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A major portion of the nucleoprotein (amino acids 67 through 300) and the glycoprotein-2 of lymphocytic choriomeningitis (LCM) virus were synthesized by using recombinant technology and were injected together with SDS twice in portions of 5 micrograms into BALB/c mice. As evidenced by diminished replication of LCM challenge virus, both proteins induced antiviral immunity, which was comparable in extent with the immunity caused by infection with LCM vaccinia recombinant viruses. Primed LCM-viral CTLs could not be demonstrated in these mice by culturing splenocytes in the presence of LCM virus, and Abs appeared slowly and in low quantities; but, after injection of large infectious doses, CTLs appeared faster and in higher numbers than in mice not previously treated with viral proteins. Depletion of CD8+ cells, but not of CD4+ cells, by treatment of mice with mAb abolished the antiviral immunity, demonstrating that protection was mediated by CD8+ T lymphocytes. Absence of CD4+ T lymphocytes before and during the period of immunization did not measurably affect the animals' antiviral immune status, indicating that activation of the CD8+ T lymphocytes was not dependent on help by CD4+ cells.

Complex adjuvant formulations have been used to introduce soluble protein antigens into the "endogenous" processing pathway and hence to elicit specific, major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) responses. We tested if simple modifications of a model protein antigen, i.e. ovalbumin (OVA), can render it immunogenic for murine class I-restricted CTL when injected into mice in soluble form. Injection of 1-100 micrograms native OVA into C57BL/6 (H-2b) mice did not stimulate a class I-restricted CTL response. In contrast, immunization of mice with 0.5 to 10 micrograms sodium dodecyl sulfate (SDS)- or deoxycholate (DOC)-denatured OVA efficiently primed CD8+ CTL specific for the well-characterized Kb-restricted OVA257-264 epitope. Gel-purified SDS-denatured OVA devoid of protein fragments and excess detergent efficiently stimulated a specific CTL response in vivo. OVA preparations denatured by heat or urea treatment were not immunogenic for murine CTL. Injection of non-treated or detergent-treated, antigenic OVA257-264 peptide into mice did not elicit a CTL response. Thus, denaturation of OVA by simple detergents such as SDS or DOC dramatically enhances its immunogenicity for class I-restricted CTL but not all modes of denaturation are equally effective.

Immunization with soluble proteins only rarely induces a specific response of CD8+ CTL. We describe experiments that demonstrate the efficient and specific in vivo priming of CTL in BALB/c mice immunized with soluble hepatitis B virus (HBV)-derived surface (S) protein. A single (s.c., i.p. or i.v.) injection of a low dose (30 ng to 3 micrograms per mouse) of recombinant S protein particles without adjuvants induced a CTL response. This specific cytotoxic response was read out against a panel of different S protein-expressing transfected mouse cell lines. Effector cells of this response were Ld-restricted, CD3+ CD4- CD8+ CTL. H-2d/Ld+ (BALB/c, C.B-17) mice were responders; H-2d/Ld- (dm2) mutant mice and H-2b (C57BL/6) mice were nonresponders. Injections of various dosages of a S protein-derived, immunogenic, synthetic peptide into BALB/c mice by various routes did not prime CTL. After incorporation of S protein particles into IFA or aluminum hydroxide, these protein Ag lost their ability to specifically stimulate CTL in vivo. After priming of mice with S protein emulsified in IFA or adsorbed to aluminum hydroxide boost injections with native S protein particles were inefficient in stimulating a specific CTL response. These findings are of relevance for the design of synthetic subunit vaccines for which specific stimulation of CD8+ T effector functions is desired.

Zusammenfassung Proben von Weizenkleie und Weizen aus Chargen, die hauptsachlich fur Futterungszwecke bestimmt waren, wurden in zwei Jahren in einem Gebiet Sudwestdeutschlands gezogen und auf Ergosterin (ERG), einen Parameter fur die von Pilzen gebildete Biomasse, sowie auf die Fusarientoxine Deoxynivalenol (DON) und Zearalenon (ZEA) untersucht. ERG, DON bzw. ZEA wurden in 100, 100 bzw. 71% der Kleieproben, und in 100, 74 bzw. 18% der Weizenproben gefunden. Der mittlere Gehalt von ERG, DON bzw. ZEA lag bei 11,3 mg/kg, 788 μg/kg, bzw. 20 μg/kg in der Kleie, und bei 4,7 mg/kg, 204 μg/kg bzw. 11 μg/kg im Weizen. Die Unterschiede zwischen Kleie und Weizen sind signifikant (p < 0,01). Der Gehalt an DON lag in 24% der Kleie und 3% der Weizenproben uber 1 mg/kg, der Gehalt an ZEA in 3% der Kleie- und 1% der Weizenproben uber 60 μg/kg. Es wird empfohlen, Weizenkleie in Futterrationen fur Schweine und bei der menschlichen Ernahrung restriktiv einzusetzen.

DNA-based vaccination efficiently primes MHC-restricted T-cell responses. This technique specifically stimulates MHC-II-restricted CD4(+) T-cell responses and MHC-I-restricted CD8(+) T-cell responses against "strong" (immunodominant) or "weak" (subdominant or cryptic) epitopes of intracellular, secreted or membrane-associated protein antigens. In many experimental systems, T-cell-mediated effector functions have the potential to control tumor growth. In particular MHC-I-restricted cytotoxic T lymphocytes (CTL) can reject tumors. This has been shown using either defined tumor-associated antigens (TAA), or viral antigens containing well-defined, MHC-binding and CTL-stimulating epitopes that are expressed by transfected tumor cells.

