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PubMed1984

Restoration of allograft responsiveness in B rats. IV. The divergent migratory behavior of lymphocyte populations mediating cardiac allograft rejection.

The T lymphocyte-deprived (B) rat, produced by X-radiation and bone marrow reconstitution of adolescent thymectomized animals, exhibits a true immunological deficit and are unable to reject histoincompatible heterotopic cardiac allografts. A comprehensive survey of lymphocyte traffic in B recipients was performed to correlate the differential potency of specifically sensitized lymphocyte populations mediating re-establishment of immune responsiveness toward the graft, with their migratory and recirculatory behavior. 111In-oxine-labeled thoracic duct lymphocytes (TDL) were retained in the peripheral blood and migrated from nonlymphoid organs to lymph nodes of B recipients in higher proportion than any other lymphoid population, particularly splenic lymphocytes (SL). Although all cell groups but TDL were sequestered in the spleen in equal and relatively large numbers, no differences were found between the lymphocyte populations tested in their capacity to accumulate in the grafts. In contrast, an increased avidity in the allograft of 125IUdR-labeled TDL and lymph node (LNL) lymphoblasts, as compared to 125IUdR-labeled SL, resembles closely the results of functional studies of the differential potency of adoptively transferred cells. We assume that specific cellular interactions induced by the accumulated 125IUdR-labeled cells invoke nonspecific mechanisms for the recruitment of other uncommitted 111In-labeled lymphocytes which recirculate between blood and lymph and localize indiscriminately in the allograft amplifying its rejection. The latter lymphocytes can be "armed" by adherent cells residing in the lymphoid organs of graft recipients, particularly spleen, and subsequently increase the penetration of the foreign tissue. When radiolabeled lymphocytes were traced in B recipients experiencing rejection of their allografts following transfer of sensitized cells plus lymphokine, their migration patterns as well as blastogenic response in B hosts were similar to those observed during acute rejection of cardiac allografts in unmodified hosts. Thus the similarities between the rejection network brought by alloimmune cells into otherwise unresponsive animals and immunocompetent animals able to reject their grafts are stressed.

Animals↗
PubMed1987

Heterogeneity of intercellular adhesion in rat liver cells in culture.

The intercellular homotypic adhesive properties of 14 clones derived from a nontumorigenic rat liver epithelial cell line (LEC), derived from neonatal Fischer rats, were examined and compared to those of the hepatoma H4-II-E cell line. Each clone was assayed also for the degree of chromosomal aneuploidy and the ability to grow in soft agar. Over 100-fold differences in adhesive properties were observed among the clones, but no correlation was observed between the degree of aneuploidy in the clones and intercellular adhesive properties. The parent LEC cell line and the clones derived from it were unable to grow in soft agar. The H4-II-E cells showed negligible capacity to reaggregate after dissociation into single cells and these cells readily formed colonies in soft agar. Many of the LEC clones were similar to the H4-II-E cells in their adhesive properties, which suggests that reduced cell-to-cell adhesiveness per se is not a necessary prerequisite of epithelial cells to be able to grow independent of anchorage. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of concanavalin A (Con A)-binding glycoproteins in the "most adhesive" clone 67 and the "least adhesive" clone 201 showed markedly elevated amounts of acidic 105 and 67-kDa glycoproteins in clone 67. Proteins with similar migration patterns in 2D-PAGE have previously been reported to participate in specific homotypic intercellular adhesion of liver cells. The Con A-binding glycoprotein pattern in H4-II-E cells was markedly different from that of LEC cells with a set of six proteins missing and nine proteins appearing new in the H4-II-E cells. It is suggested that, in addition to identifying known epithelial cell polypeptides, systematic screening of cell surface-associated glycoproteins in normal and transformed epithelial cells in vitro and in vivo may lead to identification of novel polypeptides intimately associated with the transformed phenotype.

Aneuploidy↗
PubMed1988

Plasmodium vivax: karyotype polymorphism of field isolates.

Pulse-field gradient electrophoresis (PFG) has been applied to the karyotype analysis of Plasmodium vivax isolates obtained directly from infected patients in Sri Lanka. Detection of separated chromosomes was performed either by ethidium bromide staining of gels or by hybridization with a telomer specific probe. Each of the 15 different isolates examined exhibited a different chromosome migration pattern, indicating that a high level of polymorphism prevailed in wild populations of P. vivax. Chromosome size variation was further confirmed using a P. vivax chromosome-specific probe which also demonstrated that, in each isolate, the parasite population appeared to be homogeneous. These observations were made directly on parasites from infected blood, without the necessity for culture amplification, indicating that PFG can be used on a large scale for the epidemiological analysis of wild parasite populations.

