Inhibition of human immunodeficiency virus type 1 (HIV) gag gene expression by an antisense oligodeoxynucleotide phosphorothioate.

We have employed a cos-like monkey kidney cell line (B4.14) transfected with plasmids pCMVgag-pol-rre-r (containing the HIV gag and pol genes) and pCMVrev (containing the HIV rev gene), as a model to investigate whether antisense constructs could interfere with specific HIV gene and protein expression. We utilized an antisense construct (GP12A) directed against a non-regulatory region of the HIV genome, to transfect cells that expressed the above-mentioned HIV genes. Our results show that GP12A was able to attenuate levels of relevant HIV mRNA and gag proteins in the absence of cytotoxic effects.

Antiviral activity and protection of cells against human immunodeficiency virus type-1 using an antisense oligodeoxyribonucleotide phosphorothioate complementary to the 5'-LTR region of the viral genome.

A COS-like monkey kidney cell line stably transfected with the plasmids pCMVgagpol-rre-r with the gag and pol genes, and pCMV rev with the rev gene of HIV-1 derived from the cDNA clone BH10, was used as a model for assessing the effectiveness of antisense (AS) constructs, A 20-mer oligodeoxyribonucleotide (oligo) phosphorothioate sequence (5'-CCG CCC CTC GCC TCT TGC CG) complementary to a portion of the 5'-long terminal repeat (5'-LTR) of the HIV-1 genome was tested for its inhibitory effects on the biologically important processes of HIV-1 replication and proliferation. We observed a concentration-dependent inhibition of HIV protein synthesis. Desitometric analysis of data from Western blot analysis showed sequence-specific and concentration-dependent oligo inhibition of p24 viral core antigen formation in the low-microM range. When lipofectin was used as a delivery vehicle, a markedly increased potentiation of the AS activity of the sequence was observed at a lower concentration (0.1 microM), following a 24-h preincubation. The AS construct specifically inhibited intracellular p24 production in chronically HIV-1-infected cells of lymphoid origin (H9/IIIB cells) by 95%, resulting in a 15-fold inhibitory effect relative to a similar sequence thiolated at only seven single-base positions. A concentration-dependent attenuation in the reverse transcriptase activity and a reduction in viral p24 level was observed in the culture supernatant of AS-pretreated HIV-1-infected phytohemagglutinin A-stimulated human cord blood mononuclear cells. Incubation of a HIV-1-infected lymphoid cell line with AS sequence resulted in a marked reduction in syncytium formation, and therefore protected cells from the cytopathic effects of the virus. Furthermore, the AS oligo did not appear to be cytotoxic in cell growth rate and colony-forming ability assays. The AS oligo described in this report is a useful new tool for the molecular analysis of HIV-1 gene expression and proliferation, and may have potential as a therapeutic agent.

Human monoclonal antibody BT32/A6 and a cell cycle-independent glioma-associated surface antigen.

The purpose of this study was to ascertain how various growth parameters may influence the labeling of SK-MG-1, a human glioma cell line, by BT32/A6, a human immunoglobulin M monoclonal antibody (MAb). By growing SK-MG-1 cells at different culture split ratios, significant trends in cell growth rate, culture viability, and cell cycle state were produced. Labeling of SK-MG-1 cells by BT32/A6, however, was shown to be unaffected by culture split ratio (p > 0.05) and is therefore independent of cell growth rate, culture viability, and cell cycle state. Using flow cytometry and fluorescence-activated cell sorting, BT32/A6 was shown to label a cell surface antigen on viable, clonogenic cells of SK-MG-1. Approximately 100% of SK-MG-1 cells were shown by flow cytometry to express the BT32/A6 antigen. The recognition of a glioma-associated, cell cycle-independent surface antigen by MAb BT32/A6 makes it a promising candidate for further studies aimed at elucidating its usefulness as an adjunct in the treatment of human malignant gliomas.

Effect of acute experimental influenza A virus pneumonia on concentration of alpha 1-acid glycoprotein in mouse serum.

