The protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets.

The unique germfree, colostrum-deprived, immunologically "virgin" piglet model was used to evaluate the ability of lactoferrin (LF) to protect against lethal shock induced by intravenously administered endotoxin. Piglets were fed LF or bovine serum albumin (BSA) prior to challenge with intravenous Escherichia coli lipopolysaccharide (LPS), and temperature, clinical symptoms, and mortality were tracked for 48 h following LPS administration. Prefeeding with LF resulted in a significant decrease in piglet mortality compared to feeding with BSA (16.7 versus 73.7% mortality, P < 0.001). Protection against the LPS challenge by LF was also correlated with both resistance to induction of hypothermia by endotoxin and an overall increase in wellness, as quantified by a toxicity score developed for these studies. In vitro studies using a flow cytometric assay system demonstrated that LPS binding to porcine monocytes was inhibited by LF in a dose-dependent fashion, suggesting that the mechanism of LF action in vivo may be inhibition of LPS binding to monocytes/macrophages and, in turn, prevention of induction of monocyte/macrophage-derived inflammatory-toxic cytokines.

Human immune response to cationized proteins. I. Characterization of the in vitro response to cationized diphtheria toxoid.

Cationization of proteins, i.e., increasing net positive charge by the substitution of carboxyl groups with positively charged residues, has been reported to enhance protein immunogenicity in animal model systems. In the present study, we have investigated the effect of cationization on the in vitro cell-mediated immune response of human mononuclear cells to diphtheria toxoid. A series of cationized DT preparations were generated by covalent modification with ethylenediamine, with pIs ranging from 4.6 to > 9.3, and tested for their ability to induce proliferation of normal human peripheral blood mononuclear cells. Cationized DT (cDT) was found to induce an antigen-specific, augmented proliferative response, relative to native antigen, which was directly proportional to the degree of cationization. Further characterization of the response to cDT demonstrated that (1) proliferative responses could be detected considerably earlier, and typically at much lower antigen concentrations, than the response to native DT; (2) the response was dependent on HLA-DR; (3) production of a number of cytokines, sp. IL-1 beta, IL-2, and IFN-gamma, was also elevated in cDT-stimulated cultures; and (4) the enhanced proliferative response to cDT could be attributed to CD4+ helper T cells. These results demonstrate that cationization of proteins enhances the ability to generate a cell-mediated immune response in humans and suggest that cationization may have utility in the design of more effective carrier proteins for human vaccines.

Human immune response to cationized proteins. II. Characterization of interaction of cationized diphtheria toxoid with human mononuclear cells.

Cationized diphtheria toxoid (cDT) has previously been shown to be more effective than the native protein as an inducer of human antigen-specific T cell responses. In the present study, biotin-labeled antigen and flow cytometric analysis were used to examine the possibility that enhanced immunogenicity of cDT may be a consequence of preferential binding to antigen-presenting cells. Strong binding of cDT, relative to native antigen, was noted for both monocytes and B cells. Characteristics of binding were similar for both cell types, including rapid saturation, temperature independence, and inhibition by unlabeled cationized proteins. Although both B cells and monocytes bound cDT, only monocytes were effective in triggering T cell proliferation, possibly as a result of slow internalization of bound antigen by B cells. Definition of the target structures of cationized proteins may allow for the design of more efficient vaccines, which would be specifically targeted to antigen-presenting cells in vivo.

Zaprionus tuberculatus: chromosome map and gene mapping by DNA in situ hybridization.

The genus Drosophila has long been used as a model of karyotype evolution, demonstrating change by paracentric inversion and occasional centric fusion of an ancestral karyotype of five rod-shaped and one "dot" chromosome. This study shows, by mapping D. melanogaster probes hybridized to polytene chromosomes of Zaprionus tuberculatus, that this ancestral pattern extends beyond the genus Drosophila. A formal polytene chromosome map of Z. tuberculatus is presented.

Flow cytometric assays for monitoring production of recombinant HIV-1 gp160 in insect cells infected with a baculovirus expression vector.

