The Escherichia coli heat-labile enterotoxin B subunit protects from allergic airway disease development by inducing CD4+ regulatory T cells.

The B subunit of E. coli heat-labile enterotoxin (EtxB) protects against the development of T helper type 1 (Th1)-mediated autoimmune pathologies in mice. Protection was transferable with splenic CD4(+) T cells and was less effective following CD25 depletion; implying a T regulatory cell (Treg)-mediated process. We hypothesized that if this were the case, then EtxB would also control a Th2-mediated disorder. We tested the effect of EtxB treatment on asthma development in ovalbumin (OVA)-sensitized mice. EtxB treatment diminished eosinophilia in bronchoalveolar lavage samples, reduced OVA-specific immunoglobulin E and interleukin 4 production locally and systemically, and reduced airway hyper-reactivity. EtxB induced a dose-dependent increase in Foxp3(+)CD4(+) T cells, and adoptive transfer of splenic CD4(+) T cells partially suppressed lung pathology. Importantly, EtxB treatment increased OVA-specific CD4(+)Foxp3(+) T cells in the lung and systemically. These data demonstrate that EtxB modulates the differentiation of allergen-specific T cells causing inducible Treg induction and preventing disease.

Genetic relationships between feral cattle from Chirikof Island, Alaska and other breeds.

The origin of cattle on Chirikof Island, off the coast of Alaska, is not well documented. We assessed genetic differentiation of cattle isolated on Chirikof Island from several breeds commonly used for commercial production in North America including breeds popularly believed to have contributed to the Chirikof Island population. A set of 34 microsatellite loci was used to genotype Angus, Charolais, Hereford, Highland, Limousin, Red Angus, Salers, Shorthorn, Simmental, Tarentaise and Texas Longhorn cattle sampled from North America and the Chirikof Island population. Resulting F(ST) statistics for these loci ranged from 0.06 to 0.22 and on average, 14% of total genetic variation was between breeds. Whether population structure was modelled as a bifurcating tree or genetic network, Chirikof Island cattle appeared to be unique and strongly differentiated relative to the other breeds that were sampled. Bayesian clustering for multiple-locus assignment to genetic groups indicated low levels of admixture in the Chirikof Island population. Thus, the Chirikof Island population may be a novel genetic resource of some importance for conservation and industry.

Protection against recurrent ocular herpes simplex virus type 1 disease after therapeutic vaccination of latently infected mice.

The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinant Escherichia coli heat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.

Genetic and demographic dynamics of small populations of Silene latifolia.

Small local populations of Silene alba, a short-lived herbaceous plant, were sampled in 1994 and again in 1999. Sampling included estimates of population size and genetic diversity, as measured at six polymorphic allozyme loci. When averaged across populations, there was very little change between samples (about three generations) in population size, measures of within-population genetic diversity such as number of alleles or expected heterozygosity, or in the apportionment of genetic diversity within and among populations as measured by F(st). However, individual populations changed considerably, both in terms of numbers of individuals and genetic composition. Some populations doubled in size between samples, while others shrank by more than 75%. Similarly, expected heterozygosity and allele number increased by more than two-fold in individual populations and decreased by more than three-fold in others. When population-specific change in number and change in measures of genetic diversity were considered together, significant positive correlations were found between the demographic and genetic variables. It is speculated that some populations were released from the demographic consequences of inbreeding depression by gene flow.

Polypeptide growth factors and phorbol ester induce progressive ankylosis (ank) gene expression in murine and human fibroblasts.