Animals↗
PubMed1992

A mechanism for NADPH inhibition of catalase compound II formation.

Catalase-bound NADPH both prevents and reverses the accumulation of inactive bovine liver catalase peroxide compound II generated by 'endogenous' donors under conditions of steady H2O2 formation without reacting rapidly with either compound I or compound II. It thus differs both from classical 2-electron donors of the ethanol type, and from 1-electron donors of the ferrocyanide/phenol type. NADPH also inhibits compound II formation induced by the exogenous one-electron donor ferrocyanide. A catalase reaction scheme is proposed in which the initial formation of compound II from compound I involves production of a neighbouring radical species. NADPH blocks the final formation of stable compound II by reacting as a 2-electron donor to compound II and to this free radical. The proposed behaviour resembles that of labile free radicals formed in cytochrome c peroxidase and myoglobin. Such radical migration patterns within haem enzymes are increasingly common motifs.

Animals↗
PubMed1992

A novel mutant (transthyretin Ile-50) related to amyloid polyneuropathy. Single-strand conformation polymorphism as a new genetic marker.

DNA sequence polymorphisms in transthyretin (TTR) genes were investigated by single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products. The amplified DNA fragments that encode each exon of the normal TTR gene showed two bands, representing the two complementary single strands of DNA. In one patient with amyloid polyneuropathy, the exon 3 DNA showed a unique, aberrant migration pattern. Direct sequencing analysis of the amplified exon 3 revealed a single base change (G-to-T), resulting in a novel amino acid substitution (Ser-50----Ile). We also present the SSCP patterns for five known Japanese TTR variants.

Amyloidosis↗
PubMed1983

Biosynthesis of a proinsulin-like molecule in the pancreas of a lizard.

The biosynthesis of insulin, in particular the occurrence of a proinsulin-like molecule (PILM), in the pancreas of the green anole, Anolis carolinensis, was investigated. The laboratory rat was used for comparison and validation of the procedures. Anolian pancreases were incubated in vitro with radiolabeled leucine or proline. Radioactivity incorporated into the acidic ethanol-soluble (AES) phase increased in an essentially linear manner with time. Selective incorporation of labeled amino acids into AES material was not enhanced by a high concentration of glucose (6 mg/ml), by the addition of glucagon, which increases cyclic AMP levels, or by the addition of cytochalasin B; yet all three conditions result in stimulated insulin secretion. Gel filtration of the AES material on columns of Bio-Gel P-30 revealed a major peak of radioactivity whose apex followed closely the apogee of porcine proinsulin. When this presumptive PILM was treated with trypsin and carboxypeptidase B, the radioactivity was shifted towards later elution volumes, but the peak of the converted anolian material was not coincidental with marker insulin. Immunopurification techniques and additional molecular filtrations resulted in the isolation of fractions with both insulin-like immunoreactivity (IRI) and radioactivity derived from tritium-labeled leucine. One peak of radiolabel was present in the region of the proinsulin standards. The level of radioactivity in the region of the insulin standards was relatively high after two days of organ culture of splenic pancreases. By polyacrylamide gel electrophoresis proteins that incorporated radiolabeled amino acids and had insulin-like immunoreactivity possessed migration patterns similar to those of mammalian proinsulin and insulin. The insulin-like component was less readily demonstrated than the proinsulin-like component and raises the possibility that anolian PILM may be a major storage form in this species. The results are consistent with the synthesis of a proinsulin-like precursor in the beta cells of the green anole. The results also provide evidence for a precursor-product relationship in the anolian beta cell.

Animals↗
PubMed1991

Computer-assisted image analysis assay of human neutrophil chemotaxis in vitro.

We have developed a computer-based image analysis system to measure in-filter migration of human neutrophils in the Boyden chamber. This method is compared with the conventional manual counting techniques. Neutrophils from healthy individuals and from patients with reduced chemotactic activity were used. The cells migrating into the filter were counted automatically at depths 20 microns apart commencing from the upper surface on the filter. The major advantages of this method are reproducibility and the counting of many fields which provides better data for statistical analysis of the results. Another advantage of the assay is that it can be used to show the migration pattern of different populations of neutrophils from both healthy individuals and patients.