DNA-based vaccination is a potent technique to prime cellular (T-cell mediated) immune responses (reviewed in 1). Many details of the priming of T-cell precursors by antigen translated from injected expression plasmid DNA are unknown. The relevant cell that is transfected in situ after DNA vaccination and that can process and present the protein in an immunogenic form has not yet been identified. Alternatively, the transfected cell may initiate 'cross-priming' in vivo by transferring processed antigen to a professional antigen-presenting cell (APC).

Nail-gun injury is commonly encountered in the emergency department. The severity of the injury depends on the specific injuries to organs and vascular structures. Computed tomography (CT) is the ideal imaging test of choice, as it can identify the more critical injuries that require immediate intervention. We present a case of self-inflicted nail-gun injury with intracranial and intracardiac nails that was optimally evaluated with CT. CT significantly changed the patient’s management, giving priority to potentially life-threatening penetrating cardiac injuries.

Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreER(T2), and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreER(T2). The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts.

After intravenous injection of 10(5) purified, lymph node (LN)-derived dm2 (H-2d/Ld-) CD4+ T cells into young C.B-17 scid/scid (severe combined immunodeficiency, SCID) mice (H-2d/Ld+), the transplanted Ld-T cells show a selective pattern of engraftment: they repopulate the spleen, the lamina propria of the small intestine and the mesenteric LN (but not other peripheral LN) of the immunodeficient host. CD4+ cells repopulating different lymphoid organs of the SCID recipient mice produce interleukin-2 (IL-2) and interleukin-4 (IL-4) in response to polyclonal stimulation in vitro. Some evidence has recently been provided that cytokines (e.g. IL-4) present at the site of antigen stimulation in vivo decisively influence the pattern of cytokines expressed by T cells activated at these sites. We therefore asked if neutralization of IL-4 by chronic treatment of SCID mice with high doses of recombinant soluble IL-4 receptor (sIL-4R) changes the IL-4 or IL-2 expression pattern of CD4+ T cells adoptively transferred into young SCID recipients. Transplanted SCID mice were chronically treated with two different, recombinant murine sIL-4R proteins. The experimental series further included groups of transplanted SCID mice treated with a recombinant human sIL-4R protein (which does not bind murine IL-4), treated with the anti-murine IL-4 monoclonal antibody (MoAb) 11B11, or non-treated. Transplanted SCID mice treated with the recombinant murine sIL-4R protein preparations displayed detectable sIL-4R serum levels, which demonstrates that the substitution therapy could maintain neutralizing serum levels of anti-IL-4 activity in SCID mice. By contrast, no serum sIL-4R levels were detectable in the sensitive ELISA readout in transplanted SCID mice which were non-treated, treated with the MoAb 11B11, or treated with the recombinant humans sIL-4R protein. The efficiency and the pattern of CD4+ T-cell engraftment, and the lymphokine-producing phenotype of the engrafted dm2 CD4+ cells, was not affected by the continuous IL-4-neutralizing treatment of mice with either the MoAb 11B11 or the soluble IL-4R preparations. Hence, in contrast to the published evidence of the dramatic effect of IL-4 on the lymphokine-producing phenotype of CD4+ T cells stimulated in vitro or in vivo, the chronic suppression in vivo of IL-4 activity (by either different sIL4-R protein constructs, or by the anti-IL-4 MoAb 11B11) did not lead to preferential engraftment of Th1-type CD4+ T cells after adoptive transfer of CD4+ T-cell populations into an immunodeficient recipient.

Micronutrients play an important role in function and health maintenance for the eye. Especially lutein, zeaxanthin and omega-3 fatty acids perform remarkable functions: lutein together with zeaxanthin forms the macular pigment, these carotenoids filter out the damaging blue light component from the sunlight as well as the ultraviolet light which leads to improved contrast sensitivity and less problems with screen glare. Furthermore, the macular pigment has antioxidant and anti-inflammatory effects. The omega-3 fatty acids also possess anti-inflammatory effects and, when converted into neuroprotectin, they protect against oxidative induced apoptosis in the retina. They are also responsible for the fluidity and supply to the photoreceptor membrane. These properties are important for the prevention and treatment of degenerative eye diseases like age-related macular degeneration. However, older people are often not sufficiently supplied of micronutrients in their diet. Because the supply of nutrients can hardly be achieved by dietary change, the additional intake in the form of food supplements is useful in this age group. Scientific studies have shown the positive effects of supplementation with micronutrients such as lutein/zeaxanthin, vitamin C, vitamin E, zinc and omega-3 fatty acids, docosahexaenoic acid and eicosapentaenoic acid (DHA and EPA). Currently available nutritional products are based in part on the ingredients of the ARED study (Age Related Eye Disease Study). According to more recent studies formulations containing lutein and omega-3 fatty acids in physiologically meaningful doses without additional beta-carotene should be preferred. 10 to 20 mg of lutein and zeaxanthin represent a safe daily dose Regarding to the context above, beta-carotene in high doses plays a minor role to the eye and is especially critical for the health of smokers. This paper summarises the functions of the presented micronutrients in the eye and can assist ophthalmologists in advising their patients.

