Jay Valinsky

Inactivation of viruses in blood with aluminum phthalocyanine derivatives

The inactivation of viruses added to whole blood and a red cell concentrate with aluminum phthalocyanine and its sulfonated derivatives was studied. A cell‐free form of vesicular stomatitis virus (VSV), used as a model, was completely inactivated (greater than 10(4) infectious units; TCID50) on treatment of whole blood with 10 microM (10 mumol/L) aluminum phthalocyanine chloride (AIPs) and visible light dosage of 88 to 176 J per cm2. At 44 J per cm2, complete VSV inactivation was achieved on raising the concentration of AIPc to 25 microM (25 mumol/L). Results at least as good were achieved on similar treatment of a red cell concentrate. Also inactivated were a cell‐associated form of VSV and both cell‐free and cell‐associated forms of human immunodeficiency virus; encephalomyocarditis virus, used as a model for non‐lipid‐enveloped viruses, was not inactivated by this procedure. This inactivation of cell‐free VSV suggests that a similar degree of inactivation could be achieved with a lower concentration of the sulfonated forms of aluminum phthalocyanine. Throughout the above studies, red cell integrity was well maintained, as judged by the absence of hemoglobin release (less than or equal to 2%) during the course of treatment or on subsequent storage. Red cell osmotic fragility was decreased on treatment of whole blood with AIPc. This study indicates that AIPc may be a promising method for the inactivation of viruses in cellular blood products.

Phorbol ester-induced differentiation of U937 cells enhances attachment to fibronectin and distinctly modulates the α5β1 and α4β1 fibronectin receptors☆

Monocyte interaction with fibronectin (Fn) involves specific cell surface receptors and results in cell attachment and differentiation. We have studied the regulation of these receptors using the promonocytic cell line U937 and its PMA-induced differentiation as a model. We recently reported that U937 cells interact with two sites in Fn, RGD and CS-1, via two independent receptors (O.C. Ferreira, A. Garcia-Pardo, and C. Bianco (1990)J. Exp. Med. 171, 351). In this study we have determined the effects of PMA on the interaction of U937 cells with both sites in Fn. PMA-U937 cells showed an enhanced attachment to Fn and to an RGD-containing 80-kDa Fn fragment. This enhancement paralleled a two- to threefold increase in the surface expression of the RGD-dependent receptor α5β1. An anti-α5β1 mAb completely inhibited cell adhesion to Fn and to the 80-kDa fragment. α5β1 receptors from untreated and PMA-treated U937 cells were isolated on 80-kDa-Sepharose columns and shown to contain a similar complex of 152125-kDa proteins, although proteins from PMA-treated cells had slightly faster mobility on SDS-gels. In contrast, the total number of PMA-U937 cells adhering to a 38-kDa Fn fragment (containing the CS-1 site) was lower when compared to that of untreated cells. This decrease was accompanied by a 50% loss of cell surface α4β1, the specific receptor for CS-1. Our results indicate that differentiation of U937 cells enhances adhesion to Fn primarily by up-regulating the α5β1 Fn receptor. PMA also induces a down-regulation of α4β1, suggesting that these two integrins play different roles during monocyte differentiation.

Lack of Evidence for HIV Type 1-Related SIVcpz Infection in Captive and Wild Chimpanzees (Pan troglodytes verus) in West Africa

Serum from 387 chimpanzees (Pan troglodytes verus), caught in the wild or bred in captivity, was tested for antibody to HIV-1 and HIV-2, using second- and third-generation enzyme immunoassays. Six samples were repeatedly positive; however, only one of these was Western blot positive. Serial sera drawn before and after the Western blot-positive samples were seronegative, and thus we conclude that this sample represented specimen contamination, or mislabeling. Thus, none of the 387 Pan troglodytes verus from West Africa were spontaneously infected with SIVcpz. Chimpanzees are known to be exquisitely susceptible to infection with HIV-1 when experimentally inoculated, and thus our findings suggest that HIV-1-related viruses do not exist in Pan troglodytes verus in the wild. As it has been convincingly shown that SIVcpz exists in wild Pan troglodytes troglodytes in Central Africa, this suggests that HIV-1 arose in Central Africa, but not in West Africa.

Fibronectin Receptors of Mononuclear Phagocytes: Binding Characteristics and Biochemical Isolation

Fibronectin receptors on mononuclear phagocytes are involved in the localization of monocytes at inflammatory sites and in the subsequent expression of macrophage-like phenotypes. In this study, we have investigated the hypothesis that proteolytically derived fragments of fibronectin may interfere with binding of fibronectin to monocytes in the extracellular matrix. We report on the reactivity of U937 cells with an 80-kDa tryptic fragment of fibronectin which contains the cell-binding domain but lacks the gelatin/collagen-binding domain. U937 cells attached to surfaces coated with the 80-kDa fragment as well as with intact fibronectin. Preincubation of the cells with the 80-kDa fragment inhibited attachment to both surfaces while intact fibronectin had little or no inhibitory effect. The Ki for inhibition of attachment (0.5 microM) was consistent with the Kd for binding of the 3H-labeled 80-kDa fragment (0.34 microM) to U937 cells in suspension. There were 4-5 x 10(5) 80-kDa binding sites per cell. The relatively high affinity of the 80-kDa fragment for the monocyte surface permitted the isolation and characterization of fibronectin-binding proteins from U937 cells and peripheral blood monocytes by affinity chromatography. When octylglucoside lysates of lactoperoxidase iodinated cells were applied to 80-kDa-Sepharose columns, a polypeptide complex of 152/125 kDa was eluted with the synthetic peptide GRGDSPC, but not with GRGESP. This complex resolved into a single diffuse band of 144 kDa upon reduction. Binding of the protein complex to the affinity column required divalent cations. The complex bound to wheat germ agglutinin and could be specifically eluted by N-acetylglucosamine. Similar cell-surface proteins were isolated from peripheral blood monocytes.

