DNA-MVA-protein vaccination of rhesus macaques induces HIV-specific immunity in mucosal-associated lymph nodes and functional antibodies.

Successful future HIV vaccines are expected to generate an effective cellular and humoral response against the virus in both the peripheral blood and mucosal compartments. We previously reported the development of DNA-C and MVA-C vaccines based on HIV-1 subtype C and demonstrated their immunogenicity when given in a DNA prime-MVA boost combination in a nonhuman primate model. In the current study, rhesus macaques previously vaccinated with a DNA-C and MVA-C vaccine regimen were re-vaccinated 3.5years later with MVA-C followed by a protein vaccine based on HIV-1 subtype C envelope formulated with MF59 adjuvant (gp140Env/MF59), and finally a concurrent boost with both vaccines. A single MVA-C re-vaccination elicited T cell responses in all animals similar to previous peak responses, with 4/7 demonstrating responses >1000 SFU/106 PBMC. In contrast to an Env/MF59-only vaccine, concurrent boosting with MVA-C and Env/MF59 induced HIV-specific cellular responses in multiple mucosal associated lymph nodes in 6/7 animals, with high magnitude responses in some animals. Both vaccine regimens induced high titer Env-specific antibodies with ADCC activity, as well as neutralization of Tier 1 viruses and modest Tier 2 neutralization. These data demonstrate the feasibility of inducing HIV-specific immunity in the blood and mucosal sites of viral entry by means of DNA and poxvirus-vectored vaccines, in combination with a HIV envelope-based protein vaccine.

Robust immunity to an auxotrophic Mycobacterium bovis BCG-VLP prime-boost HIV vaccine candidate in a nonhuman primate model.

We previously reported that a recombinant pantothenate auxotroph of Mycobacterium bovis BCG expressing human immunodeficiency virus type 1 (HIV-1) subtype C Gag (rBCGpan-Gag) efficiently primes the mouse immune system for a boost with a recombinant modified vaccinia virus Ankara (rMVA) vaccine. In this study, we further evaluated the immunogenicity of rBCGpan-Gag in a nonhuman primate model. Two groups of chacma baboons were primed or mock primed twice with either rBCGpan-Gag or a control BCG. Both groups were boosted with HIV-1 Pr55(gag) virus-like particles (Gag VLPs). The magnitude and breadth of HIV-specific cellular responses were measured using a gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay, and the cytokine profiles and memory phenotypes of T cells were evaluated by polychromatic flow cytometry. Gag-specific responses were detected in all animals after the second inoculation with rBCGpan-Gag. Boosting with Gag VLPs significantly increased the magnitude and breadth of the responses in the baboons that were primed with rBCGpan-Gag. These responses targeted an average of 12 Gag peptides per animal, compared to an average of 3 peptides per animal for the mock-primed controls. Robust responses of Gag-specific polyfunctional T cells capable of simultaneously producing IFN-γ, tumor necrosis alpha (TNF-α), and interleukin-2 (IL-2) were detected in the rBCGpan-Gag-primed animals. Gag-specific memory T cells were skewed toward a central memory phenotype in both CD4(+) and CD8(+) T cell populations. These data show that the rBCGpan-Gag prime and Gag VLP boost vaccine regimen is highly immunogenic, inducing a broad and polyfunctional central memory T cell response. This report further indicates the feasibility of developing a BCG-based HIV vaccine that is safe for childhood HIV immunization.

Abrogation of contaminating RNA activity in HIV-1 Gag VLPs.

BACKGROUND: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. METHODS: HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. RESULTS: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/µg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. CONCLUSIONS: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice.

The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine.

BACKGROUND: One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1) and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1), an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. RESULTS: The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap) alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. CONCLUSIONS: We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.

HIV-1 sub-type C chimaeric VLPs boost cellular immune responses in mice.