Polypeptide growth factors promote cellular proliferation by binding to specific plasma membrane-anchored receptors. This interaction triggers the phosphorylation of signal transducing molecules and the transcriptional activation of numerous genes. We have used a differential display approach to identify fibroblast growth factor (FGF)-1-inducible genes in murine NIH 3T3 fibroblasts. Here we report that one of these genes encodes ank, a type IIIa transmembrane protein reported to function in cells as an inorganic pyrophosphate transporter. FGF-1 induction of ank mRNA expression is first detectable at 2 h after growth factor addition and is dependent on de novo RNA and protein synthesis. Ank gene expression is also upregulated after treating quiescent fibroblasts with several other mitogenic agents (e.g., calf serum or platelet-derived growth factor-BB) or the tumor promoter phorbol 12-myristate 13-acetate. Furthermore, in comparison to parental NIH 3T3 cells, oncogene-transformed NIH 3T3 cells constitutively express elevated levels of ank mRNA. FGF-1 also increases ank gene expression in non-immortalized human embryonic lung fibroblasts. Finally, the murine and human ank genes are expressed in vivo in a tissue-specific manner, with highest levels of mRNA expression found in brain, heart, and skeletal muscle. These results indicate that ank is a growth factor-regulated delayed-early response gene in mammalian cells, and we propose that increased ank expression during cell cycle progression may be necessary to maintain proper intracellular pyrophosphate levels during conditions of high cellular metabolic activity.

Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant.

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.

The mitogen-inducible Fn14 gene encodes a type I transmembrane protein that modulates fibroblast adhesion and migration.

The binding of polypeptide growth factors to their appropriate cell surface transmembrane receptors triggers numerous biochemical responses, including the transcriptional activation of specific genes. We have used a differential display approach to identify fibroblast growth factor-1-inducible genes in murine NIH 3T3 cells. Here, we report that the fibroblast growth factor-inducible-14 (Fn14) gene is a growth factor-regulated, immediate-early response gene expressed in a developmental stage- and adult tissue-specific manner in vivo. This gene, located on mouse chromosome 17, is predicted to encode an 129-amino acid type Ia membrane protein with no significant sequence similarity to any known protein. We have used two experimental approaches, direct fluorescence microscopy and immunoprecipitation analysis of biotinylated cell surface proteins, to demonstrate that Fn14 is located on the plasma membrane. To examine the biological consequences of constitutive Fn14 expression, we isolated NIH 3T3 cell lines expressing variable levels of epitope-tagged Fn14 and analyzed their phenotypic properties in vitro. These experiments revealed that Fn14 expression decreased cellular adhesion to the extracellular matrix proteins fibronectin and vitronectin and also reduced serum-stimulated cell growth and migration. These results indicate that Fn14 is a novel plasma membrane-spanning molecule that may play a role in cell-matrix interactions.

Induction of mucosal immunity against herpes simplex virus type 1 in the mouse protects against ocular infection and establishment of latency.

Immune responses were assessed after intranasal immunization of mice with a mixture of herpes simplex virus type 1 (HSV-1) glycoproteins with cholera toxin and its B subunit as adjuvant. Antigen-specific serum antibodies, which were largely IgG with IgG1 the major subclass, neutralized virus in vitro with a titer equivalent to that elicited by active infection. Significant levels of antigen-specific IgA were found in mucosal fluids of the eye as well as the vagina. Lymphocytes from draining lymph nodes showed secondary proliferative responses when cultured with HSV-1 in vitro, in immunized mice only, with the production of interleukin-2, interferon-gamma, interleukin-4, and interleukin-5. After ocular challenge, immunized mice were protected against the development of severe eye disease, zosteriform spread, or encephalitis, whereas the incidence of clinical symptoms in mock-immunized mice was 83%, 74%, and 52%, respectively. Finally, the incidence of latency was reduced from 88% to 13% after intranasal immunization.

Enhancement of the immune response to non-replicating herpes simplex virus type-1 preparations by mucosal administration in the presence of cholera toxin.

Different immunization regimes were compared in order to enhance the immune response following mucosal administration of non-replicating HSV-1 preparations to mice. The serum anti-HSV Ig response following intragastric administration of heat or UV inactivated HSV-1 strain SC16 was compared with that elicited by an attenuated derivative of SC16 (TKDM21). The highest response followed immunization with TKDM21 and this was markedly enhanced by repeated intragastric administration, reaching ca 35% of that elicited following a cutaneous infection with live virus. Repeated doses of killed virus produced only a minimal increase in the response even when given intranasally (i.n.). However, cholera toxin and its B-subunit with UV-inactivated virus or a mixture of purified viral glycoproteins enhanced the anti-HSV response after i.n. immunization and produced antibody levels equivalent to those following intragastric delivery of live TKDM21.

Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin.

We demonstrate that the receptor binding moiety of Escherichia coli heat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis. Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund's adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints. A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82%. The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins. The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect. Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon gamma levels. Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio. These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response. This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease.

A phylogeny for the African treefrog family Hyperoliidae based on mitochondrial rDNA.

Mitochondrial 12S rRNA sequences were used to construct a phylogeny for the African treefrog family, Hyperoliidae. The sequences were aligned using the well-established mt 12S rRNA secondary structure as a map to designate stem and loop positions. Conservation of secondary structure permits a more accurate basis for assessing homologies than does primary sequence alone. The molecular trees showed many similarities to the morphological trees constructed previously. Species within a genus always grouped together. The genera Hyperolius, Heterixalus, Afrixalus, and Kassina were resolved, but not with statistical significance. Leptopelis was the basal group. The analysis shows clearly that Phylyctimantis groups with Kassina as it did in Drewes' morphological tree and that Tachycnemis groups closely with Heterisxalus, a relationship not suggested by the morphological data. Character weighting (including compensatory base changes), mitochondrial trees vs gene trees, and the biogeography of the group are discussed.

The bidirectional relationship between achievement and externalizing behavior problems of students with learning disabilities.

The current study examined the bidirectional relationship between academic achievement and externalizing behavior problems of adolescents with learning disabilities. Forty-three students attending a residential school were assessed for externalizing behavior problems via parent and teacher reports on the Child Behavior Checklist and the Children's Attention and Adjustment Survey. The Wide Range Achievement Test-Revised was used to estimate academic achievement in reading, spelling, and arithmetic. Achievement measures did not predict externalizing behavior problems 1 to 2 years later, although verbal IQ predicted parent reports. Teacher reports of externalizing behavior problems predicted reading and spelling achievement scores 1 year later, and parent reports of externalizing behavior problems predicted changes in reading achievement. There was evidence that attentional problems were the component of externalizing behavior accounting for these relations, consistent with the explanation that inattentive students have difficulty achieving in an intensive learning environment.

Expression of the large plastid gene, ORF2280, in tomato fruits and flowers.

The expression of ORF2280, a large plastid gene of unknown function, was examined in tomato leaves, in a developmental series of tomato fruits, and in tomato flowers. Western blots indicated that much more ORF2280 protein is present in fruits and flowers than in leaves. The most abundant proteins detected, 68 and 59 kDa, are present in about equal amounts in fruits of all stages; they are even more abundant in flowers. A 170-kDa ORF2280 protein is also present in fruit of all stages; it is most abundant in small green fruit. The presence of higher levels of ORF2280 proteins in tomato fruits and flowers indicates that it may have a specialized function in these nonphotosynthetic tissues.

Synteny mapping in the horse using horse-mouse heterohybridomas.

In a study of 35 horse-mouse heterohybridoma cell lines, synteny in the horse was found between LDHB, PEPB and IGF1 and between NP, MPI and IDH2. A synteny between ADA and PEPC was also indicated. The loci for horse immunoglobulin light chain (IgL) genes and for LDHA were independent.

Genetic and antigenic analysis of an equine influenza H 3 isolate from the 1989 epidemic.

The haemagglutinin (HA) gene from the equine influenza H3N8 isolate Suffolk/89 has been cloned by reverse transcription and polymerase chain reaction amplification. The nucleotide sequence of the HA gene was determined from two independently cloned copies of the gene and was found to be most closely related to recent American isolates supporting the idea that most isolates of equine H3N8 are evolving as a single lineage. When the predicted amino acid sequence of the Suffolk/89 HA was examined, changes had taken place in at least four of the major antigenic sites, A, B, C, and D when compared to the sequences of the isolates used in the current vaccines (Miami/63 and Fontainebleau/79). Surprisingly, when the Suffolk/89 isolate was tested in haemagglutination inhibition (HI) assays with a panel of six mouse monoclonal antibodies, no differences were observed between the Suffolk/89 and the Fontainebleau/79 isolates, suggesting that this panel of monoclonal antibodies may recognise a limited subset of the major antigenic sites. Three anti-HA horse heterohybridoma monoclonals were able to distinguish between the Suffolk/89 and Fontainebleau/79 viruses, demonstrating that the horse does recognise these isolates as being antigenically different. The results of the work suggest that the isolates used in current equine influenza vaccines may need updating.