Chemotaxis, Leukocyte↗
PubMed1984

Estrogen stimulates cell proliferation and the increase of a 52,000 dalton glycoprotein in human breast cancer cells.

In an estrogen supersensitive variant of the MCF-7 cell line, CG-5, estrogen was found to stimulate the labelling of a glycoprotein released into the culture medium which has the same electrophoretic migration pattern as that previously reported in MCF-7 cells (Biochem. Biophys. Res. Commun., 90: 410-416, 1979). To test the possibility that the 52 K is a marker of estrogen-dependent breast cancer cell proliferation, we have correlated the effect of estrogen and antiestrogen on protein labelling and cell proliferation under different experimental conditions. In cells cultured in the presence of 5% charcoal-treated fetal calf serum, physiological concentrations (0.1-1 nM) of estradiol stimulated in a dose- and time-related fashion both 52 K labelling and cell proliferation. However at high concentrations (10-100 nM) estrogen decreased 52 K labelling while it still stimulated cell proliferation. Concentrations of the tamoxifen derivative, 4-hydroxytamoxifen, which effectively prevented estrogen-stimulated cell proliferation also blocked estrogen-stimulated increase of 52 K labelling. Time-course experiments suggest that the estrogen-stimulated increase of 52 K labelling (detectable after 22 h of hormone exposure) precedes the effect of cell proliferation (detectable after 3 days of hormone exposure). In cells cultured under serum-free conditions there was no effect of estradiol at any of the concentrations and times used on either 52 K labelling or cell proliferation.

Breast Neoplasms↗
PubMed1992

Familial Creutzfeldt-Jakob disease in Chile is associated with the codon 200 mutation of the PRNP amyloid precursor gene on chromosome 20.

We have found the codon 200Lys mutation in 6 Chilean CJD families, including a family in the rural case cluster in Chillán. Thus, all 3 of the known clusters of CJD, in Slovakia, Libyan-born Israeli Jews, and Chile, are linked to the presence of the same mutation. The phenotypic features of the disease in these families are similar to those reported for other clustered or individual families elsewhere in the world. The heterogeneous genetic composition of the Chilean population interpreted in light of historical migration patterns suggests that the mutation may have entered Chile by Jewish emigration from Spain.

Adult↗
PubMed1988

The study of multi-stage carcinogenesis in retinoblastoma and familial polyposis coli patient-derived skin fibroblast cell culture systems.

Carcinogenesis is considered to be a multi-step process comprising 'initiation', 'promotion' and 'conversion' events. Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited 'initiation' event which is present in every cell. Experiments involving skin fibroblasts from FPC patients showed certain of these cells to grow in semi-solid medium following treatment with the complete carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone. When tested prior to the commencement of the experiments the FPC patient cell populations had shown no strong predisposition to malignant transformation as assessed by increased saturation densities, reduced serum requirements, altered migration patterns in collagen gels, anchorage-independent growth and tumourigenicity in nude mice. Following carcinogen or promoter treatment, apart from exhibiting low-level frequencies of anchorage-independent growth, the cells appeared no more transformed than they were before. Parallel cytogenetic studies showed TPA to increase both tetraploidy and the chromosome-aberration frequency during the course of these transformation studies. However, the FPC cell clones induced by TPA to grow in semi-solid medium were, at best, considered to be only partially transformed when their properties were compared with those of tumour-derived cell lines.

Adenomatous Polyposis Coli↗
PubMed1996

A mutation in the p53 tumor suppressor gene of AHH-1 tk+/- human lymphoblastoid cells.

Loss-of-function mutations in the p53 tumor suppressor gene result in an altered response to DNA-damaging agents. Included in the mutant p53 phenotype are the loss of the G1 checkpoint and delayed apoptotic cell death, characteristics we have consistently observed in the AHH-1 tk+/- cell line following exposure to DNA-damaging agents. In order to determine the functional status of p53 in the AHH-1 tk+/- cell line, molecular analysis (single-strand conformational polymorphism [SSCP] and sequence analysis) was performed on exons 5-9 of the p53 gene. In addition, the status of the p53 gene in the closely related lymphoblast line, MCL-5, which, in our hands, has a much higher spontaneous rate of apoptosis than AHH-1 tk+/-, was also determined by molecular analysis. Initial SSCP analysis of AHH-1 tk+/- revealed an abnormal migration pattern of exon 8 when compared to a wild-type control. Subsequent sequence analysis indicated that a base-pair substitution (CGG-->TGG) mutation had occurred at codon 282, a reported "hot spot' for 5-methylcytosine mutations in the human p53 gene. Neither SSCP nor sequence analysis of exons 5-9 of MCL-5 indicated any differences from wild-type DNA. These results suggest that the lack of a G1 arrest and the delayed entrance into apoptosis observed in chemically-exposed AHH-1 tk+/- cells are, at least partially, accounted for by a loss-of-function mutation in the p53 gene.