A baculovirus expression system for the HIV-1 envelope glycoprotein gp160 has been used as a model for development of flow cytometric assays for monitoring production of cell-associated recombinant antigen. Using monoclonal antibodies to the transmembrane (gp41) or envelope (gp120) portion of gp160, gp120, but not gp41, could be reproducibly detected on the surface of insect cells 48 h after infection with the recombinant baculovirus. In contrast, fixation and permeabilization of infected cells prior to staining, to allow access of monoclonal reagents to the intracellular compartment, markedly improved the sensitivity of detection, with reactivity to both monoclonal antibodies observed at 24 h post-infection. Specificity of the intracellular immunofluorescence was verified by demonstrating that the appropriate native or recombinant HIV-1 protein blocked reactivity of monoclonal antibody with infected cells. In addition, it was observed that production of gp160 following baculovirus infection was associated with a marked increase in the 90 degrees light scatter of insect cells, as determined by flow cytometry, and that this correlated with the kinetics of cell-associated gp160 production as determined by immunofluorescence. These procedures should be of great utility for routine monitoring of recombinant proteins produced in insect cells in response to infection with recombinant baculovirus.

In situ hybridization analysis of chromosomal homologies in Drosophila melanogaster and Drosophila virilis.

Twenty-four biotin-labeled recombinant-DNA probes which contained putative unique-sequence Drosophila melanogaster DNA were hybridized to larval salivary-gland chromosomes of D. melanogaster and Drosophila virilis. All probes hybridized to D. melanogaster chromosomes at the expected sites. However, one probe hybridized to at least 16 additional sites, and one hybridized to one additional site. Thirteen probes hybridized strongly to D. virilis chromosomes, four hybridized weakly and infrequently, and seven did not hybridize. Probes representing two multigene families (beta-tubulin and yolk-protein) hybridized as would be expected if all sites had been conserved in the two species on the same chromosomal elements. The multiple hybridization sites of a third probe which may represent a multigene family were also conserved. The results were consistent with H.J. Muller's proposal that chromosomal elements have been conserved during evolution of this genus.

Analysis of cell-associated recombinant hepatitis B surface antigen by flow cytometry.

Murine L cell lines secreting recombinant hepatitis B surface antigen (rHBsAg) of either the Adw or Ayw subtype were used as a model system to develop procedures for analysis of cell-associated HBV antigens by flow cytometry. Only weak membrane immunofluorescence was observed when viable Ad or Ay cells were reacted with monoclonal antibodies (MAbs) to either subtype specific or the common group specific "a" determinant of rHBsAg. Following fixation and permeabilisation to allow access of MAbs to the intracellular compartment, specific reactivity of cells with both anti-"a" and subtype specific MAbs was readily demonstrated by flow cytometric analysis. Comparison of the fluorescence histograms produced by analysis of Ad and Ay producing cells with the anti-"a" MAb demonstrated an increased proportion of cells with high levels of intracellular rHBsAg in the Ay line. The results of these studies demonstrate that flow cytometric analysis with MAbs is a useful tool for characterizing the expression of viral antigens at the cellular level. The application of this technique to monitoring the production of native viral proteins following in vitro infection should provide valuable insights into the process of viral replication.

Inhibition of interleukin 2 production and expression of the interleukin 2 receptor by plasma from acquired immune deficiency syndrome patients.

Plasmas from acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC) patients were screened for their ability to inhibit mitogen-induced proliferation of normal human lymphocytes. Plasmas from 67% of the individuals examined contained significant suppressive activity. Additional studies on the mechanism of action of the plasma inhibitor demonstrated that it functions as a nonlymphotoxic inhibitor of interleukin 2 production by stimulated human lymphocytes, and that this activity is accompanied by suppression of expression of the cell surface receptor for interleukin 2. A more detailed understanding of the action of this activity may aid in the design of therapy to minimize the contribution of this agent to the immune anergy observed in these patients.

Effect of dimethyldioctadecylammonium bromide induced macrophages on malignant cell proliferation.

