Antigenicity of mouse interferons: distinct antigenicity of the two L cell interferon species

Antibodies were raised in rabbits against Newcastle disease virus-induced L cell interferon of high purity, and against each of its two major species, F(24K) and S(36K) interferons. The two interferons were found antigenically distinct. Thus, the anti-F and anti-S sera failed to neutralize appreciably the antiviral, as well as the cell growth-inhibiting, activity of the heterologous interferon. Heterologous reactions were also undetectable in a modified neutralization test, in which secondary antibody against rabbit γ-globulin was used to remove, before the assay for the residual interferon, any interferon-antibody complexes that might remain biologically active. Affinity chromatography of interferons on immobilized antibodies also showed antigenic distinctness of F and S interferons. Poly (I) ·poly (C)-induced L cell interferon and Newcastle disease virus-induced C₂₄₃ cell interferon also consisted of two distinct species which are antigenically similar to F and S.     

Characterization of two distinct molecular populations of type I mouse interferons.

The molecular heterogeneity of acid-stable (Type I) mouse interferons induced in C243 cells by Newcastle disease virus was analysed by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions, and the profiles of antiviral activities obtained were characterized biologicaly in mouse cells and in heterologous (guinea-pig) cells. Two bands of activity, A and B, were consistently present in all interferon preparations tested: under reducing conditions, the activity in all fractions of band A (with a peak of activity at about 38000 daltons) was uniformly increased, while that of band B (with a peak at about 22000 daltons) was uniformly diminished. All the active fraction in band A had only slight activity (less than 10% of homologous titres) on guinea-pig cells, whereas all those in band B were significantly more active an guinea-pig cells than on homologous L cells. Thus, mouse type I interferon preparations contain two molecular populations of interferons that can be distinguished physically (by size), biochemically (by the effect of reduction on reactivation from SDS) and biologically (by activity in heterologous cells).     

Distinct molecular species of interferons

Interferons induced in mouse L₉₂₉ cells could be renatured from the protein-dissociating system of sodium dodecyl sulfate (SDS), mercaptoethanol, and urea after boiling at 100°. Therefore, SDS-polyacrylamide gel electrophoresis under reducing conditions was used to analyze mouse L cell interferons. Preparations, crude or partially purified, contained two well-separated molecular species of interferon: a major component (about 90% of the total activity) at 38,000 daltons and a minor component (about 10% of the total activity) at 22,000 daltons. That the smaller component was not a subunit of the larger component was evident from the finding that, while the amount of activity of the smaller species recoverable was essentially unchanged whether the preparations were reduced or not, the amount of activity of the larger species recoverable was greatly increased (about 10-fold), rather than decreased, under reducing conditions. Also, when the interferon eluted from unreduced gels at 38,000 daltons was boiled in SDS, mercaptoethanol, and urea, latent activity was “unmasked” giving an increase of about 10-fold in activity. The data indicate that mouse L cell interferon preparations contain two molecular species of interferon: a smaller species renaturable with or without reduction and a larger and predominant species requiring disruption of disulfide bonds for efficient renaturation.     

Antigenicity of low molecular weight surfactant species.

The authors tested the antigenicity of human lung surfactant isolated from amniotic fluid. Mice and rabbits were immunized. Rabbit polyclonal antisera to these surfactant preparations were absorbed with normal human plasma proteins. Polyclonal antisera reacted with both high molecular weight (35 kd) surfactant apoprotein and to lower molecular weight species, both 18 kd and 9 kd. Mice were used to generate monoclonal antibodies to surfactant. Enzyme-linked immunosorbant assay was used to identify five monoclonal antibodies that reacted with surfactant. By Western blot analysis, all of these recognized a low molecular weight surfactant species (9 kd) that could be either SP-B or SP-C. One reacted with a 37 kd protein in the surfactant preparation, consistent with SP-A. One monoclonal antibody also recognized a higher molecular weight species (44 kd) of unknown origin. The ability of antisera and monoclonal antibodies to inhibit the functional activity of surfactant was assayed using a pulsating bubble surfactometer. Rabbit polyclonal antisera inhibited initial surface adsorption to equilibrium surface tension and increased the minimum surface tension after 1 and 5 minutes of initiation of pulsations. This inhibitory activity of the antisera was noted in divalent F(ab')2 fragments. Monovalent F(ab) fragments and control normal rabbit sera did not inhibit surfactant function in this assay. Of the anti-surfactant monoclonal antibodies that reacted with surfactant by ELISA and Western blot, three inhibited its capacity to lower surface tension on the pulsating bubble apparatus. The other two monoclonal antibodies showed no functional inhibitory activity. It is concluded that both the 35 kd SP-A and the 9 kd proteins of human surfactant are highly immunogenic and partially crossreactive. Resulting antibodies could alter the ability of surfactant to perform its physiologic function, ie, to lower surface tension.     

Synthesis of two distinct interferons by human fibroblasts.