Apoptosis↗
PubMed1986

The schistosome egg granuloma: immunopathology in the cause of host protection or parasite survival?

The granulomatous inflammatory response induced by schistosome eggs entrapped in the microvasculature of host tissues is considered responsible for much of the symptomatology of schistosomiasis. However, the evolutionary role of the egg granuloma in the host-parasite relationship is not yet well defined. Some evidence indicates that the lesion may protect the host, either by shielding tissues against toxic egg products, or by interfering with the migration patterns of secondary infections, and thereby non-specifically contributing to the host's acquired "immunity". We here review earlier work concerned with the role of the egg granuloma in the host-parasite relationship in schistosomiasis, and we present new experimental evidence to suggest that the function of this cell-mediated immune response might, in addition to its putative host protective function, facilitate the extravasation of parasite eggs in the mesenteries, and thereby contribute directly to the continuation of the schistosome life-cycle.

Animals↗
PubMedDate not supplied

Lack of effect of ivermectin on prepatent guinea-worm: a single-blind, placebo-controlled trial.

The effect of ivermectin on prepatent guinea-worm was tested in a single-blind placebo-controlled trial; 400 adults were randomly allocated to a single dose of ivermectin (150 micrograms/kg) or placebo. Fifty-four of the 385 participants who were followed for 15 months developed a total of 69 emergent guinea-worms. There was no significant difference in the proportion of persons with emergent guinea-worms between the 2 treatment groups; 58% appeared in males. 80% of emergent guinea-worms were located below the knee. Migration of guinea-worms in the tissues was not affected. It is concluded that ivermectin has no effect on prepatent guinea-worms nor does it disturb their migration pattern. No adverse reaction to treatment was seen. It appears that ivermectin can be used safely as mass chemotherapy against onchocerciasis and lymphatic filariasis in areas where guinea-worm is also endemic.

Adolescent↗
PubMed1987

Reduction of neutral gene flow due to the partial sterility of heterozygotes for a linked chromosome mutation.

The effect of linkage between a chromosome mutation producing partially sterile heterozygotes and a neutral locus in reducing the gene flow at the neutral locus is studied using a two-population deterministic model. Chromosome mutations are more efficient in reducing gene flow with low migration rates than with high ones. The interaction between high values of partial heterozygote sterility and low recombination rates can produce, in the low migration pattern, a drastic reduction of gene flow. Nevertheless, since only chromosome mutations with low values of partial heterozygote sterility are likely to be involved in chromosomal speciation, a significant reduction of gene flow will probably occur only for a very limited part of the genome. Therefore, a single chromosome mutation is unlikely to play a primary role in speciation.

Biological Evolution↗
PubMed1983

Cross antigenicity among enteroviruses as revealed by immunoblot technique.

Antigenic relationships of various human and two animal picornaviruses were investigated by the immunoblotting ("Western blot") technique. The viruses included all coxsackievirus B types (1-6), poliovirus types 1-3, several strains of echovirus 11, EMC virus, and FMDV. Antisera included human sera and sera from rabbits hyperimmunized with either purified picornaviruses, viral structural polypeptides (VP8), boiled or "sample-boiled" virions. Group-specific reactions of various extent were observed among the human picornaviruses, but not with EMC virus. These reactions were obtained with human sera (whole serum, IgG- and IgM-fraction) as well as with "monospecific" (neutralization test) rabbit antisera. Among cross reacting polypeptides VP1 was predominant with the notable exception of coxsackie B4, where VP1 (defined according to cleavage pattern) migrates in our gel system as second largest polypeptide. Antisera prepared vs VP1 had neutralizing activity as demonstrated with five different echovirus 11 strains (titers up to 2000). Antisera vs VP1 (and other VP8) exhibited cross-reactivity in the immunoblots. Antisera to the three poliovirus types (and to certain echovirus 11 strains) showed a surprisingly narrow cross-reacting spectrum which--in the case of poliovirus--could not be broadened by additional hyperimmunization of the rabbits with heated poliovirus 2. The significance of these results for a diagnostic ELISA in patients with picornavirus infections is dealt with.

