Cytomegalovirus-infected cell polypeptides immune-precipitated by sera from children with congenital and perinatal infections.

Congenital or perinatally acquired human cytomegalovirus (CMV) infections in children may be symptomatic or asymptomatic. In this study, we characterized the electrophoretic properties of CMV-infected cell polypeptides immune-precipitated by sera from children with different types of CMV infections from birth to 4 years of age. Sodium dodecyl sulfate-polyacrylamide gel analysis of immune precipitates formed with radiolabeled extracts of cells infected with CMV strain AD169 showed the following. (i) Electrophoretic profiles of CMV polypeptides immune-precipitated by sera from children with perinatal and congenital infections were similar. At least 11 polypeptides with apparent molecular weights of 150,000, 140,000, 110,000, 100,000, 74,000, 66,000, 50,000, 49,000, 34,000, 25,000, and 20,000 were precipitated. Antibody titer in anticomplement immunofluorescence tests and virus titer in urine correlated with the intensity of polypeptide profiles in autoradiograms. (ii) The initial immune response of children with symptomatic congenital infections was delayed as compared to that of children with asymptomatic congenital and perinatal CMV infections. Sera obtained serially from symptomatic children for years after birth continued to precipitate CMV polypeptides, whereas sera from children with subclinical congenital infections precipitated lesser amounts over time. (iii) Immune precipitates obtained with sera from CMV-infected patients and with monoclonal antibodies to CMV contained polypeptides with comparable electrophoretic and immunological properties.     

Monoclonal antibodies to human cytomegalovirus: three surface membrane proteins with unique immunological and electrophoretic properties specify cross-reactive determinants

Seventy-seven clones of hybridomas selected for reactivity by immunofluorescence with human cytomegalovirus (CMV)-infected cells were produced by fusing mouse myeloma cells with the spleen cells of mice immunized with CMV strain AD169. The clones were classified into seven groups on the basis of the electrophoretic properties of the polypeptides immune precipitated from extracts of CMV-infected cells. Studies on the three groups of monoclonal antibodies directed against CMV surface membrane antigens showed the following. Clones in each group were differentiated by immunoglobulin subclass, neutralizing activity, and reactivity with the antigenic domains of proteins exposed on the surface membranes of intact CMV-infected cells. Monoclonal antibodies in each group precipitated one slowly migrating protein and multiple faster migrating forms which shared antigenic determinants. The first group of monoclonal antibodies precipitated four glycosylated polypeptides with apparent molecular weights of 130,000, 110,000, 100,000, and 60,000. Monoclonal antibody CH51 of this group neutralized infectious virus but failed to react with antigenic domains on the surfaces of infected cells. The second group of monoclonal antibodies precipitated four polypeptides with apparent molecular weights of approximately 66,000, 55,000, 50,000, and 46,000. Monoclonal antibodies CH65 and CH134 in this group had neutralizing activity and reacted with antigenic domains of proteins exposed on the surface of CMV-infected cells. The third group of monoclonal antibodies precipitated four polypeptides with apparent molecular weights of 49,000, 48,000, 34,000, and 25,000. Serological analysis of 15 naturally occurring CMV strains with a panel of monoclonal antibodies to surface membrane proteins showed that the antigenic determinants reactive with the antibodies tested were conserved in all of the strains. Monoclonal antibodies to surface membrane proteins on CMV-infected cells may prove to be valuable reagents for identification of virus isolates.     

Comparison of immunofluorescence, enzyme immunoassay, and Western blot (immunoblot) methods for detection of antibody to human T-cell leukemia virus type I.