In mice, the mean serum concentration of the acute-phase reactant alpha 1-acid glycoprotein increased 34-48% over 14 days following experimental induction of pneumonitis by intranasal inoculation of influenza A virus. Inoculation of undiluted (hemagglutination titer 640) and 10(-1) dilution of virus was followed by development of maximum concentrations of alpha 1-acid glycoprotein in serum at seven days, of 334 micrograms/ml, compared to a concentration in control mice inoculated with irradiated inactivated virus of 225 micrograms/ml (P = 0.002). Infection with 10(-2) virus yielded a peak serum alpha 1-acid glycoprotein of 301 micrograms/ml at four days, 34% higher than in control mice at four days (P = 0.04). There were no differences in alpha 1-acid glycoprotein concentrations among virus-infected mice. Influenza A virus pneumonitis was confirmed histologically, by virus isolation, and by serologic testing, but no inoculum-dependent differences were observed. On day 7, there was a direct relationship demonstrated between the severity of pneumonitis evaluated histologically and the serum alpha 1-acid glycoprotein concentration (r = 0.50; P less than 0.02). Influenza A pneumonia in mice is associated with increased concentrations of alpha 1-acid glycoprotein in serum; the increase may be directly related to the severity of the pulmonary inflammation.

Metabolic engineering of Escherichia coli to enhance phenylalanine production.

The global regulatory system of Escherichia coli, carbon storage regulator (Csr), was engineered to increase the intracellular concentration of phosphoenolpyruvate. We examined the effects of csrA and csrD mutations and csrB overexpression on phenylalanine production in E. coli NST37 (NST). Overexpression of csrB led to significantly greater phenylalanine production than csrA and csrD mutations (2.33 vs 1.67 and 1.61 g l(-1), respectively; P < 0.01). Furthermore, the overexpression of csrB was confirmed by the observed increase in csrB transcription level. We also determined the effect of overexpressing transketolase A (TktA) or glucose-6-phosphate dehydrogenase (Zwf) in NST and the csrA mutant of NST (NSTCSRA) on phenylalanine production. The NSTCSRA strain overexpressing TktA (NSTCSRA [pTktA]) produced significantly more phenylalanine than that of Zwf (2.39 vs 1.61 g l(-1); P > 0.01). Furthermore, we examined the effect of overexpressing TktA, 3-deoxy-D: -arabino-heptulosonate-7-phosphate synthase (AroF(FR)), and chorismate mutase/prephenate dehydratase (PheA(FR)) together in NSTCSRA (NSTCSRA [pTkaFpA]). It is interesting to note that NSTCSRA [pTkaFpA] produced significantly less phenylalanine than both NSTCSRA [pTktA] and NST overexpressing csrB (NST [pCsrB]) (1.84 vs 2.39 and 2.33 g l(-1), respectively; P < 0.01). Thus, csrB overexpression or csrA mutation in combination with tktA overexpression was more effective than previous approaches that targeted the glycolytic or aromatic pathway enzymes for enhancing phenylalanine production.

Chromatographic methods of fractionation.

Chromatography's functional versatility, separation efficiency, gentle non-denaturing separating process and ease of automation and scale-up make it attractive for industrial scale protein purification. The Winnipeg Rh Institute's new Plasma Fractionation facility is an example of the use of chromatography for the large scale purification of plasma protein fractions. The fractionation facility has a capacity to process 800 litres of plasma per batch into blood clotting factor VIII and IX, albumin and intravenous immune serum globulin (i.v. ISG). Albumin and i.v. ISG are purified using ion exchange columns of DEAE-Sepharose (230 litre size), DEAE-Biogel (150 litre size) and CM-Sepharose (150 litre size). The chromatographic process is automated using a Modicon 584 Programmable Logic Controller to regulate valves, pumps and sensors which control plasma flow during fractionation. The stainless steel tanks and piping are automatically cleaned-in-place. The high degree of automation and cleaning provides efficient operation and sanitary processing. Chromatographic methods (DEAE-Sepharose and metal chelation) are also being used at the pilot scale to purify the human blood products superoxide dismutase and hemoglobin from outdated red blood cells. Characterization of the protein fractions produced by chromatography has shown them to be of equal or higher quality than fractions produced by other techniques.