Individual Donor Nucleic Acid Amplification Testing for Detection of West Nile Virus

ABSTRACT We have developed an economical, high-throughput nucleic acid amplification test (NAT) for blood-borne viruses, suitable for use in the screening of plasma samples from individual blood donors. This assay system includes a semiautomated procedure, using 96-well glass fiber plates for the extraction of viral nucleic acids from plasma and “universal beacon” technology which permits the detection of all genotypes of highly variable viruses (e.g., human immunodeficiency virus and hepatitis C virus). In this detection system, two fluorescent- detection technologies were employed successfully in a single tube: molecular beacon for West Nile virus (WNV) detection using a 6-carboxyfluorescein fluorophore and TaqMan for internal control detection using a VIC fluorophore. To establish proof of concept, we focused on the development of a robust individual donor NAT for WNV. The assay showed no reactivity to 15 other viruses tested or to 420 blood donor samples from the WNV pre-epidemic season. No cross-contamination was observed on an alternating positive-/negative-well test. The sensitivity (limit of detection, 95%) of the assay for WNV is between 3.79 and 16.3 RNA copies/ml, depending on which material was used as a standard. The assay detected all positive blood donation samples identified by the Roche WNV NAT. The assay can be performed qualitatively for screening and quantitatively for confirmation.

T cell immunophenotypes and DR antigen expression in intravenous drug users: relationship to human immunodeficiency virus serology

Thirty-six intravenous drug users were studied for peripheral blood mononuclear cell (PBMC) immunophenotypes and human immunodeficiency virus (HIV) serological profiles. This population has a high risk for developing HIV infection. Half (18/36) were HIV antibody (Ab) negative (-) and half were positive (+). Total T lymphocytes (CD3+ and CD2+) were not different between HIV Ab-negative and HIV-positive groups. Unactivated T(CD3+DR-) cells/mm3 were less (p = 0.003) in HIV Ab-positive patients (1,467 +/- 628) compared to HIV Ab-negative patients (2,190 +/- 695). T-helper (CD4+) cells/mm3 were also less in HIV Ab-positive patients (762 +/- 344 vs. 1,161 +/- 419, p = 0.005). The most significant difference was in activated T lymphocyte CD3+DR+) percentages where the mean was 9.6% in those HIV Ab-positive compared to 3.8% in seronegatives (p less than 0.001). Preliminary studies showed that in vitro naloxone treatment of PBMC had no effects on immunophenotypic expression except for CD3+DR+ lymphocytes, where a significant reduction was observed in the HIV Ab-positive group (p = 0.022) but not in the HIV ab-negative group. These findings suggest that in certain populations, activated T cells may be an early manifestation of HIV infection.

Close Relationships between Neopterin and Beta-2-Microglobulin Levels in Intravenous Drug Abusers

Serum from 36 intravenous drug abusers without acquired immunodeficiency syndrome (AIDS) or AIDS-related complex were tested for concentrations of neopterin and beta 2-microglobulin. The seroprevalence of human immunodeficiency virus (HIV) antibody in this group was 50%. Previous studies of this group showed that the HIV antibody positive patients had significant increases in HLA-DR expression on peripheral blood lymphocytes and increases in serum soluble CD8 antigen. Both neopterin and beta 2-microglobulin concentrations were significantly higher in the HIV antibody seropositive patients compared to the seronegative patients (p = 0.001 and p = 0.005, respectively). A highly significant positive correlation between neopterin and beta 2-microglobulin was found for the seropositive patients (r = 0.8879, p less than 0.0001) as well as for the entire group (r = 0.6054, p = 0.0002). Significant positive correlations were also found between neopterin or beta 2-microglobulin and the percent DR + T cells and CD8 antigen levels, although these correlations were not as significant as that observed between neopterin and beta 2-microglobulin. No relationships were found between neopterin or beta 2-microglobulin and total CD4 cell concentrations or CD4/CD8 ratios. These data demonstrate the significant interrelationships between various immune activation markers in a population at risk for developing AIDS.

The integration of high-throughput testing of blood donors for cardiovascular disease risk assessment and prevention

BACKGROUND AND OBJECTIVES Some blood centers provide health screening as a public health measure and to encourage donation. The goal of the current study was to provide cardiovascular disease (CVD) screening to donors using high-throughput testing and web-based communications. MATERIALS AND METHODS CVD risk screening was offered to donors at selected mobile drives in a large metropolitan area. Risk factors were determined by donor questionnaire, laboratory testing (total cholesterol, HDL levels and hemoglobin A1c), and blood pressure measurement. Results were reported to participants via mail and website. A 60-day follow up web-based survey was sent to participants via email to assess the impact of the program on donors behavior. RESULTS 9435 donors, 17-75 years old participated with the following risk factors: 61.3% BMIs>25, 28.8% high total cholesterol, and 31.4% lower than recommended HDL levels. 25.3% of donors that responded to the follow up survey went to see their health care provider based on screening results and 9% of these received new or modified treatment. CONCLUSION In our sample, blood donors are healthier than the general population, but many still have CVD risk factors, particularly obesity. CVD screening can be successfully used to make donors aware of this important health information and some donors act on this information.