Immunity to Trichinella spiralis infection in vitamin A-deficient mice.

Vitamin A-deficient (A-) mice make strikingly poor IgG responses when they are immunized with purified protein antigens. Previously, we showed that A- T cells overproduce interferon gamma (IFN-gamma), which then could inhibit interleukin 4 (IL-4)-stimulated B cell IgG responses. To determine if the altered IFN-gamma regulation pattern and its immunological consequences would extend to a natural infection, we studied mice infected with the parasitic helminth Trichinella spiralis. The course of the infection was similar in A- and A-sufficient (A+) mice. These mice did not differ with respect to newborn larvae/female/hour produced in the intestine, or muscle larvae burden 5 wk postinfection. They also did not differ in the intestinal worm expulsion rate until day 15, when A- mice still harbored parasites, whereas A+ mice had cleared intestinal worms. Vitamin A deficiency reduced both the frequency of B lymphocytes secreting IgG1 antibodies to parasite antigens, and the bone marrow eosinophilia associated with helminth infection. The cytokine secretion patterns in infected mice were consistent with these observations and with previous studies. Mesenteric lymph node cells from infected A- mice secreted significantly more IFN-gamma, and significantly less IL-2, IL-4, and IL-5 than infected A+ controls. A- splenocytes secreted significantly more IFN-gamma, and equivalent amounts of IL-2, IL-4, and IL-5 compared with A+ controls. Interestingly, CD4-CD8- cells secreted the majority of the IL-4 produced in the spleen. The IL-2, IL-4, and IL-5 steady-state transcript levels correlated with secreted protein levels, but IFN-gamma transcripts did not. Although they secreted more protein, A- cells contained fewer IFN-gamma transcripts than A+ cells. These results suggest two vitamin A-mediated regulation steps in IFN-gamma gene expression: positive regulation of IFN-gamma transcript levels, and negative regulation posttranscriptionally. The essentially unaltered outcome of T. spiralis infection in vitamin A-deficient mice probably reflects a balance between cellular and humoral responses. The IFN-gamma overproduction might have a positive effect on the gut inflammatory response, but the decrease eosinophilia, cytokine production in mesenteric lymph node, and IgG1-secreting cell frequency might have a negative effect on T. spiralis immunity.

Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes.

The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231. Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed.

Diet, prolactin, and breast cancer.

Relationships between dietary nutrients and plasma prolactin concentration were studied in 249 women with a history of nonskin cancers among first-degree female relatives. For each quintile of nutrient density, the odds ratio (OR), relative to the lowest quintile, of having an elevated (above the median) prolactin concentration was estimated by logistic regression, taking into account parity, menopausal status, and current tobacco-smoking habits. For nutrient densities estimated from 24-h recall data there was a significant positive association between plasma prolactin concentration and increasing saturated fatty acid intake; the OR of elevated prolactin in the top quintile was 3.1 [95% confidence interval (CI) 1.2-8.1] and there was a negative association with vitamin C [OR in the top quintile 0.28, (95% CI 0.10-0.78)]. For usual nutrient densities (estimated by quantitative food frequency questionnaire) there was a statistically significant trend (P = 0.04) toward lower prolactin concentrations with increasing sodium density, and a marginally significant positive trend (P = 0.07) with increasing dietary density of refined sugars.

The EGR1 protein contains a discrete transcriptional regulatory domain whose deletion results in a truncated protein that blocks EGR1-induced transcription.

Egr-1 is a ubiquitous immediate-early gene whose expression is induced by a wide range of different stimuli. A requirement for egr-1 expression has been demonstrated in pathways leading to both proliferation and differentiation, suggesting that egr-1 is a critical intermediary in determining the long-term cellular response to a stimulus. To determine how egr-1 coordinates a cellular response to receptor-mediated stimulation, we have developed a transient cotransfection assay to map functional domains in the EGR1 protein. We localized an activation domain to a serine/threonine/proline-rich region between amino acids 174 and 270. Using this information, we designed a mutant that lacks this activation domain, but retains the DNA-binding domain. When cotransfected into fibroblasts with an EGR1-dependent reporter, this mutant inhibited the transcriptional activity of both endogenous EGR1, as well as exogenously expressed, wild-type EGR1 protein. These data demonstrate that the activation domain of EGR1 is critical for the activity of the protein, and that a mutant lacking this domain can dominantly inhibit wild-type EGR1 function.