Studies on incorporation of mannose into rat alpha 1-acid glycoprotein: evidence for precursor forms in rough membranes.

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and alpha 1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and alpha 1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of alpha 1-acid glycoprotein by isolation of a high mannose containing precursor species of alpha 1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of alpha 1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of alpha 1-acid glycoprotein with extensions of the polypeptide chain may also exist.

Column ion exchange chromatographic production of human immune serum globulin for intravenous use.

Column ion exchange chromatographic processes were developed for the production of immune serum globulin (ISG) for intravenous use. Sequential chromatography on two cross-linked agarose gel anion exchangers yielded the same immunoelectrophoretic pure ISG as a single column DEAE-Sephadex procedure with the advantage of repeated 'in column' cycling. ISG with a low content of aggregated IgG and low anticomplementary activity was prepared from plasma with 53-69% efficiency. The ISG prepared by these processes was characterized with respect to the content of IgA, prekallikrein activator (PKA), plasminogen, fragmentation, and heavy chain subclass distribution.

Sequence-specific inhibition of gene expression by a novel antisense oligodeoxynucleotide phosphorothioate directed against a nonregulatory region of the human immunodeficiency virus type 1 genome.

Previous studies have demonstrated that oligodeoxynucleotide phosphorothioates complementary to human immunodeficiency virus type 1 (HIV-1) RNA are more nuclease resistant and are effective inhibitors of HIV-1 replication than their unmodified counterpart. In this study, antisense oligodeoxynucleotide sequences were evaluated for therapeutic potential in the treatment of HIV infections. The use of HIV-infected lymphocytes to test the efficacy of a drug is very complex, and therefore it is difficult to draw conclusions about the mechanism. We used a COS-like Monkey kidney cell line (CMT3) stably transfected with plasmids pCMVgagpol-rre-r (containing gag and pol genes) and pCMVrev (containing the rev gene of HIV-1), derived from cDNA clone BH10, as a model. A biologically active provirus that transcribes and translates their nucleotide sequences into viral proteins p24, p39/41, p55, and p160 was generated. Sequence-specific and dose-dependent inhibition of HIV-1 viral protein synthesis and significant inhibition at the mRNA level were demonstrated by antisense construct GPI2A, directed against a nonregulatory region of the HIV-1 genome. Also, our studies demonstrated enhancement of the antisense effect through encapsulation in a cationic lipid preparation. The observed attenuation of HIV-1 mRNA levels suggests that, at least in part, the mechanism of action of GPI2A was at the transcript level. Further studies have also shown antiviral activity of this construct as determined by the reverse transcriptase assay using acutely and chronically infected cells of lymphoid origin (H9 cells). Toxicological studies involving cell growth characteristics, colony-forming ability, effects on cellular proteins, specific activities of labeled proteins, and DNA synthesis in cell culture showed no cytotoxic effects of GPI2A.

Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography.

Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

A calorimetric study of human CuZn superoxide dismutase.

Structural alterations, as manifested by thermal transitions, caused by removal or binding of metal ions to human and bovine CuZn superoxide dismutases (SODs) were investigated by differential scanning calorimetry. Although holo forms of the two mammalian enzymes exhibited irreversible thermal transitions (delta Hcal. = 27.7 J/g and Td = 104 degrees C for bovine SOD; delta Hcal. = 23.6 J/g and Td = 101 degrees C for human SOD), only the bovine apoenzyme showed the presence of a less thermostable form (delta Hcal. = 10.7 J/g and Td = 63 degrees C). These observations suggested that human apo-SOD had considerably less conformational order than bovine apo-SOD. Reconstitution of human and bovine apoenzymes with Cu2+ and Zn2+ resulted in recovery of thermodynamic parameters and specific activity. Binding of Zn2+ alone to human apo-SOD resulted in the formation of two distinct structural units, detectable by differential scanning calorimetry, which underwent conformational disorder at 82 and 101 degrees C respectively. Saturation of binding sites with both Zn2+ and Cu2+ appeared to stabilize the enzyme structure further as shown by elimination of the low-temperature transition and the appearance of another thermal transition at a higher temperature.

