Interactions of human cytomegalovirus with human fibroblasts

Vonka, Vladimir (Baylor University College of Medicine, Houston, Tex.), and Matilda Benyesh-Melnick. Interactions of human cytomegalovirus with human fibroblasts. J. Bacteriol. 91:213-220. 1966.-Virus attachment of human cytomegalovirus to human embryo lung fibroblasts was found to be temperature-independent, from 4 to 37 C. Prolonged incubation at 4 C, however, resulted in inactivation of a high proportion of attached virus. Virus penetration seemed to be temperature-dependent, occurring at 37 C but not at 4 C. Detailed studies of the growth curve of the virus were made. Cell-associated virus preceded the appearance of virus in the fluid phase by 2 to 5 days. Complement-fixing antigen could be detected, but only when the cytopathic effect was advanced, and it was demonstrable only in the cell-associated fraction. Under methyl cellulose, decreasing the bicarbonate concentration in the overlay from 0.225 to 0.15% resulted in marked increase in plating efficiency with all strains tested. However, varying the concentration of bicarbonate from 0.3 to 0.15% in fluid medium did not influence the growth of virus.     

Tracking down human contamination in ancient human teeth

DNA contamination arising from the manipulation of ancient calcified tissue samples is a poorly understood, yet fundamental, problem that affects the reliability of ancient DNA (aDNA) studies. We have typed the mitochondrial DNA hypervariable region I of the only 6 people involved in the excavation, washing, and subsequent anthropological and genetic study of 23 Neolithic remains excavated from Granollers (Barcelona, Spain) and searched for their presence among the 572 clones generated during the aDNA analyses of teeth from these samples. Of the cloned sequences, 17.13% could be unambiguously identified as contaminants, with those derived from the people involved in the retrieval and washing of the remains present in higher frequencies than those of the anthropologist and genetic researchers. This finding confirms, for the first time, previous hypotheses that teeth samples are most susceptible to contamination at their initial excavation. More worrying, the cloned contaminant sequences exhibit substitutions that can be attributed to DNA damage after the contamination event, and we demonstrate that the level of such damage increases with time: contaminants that are >10 years old have approximately 5 times more damage than those that are recent. Furthermore, we demonstrate that in this data set, the damage rate of the old contaminant sequences is indistinguishable from that of the endogenous DNA sequences. As such, the commonly used argument that miscoding lesions observed among cloned aDNA sequences can be used to support data authenticity is misleading in scenarios where the presence of old contaminant sequences is possible. We argue therefore that the typing of those involved in the manipulation of the ancient human specimens is critical in order to ensure that generated results are accurate.     

Characterization of human beta-interferon-binding sites on human cells

Radioiodinated recombinant human beta-interferon (rHuIFN beta Ser), with almost full (greater than 90%) biological activity, was used to study the binding of human beta-interferon to Daudi cells. Specific binding was not observed with less biologically active (less than or equal to 10%) radioiodinated interferon. The bound radioiodinated interferon was shown to compete with human beta-interferon (HuIFN beta), rHuIFN beta Ser, human alpha-interferon (HuIFN alpha) and with human gamma-interferon (HuIFN gamma). Scatchard plot analyses suggest the presence of about 10,000 binding sites for HuIFN beta/Daudi cell. About 6,600 of these sites can be blocked by HuIFN alpha and 3,700 sites can be blocked by HuIFN gamma. The apparent Kd for HuIFN beta is 2.7 nM. The apparent Kd values for HuIFN alpha and HuIFN gamma are 3.7 and 1.1 nM, respectively. It was possible to demonstrate the cross-linking of HuIFN beta to two macromolecular components of Mr = 128,000 and 103,000. We propose the existence of at least two binding sites for HuIFN beta in Daudi cells, one site recognizing both HuIFN beta and HuIFN gamma, the other site recognizing both HuIFN beta and HuIFN alpha. Each site is capable of recognizing only HuIFN gamma or HuIFN alpha.     

Binding of human carbonic anhydrase to human hemoglobin

The ability of human carbonic anhydrases to interact with human CO-hemoglobin have been studied with the counter-current distribution technique in aqueous/aqueous biphasic systems. The experimental results show that human carbonic anhydrase II interacts with human CO-hemoglobin whereas human carbonic anhydrase I does not. THe interaction between CO-hemoglobin and carbonic anhydrase II was quantified using the theoretical model developed previously for one-to-one interacting systems. [Backman, L. and Shanbhag, V.P. (1979) J. Chromatogr. 171, 1-13]. The apparent association constant was estimated to be 4.1 x 10(5) l mol-1 at pH 8.0 and 21 degrees C for the association of carbonic anhydrase II and CO-hemoglobin.     