Identification of Bacillus anthracis by API tests.

API and morphological tests were examined for their ability to distinguish between 37 Bacillus anthracis strains (virulent and avirulent) and 194 strains of closely related Bacillus species (B. cereus, B. mycoides and B. thuringiensis). In addition, 34 strains of B. anthracis and four of B. cereus were tested by several other methods that included capsule formation, ability to grow on a selective medium, and sensitivity to phage. It was found that virulent strains of B. anthracis were easily separated from the closely related Bacillus species by most of the test methods; but separation of slightly virulent and avirulent strains of B. anthracis from the closely related species could be done only by API and phage-sensitivity tests.

GM crops and the rat digestive tract: a critical review.

The aim of this review is to examine the relationship between genetically modified (GM) crops and health, based on histopathological investigations of the digestive tract in rats. We reviewed published long-term feeding studies of crops containing one or more of three specific traits: herbicide tolerance via the EPSPS gene and insect resistance via cry1Ab or cry3Bb1 genes. These genes are commonly found in commercialised GM crops. Our search found 21 studies for nine (19%) out of the 47 crops approved for human and/or animal consumption. We could find no studies on the other 38 (81%) approved crops. Fourteen out of the 21 studies (67%) were general health assessments of the GM crop on rat health. Most of these studies (76%) were performed after the crop had been approved for human and/or animal consumption, with half of these being published at least nine years after approval. Our review also discovered an inconsistency in methodology and a lack of defined criteria for outcomes that would be considered toxicologically or pathologically significant. In addition, there was a lack of transparency in the methods and results, which made comparisons between the studies difficult. The evidence reviewed here demonstrates an incomplete picture regarding the toxicity (and safety) of GM products consumed by humans and animals. Therefore, each GM product should be assessed on merit, with appropriate studies performed to indicate the level of safety associated with them. Detailed guidelines should be developed which will allow for the generation of comparable and reproducible studies. This will establish a foundation for evidence-based guidelines, to better determine if GM food is safe for human and animal consumption.

Abnormal regulation of IFN-gamma secretion in vitamin A deficiency.

T lymphocytes from vitamin A-deficient (A-) mice show a decreased ability to stimulate B lymphocytes for Ag-specific secondary IgG1 responses in vivo and in vitro. Experiments reported here traced the molecular basis for this functional defect to an overproduction of IFN-gamma by A- CD4+ T cells compared with cells from A-sufficient (A+) mice. Secretion of IL-2 and IL-4 by cells from A- and A+ mice was equivalent. Retinoic acid supplementation in vitro decreased IFN-gamma secretion from A- T cells, indicating that IFN-gamma production is retinoid-responsive. Adding IFN-gamma neutralizing antibodies to cultures established with cells from immune A- mice substantially increased IgG1 production, whereas IL-4 addition moderately increased IgG1 production. Adding retinoic acid to the cultures either at initiation, or 48 h later, fully restored IgG1 production by A- cultures to the level of A+ control cultures. These results are consistent with a role for vitamin A in negatively regulating IFN-gamma secretion.

Anthrax: the disease in relation to vaccines.

The authors trace the origins and history of anthrax and anthrax vaccines. They describe the aetiology and pathogenesis of the disease and the variety of symptoms which result from infection. The authors relate the early work performed by Pasteur, the development of existing vaccines and the efficacy of these vaccines, and predict the type of non-living vaccines which may be used to combat anthrax in the future.

Characterization of a helper T lymphocyte defect in vitamin A-deficient mice.