Several approaches have been explored to eradicate HIV; however, a multigene vaccine appears to be the best option, given their proven potential to elicit broad, effective responses in animal models. The Pr55Gag protein is an excellent vaccine candidate in its own right, given that it can assemble into large, enveloped, virus-like particles (VLPs) which are highly immunogenic, and can moreover be used as a scaffold for the presentation of other large non-structural HIV antigens. In this study, we evaluated the potential of two novel chimaeric HIV-1 Pr55Gag-based VLP constructs - C-terminal fusions with reverse transcriptase and a Tat::Nef fusion protein, designated GagRT and GagTN respectively - to enhance a cellular response in mice when used as boost components in two types of heterologous prime-boost vaccine strategies. A vaccine regimen consisting of a DNA prime and chimaeric HIV-1 VLP boosts in mice induced strong, broad cellular immune responses at an optimum dose of 100 ng VLPs. The enhanced cellular responses induced by the DNA prime-VLP boost were two- to three-fold greater than two DNA vaccinations. Moreover, a mixture of GagRT and GagTN VLPs also boosted antigen-specific CD8+ and CD4+ T-cell responses, while VLP vaccinations only induced predominantly robust Gag CD4+ T-cell responses. The results demonstrate the promising potential of these chimaeric VLPs as vaccine candidates against HIV-1.

Use of the piggyBac transposon to create HIV-1 gag transgenic insect cell lines for continuous VLP production.

BACKGROUND: Insect baculovirus-produced Human immunodeficiency virus type 1 (HIV-1) Gag virus-like-particles (VLPs) stimulate good humoral and cell-mediated immune responses in animals and are thought to be suitable as a vaccine candidate. Drawbacks to this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used piggyBac transposition as a novel method to create transgenic insect cell lines for continuous VLP production as an alternative to the baculovirus system. RESULTS: Transgenic cell lines maintained stable gag transgene integration and expression up to 100 cell passages, and although the level of VLPs produced was low compared to baculovirus-produced VLPs, they appeared similar in size and morphology to baculovirus-expressed VLPs. In a murine immunogenicity study, whereas baculovirus-produced VLPs elicited good CD4 immune responses in mice when used to boost a prime with a DNA vaccine, no boost response was elicited by transgenically produced VLPs. CONCLUSION: Transgenic insect cells are stable and can produce HIV Pr55 Gag VLPs for over 100 passages: this novel result may simplify strategies aimed at making protein subunit vaccines for HIV. Immunogenicity of the Gag VLPs in mice was less than that of baculovirus-produced VLPs, which may be due to lack of baculovirus glycoprotein incorporation in the transgenic cell VLPs. Improved yield and immunogenicity of transgenic cell-produced VLPs may be achieved with the addition of further genetic elements into the piggyBac integron.

A prime-boost immunisation regimen using recombinant BCG and Pr55(gag) virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons.

Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South African HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55(gag) virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses. These responses were predominantly CD8+ T cell-mediated and recognised similar epitopes as those targeted by humans with early HIV-1 subtype C infection. In addition, a Gag-specific humoral response was elicited. These data support the development of HIV-1 vaccines based on rBCG and Pr55(gag) VLPs.

Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice.

BACKGROUND: Recombinant Salmonella vaccine vectors may potentially be used to induce specific CD4+ T cell responses against foreign viral antigens. Such immune responses are required features of vaccines against pathogens such as human immunodeficiency virus type 1 (HIV-1). The aim of this study was to investigate the induction of systemic HIV-1-specific CD4+ T helper (Th) responses in mice after oral immunization with a live attenuated Salmonella vaccine vector that expressed HIV-1 subtype C Gag. Groups of BALB/c mice were vaccinated orally three times (4 weeks apart) with this recombinant Salmonella. At sacrifice, 28 days after the last immunization, systemic CD4+ Th1 and Th2 cytokine responses were evaluated by enzyme-linked immunospot assay and cytometric bead array. HIV-1 Gag-specific IgG1 and IgG2a humoral responses in the serum were determined by enzyme-linked immunosorbent assay. RESULTS: Mice vaccinated with the recombinant Salmonella elicited both HIV-1-specific Th1 (interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha)) and Th2 (interleukin-4 (IL-4) and interleukin-5 (IL-5)) cytokine responses. The vaccine induced 70 (IFN-gamma) spot-forming units (SFUs)/10e(6) splenocytes and 238 IL-4 SFUs/10e(6) splenocytes. Splenocytes from vaccinated mice also produced high levels of Th1 and Th2 cytokines upon stimulation with a Gag CD4 peptide. The levels of IFN-gamma, TNF-alpha, IL-4 and IL-5 were 7.5-, 29.1-, 26.2- and 89.3-fold above the background, respectively. Both HIV-1 Gag-specific IgG1 and IgG2a antibodies were detected in the sera of vaccinated mice. CONCLUSION: The study highlights the potential of orally-delivered attenuated Salmonella as mucosal vaccine vectors for HIV-1 Subtype C Gag to induce Gag-specific CD4+ Th1 and Th2 cellular immune responses and antibodies which may be important characteristics required for protection against HIV-1 infection.