Aggregation of biopharmaceuticals in human plasma and human serum

Analytical methods based on light microscopy, 90° light-scattering and surface plasmon resonance (SPR) allowed the characterization of aggregation that can occur when antibodies are mixed with human plasma. Light microscopy showed that aggregates formed when human plasma was mixed with 5% dextrose solutions of Herceptin^® (trastuzumab) or Avastin^® (bevacizumab) but not Remicade^® (infliximab). The aggregates in the plasma-Herceptin^®-5% dextrose solution were globular, size range 0.5–9 μm, with a mean diameter of 4 μm. The aggregates in the plasma-Avastin^®-5% dextrose samples had a mean size of 2 μm. No aggregation was observed when 0.9% NaCl solutions of Herceptin^®, Avastin^® and Remicade^® were mixed with human plasma. 90° light-scattering measurements showed that aggregates were still present 2.5 h after mixing Herceptin^® or Avastin^® with 5% dextrose-plasma solution. A SPR method was utilized to qualitatively describe the extent of interactions of surface-bound antibodies with undiluted human serum. Increased binding was observed in the case of Erbitux^® (cetuximab), whereas no binding was measured for Humira^® (adalimumab). The binding of sera components to 13 monoclonal antibodies was measured and correlated with known serum binding properties of the antibodies. The data presented in this paper provide analytical methods to study the intrinsic and buffer-dependent aggregation tendencies of therapeutic proteins when mixed with human plasma and serum.     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

Characterization of human megakaryocytic colony formation in human plasma

We have analysed the contribution to megakaryocyte colony formation in methylcellulose made by human plasma, serum, media conditioned by phytohemagglutinin (PHA) stimulated leukocytes (PHA-LCM), erythropoietin (EPO) preparations, and platelets. The culture system was used as a bioassay for megakaryocyte colony stimulating activity (Meg-CSA) in plasma samples of patients with perturbed megakaryocytopoiesis. Preparations of heparinized platelet-poor plasma yielded the most consistent results. Platelet-poor plasma of normal subjects will at best facilitate the occasional growth of small megakaryocyte colonies. Colony frequency and size are reproducibly enhanced in the presence of PHA-LCM as a source of exogenous Meg-CSA. Commercially available EPO preparations may vary in their content of activities that influence megakaryocyte colony formation. Addition of these preparations to cultures that contain plasma and PHA-LCM usually does not enhance colony formation. In contrast to platelet-poor plasma, platelet rich plasma and serum are less supportive of megakaryocyte colony growth. It is suggested that this loss of activity may be related to the release of inhibitors by activated platelets or alternatively caused by absorption of activities by platelets. Plasma samples from patients with megakaryocytopoietic dysfunction may contain components that promote colony formation without addition of PHA-LCM or EPO. This phenomenon is consistently observed for patients with severe aplastic anemia and bone marrow transplant recipients after completion of their ablative preparative regimen.     

Inhibition of human factor IXa by human antithrombin.

A procedure is presented for the purification of Factor IX from human plasma. The final product is homogeneous as judged by disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Furthermore, it is completely free of other coagulation component activities. Factor IX is converted to its enzymatically active form by the addition of small quantities of Factor IXa in the presence of calcium ions. This activated species is added to purified antithrombin-heparin cofactor and the interaction is studied in the presence and absence of heparin. Antithrombin-heparin cofactor is found to be a progressive, time-dependent inhibitor of Factor IXa and neutralizes approximately 57% of this enzyme's proteolytic activity within 30 min. The addition of heparin dramatically accelerates the rate of this interaction with virtually complete inhibition of Factor IXa occurring within 15 s. Sodium dodecyl sulfate gel electrophoresis of reduced and nonreduced proteins indicates that antithrombin-heparin cofactor functions as a potent inhibitor of Factor IXa by forming an undissociable complex with the enzyme which is stable in the presence of denaturing or reducing agents (or both). This complex represents a 1:1 stoichiometric combination of enzyme and inhibitor. Heparin increases the rate of formation of this complex without affecting its dissociability or stoichiometry.     

Sensitivity of human germ-cell-tumor cell lines to human interferons

We examined the sensitivity of four human germ-cell-tumor cell lines exhibiting different stages of differentiation to human interferons (IFNs) in vitro. The cell lines were derived from two embryonal carcinomas (NEC 8 and NEC 14), a choriocarcinoma (IMa), and a yolk-sac tumor (HUOT). Treatment with poly I:C induced IFN production in IMa and HUOT cells, but not in NEC-8 and NEC-14 cells. In the two embryonal-carcinoma cell lines, the addition of IFN-alpha, IFN-beta, and IFN-gamma did not prevent infection by vesicular stomatitis virus and encephalomyocarditis virus. Also, in these two lines, 2-5A synthetase was not induced by the addition of IFN-alpha. In contrast, both IMa and HUOT showed sensitivity to the antiviral action of IFN-alpha and IFN-beta against the two viruses, and 2-5A synthetase was induced by IFN-alpha. IFNs added at doses of up to 1000 IU/ml had no antiproliferative effect on NEC 8, NEC 14, and HUOT, whereas colony formation by IMa cells was greatly suppressed by all three forms of IFN. These results indicate that the production of and sensitivity to IFN are developmentally regulated and are related to the level of differentiation of human germ-cell stem cells.     