Relative levels of inhibition of p24 gene expression by different 20-mer antisense oligonucleotide sequences targeting nucleotides + 1129 to + 1268 of the HIV-1 gag genome: an analysis of mechanism.

GPI2A is a 20-mer antisense oligonucleotide sequence that is complementary to a region of the HIV-1 gag gene. An analysis of viral core antigen p24 protein synthesis inhibition was performed with cells expressing HIV-1 proteins, following treatment with GPI2A or eight other unique antisense constructs designed to bind to regions of the gag gene, at positions that 5' or 3' flank the GPI2A target site. GPI2A was found to be the most effective construct, indicating that the GPI2A target region is a particularly sensitive site for antisense activity. An analysis of energy-related parameters important in complementary duplex formation was performed for each antisense construct. Also, the potential of each antisense sequence to exhibit self-complementarity or to self-dimerize was assessed. The results from these analyses provided an explanation for the high specificity and the superior inhibitory characteristics of GPIA when compared to the eight other antisense oligonucleotides. GPI2A exhibited the second most favorable energy-related characteristics for hybridization reactions, and most importantly, unlike the other eight antisense sequences, it did not show the potential to self-complement or to dimerize. The results of this study and a previous investigation of sequence specificity requirements for GPI2A inhibition of HIV-1 gene expression provide strong evidence for an antisense mode of action for this oligonucleotide construct, a useful tool for analysis of viral gene expression and perhaps a potential therapeutic agent.

WinRho: Rh immune globulin prepared by ion exchange for intravenous use.

An Rh immune globulin [Rh IgG] for intravenous use, WinRho, has been prepared by the Winnipeg Rh Institute by a modification of the ion-exchange column method of Hoppe and colleagues. When administered to Rh-negative male and nonpregnant female volunteers WinRho was found to be nonpyrogenic, nontoxic, safe and protective against Rh alloimmunization. In a clinical trial with 240 microgram given at about 28 weeks' gestation and 120 microgram given after delivery to Rh-negative women at risk of Rh immunization WinRho was effective in preventing Rh immunization. Of the 870 women carrying Rh-positive fetuses who were treated with WinRho during pregnancy and were not tested several months after delivery 14 would have shown evidence of Rh immunization by the time of delivery if WinRho had been ineffective; none showed such evidence. Of the 1122 women carrying Rh-positive fetuses who were retested 4 to 6 months after delivery 83 would have shown evidence of Rh immunization at that time if WinRho had been ineffective; only 1 showed such evidence. The efficiency of yield of anti-D with the modified method of production, the fct that it can be given intravenously (a route that causes the patient less discomfort and immediately results in high anti-D levels) and the lower levels of contaminating IgA and IgM make WinRho the preparation of choice for preventing Rh immunization.

Partial characterization of alpha 1-acid glycoprotein isolated from mouse serum.

AGP was purified from mouse serum by perchloric acid treatment and CM-Sepharose chromatography. Induction of inflammation with turpentine resulted in a 10-fold increase in the serum level of mouse AGP, indicating mouse AGP is an acute phase reactant. Biochemical characterization of mouse AGP indicated similarity with human and rat AGP.

Production and characterization of human-human and human-mouse hybridomas secreting Rh(D)-specific monoclonal antibodies.