Murine peritoneal macrophages elicited by dimethyldioctadecylammonium bromide (DDA), which is a potent immunologic adjuvant, were examined for cytotoxic and growth inhibiting activity for malignant cells. DDA macrophages had no cytolytic activity for murine B16BL-6 melanoma or human SMS-SB pre-B leukemia cells even in the presence of up to 1 microgram bacterial endotoxin (lipopolysaccharide, LPS)/ml. However, they exhibited a variable inhibitory effect on the growth of several lines of leukemia cells. The number of SMS-SB and human NALL cells remained essentially static in the presence of DDA macrophages while they increased significantly when cultured with resident macrophages. In contrast, L1210 cells increased 5-8-fold in the presence of macrophages elicited either by DDA or the inflammatory agent proteose peptone (PP). Although DDA macrophages retarded L1210 growth relative to PP macrophages, both populations responded to LPS in a comparable dose dependent manner to become essentially cytostatic at 1 microgram LPS/ml.

Counter inhibitor: a low molecular weight cytokine derived from human leukocyte dialysates reverses antigen dependent PMN and macrophage migration inhibition.

A low molecular weight component, termed counter inhibitor (CI), has been partially purified from human dialyzable leukocyte extracts. Addition of CI to either a direct leukocyte or macrophage migration inhibition system results in reversal of antigen-induced migration inhibition. CI activity requires the presence of antigen for expression, but does not require that the donor of the CI be immune to the antigen used in the migration inhibition assay. Reversal of migration inhibition by CI appears to be a consequence of its ability to prevent PMNs or macrophages from responding to lymphokines which induce migration inhibition.

Modulation of human T cell production of migration inhibitory lymphokines by cytokines derived from human leukocyte dialysates.

Human leukocyte dialysates contain components capable of amplifying cutaneous delayed-type hypersensitivity (DTH) reactions. In the present study, two such amplifiers, both less than 3500 m.w., were partially purified from human leukocyte dialysates by gel filtration on Sephadex G-10 followed by high pressure reverse-phase liquid chromatography. These amplifiers of DTH were examined for their effects on production of the migration inhibitory lymphokines leukocyte migration inhibition factor (LIF) and macrophage migration inhibition factor (MIF). The amplifiers were found to increase LIF and MIF production by antigen- or alloantigen-stimulated human peripheral blood lymphocytes in a dose-dependent fashion. Further analysis demonstrated that although antigen-stimulated T4 and T8 cell subpopulations could produce LIF activity under the assay conditions employed, amplification of lymphokine production by modulator was only observed with the T4 subset.

Studies with a human plasma-derived immunosuppressive, anti-lymphoma factor.

A low-molecular-weight (1,400) factor isolated from a human plasma alpha-globulin concentrate by acid-salt dissociation and ultrafiltration inhibits proliferation of mitogen-stimulated T cells and L1210 leukemia cells. The factor (UM05R) inhibits DNA, RNA, and protein synthesis in sensitive cells, acts in G1 of the cell cycle, and appears to suppress mitogen-responsive T cells without an accessory cell requirement. UM05R activity is enhanced by known cAMP-elevating agents and by sulfhydryl compounds. The results of the present study are consistent with the hypothesis that the plasma-derived agent inhibits lympho-proliferation as a result of elevation of intracellular cAMP.

Tissue localization of esterase-5 in Drosophila pseudoobscura.

Disc gel electrophoresis of dissected adults of Drosophila pseudoobscura showed that most of the esterase-5 activity was in the head (36%) and thorax (51%), with little activity in the abdomen (13%). No activity was found in digestive tissues, reproductive tissues, or nervous tissues. Most of the est-5 activity (61%) was found to be associated with hemolymph. However, the eyes contained 39% of the total est-5 activity. These results were supported by spectrophotometric assays of esterase activity in crude extracts of eyes and whole flies without eyes which showed that 27% of the total est-5 activity was in the eyes.

Inhibition of lymphoproliferation by dipyridamole.

Dipyridamole (DP, Persantin) was examined for its effects on the proliferation of mitogen-stimulated murine splenocytes and L1210 leukemia cells. In keeping with its reported activity as an inhibitor of nucleoside transport, DP inhibited incorporation by lymphoid cells of labeled thymidine and uridine ino macromolecules. That this inhibition resulted from activities in addition to suppression of nucleoside transport was verified by measured decreases of cellular DNA and viable cell numbers. In addition, protein synthesis was also decreased as indicated by labeled valine incorporation and total protein content of the cells. The rapid accumulation of cAMP in phytohemagglutinin-stimulated splenocytes in the presence of DP may provide an explanation for the anti-proliferative effect of DP on lymphoid cells.