Vitamin A-deficient mice exhibited sharply decreased IgG1 responses to protein Ag in vivo and in vitro. We traced the cellular basis for these impaired responses to a functional defect in A-deficient Th. Ag-presenting cells and B cells from A-deficient mice showed no functional defects. The A-deficient T cells proliferated normally in response to Ag but failed to provide the B cell stimulus for Ag-specific IgG1 responses. The A-deficient mice had fewer Th by limiting dilution analysis, and retinyl acetate supplementation in vitro restored Th frequency to control levels. Because added retinyl acetate reversed the Th functional block, these vitamin A-deficient T cells must be primarily defective in activation, not in establishment of immunologic memory.

Retinoid repletion of vitamin A-deficient mice restores IgG responses.

Vitamin A-deficient (A-) mice produce poor IgG antibody responses due to a helper T cell dysfunction. We performed retinoid repletion studies to determine the minimum dietary retinyl acetate dose and the most active retinoid for supporting immune function. Dietary retinyl acetate repletion at 2 (R2 group) or 4 (R4 group) microgram/g diet restored serum retinol in A- mice to vitamin A-sufficient (A+) control levels within 24 h. However, in R4 mice, liver retinyl palmitate was restored about twofold faster than in R2 mice; liver retinyl palmitate reached A+ control levels by d 30 in R4 mice but not in R2 mice. We challenged the mice with antigen 24 h post repletion; the R4 mice gave an IgG1 response equal to that of A+ controls, but the R2 mice were comparable with the A- controls. We also compared four retinoids for IgG1 response restoration in vitro; 1 nmol/L retinoic acid fully repleted A- cell IgG1 responses and helper T cell frequencies to the unsupplemented A+ control levels. Retinoic acid was at least 10-fold more active than retinyl acetate or retinaldehyde, and 100-fold more active than retinol. Collectively, our results suggest that retinoic acid is probably the physiologically important metabolite for sustaining IgG immune responses in vivo. We discuss the possible relationship between liver retinyl palmitate levels and availability of retinoic acid to support immune function.

Development of antibodies to protective antigen and lethal factor components of anthrax toxin in humans and guinea pigs and their relevance to protective immunity.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in serum to the protective antigen (PA) and lethal factor (LF) components of anthrax toxin. Current human vaccination schedules with an acellular vaccine induce predictable and lasting antibody titers to PA and, when present in the vaccine, to LF. Live spore vaccine administered to guinea pigs in a single dose conferred significantly better protection than the human vaccines (P less than 0.001), although they elicited significantly lower (P less than 0.0005) anti-PA and anti-LF titers at time of challenge with virulent Bacillus anthracis. Substantial anti-PA and anti-LF titers may not, therefore, indicate solid protective immunity against anthrax infection. The ELISA system was also shown to be capable of detecting anti-PA and anti-LF antibodies in the sera of individuals with histories of clinical anthrax. The advantage of ELISA over the Ouchterlony gel diffusion test and indirect microhemagglutination assay are demonstrated. There was a highly significant degree of correlation between ELISA and the indirect microhemagglutination assay (P less than 0.0005); but ELISA was markedly superior in terms of reproducibility, reliability, specificity, and simplicity in performance and stability of the bound antigen.

A single autosomal gene defect severely limits IgG but not IgM responses in B lymphocyte-deficient A/WySnJ mice.

