produced 7 to 42 times more fluorescent foci than MRC-5 or rhabdomyosarcoma cells did. Centrifugation enhanced the infectivity of BKV in the SVA. To define the clinical utility of SVA, urine specimens from organ transplant patients were tested. Of 27 patients, 4 (15%) were found to be positive by SVA. SVA offers a simple and rapid method for detection of BKV that can be of use in clinical studies of this virus.     

The HLA genetic constitution of the Bushmen (San)

The HLA-A, -B, and -C antigens of 290 and the DR antigens of 212 !Kung San individuals were characterized. The most frequent antigens were HLA-A30 gene frequency (gf) = 0.193, Bw58 (gf = 0.303), Cw6 (gf = 0.327), DR4 (gf = 0.273), and DQw3 (gf = 0.553). An unexpected finding was the low frequency of the classic African black antigen Bw42 (gf = 0.004). Marked differences as well as similarities in HLA gene frequencies were observed between the San and the South African Negroes, supporting the view that they had a common origin and were then separated for a very long time. During this period differences developed as a result of selective advantage in the Negroes following the pastoralist-agriculturalist way of life as opposed to the hunter-gatherer way of life. The picture is further complicated by the fact that gene flow, mostly from the San to the southern African Negroes, took place when they met again a few hundred years ago. The data also illustrate HLA haplotypes, linkage disequilibria, and four-locus haplotypes not previously seen in other human populations. The most frequent four-locus haplotype in the San, HLA-Aw43, Cw7, B7, DRw6 was also different from A30, Cw2, Bw42, DR3, the most common among the South African Negroes.     

Tumor necrosis factor alpha is not implicated in the genesis of experimental autoimmune gastritis

Experimental autoimmune gastritis (EAG) characterised by mononuclear cell infiltrate, parietal and zymogenic cell destruction and circulating autoantibodies to gastric H(+)/K(+)ATPase is an animal model for human autoimmune gastritis, that leads to pernicious anaemia. We have previously shown that Fas has a role in initiating damage to target cells in EAG. Here we used three strategies to examine the role of TNFalpha in this disease. We administered neutralising anti-TNFalpha antibody either as a single injection or as twice weekly injections for 8 weeks to mice subjected to neonatal thymectomy-induced EAG. To address the role of apoptotic signals through TNFR1, TNFR1 deficient mice were either neonatally thymectomised or crossed to PC-GMCSF transgenic mice that spontaneously develop EAG. Neonatally thymectomised mice treated with anti-TNFalpha antibody developed destructive gastritis and autoantibodies to gastric H(+)/K(+)ATPase similar to control mice. Following either neonatal thymectomy or crossing to PC-GMCSF transgenic mice, TNFR1 deficient mice developed autoantibody-positive destructive gastritis at similar frequency compared with wild type and heterozygous littermates. Our observations that neutralisation of TNFalpha and absence of TNFR1 has no discernible effect on development of EAG suggest that TNFalpha is not required for mucosal cell damage or development of autoimmune gastritis. While blocking TNFalpha activity has therapeutic benefit in certain autoimmune diseases, this is not the case for EAG.     

Characterization of a precipitating antigen detected in the serum of patients with viral hepatitis

During a search for the aetiological agent of non-A non-B hepatitis, a precipitating antigen was detected in the sera of some patients during the acute phase of their illness. The antigen was detected by agar gel diffusion using antibody from convalescent sera obtained from patients with non-A non-B hepatitis, and from haemophiliac sera. The antigen was usually detected early in the patient's illness, disappearing as liver function tests returned to normal. In some patients specific antibody appeared during the convalescent phase of the disease. The antigen does not appear to be specific for non-A non-B hepatitis, as it could be detected with similar frequency in patients with hepatitis A or hepatitis B and some patients with other liver disorders. Biochemical and biophysical studies suggest that the antigen is probably an abnormal lipoprotein produced as a result of acute liver damage.     

Sodium currents in mammalian muscle

1. A method is described which allows the approximate computation of membrane current from measurements with three electrodes in the mid-region of a muscle fibre.     

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.     

The So-Called Borderline Patient: Aetiology,Diagnosis, and Treatment

This short paper examines the relationship between borderline psychopathology and various theories regarding the aetiology of this disorder. In the formation of borderline personality organization, distinct structural alterations in personality development are thought to arise from both genetic/neurobiological and environmental/trauma factors. We concur that these variables are instrumental in the formation of borderline personality organization. However, we believe that genetic/neurobiological variables are more closely related to developmental deficits, whereas environmental/trauma factors are primarily associated with either arrested development or regressive phenomenon. Regardless of aetiology, the resultant borderline personality organization disorders may present with comparable symptoms. Further, we hypothesize that the prognosis for response to treatment is related primarily to whether the borderline disorder arises from developmental deficits, arrested development, or regressive phenomena. Diagnostic indicators and treatment considerations for each of the borderline aetiologies are presented.