Antibodies to human immunodeficiency virus in human sera induce cell-mediated lysis of human immunodeficiency virus-infected cells

The capacity of human immunodeficiency virus (HIV) antibody-positive sera from homosexually active men without acquired immune deficiency syndrome to lyse the HIV-infected T cell lines MOLT-4f and CCRF-CEM (CEM) in cooperation with lymphocytes from normal donors was investigated. Twenty-seven HIV antibody-positive sera, most of which enhanced the killing of HIV-infected MOLT-4f and CEM target cells by normal mononuclear cells were studied in detail. HIV antibody-positive sera resulted in lysis at dilutions as high as 1/10,000. HIV antibody-negative sera did not augment lysis of infected target cells. In addition, lysis of uninfected targets was not enhanced in the presence of HIV antibody-positive sera. Because fractionation of the HIV antibody-positive sera on a protein A affinity column resulted in recovery of the activity from the IgG fraction, the extra cytotoxic activity mediated by nonimmune cells in the presence of immune sera appears to be antibody-dependent. Furthermore, the cytotoxic effector cells were in the nonrosetting fraction of lymphocytes and expressed Leu-11 (cluster designation (CD)15) antigens, which is characteristic of cells participating in antibody-dependent cellular cytotoxicity reactions. The antibody specificity of the sera, determined by radioimmunoprecipitation, provides evidence that antibody-dependent cellular cytotoxicity can occur even when there are no detectable antibodies directed against gag proteins. Sera which lacked detectable antibodies to the envelope protein gp120 by radioimmunoprecipitation did not mediate antibody-dependent cellular cytotoxicity.     

Isolation of human monoclonal antibodies that neutralize human rotavirus

A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones-1-2H, 2-3E, and 2-11G-were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 micro g/ml.     

Interaction of human plasmin with human alpha 2-macroglobulin

The steady-state kinetic parameters of plasmin and the alpha 2-macroglobulin (alpha 2M)-plasmin complex toward the chromogenic substrate Val-Leu-Lys-p-nitroanilide (S-2251), in the presence and absence of plasmin competitive inhibitors, have been determined. At pH 7.4 and 22 degrees C, the Km values for plasmin and alpha 2M-plasmin for S-2251 were 0.13 +/- 0.02 mM and 0.3 +/- 0.03 mM. The kcat of this reaction, when catalyzed by alpha 2M-plasmin, was 6.0 +/- 0.5 s-1, a value significantly decreased from the kcat of 11.0 +/- 1.0 s-1, determined when free plasmin was the enzyme. KI values for benzamidine of 0.50 +/- 0.05 mM and 0.23 +/- 0.02 mM were obtained for S-2251 hydrolysis, as catalyzed by alpha 2M-plasmin and plasmin, respectively. When leupeptin was the competitive inhibitor, KI values of 5.0 +/- 0.65 microM and 1.0 +/- 0.1 microM were obtained when alpha 2M-plasmin and plasmin, respectively, were the enzymes employed for catalysis of S-2251 hydrolysis. The comparative rates of reaction of the peptide inhibitor Trasylol (Kunitz basic pancreatic inhibitor) with plasmin and alpha 2M-plasmin were also determined. A concentration of Trasylol of at least 3 orders of magnitude greater for alpha 2M-plasmin than for free plasmin was required to observe inhibition rates on comparable time scales.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant human interferon-gamma inhibits formation of human osteoclast-like cells

Interferon-gamma (IFN-gamma) inhibits osteoclastic bone resorption in vitro, but the mechanism responsible for this inhibition is unknown. We have used a long-term human marrow culture system that forms multinucleated cells (MNC) with osteoclast characteristics to test the effect of recombinant human IFN-gamma on MNC formation. The addition of 1,25-dihydroxy-vitamin D3 (1,25D3) at 10(-8) M to these cultures significantly increased both MNC formation and the number of nuclei per MNC. IFN-gamma at 100 U/ml strongly inhibited both of these effects of 1,25D3 in this system. IFN-gamma significantly inhibited MNC formation at very low concentrations (4 U/ml), with 10 U/ml inhibiting 1,25D3-stimulated MNC formation by 50%. In contrast, 100 U/ml of IFN-gamma were required to inhibit the growth of granulocyte-macrophage colony-forming cells, the probable progenitor for MNC, by 50%. Treatment of cultures with IFN-gamma for only the first or last week of culture significantly inhibited MNC formation stimulated by 1,25D3. Autoradiographic studies with [3H]thymidine showed that IFN-gamma did not inhibit proliferation of precursors for MNC. Additionally, IFN-gamma inhibited MNC formation stimulated by parathyroid hormone or interleukin 1. These results suggest that IFN-gamma inhibits MNC formation, and that IFN-gamma inhibits bone resorption in part by inhibiting osteoclast formation.