A total of 3,349 serum samples were screened by the immunofluorescence (IF) method for antibody to human T-cell leukemia virus type I (HTLV-I). Only 9 of 2,409 specimens from selected individuals, blood bank donors, patients with encephalitis-meningitis, and human immunodeficiency virus antibody-positive homosexual or bisexual men were reactive by IF. In addition, 940 serum samples from intravenous drug abusers were tested by IF and also by an HTLV-I enzyme immunoassay (EIA) method. Of these, 222 (24%) were positive for both HTLV-I and HTLV-II antigens by IF, and 191 of these 222 were also reactive in the HTLV-I EIA. Of the 31 IF-positive, EIA-negative serum samples, 20 exhibited optical density readings greater than or equal to 70% of the positive cutoff in the EIA, and 29 samples reacted with 1 or more bands in the Western blot (immunoblot) test. An additional 10 specimens that were EIA negative reacted only with HTLV-I by IF. Differences in staining morphology and in reactions on HTLV-I and HTLV-II antigens before and after absorption of the serum specimens with HTLV-I and HTLV-II-infected cell pellets revealed six distinct serological patterns by IF. These results indicate that infections by HTLV-I or by another closely related retrovirus(es) occur in California. Further studies utilizing statistically valid sampling methods are needed to estimate true prevalence rates among various groups. IF and Western blot tests should supplement the EIA method to maximize sensitivity and specificity of test procedures.     

Imaging tumor angiogenesis with fluorescent proteins

We have developed three unique mouse models to image angiogenesis with fluorescent proteins, which are described in this review. First, we have adapted the surgical orthotopic implantation (SOI) model to image angiogenesis of human tumors labeled with green fluorescent protein (GFP) transplanted in nude mice. The nonluminous induced capillaries are clearly visible by contrast against the very bright tumor fluorescence examined either intravitally or by whole-body imaging in real time. Intravital images of an SOI model of human pancreatic tumors expressing GFP visualized angiogenic capillaries at both primary and metastatic sites. Whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period in an SOI model of human breast cancer expressing GFP. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold and enabling vessels to be externally visualized in GFP-expressing tumors growing on internal organs. The second model utilizes dual-color fluorescence imaging, effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice that express GFP in all cells. This dual-color model visualizes with great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis. The GFP-expressing tumor vasculature, both nascent and mature, are readily distinguished interacting with the RFP-expressing tumor cells. Using a spectral imaging system based on liquid crystal tunable filters, we were able to separate individual spectral species on a pixel-by-pixel basis. Such techniques non-invasively visualized the presence of host GFP-expressing vessels within an RFP-labeled orthotopic human breast tumor by real-time whole-body imaging. The third model involves a transgenic mouse in which the regulatory elements of the stem cell marker nestin drive GFP. The nestin-GFP mouse expresses GFP in areas of the brain, hair follicle stem cells, and in a network of blood vessels in the skin interconnecting hair follicles. RFP-expressing tumors transplanted to nestin-GFP mice enable specific visualization of nascent vessels in skin-growing tumors such as melanoma. Thus, fluorescent proteins expressed in vivo offer very high resolution and sensitivity for real-time imaging of angiogenesis.     

Comparison of MICs of ceftiofur and other antimicrobial agents against bacterial pathogens of swine from the United States, Canada, and Denmark.