One of the goals of NAPAP-sponsored research on the effects of acidic deposition on carbonate stone has been to quantify the incremental effects of wet and dry deposition of hydrogen ion, sulfur dioxide and nitrogen oxides on stone erosion. Test briquettes and slabs of freshly quarried Indiana limestone and Vermont marble have been exposed to ambient environmental conditions in a long-term exposure program. Physical measurements of the recession of test stones exposed to ambient conditions at an angle of 30° to horizontal at the five NAPAP materials exposure sites range from ∼ 15 to ∼ 30 μm yr−1 for marble, and from ∼ 25 to ∼ 45 μm yr−1 for limestone, and are approximately double the recession estimates based on the observed calcium content of run-off solutions from test slabs. The difference between the physical and chemical recession measurements is attributed to the loss of mineral grains from the stone surfaces that are not measured in the run-off experiments. The erosion due to grain loss does not appear to be influenced by rainfall acidity, however, preliminary evidence suggests that grain loss may be influenced by dry deposition of sulfur dioxide between rainfall events. Chemical analyses of the run-off solutions and associated rainfall blanks suggest that ∼ 30% of erosion by dissolution can be attributed to the wet deposition of hydrogen ion and the dry deposition of sulfur dioxide and nitric acid between rain events. The remaining ∼ 70% of erosion by dissolution is accounted for by the solubility of carbonate stone in rain that is in equilibrium with atmospheric carbon dioxide (“clean rain”). These results are for marble and limestone slabs exposed at an angle of 30° from horizontal. The relative contribution of sulfur dioxide to chemical erosion is significantly enhanced for stone slabs having an inclination of 60° or 85°. The dry deposition of alkaline particulate material has a mitigating effect at the two urban field exposure sites at Washington, DC, and Steubenville, OH.

This paper discusses tritium removal from a self-cooled Pb-17Li blanket of a fusion reactor, permeation into an intermediate NaK loop and precipitation of the tritide in a cold trap. First experiments on the kinetics of hydride precipitation showed that low supersaturation concentrations are obtained at low concentration ranges, these values are obtained after a very short cold trap loading period. Both results meet essential requirements for fusion blanket cold traps. Theoretical work has shown that two-dimensional calculations (including buoyancy effects) of the temperature, velocity and concentration distributions result in precipitation distributions which differ significantly form these obtained with 1d-models currently used to develop mass transfer relationships.

SUMMARY We have investigated the effect of a bolus injection of atracurium 0.6mg kg−1 on the cardiovascular system in 16 patients undergoing aortocoronary bypass surgery. H1- and H2-receptor antagonists were administered to eight patients before the neuromuscular blocker. A standard anaesthetic was used comprising fentanyl, flunitrazepam, etomidate and enflurane. After administration of atracurium, haemodynamic changes and plasma histamine concentrations were measured at frequent intervals. In the first group, who received only atracurium, a brief but marked decrease in SVR and MAP occurred, accompanied by an increase in Cl, together with a marked increase in plasma concentration of histamine. In the second group, who received H1 and H2-receptor block, there was no decrease in MAP and only a small increase in plasma histamine concentration. However, there were significant changes in SVR and Cl similar to those in the atracurium group.

In fusion reactors, Be is a candidate material for both ceramic breeder blankets in the form of pebbles and as plasma facing components (PFC) at the First Wall in the form of tiles.

A potential tritium removal technique for a fusion reactor with a self-cooled Pb-17Li blanket consists of tritium permeation into an intermediate NaK loop and the precipitation of tritide in a cold trap. Tritium is recovered by thermal decomposition of the tritide under vacuum. Hydrogen recovery experiments (tritium simulated by hydrogen) were performed in the temperature range between 280 and 400°C with complete wire mesh packed cold traps or single packings. The recovery rate coefficients obey pseudo first order reaction kinetics and are similar to those found in previous experiments. No difference for the two types of experiments were observed. Analysis of the hydride crystal showed that the crystals are of the octahedral type and consist only of potassium hydride.

Tritium recovery by cold trapping in an intermediate NaK loop is considered for a self-cooled Pb-17Li blanket. The transient build-up of tritium inventory in the Pb-17Li and NaK loops is calculated for a test-plug to be used in NET. A steady-state tritium inventory of about two gramms is assessed for the blanket system including the cold traps. Hydrogen separation and release experiments in cold traps are going to start. Calculations of the temperature, velocity and concentration distributions in cold traps point to the influence of natural convection in mesh packed cold traps. First hydrogen release experiments were performed with hydride powder and hydride crystals. Different release mechanisms were observed during recovery from NaK which suggest that tritium recovery from drained cold traps should be preferred.

A blanket design concept using Pb17Li eutectic alloy as both breeder material and coolant is described. Such a self-cooled blanket for the boundary conditions of a DEMO-reactor is under development at the Kernforschungszentrum Karlsruhe (KfK) in the frame of the European blanket development program. Results of investigations in the areas of design, neutroncs, magneto-hydrodynamics, thermo-mechanics, ancillary loop systems, and safety are reported. Based on recent progress, it can be concluded that the boundary conditions of a DEMO-reactor can be met, tritium self-sufficiency can be obtained without using beryllium as an additional neutron multiplier, and tritium inventory and permeation are acceptably low. However, to completely judge the feasibility of the proposed concept, further studies are necessary to obtain a better understanding of the magneto-hydrodynamic phenomena and their effects on the thermal-hydraulic performance of a fusion reactor blanket.

