Protective immunity against<Emphasis Type="Italic"> Toxoplasma gondii</Emphasis> in mice induced by a chimeric protein rSAG1/2

The aim of this study was to test the protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2. The PCR-amplified SAG2 fragment (558 bp) digested with the restriction enzyme XhoI was inserted into the XhoI site of plasmid pGEX-6p-1, termed pGexSAG2. The PCR-amplified SAG1 fragment (1,008 bp) digested with restriction enzymes EcoRI and XhoI was cloned into the EcoRI/XhoI sites of a separate plasmid pGEX-6p-1, termed pGexSAG1. The SAG2 fragment (HindIII/HindIII) excised from pGexSAG2 was inserted into the HindIII site of pGexSAG1 and a chimeric vector constructed, pGexSAG1/2. The fusion proteins GST-SAG1/2, GST-SAG1 and GST-SAG2 were expressed in BL21 Star (DE3) Escherichia coli and purified by GSTrap FF columns. After removing the GST tag, the recombinant proteins rSAG1/2, rSAG1 and rSAG2 were independently collected and injected into different groups of mice to evaluate their protective capability. The highest proliferation of splenocytes stimulated with tachyzoite sonicate antigen (TsoAg) was observed in BALB/c mice which had received two intraperitoneal injections of rSAG1/2. Maximum production of IFN- was also found in the culture supernatants of TsoAg-stimulated splenocytes from rSAG1/2-immunized mice. Finally, 73% (11/15) of mice immunized with rSAG1/2 survived at least 28 days after a lethal challenge of 1×10³ live tachyzoites which killed all non-immunized mice within 10 days. Moderate survival rates were observed in mice immunized with either rSAG1 (60%) or rSAG2 (53%). These results show that the chimeric protein rSAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice.     

In vivo and in vitro activity of roxithromycin againstToxoplasma gondii in mice

Roxithromycin protected mice against a lethal infection with the extremely virulent RH strain and the CS6 strain ofToxoplasma gondii. Therapy initiated 2 h after infection with 2×10³ or 2×10⁴ RH or C56 tachyzoites protected more than 80% of the mice, compared with 0% of untreated controls (p < 0.001). Paradoxically,Toxoplasma gondii was isolated more frequently (> 80%)in treated mice surviving infection with the less virulent strain (CS6) than those surviving infection with the more virulent RH strain (< 25%). Furthermore, in vitro studies revealed that roxithromycin at dosages up to 250 g/ml had no effect on the proliferation ofToxoplasma gondii in murine peritoneal macrophage cultures.     

Immunogenic characterization of the chimeric surface antigen 1 and 2 (SAG1/2) of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> expressed in the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-γ, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.005), and their survival time increased significantly compared to the negative control.     

Human T-cell clones againstToxoplasma gondii: Production of interferon-γ, interleukin-2, and strain cross-reactivity

The soluble fraction from a sonicate ofToxoplasma gondii tachyzoites (termed F3) was shown to induce dose-dependent blastic transformation of peripheral-blood mononuclear cells (PBMC) from seropositive individuals only and was used to isolate a panel of T-cell clones from the PBMC of an immune donor. Proliferation assays using F3 showed that 15 (14 CD4⁺ and 1 CD8⁺) of the 18 isolated clones were specific forT. gondii. In response to antigen stimulation, 5 of the 15 clones produced detectable levels of interleukin-2 (IL-2, 0.2–15 u/ml) and 9 clones produced significant levels of interferon- (IFN-, 17.5–1400 IU/ml). Seven of the 7 T-cell clones tested reacted with two differentToxoplasma strains (RH and Wiktor). When used as antigen-presenting cells, an autologous B-lymphoblastoid cell line could efficiently present the antigen to only three of the six T-cell clones tested. This study identifies and characterizes cellular probes that could be useful for future vaccine design.     