Human interferon species Le and F can be distinguished on the basis of their distinct antigenic, biological, and physicochemical characteristics. Until now, Le interferon was thought to be produced only in cells of lymphoid origin. This study demonstrates that cultures of human fibroblast cell strains GM-258 and FS-4 produce both F and Le interferons. Le interferon constituted up to about 20% of the total interferon activity produced in fibroblast cultures after stimulation with Newcastle disease virus or vesicular stomatitis virus. In contrast, only F interferon was detected in preparations obtained after induction with polyinosinate-polycytidylate. These results indicate that the Le interferon gene is inducible in cells of nonlymphoid origin and that the expression of this gene depends on the nature of the inducing agent.     

Distinct molecular species of human interferons: requirements for stabilzation and reactivation of human leukocyte and fibroblast interferons.

Human fibroblast interferon preparations were completely stabilized to 100 degrees C by sodium dodecyl sulphate (SDS) in the presence of mercaptoethanol, but only a minor fraction of their activities were stabilized by SDS without mercaptoethanol. On the contarary, human leukocyte interferon preparations were completely stabilized to 100 degrees C by SDS in the absence of mercaptoethanol, but only a minor fraction of their activities were stabilized by SDS in the presence of mercaptoethanol. Furthermore, human fibroblast interferon preparations whose activities had been destroyed by boiling at 100 degrees C were completely reactivated by SDS under reducing conditions, but only a minor part of their activities were restored by SDS in the absence of reduction. On the contrary, human leukocyte interferon preparations whose activities had been destroyed by boiling at 100 degrees C were completely reactivated by SDS in the absence of reduction, but only a minor part of their activities were restored by SDS under reducing conditions. These data suggest that there are distinct molecular species of human interferons.     

Antigenicity of lateral flagella of Vibrio parahaemolyticus: existence of two distinct antigenic determinants in a flagellum.

Cells of Vibrio parahaemolyticus treated with Formalin agglutinated with anti-lateral flagella antiserum. On the basis of agglutination tests, antigens of lateral flagella were divided into three groups. HL1, HL2, and HL3. However, in gel diffusion tests, flagellins prepared from strains belonging to different groups showed a common antigenicity. It is assumed that these results are due to the existence of two distinct antigenic determinants in the lateral flagella. One of them exists on the surfaces of the flagella and is responsible for H-agglutination, and the other exists inside the flagella and is exposed when the flagella are solubilized to flagellin monomers.     

Bordetella pertussis and Bordetella parapertussis: two immunologically distinct species.

Bordetella pertussis and Bordetella parapertussis are closely related species. Both are responsible for outbreaks of whooping cough in humans and produce similar virulence factors, with the exception of pertussis toxin, specific to B. pertussis. Current pertussis whole-cell vaccine will soon be replaced by acellular vaccines containing major adhesins (filamentous hemagglutinin and pertactin) and major toxin (pertussis toxin). All of these factors are antigens that stimulate a protective immune response in the murine respiratory model and in clinical assays. In the present study, we examined the protective efficacies of these factors, and that of adenylate cyclase-hemolysin, another B. pertussis toxin, against B. parapertussis infection in a murine respiratory model. As expected, pertussis toxin did not protect against B. parapertussis infection, since this bacterium did not express this protein, but the surprising result was that none of the other factors were protective against B. parapertussis infection. Furthermore, B. parapertussis adenylate cyclase-hemolysin, although it protected against B. parapertussis infection, did not protect against B. pertussis infection. Despite a high degree of homology between both B. pertussis and B. parapertussis species, no cross-protection was observed. Our results outline the fact that, as in other gram-negative bacteria, Bordetella surface proteins vary immunologically.     

Structure of Ia antigens from the rat. Mouse alloantisera demonstrate at least two distinct molecular species

Ia antigens isolated from spleen cells of rats and mice are composed of two polypeptide chains, designated α and β. Mouse alloantisera specific for the I-A^k and I-E^k subregions react with two distinct groups of rat Ia antigens, designated A-like and E- like, respectively. Two-dimensional gel electrophoresis and peptide map analysis demonstrate that the A-like antigens of rat are distinct from the E-like antigens. Both rat Ia antigens react with alloantiserum produced in rats congenic for the major histocompatibility complex (MHC). These results demonstrate for the first time that two distinct Ia antigens are present in the rat. Accordingly, the rat, like the mouse, may have Ia antigens encoded by at least two sub regions of the rat MHC. The existence of multiple Ia gene products in rats is revealed by chemical techniques even in the absence of formal genetic evidence of more than one I subregion in the rat.     

Action of different gamma interferons on mouse leukemic cells resistant to alpha and beta interferons

Phytohemagglutinin-induced interferon-gamma (IFN-gamma) was reported to act on mouse leukemic L1210 R cells resistant to IFN-alpha and -beta. Results reported here show that these cells are also sensitive to various preparations of murine IFN-gamma derived from different sources and purified to different degrees and that lymphokines present in the preparations are not involved in the antiviral effect of IFN. In addition, IFN-gamma preparation increases concanavalin A binding to L1210 S and L1210 R cells indicating that the sensitivity of L1210 R cells to IFN-gamma is not limited to its antiviral effect.     