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The 38-year-old index patient was examined by visual acuity testing, perimetry, dark adaptometry, funduscopy, electroretinogram (ERG), and multifocal ERG. She was screened for mutations in exons 2-5 and exon/intron boundaries of the 11- cis retinol dehydrogenase gene by direct sequencing.Visual acuity was 1.0, but perimetry revealed paracentral scotomas associated with reading problems. The optic discs were normal. After 45 min of darkness there was nearly no increase of light sensitivity. After 30 min of dark adaptation, the scotopic ERG showed reduced amplitudes, but after 60 min a nearly normal level was reached. The 30-Hz flicker response of the cone ERG showed borderline implicit times, but no reduction of amplitudes. However, multifocal ERG clearly disclosed a paracentral amplitude reduction as the reason for the visual field defects. The fundus was typical for fundus albipunctatus. The patient is a compound heterozygote carrying a Ile33Asn and a Arg157Trp mutation.The paracentral visual field defects were due to cone dysfunction. So far the patient exhibits no cone dystrophy.

A 67-year-old man presented with a 6-week history of slowly progressive muscle pain and generalized muscular weakness. Neurological examination showed symmetrical proximal weakness (MRC grade 4/5).Laboratory tests revealed an elevated CK level of 124 U/l (normal80). Needle EMG showed pathological spontaneous activity in proximal muscle groups. A diagnostic muscle biopsy taken from the left deltoid muscle revealed a characteristic infiltrate of PAS-positive and acid phosphatase-positive macrophages. Electron microscopic analysis of muscle tissue failed to demonstrate aluminium hydroxide inclusion. Serological analysis excluded antibodies against HAV and HBV but was compatible with a previous tetanus vaccination (antibody titre of 2.3 IE/ml).A 2-year treatment with steroids and azathioprine led to complete recovery of muscle strength without relapse after discontinuing the immunosuppression.Macrophagic myofasciitis (MMF) is a rare inflammatory muscle disorder characterized by a characteristic infiltration of muscle tissue by PAS-positive macrophages, which is caused by pathological persistence of vaccine-derived aluminium hydroxide. The diagnosis can only be established by an open muscle biopsy from the muscle that was used for intramuscular vaccination.

CD11c+ (F4/80– CD68– ) dendritic cells (DC) in the colonic lamina propria (cLP) of normal and immunodeficient (RAG1– /– ) C57BL/6 (B6) mice show high surface expression of MHC class I/ II molecules and CD1d, and low surface expression of CD40, CD80, CD86 costimulator molecules. CD4+ § g T cells from normal or MHC class II-deficient B6 mice transferred into congenic RAG1– /– hosts induce a progressive, lethal colitis. Concomitant with colitis development, DC in the inflamed cLP increase in number and up-regulate surface expression of CD1d, MHC class II molecules and CD40, CD80, CD86 costimulator molecules. cLP DC from non-transplanted (healthy) and transplanted (diseased) mice produce similar amounts of IL-12 p70 and IL-10 in response to CD40 signaling, but the inducible IL-12 p40 release is 5– 15-fold higher in mice with colitis than in non-transplanted mice. Binding of IL-12 p40 to p19 generates IL-23. Freshly isolated cLP lymphocytes (cLPL) from transplanted, diseased mice express 3– 10-fold more p19 transcripts than cLPL from non-transplanted, healthy mice. p19 expression by cLPL is further up-regulated in response to CD40 ligation. Freshly isolated cLP DC from transplanted mice with colitis (but not from non-transplanted controls) stimulate IFN- + (but not IL-4 or IL-13) release by co-cultured NKT cells. In colitis, DC accumulate in the cLP, show an activated surface phenotype, up-regulate IL-12 p40 and p19 expression, and ‘ spontaneously’ stimulate NKT-like cells. cLP DC may be interesting targets for novel therapeutic approaches to modulate mucosal T cell responses in situ.

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10–100 ng/mouse) of α-galactosyl ceramide (αGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following αGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to αGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-γ response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG−/− or HBs-tg/RAG−/−) hosts via the portal vein elicited IFN-γ responses of liver NK cells in situ. IFN-β down-regulates the pathogenic IL-12/IFN-γ cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-β suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (αGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-γ response of the specifically activated liver NKT cells. In vivo, IFN-β attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.

Mutations of the human telethonin gene have recently been shown to cause limb girdle muscular dystrophy type 2G in three Brazilian families. The mRNA has been shown to be dynamically regulated in animals, however, the fate of the protein in human muscle is unknown. In order to assess the expression of telethonin in more frequently encountered myopathological conditions we generated and characterized a rabbit antiserum raised against the C-terminal end of telethonin by immunoblotting and immunogold EM. Indirect immunofluorescence analysis of a wide variety of neuromuscular disorders including dystrophinopathies, metabolic myopathies, denervation disorders, congenital and inflammatory myopathies revealed that the characteristic Z-band staining of telethonin was preserved in all disease entities included in our study. However, a reduced telethonin immunoreactivity was observed in up to 10% of type II fibers in 10 cases of neurogenic atrophy. A decreased telethonin staining was more frequently observed in early stages of fiber atrophy than in type II fibers displaying normal or highly atrophic fiber diameters. Hence, not only the telethonin transcript is rapidly downregulated in denervated muscle but the protein itself undergoes dynamic changes while its known sarcomeric binding partner titin remains unaltered. Beyond its role as a static component of Z-bands, these findings indicate that telethonin protein levels seems to be at least in part regulated by neuronal activity and is thus linked to the dynamic control of myofibrillogenesis and muscle turnover in human skeletal muscle.