<Emphasis Type="Italic">Sarcocystis neurona</Emphasis> major surface antigen gene 1 (<Emphasis Type="Italic">SAG1</Emphasis>) shows evidence of having evolved under positive selection pressure

The major surface antigen gene 1 (SAG1) is conserved among members of Sarcocystidae and may play an important role in parasite pathogenesis. Additionally, generation and selection of different antigenic variants of SAG1 has the potential for inclusion in a subunit vaccine or in the development of a diagnostic assay. In this study, patterns of nucleotide polymorphism were used to test the hypothesis that natural selection promotes diversity in different parts of SAG1 of Sarcocystis neurona. Nucleotide and amino acid sequence analysis of SAG1 from multiple S. neurona isolates identified two alleles. Sequences were identical intra-allele and highly divergent inter-alleles. Also, phylogenetic reconstruction showed sequences clustering into two clades. Tajimas and Fu and Lis neutrality tests indicated that selection is more likely to be acting on SAG1. Moreover, a sliding window analysis based on the ratio of silent substitutions to amino acid replacements provided strong evidence that two short segments in the central and 3 domain of SAG1 have been under positive selection in the divergence of the two alleles, suggesting that it may be important for the evasion of host immune responses and would be a suitable target for vaccine development.This revised version was published online in November 2004. In the Abstract an the Results section 5 has been corrected to 3.     

Posttranslational modifications of α-tubulin of Toxoplasma gondii

The posttranslational modifications of -tubulin of Toxoplasma gondii were characterized by antibodies and biochemical analysis of the carboxy-terminal peptide. -Tubulin is acetylated and glutamylated. Side chains with up to three glutamate residues are linked to Glu445 of T. gondii -tubulin. The data suggest that the site of glutamylation on -tubulin is conserved over a broad range of species.     

The Effects of Epiphyseal Peptides on the Release of Immunoglobulins in Peyer's Patches in Rats in Vitro

The effects of epiphyseal peptides (1 g/ml) on the release of immunoglobulins into the incubation medium by isolated Peyer's patches from non-immunized mice and mice immunized orally against ovalbumin were studied during 40-min incubations. The possibility that epiphyseal peptides act on adrenoreceptors of cells in secondary lymphoid organs in the small intestine was assessed using - and -adrenoreceptor blockers, i.e., phentolamine HCl (0.02 mg/ml) and anaprilin (0.06 mg/ml) respectively. Basal levels of secretory activity in control Peyer's patches from immunized rats were 2.4 times (p < 0.01) greater than for the lymphoid organs of non-immunized animals. The effects of epiphyseal peptides on the secretory activity of antibody-forming cells depended on the functional state of Peyer's patches. Application of epiphyseal peptides led to a 35% increase (p < 0.05) in the release of immunoglobulins from Peyer's patches in non-immunized rats and a 25% decrease (p < 0.05) in the release of antibody into the incubation medium from the lymphoid organs of immunized animals. These data lead to the suggestion that the activatory effect of epiphyseal peptides on antibody-forming cells in Peyer's patches from non-immunized animals is associated with -adrenoceptors, while their inhibitory action on immunoglobulin release by the small intestine lymphoid organs from immunized animals is not mediated via adrenoceptors.     

Comparative evaluation of immunization with recombinant protein and plasmid DNA vaccines of fusion antigen ROP2 and SAG1 from <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in mice: cellular and humoral immune responses

The aim of this work was to evaluate immune responses in BALB/c mice vaccinated subcutaneously by recombinant protein, or intramuscularly by plasmid DNA with fusion antigen of rhoptry protein 2 (ROP2) and major surface protein 1 (SAG1) from Toxoplasma gondii (T. gondii). BALB/c mice were immunized with one of three different antigen formulations respectively, which were rROP2-SAG1, pcROP2-SAG1, and pcROP2-SAG1 boosted with rROP2-SAG1. The production of IgG, IgG subclasses, lymphoproliferation, and level of gamma interferon (IFN-γ) were detected after vaccination. The animals vaccinated with rROP2-SAG1 quickly developed specific anti-TLA (T. gondii lysate antigen) antibodies, which continued to rise after immunization. However, production of IgG against TLA in mice vaccinated with pcROP2-SAG1 was relatively slow and maintained a high level after reaching plateau. There are more vigorous specific lymphoproliferative responses observed in mice of group rROP2-SAG1 than in pcROP2-SAG1. Immune responses in mice of group pcROP2-SAG1 boosted with rROP2-SAG1 were similar to the protein immunization group. Three immunization procedures resulted in a similar level of IFN-γ production. Our results indicate that BALB/c mice vaccinated by three immunization procedures induce similar humoral and cellular immunity against infection of T. gondii. Mice immunized with recombinant protein rROP2-SAG1 produce more humoral immune responses than mice immunized with other antigen formulations.     