Molecular arguments for splitting of Schistosoma intercalatum, into two distinct species

The taxonomic status of the two known strains of Schistosoma intercalatum, the Lower Guinea strain (originating from Edea, Cameroon) and the Zaire strain (originating from Kinshasa, Democratic Republic of Congo, formerly Zaire) was examined using random amplified polymorphic DNA (RAPD) markers. Two additional species within the S. haematobium group, S. haematobium and S. mattheei, were included in the study. DNA was extracted from four male and four female worms of each species and strain under investigation. In all, 13 primers gave reproducible and informative marker patterns; the monomorphic bands in all the males and females of each sample were scored, and 138 bands were included in the final analysis. Overall, 14 RAPD fragments were shared by all the schistosomes studied, and 19 RAPD fragments were considered to be sex markers. Only 22% (20/91) of the RAPD fragments were shared between S. intercalatum Zaire and S. intercalatum Cameroon. The mean values recorded for the Nei and Li's genetic distances between S. haematobium and S. mattheei and between S. intercalatum Zaire and S. intercalatum Cameroon were 0.546 and 0.596, respectively. A principal component analysis and one-way analysis of variance (ANOVA/MANOVA) showed a significant separation between S. intercalatum Zaire and S. intercalatum Cameroon. The data support the hypothesis that S. intercalatum Zaire and S. intercalatum Cameroon are distinct species. Additional molecular-biology studies are in progress that involve the use of nuclear and mitochondrial markers to confirm the extent of the genetic divergence prior to the establishment of final decision on the taxonomic status of the two strains of S. intercalatum. Received: 6 June 2000 / Accepted: 11 July 2000     

Establishment and characterization of two distinct malignant mesothelioma cell lines with common clonal origin

We describe two newly established malignant mesothelioma (MM) cell lines derived from a pleural effusion of a male. One cell line, designated as MM-Z03E, reveals an epithelioid cobblestone morphology, while the second one, designated as MM-Z03S and subcloned after in vivo selection, exhibits a sarcomatoid storiform growth pattern. Both cell lines showed the immunologic profile characteristic for MM (i.e., expression of cytokeratin, CK18, calretinin, and vimentin in both phenotypes). Cytogenetics, multicolor fluorescence in situ hybridization, comparative genomic hybridization, and oligonucleotide array CGH were performed on both cell lines. Aberrations shared by both cell lines included chromosomal losses of 1q34 approximately qter, 4, 9p, 10p, 13, 14, 16q, 18, and 22, as well as a complex structural aberration involving chromosome 17. Aberrations exclusive to MM-Z03E included gains of 3q11q27 and 5p, while gain of 9q and losses of 3q27qter, 11q, and 18 in MM-Z03S were exclusive to MM-Z03E. Both cell lines were able to develop solid transplant tumors in nude mice within 16 weeks, and immunophenotyping of tumor xenografts revealed an overall retained expression profile of the markers used. Remarkably, one xenograft from MM-Z03E revealed overexpression of p53 and widely invasive growth. In conclusion, both cell lines are useful in vivo and in vitro model systems to study the underlying genetic mechanisms of biphasic differentiation in MM, which can be of certain value considering the increasing relevance of assessing MM tumor biology for the clinical management of this disease.     

Demonstration of two immunologically distinct xenotropic type C RNA viruses of mouse cells

Several independent type C virus isolates were obtained by transplantation of human tumor cell lines into immunosuppressed NIH Swiss mice. Each isolate appeared to be of mouse origin as determined by the antigenicity of its major virion polypeptide, p30. The virus isolates were indistinguishable from each other in serologic and host range properties and also closely resembled BALB:virus-2, one endogenous virus of $\text{BALB}\text{c}$ mouse cells. However, in immunoassays for a highly type-specific virion polypeptide, designated p12, every isolate was found to differ from BALB: virus-2. These results provide evidence for the existence of immunologically distinct xenotropic viruses of mouse cells.     

Differences in mouse interferons according to cell source and mode of induction.

Mouse interferon induced by ultraviolet-irradiated Newcastle disease virus or polyriboinosinic-polyribocytidylic acid in T lymphocytes, B lymphocytes, macrophages, and primary mouse embryonic cell culture was studied. Irrespective of the inducer, interferons produced by T or B lymphocytes were relatively heat stable and of low antigenicity when reacted with antiserum against L-cell interferon (ALI), whereas interferons produced by macrophages and mouse embryo cells were heat labile and of high antigenicity against ALI. Mouse interferons induced by ultraviolet-irradiated Newcastle disease virus were separated into three components by chromatography on CH-Sepharose 4B. Interferons produced by T and B lymphocytes consisted primarily of component 1 (unbound fraction), whereas interferons produced by macrophages or mouse embryo cells consisted primarily of component 3 (eluted by 0.5 M NaCl). Component 1 was heat stable and of low antigenicity against ALI, properties characteristic of T- and B-cell interferon. Components 2 and 3 were heat labile and of high antigenicity against ALI, properties characteristic of macrophage and mouse embryo cell interferon. In contrast, interferon induced in mice sensitized with BCG differed from these interferons induced in B cells, T cells, macrophages, and fibroblasts in being extremely acid labile and nonreactive against ALI.