We have used the hepatitis B virus system in mice to investigate under which conditions the different DNA delivery techniques used in DNA vaccination introduce a T2 or T1 bias into the responses they elicit. Priming mice by an intramuscular injection of 100 microg plasmid DNA stimulated long-lasting T1 immune responses that included specific activation of potent MHC-1-restricted CTL responses. In contrast, priming mice with an intradermal injection of a low dose of plasmid DNA (0.1-1 microg/mouse) stimulated long-lasting T2 immune responses but no CTL responses. This polarization (spectrum of immune effector specificities) of immune responses elicited by plasmid DNA vaccines is determined by different factors, e.g. (i) the vaccination technique used; (ii) the age of the immunized animal; (iii) the condition of the local tissue environment to which DNA is delivered; (iv) the co delivery of immune-modulating factors; and (v) the type of antigen encoded by the plasmid DNA vaccine. DNA vaccination offers the possibility of constructing endogenous, chimaeric antigens with enhanced, hsp-facilitated expression, enhanced immunogenicity for B cells, and alternative, TAP-independent processing for MHC-1 restricted peptide presentation.

A prominent subset of the hepatic innate immune system is α-galactosylceramide (αGalCer)-reactive, (CD4+ and CD4−CD8−) CD1d-restricted NKT cells. We investigated in C57BL/6 (B6) mice which hepatic cell type stimulates hepatic NKT cell activation. Surface expression of CD1d but not CD40, CD80, or CD86 costimulator molecules was detected in hepatocytes. Pulsed in vitro or in vivo with αGalCer, hepatocytes triggered IL-4 release by liver NKT cells but required exogenous IL-12 to trigger IFN-γ release by NKT cells. Liver dendritic cells (DC) isolated from nontreated mice showed low surface expression of MHC, CD1d, and CD40, CD80, or CD86 costimulator molecules that were strikingly up-regulated after αGalCer injection. Although liver CD11c+ DC displayed lower CD1d surface expression than hepatocytes, they were potent stimulators of IFN-γ and IL-4 release by liver NKT when pulsed with αGalCer in vitro or in vivo. Liver DC are thus potent stimulators of proinflammatory cytokine release by NKT cells, are activated themselves in the process of NKT cell activation, and express an activated phenotype after the NKT cell population is eliminated following αGalCer stimulation.

Scid mice transplanted with CD4(+) T cells from congenic donor mice develop a chronic and lethal inflammatory bowel disease (IBD) 2-3 months post-transplantation. In the present study we have investigated the response of CD4(+) T cells from scid mice with colitis against fecal extracts. Our results show that in contrast to CD4(+) T cells from normal BALB/c mice, CD4(+) T cells from scid mice with colitis proliferate strongly in response to antigen-presenting cells (APC) pulsed with fecal extracts. The IBD-associated T cells did not respond to either extracts from food antigens or fecal extracts from germ-free mice, which indicates that they recognize bacterial antigens in the fecal extracts. CD4(+) T cells isolated from the colonic lamina propria of scid mice 3 weeks post transplantation also responded vigorously to fecal extracts, demonstrating that reactive CD4(+) T cells are present in the gut mucosa of transplanted scid mice prior to clinical manifestations of IBD. CD4(+) T cells activated by fecal extracts produced high amounts of IL-2 and IFN-gamma, intermediate amounts of IL-4 and low amounts of IL-10, consistent with a Th1 profile. The proliferative reactivity towards fecal extracts was restricted by MHC class II molecules and dependent on antigen processing, as the response could be blocked by anti-MHC class II antibodies or a short fixation of the APC. This study demonstrates that class II-restricted CD4(+) Th1 cells, which recognize enteric bacterial antigens, infiltrate the gut mucosa and spleen of transplanted scid mice prior to and during the course of colitis.

Clinical observations have shown histamine-typical cardiopulmonary reactions immediately after application of heparin. Heparin is produced from animal tissues, rich in mast cells which contain both heparin and histamine. Heparin in clinical use might therefore still contain considerable amounts of histamine. After mixing heparin with human blood,in vitro, an increase in plasma histamine concentration was observed. Additionally, histamine, in various concentrations, could be detected in different heparin batches and separations.

Inflammatory bowel disease in scid mice is initiated by transplantation of CD4 + T-cells from immunocompetent syngenic donor mice. As the disease progresses, immunoglobulin (Ig)-containing cells appear in the gut lamina propria, suggesting that locally accumulating Ig may play a role in disease development. In the present work we have investigated the relationship between disease progression and patterns or levels of Ig isotypes in the feces of scid mice suffering from an ongoing colitis. The data clearly showed that the severity or progression of the disease did not influence the levels of IgA, IgG1, IgG2a, IgG2b, and IgG3, whereas the level of fecal IgM increased during the course of colitis. The presence of the serum protein α-1-antitrypsin in fecal extracts from diseased mice suggests that some of the fecal Ig has leaked through the inflamed epithelial membrane into the gut lumen. Finally, Ig-containing cells were observed in mesenteric lymph nodes and in the spleen, suggesting that the fecal Ig is produced both systemically and locally in the gut wall. In conclusion, the present results demonstrate that the level of IgM increases as colitis progresses. Also, the five remaining major Ig isotypes are increased in the gut lumen of scid mice with colitis, but the individual Ig types vary randomly during the course of the disease. Thus, it is unlikely that immunoglobulins are involved in the immunopathogenesis of this model of colitis.

Humoral and cellular immune responses to protein antigens can be efficiently primed by nucleic acid or DNA vaccination. In DNA-based vaccination, immunogenic proteins are expressed with correct posttranslational modification, conformation or oligomerization; this ensures the integrity of epitopes that stimulate neutralizing antibody (B cell) responses. DNA (or RNA) immunization is exceptionally potent in stimulating T cell responses because antigenic peptides are efficiently generated in (endogenous or exogenous) processing pathways (without interference by viral proteins) from intracellular or extracellular protein antigens expressed after transient in vivo transfection. Both features are difficult to achieve with recombinant subunit vaccines produced in eukaryotic or prokaryotic expression systems. The current state of vector designs, strategies for delivery of DNA vaccines, priming humoral and cellular immune responses by DNA vaccines, experimental strategies facilitated by DNA vaccines, unique advantages of DNA vaccination, experience of DNA vaccination in preclinical animal models and clinical trials, and potential risks of DNA vaccination are discussed. Excellent reviews on DNA-based vaccination have been published recently [1-3].