Antigen-stimulated B lymphocytes either differentiate into IgM-secreting plasma cells or into memory B cells that secrete other immunoglobulin isotypes upon antigen restimulation. The mechanisms that generate and maintain memory B cells are poorly understood. Previously, we described a severe B lymphocyte deficiency in adult strain A/WySnJ mice compared to subline A/J. Here we show that the single, autosomal co-dominant locus responsible for the deficiency also diminishes IgG-secreting B cell formation without interfering with IgM-secreting plasma cell differentiation. A/WySnJ secondary IgG1 responses to the protein antigens hemocyanin, bovine gamma-globulin, ovalbumin, lysozyme and beta-galactosidase were 6- to 50-fold lower than A/J responses. The defect also decreased secondary IgG2a and IgG3 responses, and primary IgG1 and IgG2a responses. The reduced A/WySnJ secondary IgG1 response was not due to differential response kinetics or dose responsiveness, and could not be augmented to A/J levels by repeated immunizations. Serum IgG1, IgG2a and IgG3 levels from nonimmune A/WySnJ mice were similarly reduced. The secondary IgG1 response and splenic B cell percentage showed significant positive correlation (r = 0.72) in F2 mice, suggesting that a single locus controlled both traits. In contrast, A/WySnJ mice made good primary IgM responses to hemocyanin, beta-galactosidase, and the thymus-independent antigen trinitrophenyl-Ficoll. The A/WySnJ splenic adherent cells were competent in antigen-presenting function, and A/WySnJ immune T cells proliferated in response to antigen and provided the requisite B cell stimulatory signals for an IgG1 response. Together, our results suggest that A/WySnJ mice have a genetic lesion that causes a selective IgG immune response dysfunction. The absence of IgG-secreting cell precursors or a defect in precursor activation or differentiation are two possible mechanisms which could precipitate a selective IgG response dysfunction. We propose that the defective A/WySnJ and normal A/J strain pair offer the opportunity to use a natural genetic variation as a tool to investigate B lymphocyte development and function.

Immature stage B cells enter but do not progress beyond the early G1 phase of the cell cycle in response to antigen receptor signaling.

In contrast to mature B cells, immature stage B cells do not proliferate following Ag receptor cross-linking with anti-Ig Abs. To determine where in the cell cycle immature B cells arrest, we have examined the expression of specific G, cell cycle regulators. Following surface IgM (sIgM) cross-linking on mature B cells, we observed increased expression of the early G1 kinase, cyclin-dependent kinase 4 (cdk4), and one of its regulatory subunits, cyclin D2. Mature B cells also showed increased expression of components required for G1/S transition, including cyclin E and cdk2. Whereas immature stage B cells increased expression of cyclin D2 and cdk4 after anti-IgM stimulation, unlike mature stage B cells they failed to express cyclin E and cdk2. Expression of cyclin D2 and cdk4 indicates that these cells can exit G0 and enter the initial G1 phase following sIgM ligation. Interestingly, IL-4, which by itself does not stimulate proliferation of immature B cells, induced expression of cyclin E and cdk2. These latter results suggest that IL-4 complements sIgM, signaling for proliferation by increasing the basal levels of late G1 cell cycle regulators. Consistent with this idea, IL-4 synergizes with anti-Ig Abs to promote cell cycle progression and proliferation of immature B cells. Finally, c-myc, a transcriptional regulator of some members of the cell cycle machinery, is not induced following sIgM cross-linking of immature cells. This lack of inducible expression contrasts with that seen in mature stage B cells, and in immature stage cells stimulated to proliferate with LPS. These results suggest that c-myc may be a component of the signaling pathway that induces cyclin E and cdk2 expression.

Viral haemorrhagic disease of rabbits and human health.

Viral haemorrhagic disease of rabbits (VHD), a potential biological control for wild rabbits in Australia and New Zealand, escaped from quarantined field trials on Wardang Island and spread to the mainland of Australia in October 1995. This study looked for any evidence of infection or illness in people occupationally exposed to the virus. Two hundred and sixty-nine people were interviewed and 259 blood samples were collected. Exposures to VHD-infected rabbits ranged from nil to very high. No VHD antibodies were detected in any of the 259 sera when tested by VHD competitive enzyme immunoassay, which had been validated with 1013 VHDV-specific antibody negative sera. A questionnaire designed to elicit symptoms of disease in a range of organ systems found no significant differences between illness in those exposed and those not exposed to VHD, nor could an association be found between exposure and subsequent episodes of illness. The findings are consistent with the view that exposure to VHD is not associated with infection or disease in humans.