The production of equine monoclonal immunoglobulins by horse-mouse heterohybridomas.

Studies were carried out to determine the optimum conditions for the production of equine monoclonal antibodies (MAbs). Lymphocytes from ponies immunised with influenza A equine 2 virus, isolate A/Equine/Newmarket/79 (H3N8) were fused with mouse myeloma (NSO) cells and with horse-mouse heterohybridomas made aminopterin-sensitive by selective growth in 8-azaguanine. Although all fusions initially resulted in heterohybridoma colonies that secreted equine immunoglobulin, many of these were unable to maintain secretion for longer than a few weeks. Increasing the time between immunisation and the booster injection of Newmarket/79 virus, the inclusion of Freund's incomplete adjuvant and the use of an aminopterin-sensitive primary heterohybridoma as the fusion partner, improved the production of HIg-secreting heterohybridomas. After two clonings eight cell lines were established which maintained anti-Newmarket/79 antibody secretion for over a year. FACS analysis of the cell lines provided a useful means of predicting breakdown of MAb secretion by the cell lines, thus enabling re-cloning to be carried out in time.

Survey of plastid RNA abundance during tomato fruit ripening: the amounts of RNA from the ORF 2280 region increase in chromoplasts.

A comprehensive survey of the levels of plastid RNAs at progressive stages of tomato fruit ripening was conducted by hybridizing total RNA with labeled Pst I fragments that cover almost the entire tomato plastid genome and with gene-specific probes. Two different cultivars of tomato (Lycopersicon esculentum Mill.) were examined, Traveler 76 and Count II. One of the tomato probes, P7, revealed a pronounced increase in the amount of an 8.3 kb RNA in ripe fruit. The homologous region of the tobacco plastid genome contains several genes for ribosomal proteins and a large unidentified open reading frame (2280 codons). Little change was observed in the levels of many transcripts during ripening. However, in some cases (e.g. psbA and psbC/D) the amount of RNA decreased during ripening of Count II but showed little or no change in Traveler 76. The contrast between Traveler 76 and Count II tomatoes shows that the level of plastid transcripts can vary substantially during fruit ripening with no obvious effect on the chloroplast to chromoplast transition. The large RNA from the P7 region may encode a protein that functions predominantly in chromoplasts.

Cancer and humiliation: The "catch 22" of disease.

In the author's own words: I am a cancer patient involved in an agonizing struggle against disease. I decided to write about the humiliating aspects of cancer because I have experienced them firsthand. I know them all too well. I have incorporated my feelings about the humiliation of disease ... hoping they may help the reader better understand.

Two sex-linked loci in the leopard frog, Rana pipiens.

Crosses involving one heterozygous parent were performed to test the inheritance of enzymes in the leopard frog, Rana pipiens. After metamorphosis, offspring were sexed and tissue extracts from them were analyzed by gel electrophoresis. Enzyme genotype and sex showed independent assortment for 10 of 12 enzymes heterozygous in the male parent. However, among the offspring of males heterozygous for peptidase C (Pep-C) or superoxide dismutase 1 (SOD-1), male progeny tend to inherit one allele, whereas female progeny tend to inherit the other. Data from several different crosses yield recombination frequencies of 8.6% between sex and SOD-1, 6.9% between SOD-1 and Pep-C and 12.1% between sex and Pep-C. When the female parent is heterozygous for these enzymes no significant difference is seen, in the offspring, between male and female homozygotes and heterozygotes. These results confirm that males are the heterogametic sex in R. pipiens and suggest that sex is determined by a small number of genes on otherwise identical X and Y chromosomes.