Human monoclonal antibodies specific for the Rh(D) antigen were produced by cell lines generated by the fusion of pooled Epstein-Barr virus (EBV)-transformed B-cell lines secreting Rh(D) antibodies with the murine myeloma cell line NS.1 or with the human lymphoblastoid cell line HOA.1. The selection of hybrids was achieved in RPMI 1640 medium containing HAT and ouabain. Higher fusion efficiency was obtained with the NS.1 cell line; however, the hybrids with HOA.1 exhibited a greater clonal stability. The products of four clones (three human-human and one human-mouse) that consistently secreted antibodies for over 11 months were tested for specificity with a panel of red cells of various Rh phenotypes. The supernatants of all four clones showed anti-Rh(D) specificity but failed to react with the red cell Du phenotypes categorized as DV(Dw+) and DVI. Two of the three human-human clones secreted IgM(lambda) and the third IgG(kappa). The human-mouse clone produced IgG(kappa) antibody.

Intravenous human rabies immunoglobulin for post-exposure prophylaxis: serum rabies neutralizing antibody concentrations and side-effects.

The beneficial effect of passive immunization for post-exposure rabies prophylaxis is associated with the appearance of serum neutralizing antibody (SNA) earlier than occurs with vaccine alone. We compared the SNA response and the side-effects in 30 previously unimmunized healthy volunteers given a commercially available human rabies immunoglobulin (HRIG) intramuscularly (i.m.) or an experimental HRIG prepared by DEAE Sephadex column chromatography, intravenously (i.v.) with or without human diploid-cell culture rabies vaccine (HDCS). The subjects were divided into five equal groups: HDCS alone, HDCS + i.m. HRIG 20 IU/kg (currently recommended), i.v. HRIG alone 15 IU/kg, HDCS + i.v. HRIG 15 IU/kg or HDCS + HRIG 5 IU/kg i.v. plus 10 IU/kg i.m. to simulate local bite wound infiltration. HDCS, 1.0 ml, was injected subcutaneously (s.c.) on days 0, 3, 7, 14 and 28. Only local discomfort at injection sites was observed without differences between groups. SNA was demonstrated in all HRIG recipients at day 1, but the concentrations were higher in those receiving it intravenously. No difference in the SNA response to vaccine was observed between the i.v. and i.m. HRIG groups given the same vaccine lot. It would appear that i.v. HRIG 15 IU/kg can be substituted for i.m. HRIG 20 IU/kg for post-exposure prophylaxis. Since the current regimen is almost 100% protective, there is no way of proving that i.v. HRIG 15 IU/kg is more efficacious. The immediate SNA level and economy are the chief advantages of i.v. HRIG 15 IU/kg.

Immunoreactivity of human MAb BT32/A6 with neuroepithelial tumors.

The present study was undertaken to determine the pattern of immunoreactivity of BT32/A6, a human IgM monoclonal antibody (MAb), with the following histological panels: 1) 30 human and non-human cell lines, 2) 32 normal human tissues, and 3) 28 tumors of central neuroepithelial origin (16 astrocytic; 11 non-astrocytic). Antibody BT32/A6 recognizes a surface and cytoplasmic antigen present on a variety of human tumor cell lines including gliomas, melanomas, neuroblastomas, and a few sarcomas. The antigen is present (at least focally) on 15/16 astrocytic tumor tissue sections (94%), and in some cases, on close to 100% of cells. All malignant cell types, including small anaplastic cells, giant cells, gemistocytic cells, and cells forming pseudopalisades were labeled by MAb BT32/A6. Non-astrocytic neuroepithelial tumors did not stain appreciably with MAb BT32/A6. There was weak immunoreactivity in a small subset of normal human tissues of epithelial and lymphoid origin, with the exception of adrenal cortex, which exhibited weak to moderate staining. All normal tissues of neuroectodermal and mesenchymal origin were unreactive. In conclusion, MAb BT32/A6 appears to be unique in that it recognizes a highly-expressed astrocytic tumor-associated antigen that is present on both low and high grade tumors. This makes it a strong candidate for further studies aimed at establishing its usefulness in the treatment of human astrocytic tumors.