We describe a 52-year-old woman who presented with severe diarrhea, nausea, intermittent abdominal pain and weight loss of 18 kg within ten months. Jejunal and duodenal ulcers were detected by endoscopy and multiple biopsies revealed villous atrophy of the jejunum. However, neither gliadin nor endomysium antibodies were detected and no clinical and histological improvement was achieved after gluten withdrawal. Despite strong clinical suspicion for intestinal lymphoma many unrevealing biopsies were done. The patient developed intermittent septic fever and diagnostic laparotomy revealed jejunal perforation. Partial jejunal resection was performed and histology confirmed the diagnosis of an intestinal T-cell lymphoma without celiac disease. Malabsorption and all intestinal ulcers disappeared during the course of chemotherapy (six cycles CHOP) and the patient recovered remarkably.

Hepatitis B virus (HBV) causes acute self-limiting and chronic infection in humans (24). A chronic HBV infection leads to a high risk for the development of liver cirrhosis and hepatocellular carcinoma (30, 54). The current strategy for preventing HBV infection is vaccination with hepatitis B surface antigen (HBsAg), which induces virus-neutralizing anti-HBsAg antibodies (28). Though HBsAg is a potent immunogen and induces protective immunity in the majority of vaccines, 5 to 10% of persons who receive the HBsAg vaccine failed to develop anti-HBsAg antibodies. In addition, HBV variants carrying mutations within the HBsAg can escape the neutralization of vaccine-induced anti-HBsAg and establish acute or chronic infection (3, 4, 6, 25, 29, 43). Therefore, a new vaccine strategy would be desirable to induce a multiple immune response consisting of HBV-specific T helper (Th), cytotoxic T cells (CTLs), and anti-HBsAg antibodies. HBV-specific Th and CTL responses play a pivotal role for the clearance of virus in a primary HBV infection and may control HBV persisting in unknown reservoirs in patients whose disease is resolved (1, 7, 16, 18, 26, 27, 35, 41, 42, 44, 45). The induction of HBV-specific humoral and cellular immune response by a single vaccine may overcome the nonresponsiveness of individuals to conventional HBsAg vaccines and control immune escape variants of HBV with mutations within HBsAg. DNA vaccination is a powerful method to induce antigen-specific humoral and cellular immune response (14, 56). DNA-induced immune response provides protective immunity to various viruses in animal models (2, 5, 13, 19, 22, 31, 34, 36, 51, 55, 57). Genetic vaccination to HBsAg, HBV core antigen (HBcAg), and HBV e antigen (HBeAg) was evaluated in different animal models. In mice, a single intramuscular injection of plasmids expressing HBsAg is sufficient to induce a long-lasting humoral response to HBsAg and CTL response (10, 12, 40, 50). A plasmid vaccination of chimpanzees led to the production of low anti-HBsAg antibody titers (11, 47). Recently, Triyatni et al. reported that vaccination of ducks with plasmid expressing duck hepatitis B virus (DHBV) surface antigens (DHBsAg) induced antibodies to DHBsAg (55). Anti-DHBsAg antibodies induced by DNA vaccination were able to neutralize virus in vitro. DHBV was removed more rapidly from the bloodstreams of vaccinated ducks after a challenge. Infection of hepatocytes by DHBV was limited or prevented in vaccinated ducks. Therefore, the genetic vaccination was effective to prime an anti-HBsAg antibody response in this model. The vaccination of mice with HBcAg or HBeAg was also effective for inducing specific CTL responses (33). The woodchuck (Marmota monax) model is useful to study immune response to hepadnavirus and to perform vaccination trials (8, 9, 23, 39, 48, 49, 52). Woodchuck hepatitis virus (WHV) causes acute self-limiting and chronic infection, like HBV in humans (53). The humoral immune responses to woodchuck hepatitis surface antigen (WHsAg) and core antigen (WHcAg) in acute and chronic WHV infection have the same features as those of HBV infection. Anti-WHcAg develops in woodchucks during the early phase of a primary WHV infection and persists lifelong. Anti-WHsAgs, like anti-HBsAgs, increase at the end of the viremic phase and may provide immunity to a secondary WHV infection. Recently, T-cell response to WHsAg and WHcAg in woodchucks during acute and chronic WHV infection was investigated by an in vitro assay to measure the antigen-specific proliferation of peripheral blood mononuclear cells (PBMCs) (8, 32, 38, 39). Multispecific Th response to WHcAg and WHsAg was present during acute WHV infection but absent in woodchucks with chronic WHV infection (39). Thus, the Th response to WHV in woodchucks closely resembles the HBV-specific Th response in humans (52). The woodchuck model is informative in the study of immune response induced by vaccines and virus challenge. Immunization of woodchucks with WHsAg-induced anti-WHsAg antibodies provided protection against a subsequent challenge with WHV (9). Interestingly, woodchucks immunized with WHcAg were protected against WHV challenge even though anti-WHcAg antibodies do not possess the ability to neutralize WHV (49, 52). Apparently, WHcAg induced a specific T-cell response which conferred protective immunity (39). We demonstrated that immunization with a peptide containing a T-cell epitope derived from WHcAg leads to the protection of woodchucks against WHV infection. These results emphasize the significance of T-cell response to the core antigen for control of hepadnavirus infection (16, 18). In the present study, we wanted to determine whether vaccination of woodchucks with plasmids expressing WHV proteins can induce a protective immune response to WHV. We vaccinated mice and woodchucks with plasmids expressing WHcAg and WHsAg and investigated the humoral and cellular immune response to WHcAg and WHsAg in woodchucks. The protective efficacy of plasmid vaccination was demonstrated in woodchucks in subsequent challenge experiments.