Characterization of the cellular response of spleen cells in BALB/c mice inoculated with Mycoplasma pneumoniae or the P1 protein

BALB/c mice were intranasally infected or intraperitoneally inoculated with Mycoplasma pneumoniae whole cells or were immunized with the isolated adhesin (P1 protein). Spleen cells were isolated and tested in vitro for proliferation activity after stimulation with the P1 protein and sonicated M. pneumoniae whole antigen preparations. In frequency analysis experiments the P1 protein-specific proliferative response of spleen lymphocytes increased from 1/11494 in mice immunized once to 1/3246 in eightfold-inoculated mice, demonstrating that the P1 protein is a prominent immunogen of M. pneumoniae cells. Depletion experiments showed that T and B cells are activated in a 21 relation. Fluorescence-activated cells sorting analysis revealed a shift of the CD4/CD8 ratio from 21 in control mice up to 31 in M. pneumoniae-, and to 3.41 in P1 protein-immunized mice, as well as an increase in interleukin 2 receptor-bearing cells and macrophage cell populations. The results indicate that this animal model is appropriate to study host-M. pneumoniae interactions and vaccination schedules.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Detection of an “epimastigote-like” intracellular stage ofTrypanosoma cruzi

Monoclonal antibody (mAb) DION 5.1b, derived from mice immunized withTrypanosoma dionisii, recognizes a 72/76-kD surface glycoprotein specific to the epimastigote stage ofT. dionisii andT. cruzi. None of the three other stages of theT. cruzi life cycle expresses any DION 5.1b-specific epitope. However, mAb DION 5.1b labels an intracellular form with epimastigote-like morphology that appears to be late and transient in the intracellular cycle. This result suggests that the morphological similarity between the observed epimastigote-like intracellular form in mammals and the epimastigote form in insects may extend to the antigenic pattern.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis>: expression and characterization of a recombinant protein containing SAG1 and GRA2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Toxoplasma gondii is an obligate intracellular protozoan which infects most species of warm-blooded animals and causes toxoplasmosis. Previous immunological and immunization studies have demonstrated the potential role of T. gondii antigens SAG1 and GRA2 as a vaccine candidate. In the present study, we have cloned, expressed, and purified a recombinant protein SAG1–GRA2 in Pichia pastoris. Results showed that P. pastoris was a robust system producing a large amount of highly purified and biological activity protein. BALB/c mice immunized with SAG1–GRA2 elicited stronger humoral and cellular responses in comparison to control groups. This immunization resulted in an enhanced Th1 immune response as measured by IgG2a antibody production and increased splenocyte IFN-γ production, whereas no IL-4 was detected. After a lethal challenge with the highly virulent T. gondii RH strain, a prolonged survival time in SAG1–GRA2-immunized mice was observed in comparison to control groups. Our data demonstrate that SAG1–GRA2 triggered a protective response against toxoplasmosis. Therefore, SAG1–GRA2 protein might be a good candidate for the further development of a multiantigenic vaccine.     

Mapping of human fibronectin receptorβ subunit gene to chromosome 10

Human-mouse hybrid cells were examined by indirect immunofluorescence with Mab DH12, a monoclonal antibody that recognizes the subunit of the human fibronectin receptor. Cells that expressed the antigen at their surface were sorted by FACS and karyotyped. Immunoaffinity chromatography on Mab DH12 was used to confirm the presence of the human antigen. The chromosome assignment was strengthened by isozyme analysis of markers for chromosomes 9 and 10. The results are suggestive for a 10p mapping of this subunit of the fibronectin receptor. Since the gene coding for the subunit of the VLA proteins was previously assigned to the same chromosome, our result could provide further evidence for the relationship between the subunit of the human fibronectin receptor and the VLA protein family.     