Animals↗
PubMed1989

Sequence divergence yet conserved physical characteristics among the E4 proteins of cutaneous human papillomaviruses.

Human papillomavirus (HPV) types 1, 2, and 4 together comprise the major cause of cutaneous papillomas in the general population. We have aligned the genomes of these three viruses by partial sequence analysis, and have sequenced the E4 open reading frames (ORFs) of HPV 2 and HPV 4. After expression as beta-gal fusion proteins in bacteria, antibodies raised to the putative E4 gene-products of both virus types were used to identify the native E4 proteins in naturally occurring tumors. At the primary amino acid sequence level, the E4 protein of HPV 2 was found to be most homologous with those of HPV 6 and 11 and was not closely related to those of HPV 1 or 4. Although the E4 ORF represents a region of weak homology amongst papillomaviruses, the E4 encoded proteins showed significant conservation in their physical characteristics. Like those of HPV 1, the E4 proteins of both HPV 2 and HPV 4 were found to be composed of a major low-molecular-weight doublet (16.5/18K for HPV 2, 20/21K for HPV 4, c.f. 16/17K for HPV 1) along with minor high-molecular-weight species, which probably represent dimers of the smaller proteins, (33K for HPV 2, 40K for HPV 4, c.f. 32/34K for HPV 1). The E4 products of all three virus types were multiply charged, and exhibited a characteristic migration pattern following alkaline urea gel electrophoresis. Although the levels of E4 expression in tumors induced by the different virus types was very different, this was found to correlate closely with the level of virus production characteristic of each virus type. In all three cases, E4 proteins were found to be primarily cytoplasmic, and to be associated with the distinctive cytoplasmic inclusion granules characteristic of each virus type. The poor sequence conservation between the E4 protein of HPVs 1, 2, and 4, taken alongside the ability of these viruses to infect similar histological sites, suggests that E4 may not be involved in determining tissue specificity. Our results suggest conserved physical characteristics (acidic, multiply charged, ability to form dimers) and similar site of expression may be the important factors for E4 function.

Amino Acid Sequence↗
PubMed1987

Mucosal immunity: the immunology of breast milk.

The mammary glands represent one part of the mucosal immune system, a definable, subunit of humoral and cellular immune functions in man that appears to have developed particular qualities well suited to guard our interface with the environment. As our understanding of secretory immunoglobulins and lymphocyte migration patterns continues to develop, the immunologic components found in breast milk appear increasingly likely to play a specific immunologic role in the protection of the nursing infant. The biologic basis for the observed protective effect of breast-feeding is reviewed with an emphasis on the mechanisms involved in the development and maintenance of mucosal immunity in general.

Adult↗
PubMed1983

Characterization of immunoglobulins from the brown bullhead (Ictalurus nebulosus) produced against a naturally occurring bacterial pathogen, Aeromonas hydrophila.

The primary immune response in the brown bullhead (Ictalurus nebulosus) to intramuscularly injected, chloroform-killed Aeromonas hydrophila reached an agglutination titer of 128 in 3 wk, and the response to the bacteria plus adjuvant reached a titer of 512 in 5 wk. Anti-Aeromonas hydrophila antibodies from brown bullheads were excluded from Sephadex G-200 and had an immunoelectrophoretic migration pattern toward the cathode. Cleavage of the macromolecule by 2-ME reduction and iodoacetamide alkylation yielded subunits that were resumptively heavy chains of about 50,500 daltons and light chains of about 22,500 daltons. Bullhead antibodies were assumed to be tetrameric in configuration and therefore to have an approximate molecular weight of 600,000 daltons assuming a J chain is present in both early (6 wk) and late (20 wk) immune sera. The agglutinating activity of the antibody was completely eliminated by exposure to 65 degrees C for 30 min. Immunoelectrophoresis showed a single antibody component that moved toward the cathode. The structure and molecular weight of bullhead antibodies appears similar to that observed in channel catfish and other closely related teleost fish.

Aeromonas↗