The MICs of ceftiofur and other antimicrobial agents, tested for comparison, for 515 bacterial isolates of pigs from the United States, Canada, and Denmark with various diseases were compared. The organisms tested included Actinobacillus pleuropneumoniae, Escherichia coli, Pasteurella multocida, Salmonella choleraesuis, Salmonella typhimurium, Streptococcus suis, Streptococcus dysgalactiae subsp. equisimilis, Streptococcus equi subsp. equi, and Streptococcus equi subsp. zooepidemicus. In addition to ceftiofur, the following antimicrobial agents or combinations were tested: enrofloxacin, ampicillin, sulfamethazine, trimethoprim-sulfadiazine (1:19), erythromycin, lincomycin, spectinomycin, lincomycin-spectinomycin (1:8), tilmicosin, and tetracycline. Tilmicosin was only tested against the U.S. isolates. Overall, ceftiofur and enrofloxacin were the most active antimicrobial agents tested against all isolates, with MICs inhibiting 90% of isolates tested (MIC90s) of < or = 2.0 and < or = 1.0 microgram/ml, respectively. Erythromycin, sulfamethazine, spectinomycin, and lincomycin demonstrated limited activity against all of the organisms tested, with MIC90s of > or = 8.0, > or = 256.0, > or = 32.0, and > or = 16.0 micrograms/ml, respectively. Trimethoprim-sulfadiazine was active against isolates of A. pleuropneumoniae, S. choleraesuis, S. typhimurium, P. multocida, S. equi, and S. suis (MIC90s, < or = 0.5 microgram/ml) but was less active against the E. coli strains tested (MIC90, > 16.0 micrograms/ml). Ampicillin was active against the P. multocida, S. suis, and S. equi isolates tested (MIC90s, 0.5, 0.06, and 0.06 micrograms/ml, respectively) and was moderately active against S. typhimurium (MIC90s, 2.0 micrograms/ml). However, this antimicrobial agent was much less active when it was tested against A. pleuropneumoniae, S. cholerae-suis, and E. coli (MIC90s, 16.0, > 32.0, and 32.0 micrograms/ml, respectively). Against the U.S. isolates of A. pleuropneumoniae and P. multocida, tilmicosin was moderately active (MIC90s, 4.0 and 8.0 micrograms/ml, respectively). However, this compound was not active against the remaining U.S. isolates (MIC90s, > 64.0 micrograms/ml). Differences in the MICs from one country to another were not detected with enrofloxacin, ceftiofur, or lincomycin for the strains tested, but variations in the MICs of the remaining antimicrobial agents were observed.     

Vitronectin binds to Pneumocystis carinii and mediates organism attachment to cultured lung epithelial cells.

Pneumocystis carinii attaches to alveolar epithelial cells during the development of pneumonia. Adhesive proteins found within the alveolar space have been proposed to mediate P. carinii adherence to lung cells. Vitronectin (Vn), a 75-kDa glycoprotein present in the lower respiratory tract, has substantial cell-adhesive properties and might participate in the host-parasite interaction during P. carinii pneumonia. To address whether Vn binds to P. carinii, we studied the interaction of radiolabeled Vn with purified P. carinii organisms. Vn bound to P. carinii, occupying an estimated 5.47 x 10(5) binding sites per organism, with an affinity constant, Kd, of 4.24 x 10(-7) M. Interestingly, the interaction of Vn with P. carinii was not mediated through the Arg-Gly-Asp cell-adhesive domain of Vn. Addition of Arg-Gly-Asp-Ser (RGDS) peptides did not inhibit binding. In contrast, Vn binding to P. carinii was substantially inhibited by the addition of heparin or by digesting the organisms with heparitinase, suggesting that P. carinii may interact with the glycosaminoglycan-binding domain of Vn. To determine whether Vn might enhance P. carinii attachment to lung epithelial cells, we studied the binding of 51Cr-labeled P. carinii to cultured A549 lung cells. Addition of Vn resulted in significantly increased binding of P. carinii to A549 cells, whereas a neutralizing anti-Vn serum substantially reduced the binding of P. carinii to A549 cells. These data suggest that Vn binds to P. carinii and that Vn might provide an additional means by which P. carinii attaches to respiratory epithelial cells.     

Neutralization of lymphocyte immortalization by different strains of Epstein-Barr virus with a murine monoclonal antibody.

A murine monoclonal antibody was raised against the B95-8 strain of Epstein-Barr virus (EBV), which was isolated from a case of mononucleosis after blood transfusion (Hoffman et al. Proc. Natl. Acad. Sci. U.S.A. 77:2979, 1980). We provide evidence that neutralization of immortalization by this monoclonal antibody is virus specific, since its potency was inversely related to the dose of challenge virus. Furthermore, the monoclonal antibody recognized antigens on viruses grown in human as well as in marmoset cells. We show that this monoclonal antibody neutralized three other transforming strains of EBV originating, respectively, from American patients with mononucleosis and fatal polyclonal lymphoma and from an African child with Burkitt lymphoma. However the antibody did not neutralize or detect antigens by immunofluorescence in the W91 strain of EBV. The hybridoma antibody did neutralize other EBV strains derived from the same Burkitt lymphoma cell line (Nyevu), as was the case with the W91 strain. This monoclonal antibody provides clear evidence of antigenic differences on the surface of EBVs and will ultimately prove useful in defining the antigenic site on EBV which elicits neutralizing antibody.     