Carboxy-terminally truncated Dengue 4 virus envelope glycoprotein expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> induced neutralizing antibodies and resistance to Dengue 4 virus challenge in mice

Summary. We have expressed a recombinant Dengue 4 virus envelope glycoprotein (E4rec), truncated at its C-terminus by 53 amino acids, in Pichia pastoris. The presence of E4rec was confirmed by Western-blot using anti-DEN 4 hyper immune mouse ascitic fluid. E4rec migrated during SDS-PAGE as a 64kDa protein. Treatment with endoglycosidases showed that the E protein was modified by the addition of short mannose chains and the absence of hyperglycosylation. When administered to BALB-C mice, E4rec elicited a DEN 4 neutralizing antibody response haemagglutination inhibition antibodies and specific memory T cell response. Mice immunized were also significantly protected against lethal DEN 4 virus challenge (86.6%, p<0.001).Received November 8, 2003; accepted June 7, 2003 Published online August 7, 2003     

Ultrastructure of spermiogenesis and spermatozoon of<Emphasis Type="Italic"> Leptorhynchoides plagicephalus</Emphasis> (Acanthocephala,Palaeacanthocephala), a parasite of the sturgeon<Emphasis Type="Italic"> Acipenser naccarii</Emphasis> (Osteichthyes,Acipenseriformes)

This paper describes the ultrastructure of spermiogenesis and the spermatozoon of Leptorhynchoides plagicephalus, an acanthocephalan parasite of the sturgeon Acipenser naccarii, a species which is under the threat of extinction. At the beginning, spermiogenesis in L. plagicephalus is characterized by the presence of a single centriole in the early spermatid. This centriole generates a flagellum with a 9+0 pattern. Another ultrastructural feature observed during the spermiogenesis of L. plagicephalus is the condensation of chromatin to form an intranuclear wall. The mature spermatozoon of L. plagicephalus presents a reversed anatomy, as observed in other species of the Acanthocephala. The spermatozoon is divided into two parts: an axoneme and a nucleocytoplasmic derivative. The pattern of spermiogenesis and the ultrastructural organization of the spermatozoon of L. plagicephalus are compared with information available on other acanthocephalan species. The appearance of an intranuclear wall observed during the present study represents the first record within the Acanthocephala and is unknown from other animal taxa.     

Toxoplasma gondii surface antigen-1 in sera of HIV-infected patients as an indicator of reactivated toxoplasmosis

The major surface antigen from the proliferative form ofToxoplasma gondii (P-30 or SAG-1) was chosen as a target for exploration ofToxoplasma gondii reactivation in sera from immunocompromised patients. Samples were obtained from 37 HIV-infected subjects with lymphocyte levels of CD4+ <200/mm³. The prevalence of IgG antibodies toToxoplasma gondii was 64.9 %. Ten patients had clinical symptoms of reactivated toxoplasmosis; eight of these hadToxoplasma encephalitis. The SAG-1 epitopes were found as circulating antigen in five cases with an immunocapture enzyme immunoassay (EIA). The EIA was improved with an IgG1 monoclonal antibody to SAG-1 and a streptavidinbiotin amplification. The sensitivity, specificity and positive predictive value were 30, 92 and 60 %, respectively. The SAG-1 levels were compared with different biological parameters such as HIV p24 antigen, ₂ microglobulin, CD4+ cell count and IgG antibodies toToxoplasma gondii. The levels of SAG-1 in these patients were significantly higher than those in the 75 healthy control persons with or without a chronicToxoplasma gondii infection. Therefore, SAG-1 may be involved as a marker of reactivated toxoplasmosis in HIV-infected patients.     

Gel double immunodiffusion studies with six human rhinoviruses

Summary Rabbits were immunized on six occasions during a seven month period with fluorocarbon and sucrose density gradient purified preparations of human rhinovirus types 1A, 2, 3, 4, 9, or 14. Sera collected 1 week after the final immunization formed 1 or 2 precipitin lines when reacted by immunodiffusion against fluorocarbon purified preparations of each homologous immunizing virus. Heterologous precipitin cross reactions were detected between: RV1A antigen and rabbit sera to RV2, RV9 and RV14; RV2 antigen and sera to RV1A; RV14 antigen and sera to RV3.The heterologous group or C-antigenic nature of the cross reactions was suggested by the merging of heterologous precipitin lines formed against rabbit sera with group antibody precipitin lines fromed against human sera and by the location of heterologous reactions close to the antigen well. In addition, the presence of C-antigenic particles in the fluorocarbon treated RV2 preparation was suggested by the demonstration that a subpopulation of viral particles migrated to a pH of 4.4 by isoelectric focusing.With 6 Figures     

Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice

To investigate the vaccine potential of multi-epitope vaccines against toxoplasmosis, a multi-epitope DNA vaccine, eukaryotic plasmid pcDNA3.1/T-ME expressing six antigen segments (SAG1_238–256, SAG1_281–320, GRA1_170–193, GRA4_331–345, GRA4_229–245, and GRA2_171–185) of Toxoplasma gondii was constructed. We investigated the efficacy of pcDNA3.1/T-ME with or without co-administration of a CpG-oligodeoxynucleotide (CpG-ODN) as an adjuvant to protect mice (BALB/c and C57BL/6) against toxoplasmosis. High survival rates were observed in mice immunized with pcDNA3.1/T-ME when challenged with T. gondii RH strain. Lymphocyte proliferation assays, cytokine, and antibody determinations show that mice immunized with pcDNA3.1/T-ME produced stronger humoral and Th1-type cellular immune responses compared to untreated mice or those immunized with empty plasmids. However, co-immunization with CpG-ODN resulted in impaired immune responses. Our data demonstrates that multi-epitope DNA vaccination is a potential strategy for the control of toxoplasmosis and paves the way for further investigations into producing a multi-epitope anti-T. gondii DNA vaccine. First co-author: Lin Shi This work was partially funded by National Natural Scientific Foundation of China grant 30571628.     

Intra-articular treatment of arthritis with microsphere formulations of paclitaxel: biocompatibility and efficacy determinations in rabbits

Objectives:To assess the biocompatibility of controlled release microspheres prepared from different polymeric biomaterials in various size ranges in rabbit synovial joints and based on these data, design and evaluate the efficacy of an intra-articular, paclitaxel-loaded microspheres formulation in rabbit models of arthritis. Methods:Paclitaxel-loaded microspheres of poly(lactide-co-glycolide) (PLGA), poly(L-lactic acid) (PLA) and poly(caprolactone) (PCL) were prepared in different size ranges and inflammatory responses monitored following injection into healthy rabbit joints. The efficacy of 20% paclitaxel-loaded PLA microspheres (35–105 m size range) injected intra-articularly into antigen and carrageenan induced rabbit models of arthritis was monitored. Results:Polymeric microspheres in the 35–105 m size range were biocompatible whereas smaller microspheres (1–20 m) produced an inflammatory response. Efficacy studies showed that injection of 20% paclitaxel-loaded PLA microspheres significantly reduced all measures of inflammation in the antigen arthritis rabbit model. Conclusions:Paclitaxel-loaded PLA microspheres in the 35–105 m size range, released paclitaxel in a controlled manner over several weeks, and may be a potential formulation for the intra-articular treatment of inflammation in arthritic conditions.Received 6 November 2003; returned for revision 12 January 2004; accepted by J. S. Skotnicki 8 March 2004     

Acid hydrolase activity in red and white skeletal muscle of mice during a two-week period following exhausting exercise

The activities of -glucuronidase, -N-acetylglucosaminidase, arylsulphatase, ribonuclease,p-nitrophenylphosphatase, and malate dehydrogenase together with protein content were assayed from representative mixed (m. rectus femoris), predominantly red (proximal heads ofm. vastus lateralis, m.v. medius andm. v. intermedius), and predominantly white (distal head ofm. vastus lateralis) muscle homogenates of mice during a two-week period following one single exposure to exhausting intermittent running on a treadmill. The activities of cathepsin D and -glycerophosphatase were assayed from mixed muscle only. In all three muscle types, particularly in red muscle, the activities of -glucuronidase, -N-acetylglucosaminidase, arylsulphatase, and ribonuclease progressively increased between one to five days after the exercise; thereafter the activities began to decrease, being near the control values 15 days after the exercise. In mixed muscle, cathepsin D activity increased. No corresponding changes were observed in the activities of acid phosphatases.The time course of the activity changes closely resembled that earlier found to be caused by ischaemia in rabbit muscles. It is tentatively concluded that the two treatments, exhaustive exercise and temporary ischaemia, cause similar cell injuries, and that the lysosomal system involved seems to function similarly in the post-stress recovery of the fibres from these injuries.Supported by grants from the Ministry of Education (Finland) and the Emil Aaltonen Foundation