SUMMARYScid mice transplanted either with a gut wall graft or with low numbers of purified CD4+ T cells from immunocompetent syngeneic donor mice show clinical signs of IBD 3–4 months post-transplantation. The disease is mediated by mucosa-infiltrating CD4+ TCRαβ+ T cells. The pathology of 52 individual colon segments obtained from 20 gut wall- or CD4+ T cell-transplanted diseased scid mice was evaluated by histology and the numbers of infiltrating immunoglobulin-containing cells were determined. In particular, cells positive for IgM, IgA and non-inflammatory immunoglobulin isotypes such as IgG1 and IgG2b were found to accumulate in colon segments displaying the most severe histopathology, including inflammatory cellular infiltration, epithelial hyperplasia and ulcerative lesions. Compared with colon segments of normal C.B-17 mice, the lesional scid colon shows increased levels of cells positive for the IgG classes. Faecal extracts of the CD4+ T cell-transplanted scid mice revealed the presence of all six murine immunoglobulin isotypes. Disease progression was accompanied by an increased level of excreted IgM and IgG3 and decreased levels of IgA. It is concluded that locally secreted immunoglobulins may play an immunomodulating role in the pathological changes observed in the present model of T cell-induced inflammatory bowel disease.

The murine melanoma cell line B16.F10 (H-2b) was used to study specific T cell responses that reject tumors. Stable B16 transfectants were established that express viral Ags, either the hepatitis B surface Ag (HBsAg) or the large tumor Ag (T-Ag) of SV40. B16 cells and their transfected sublines were CD40+ CD44+ but expressed no (or low levels of the) costimulator molecules CD154 (CD40L), CD48, CD54, CD80, and CD86. Surface expression of MHC class I (Kb, Db) and class II (I-Ab) molecules by B16 cells was low, but strikingly up-regulated by IFN-gamma. CD95 (Fas) and CD95 ligand (CD95L (FasL)) were "spontaneously" expressed by B16 cells growing in vitro in serum-free medium; these markers were strikingly up-regulated by IFN-gamma. B16 cells coexpressing CD95 and CD95L were irreversibly programmed for apoptosis. In vitro, noninduced B16 transfectants stimulated a specific IFN-gamma release response, but no cytolytic response (in a 4-h assay) in MHC class I-restricted CTL; in contrast, IFN-gamma-induced B16 targets were efficiently and specifically lysed by CTL. In vivo, B16 transfectants were specifically rejected by DNA-vaccinated syngeneic hosts through a T-dependent immune effector mechanism. The tumors showed evidence of massive apoptosis in vivo during the rejection process. The data suggest that CTL-derived IFN-gamma enhances an intrinsic suicide mechanism of these tumor cells in addition to facilitating lytic interactions of effectors with tumor targets.

CD4+ TCRalphabeta+ T cells from the colonic lamina propria of athymic (nude) mice were adoptively transferred into histocompatible (SCID) mice homozygous for the autosomal recessive mutation scid (severe combined immunodeficiency). Transfer of these extrathymic CD4+ T cells into SCID mice induced a pancolitis in the adoptive host. The histopathology of this inflammatory response was restricted to the colon and closely resembled human UC. CD4+ T cells infiltrating the colonic lamina propria of diseased SCID mice displayed the surface phenotype of mucosa-seeking memory/effector cells, expressed interferon-gamma (IFN-gamma), and lysed targets in a Fas (CD95)/FasL-dependent pathway. Massive accumulation of oligoclonal CD4+ T cells of athymic origin with the phenotype of Th1 memory/effector T cells in the colonic lamina propria of a histocompatible, immunodeficient host elicits a pancolitis that morphologically mimics human UC.

We demonstrate in a murine model that targeting an anti-viral T cell response to a growing tumor facilitates priming of a tumor-associated antigen (TAA)-specific, rejecting T cell response. Murine P815 mastocytoma cells grow aggressively in a syngeneic host. Transfected P815/S cells (expressing the hepatitis B surface antigen, HBsAg) also grow as subcutaneous tumors, but occasional ‘spontaneous’ rejections after transient growth are observed. Growth of P815/S tumors (but not of P815 tumors) is efficiently suppressed by a CD8+ cytotoxic T lymphocyte (CTL)-dependent immune mechanism in mice primed to HBsAg by DNA–immunization. In hosts immunized against HBsAg by DNA vaccination, HBsAg-specific CTL are generated. This specific CTL reactivity was targeted to s.c.-growing P815 tumors by intra tumor injections of either HBsAg-encoding plasmid DNA or viable P815/S cells; this treatment led to tumor rejection in 70–80% of the tumor-bearing animals. All rejecting animals showed a CD8+ CTL-dependent resistance to subsequent challenges by native, non-transfected P815 tumors. Targeting an established anti-viral (‘strong’) CTL response to a growing tumor hence is an efficient strategy to facilitate priming of a rejecting CTL response against (‘weak’) TAA in this system.

