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.     

Phosphonate-phospholipid analogues inhibit human phospholipase A2

A phosphonate-containing phospholipid (PL) analogue (Compound 1) designed as a transition-state inhibitor competively inhibits non-human extracellular PLA₂ at a mole fraction of 0.003 in the kinetic scooting mode (Jain et al., Biochem. 284135 (1989)). To further profile the activity of Compound 1, we examined its activity with purified human enzyme and in whole cell systems. Compound 1 effectively inhibited a 14 kDa human PLA₂ purified from joint synovial fluid of patients with rheumatoid arthritis using³H-AA labeledE. coli as substrate (IC₅₀=1.7M) and a high MW PLA₂ (110 kDa) isolated from the cytosol of a human monocytic cell line, U-937, which selectively hydrolyzes AA-containing PL (IC₅₀=165M). It failed to reduce A23187-induced PGE₂ or LTC₄ production by human adherent monocytes or LTB₄ release from human neutrophils which may be due, in part, to poor membrane partitioning.     

Detection of Serum Antibodies to Ovine Progressive Pneumonia Virus in Sheep by Using a Caprine Arthritis-Encephalitis Virus Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [³⁵S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.     

Fibronectin-facilitated invasion of T84 eukaryotic cells by Campylobacter jejuni occurs preferentially at the basolateral cell surface

Previous studies have indicated that the ability to bind to fibronectin is a key feature in successful cell invasion by Campylobacter jejuni. Given the spatial distribution of fibronectin and the architecture of the epithelium, this suggests the possibility that C. jejuni cell invasion might preferentially occur at the basolateral cell surface. To test this hypothesis, we examined the interaction of C. jejuni with T84 human colonic cells. When grown under the appropriate conditions, T84 cells form a polarized cell monolayer. C. jejuni translocation of a T84 cell monolayer appeared to occur via a paracellular (extracellular) route as opposed to a transcellular (intracellular) route based on the finding that a C. jejuni noninvasive mutant translocated as efficiently as its isogenic parent. Additional studies revealed that two distinct C. jejuni wild-type isolates could compete with one another for host cell receptors, whereas a C. jejuni fibronectin-binding-deficient mutant could not compete with a wild-type isolate for host cell receptors. Further, C. jejuni adherence and internalization were significantly inhibited by antifibronectin antibodies but only when cells were first treated with EGTA to expose basolateral cell surfaces. Together, these results support the theory that C. jejuni invasion occurs preferentially at the basolateral surface of eukaryotic cells.     

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.     

T-cell variant of classical Hodgkin's lymphoma with nodal and cutaneous manifestations demonstrated by single-cell polymerase chain reaction

The atypical cells of CD30(+) cutaneous lymphoproliferative disorders (CD30CLD) are commonly of T-cell origin and frequently have a similar morphology as Hodgkin or Reed-Sternberg cells of Hodgkin's lymphoma (HL). HL is one of the tumors associated with CD30CLD. Although most studies support a B-cell derivation of the tumor cells in HL, recently a few cases of classical HL with T-cell genotype have been reported. We report a patient who presented with CD30CLD whose lymph nodes showed classical HL of mixed cellularity subtype at presentation. By single-cell PCR, the same clonal gene rearrangements of the T cell receptor-beta gene locus could be assigned to the CD30(+) and CD15(+) cells of both skin and lymph node. In a lymph node biopsy specimen taken in relapse after several courses of chemotherapy, the CD30(+) tumor cells were abundant. The T cell-derived tumor cells displayed aberrant expression of the Pax-5 gene in all specimens. A common clonal origin of both CD30CLD and HL of the lymph node in the patient presented here suggests that HL with T-cell genotype exists in association with CD30CLD as well as in sporadic cases and may share clonal origin with the skin tumor.     

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.