Conditional antifolate resistance in Bacillus subtilis thyA.

Resistance to antifolates in Bacillus subtilis strains results from the presence of an antifolate resistance mutation (afo). Strains which are thyA(+)afo are unconditionally resistant to antifolates. The conditional resistance of thyA afo strains is hypothesized to be due to the thyB(+) gene product (thymidylate synthetase B) having a high K(m) for the folate substrate, thus leading to thymineless death in the presence of antifolates. An alternative model for conditional antifolate resistance was shown to be incorrect by analysis of folate metabolism in methotrexate-treated cells. Genetic analysis and studies of the response of afo(+) cells to methotrexate suggested that most, if not all, B. subtilis thymine-requiring mutants are afo. Analysis of dihydrofolate reductase from afo cells did not reveal an obvious mechanism for antifolate resistance in those cells.

Characteristics of delta 1-pyrroline-5-carboxylate reductase from Drosophila melanogaster.

1. Biochemical properties of delta 1-pyrroline-5-carboxylate reductase from d. melanogaster have been investigated. 2. The enzyme is stable below 4 degrees C. 3. the pH optimum of the enzyme is 5.7. It is rapidly inactivated below pH 5.4. 4. The Km values for NADPH and delta 1-pyrroline-5-carboxylate are 1.6 x 10-5 and 2.5 x 10-6 M, respectively. 5. the estimated molecular weight of the enzyme is 225,000. 6. the enzyme is weakly inhibited by L-proline (Ki = 0.12 M).

Ethionine-resistant mutants of the filamentous blue-green alga Plectonema boryanum.

Plectonema boryanum mutants that are resistant to ethionine are unable to incorporate ethionine into acid-precipitable material. Ethionine causes bleaching of chlorophyll in sensitive cells.

An allele-specific suppressor of white-coral in Drosophila melanogaster.

A new allele of white-coral (wco2) was isolated from Canton S after mutagenesis. Many common laboratory stocks were found to carry a suppressor gene (Su(wco2)) which alters the phenotype of wco2 flies toward wild-type. The Su(wco2) is allele-specific (it does not suppress wco), dominant, homozygous viable, located near Su(bwV1) on the right arm of chromosome 2, and shows a simple gene-dosage effect. The degree of suppression is sensitive to the genetic background. There appears to be selection for Su(wco2) in a genotype where it does not affect eye pigmentation.

Identification of poly-gamma-glutamyl chain lengths in folates of Bacillus subtilis.

Bacillus subtilis strains 168 met ile leu and 23 thy contain folates which differ from one another in the number of glutamyl residues. The folate species were identified by reductive cleavage to the corresponding p-aminobenzoylglutamyl poly-gamma-glutamates and chromatography on diethylaminoethyl-cellulose. Pteroyltriglutamate is the predominant folate type, accounting for 86 to 88% of the total. Pteroyltetraglutamate is the only other type present in appreciable quantities, accounting for 5 to 6% of the total folates. Pteroyldiglutamate and pteroylpentaglutamate are present in small amounts, accounting for 1 to 3% and 1% of the total folates, respectively. Strain 168 met ile leu contains a very small amount of pteroylmonoglutamate (less than 0.5% of the total folates), but the other strain contains none.

Deoxyribonucleic acid degradation in Bacillus subtilis during exposure to actinomycin D.

At high concentrations (10 mug/ml), actinomycin D inhibited deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis. Inhibition occurred quickly (in less than 1 min) and was complete. In strain 23 thy his, inhibition of DNA synthesis by actinomycin D was followed by partial degradation of one of the two daughter strands to acid-soluble products. Degradation began at the replication point and proceeded over a distance equal to about 12% of a chromosome in length. Actinomycin D played some essential part in degradation, since exposure of the cells to other treatments or agents which inhibit growth did not lead to the above result.