A total of 44, 40, 47, 51, and 58 barley samples for feed use were collected randomly after the 1987, 1989, 1990, 1991, and 1992 crops, respectively, from farms located in an area of southwest Germany. The sum of precipitation from May to September was high in 1987 and markedly lower in 1989-1992. Deoxynivalenol, 3-. and 15-acetyldeoxynivalenol, nivalenol, fusarenon-X, T-2 toxin, HT-2 toxin and diacetxyscirpenol were determined by gas chromatography with mass selective detection (GC-MS), zearalenone,alpha- and beta-zearalenol by GC-MS or by HPLC. Deoxynivalenol was the major toxin with incidences at 71-98% and mean contents at 42-400 microg/kg. In contrast, incidences of zearalenone, 3-acetyldeoxynivalenol, nivalenol, HT-2 toxin, and T-2 toxin were at 7-68, 7-48, 11-41, 0-9, and 2-29%, respectively; with mean contents at 3-146 microg/kg. alpha- and beta-zearalenol and diacetoxyscirpenol were not detected in any sample. 15-acetyldeoxynivalenol and fusarenon-X were assayed in samples from 1987, 1991 and 1992. 15-acetyldeoxynivalenol was detected in 30, 0 and 2% of samples, respectively, with an average content of positive samples at 8 and 4 microg/kg, fusarenon-X was not detected. Over the years, incidences and levels of toxins remained constant, decreased or increased. The correlation between the occurrence of toxins and level of precipitation is discussed.

Essential hypertension and gastrointestinal disease are two of the most common diagnoses made in the doctor's office. Since they may occur simultaneously, the differential therapeutic aspects of the respective therapies are of importance for every general practitioner. However, complex interactions between hypertension and gastrointestinal diseases often make the choice of the most suitable drug difficult. The present article provides an overview of the most common therapeutic regimens, identifies possible interactions, and should help the doctor to select the optimal treatment for the individual patient.

A total of 84, 78, 80, 80, 78, and 45 wheat samples for feed use were collected randomly after the 1987, 1989, 1990, 1991, 1992, and 1993 crops, respectively, from farms in an area of southwest Germany. The sum of precipitation from May to September varied during years, with markedly higher precipitation in 1987, compared to 1989-1993. Deoxynivalenol, 3- and 15-acetyldeoxynivalenol, nivalenol, HT-2 toxin, T-2 toxin, diacetoxyscirpenol, and fusarenon-X were determined by gas chromatography, combined with mass selective detection (GC-MS), zearalenone, alpha- and beta-zearalenol by GC-MS or HPLC. Deoxynivalenol was the major toxin, with incidences of 68-95% and mean contents at 152-1,692 micrograms/kg. In contrast, incidences of zearalenone, 3-acetyldeoxynivalenol, nivalenol, HT-2 toxin, and T-2 toxin were at 11-80, 17-60, 25-64, 0-8, and 0-41%, respectively, with mean contents between 3 and 209 micrograms/kg. alpha-zearalenol and/or beta-zearalenol were detected in five samples at contentsor = 71 micrograms/kg; diacetoxyscirpenol was not detected in any sample. 15-acetyldeoxynivalenol and fusarenon-X were assayed in samples from 1987 and 1991-1993. 15-acetyldeoxynivalenol was detected in 3-15% of samples at mean contents of 5-84 micrograms/kg; fusarenon-X was not detected. Over the years, incidences and levels of toxins remained constant, decreased or increased. The correlation between the occurrence of toxins and precipitation is discussed.

The serum Ab response and the class I-restricted CTL response of C57BL/6 (H-2b) mice to hepatitis B (pre)core Ag (HBcAg, HBeAg) was studied. Injection of HBcAg particles without adjuvants into mice efficiently primed serum Ab responses but not CTL response. We constructed the expression plasmids pCMV-1/c and pCMV-1/e in which expression of HBcAg or HBeAg was driven by cytomegalovirus immediate early region promoter sequences. Stable murine RBL5/C transfectant lines expressing HBcAg were established. Intramuscular DNA immunization with plasmid pCMV-1/c (encoding intracellularly expressed core Ag) or pCMV-1/e (encoding secreted precore Ag) efficiently primed specific serum Ab responses and CTL responses. The CTL response elicited in this system was mediated by CD4-CD8+ effector cells primed in vivo. The CTL recognized the HBcAg93-100 8-mer peptide MGLKFRQL in the context of Kb. Hence, DNA immunization with HBcAg/HBeAg-expressing plasmids, but not immunization with exogenous HBcAg particles, elicits a class I-restricted CTL response of defined epitope/restriction specificity in H-2b mice.

H-2d mice generated a major histocompatibility complex (MHC) class I (Ld)-restricted T-cell response of defined restriction and epitope specificity to the hepatitis B virus small surface antigen (HBsAg). Here, we compare different vaccination techniques that prime in vivo class I-restricted, murine cytotoxic T lymphocyte (CTL) precursors and specific serum antibody responses. CTL were efficiently primed by the injection of low doses of recombinant native HBsAg particles without adjuvants, by the injection of low doses of denatured HBsAg monomers without adjuvants, by infection with recombinant vaccinia virus carrying a HBsAg-encoding gene, or by intramuscular transfer of plasmid DNA encoding HBsAg under appropriate promoter control. The observation that the injection of 100 ng to 1 microgram of native HBsAg "virus-like particles' (VLP) without adjuvants is an exogenous antigen preparation that efficiently primes class I-restricted CTL responses was unexpected. It reveals a novel aspect of the immunogenicity of VLP for T cells.