Identification and characterization of a Candida albicans-binding proteoglycan secreted from rat submandibular salivary glands.

A previously identified Candida albicans-binding glycoprotein secreted from rat submandibular glands (RSMG) has been further purified from an aqueous RSMG extract by ion-exchange chromatography and gel filtration. Biochemical analysis of the glycoprotein revealed high levels of uronic acid and sulfate, suggesting that it was a proteoglycan. Its amino acid and carbohydrate compositions were similar to those observed for other proteoglycans and differed significantly from those of RSMG mucin, the major secretory glycoprotein of RSMG. In addition, the apparent molecular weight of the glycoprotein was reduced following treatment with either chondroitinase ABC or heparitinase, demonstrating the presence of chondroitin sulfate and heparan sulfate. On the basis of its structure and anatomical source, the glycoprotein is referred to as submandibular gland secreted proteoglycan 1 (SGSP1). SGSP1 also binds monoclonal antibody 1F9, which recognizes the human blood group A carbohydrate epitope found on RSMG mucin. Hence, SGSP1 appears to be a hybrid molecule with carbohydrate structures found in both proteoglycans and RSMG mucin. Enzymatic digestion of SGSP1, followed by its interaction with a radiolabelled C. albicans strain in a filter-binding assay, demonstrated that binding to this strain appears to be mediated primarily via the heparan sulfate side chains of SGSP1 and not via the blood group A oligosaccharide.     

The effect of an intercollegiate soccer game on maximal power performance.

The effect of a competitive soccer match on maximal power performance was assessed on 19 members of an NCAA Division III female soccer team. Performance testing occurred within 24 hours prior to the game (Pre), immediately postgame (IP), and 24 hours postgame (24P). Each subject performed a squat jump (SJ) and countermovement jump (CMJ). Comparisons between starters (n = 10) and nonstarters (n = 9) revealed no between-group differences in power performance at IP, but starters were found to have significantly lower power and force measures at 24P than nonstarters. There were significant correlations between playing time and peak force during the SJ at 24P (r = -0.47), and between playing time and peak power during the SJ at IP (r = -0.57) and 24P (r = -0.51), and during the CMJ at IP (r = -0.49). Comparisons between different positions revealed no differential fatigue patterns. Results of this study show that power performance appears to be maintained for the duration of a soccer match but declines significantly within 24 hours after the match. Position played does not appear to affect performance decrements seen at 24 hours postmatch.     

B-cell activation and allosensitization after left ventricular assist device implantation is due to T-cell activation and CD40 ligand expression

Left ventricular assist device (LVAD) implantation is frequently complicated by B-cell activation and allosensitization, posing a significant risk to successful transplant outcome. This study investigated whether B-cell hyperreactivity and alloantibody production in LVAD recipients involves T-cell dependent pathways. T-cell calcium flux and nuclear translocation of NFATc were used to determine states of T-cell activation. Flow cytometry was used to assess human T- and B-cell activation after culture with LVAD-derived biomaterial particles. Sera from LVAD recipients and controls were tested for the presence of anti-HLA antibodies, and for soluble CD40 ligand. LVAD-derived biomaterial induced rapid and sustained calcium flux into normal T cells, resulting in calcineurin-dependent nuclear translocation of NFATc. This resulted in increased T-cell expression of CD40 ligand and subsequent B-cell activation, which was reduced by inhibitors of T-cell activation (CsA or anti-CD25 mAb) or by anti-CD40 ligand mAb. LVAD recipients demonstrated higher frequencies of anti-HLA antibodies and serum levels of soluble CD40 ligand compared with heart failure controls. The results indicate that exposure of human mononuclear cells to LVAD-derived biomaterial leads to T-cell dependent B-cell activation via CD40--CD40 ligand interaction, and suggest that treatment with calcineurin inhibitors or monoclonal antibodies against either CD25 or CD40 ligand could be effective at preventing B-cell hyperreactivity and allosensitization after LVAD implantation.