An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice.

BACKGROUND: The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP) model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli beta-galactosidase alpha-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. RESULTS: High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-gamma and IL-4) immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-gamma and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-gamma produced by GFP peptide-stimulated cells was 65.2-fold above background (p < 0.05). The level of IL-4 produced by the cells was 10.4-fold above background (p < 0.05). CONCLUSION: These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

Broad, high-magnitude and multifunctional CD4+ and CD8+ T-cell responses elicited by a DNA and modified vaccinia Ankara vaccine containing human immunodeficiency virus type 1 subtype C genes in baboons.

Candidate human immunodeficiency virus (HIV) vaccine regimens based on DNA boosted with recombinant modified vaccinia Ankara (MVA) have been in development for some time, and there is evidence for improved immunogenicity of newly developed constructs. This study describes immune responses to candidate DNA and MVA vaccines expressing multiple genes (gag, RT, tat, nef and env) from HIV-1 subtype C in chacma baboons (Papio ursinus). The vaccine regimen induced (i) strong T-cell responses, with a median of 4103 spot forming units per 10(6) peripheral blood mononuclear cells by gamma interferon (IFN-gamma) ELISPOT, (ii) broad T-cell responses targeting all five vaccine-expressed genes, with a median of 12 peptides targeted per animal and without any single protein dominating the response, (iii) balanced CD4(+) and CD8(+) responses, which produced both IFN-gamma and interleukin (IL)-2, including IL-2-only responses not detected by the ELISPOT assay, (iv) vaccine memory, which persisted 1 year after immunization and could be boosted further, despite strong anti-vector responses, and (v) mucosal T-cell responses in iliac and mesenteric lymph nodes in two animals tested. The majority of peptide responses mapped contained epitopes previously identified in human HIV infection, and two high-avidity HIV epitope responses were confirmed, indicating the utility of the baboon model for immunogenicity testing. Together, our data show that a combination of DNA and MVA immunization induced robust, durable, multifunctional CD4(+) and CD8(+) responses in baboons targeting multiple HIV epitopes that may home to mucosal sites. These candidate vaccines, which are immunogenic in this pre-clinical model, represent an alternative to adenoviral-based vaccines and have been approved for clinical trials.

HIV-1 subtype C Pr55gag virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine.

A DNA vaccine expressing human immunodeficiency virus type 1 (HIV-1) southern African subtype C Gag (pTHGag) and a recombinant baculovirus Pr55gag virus-like particle prepared using a subtype C Pr55gag protein (Gag VLP) was tested in a prime-boost inoculation regimen in Chacma baboons. The response of five baboons to Gag peptides in a gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay after three pTHGag immunizations ranged from 100 to 515 spot-forming units (s.f.u.) per 10(6) peripheral blood mononuclear cells (PBMCs), whilst the response of two baboons to the Gag VLP vaccine ranged from 415 to 465 s.f.u. per 10(6) PBMCs. An increase in the Gag-specific response to a range of 775-3583 s.f.u. per 10(6) PBMCs was achieved by boosting with Gag VLPs the five baboons that were primed with pTHGag. No improvement in Gag responses was achieved in this prime-boost inoculation regimen by increasing the number of pTHGag inoculations to six. IFN-gamma responses were mapped to several peptides, some of which have been reported to be targeted by PBMCs from HIV-1 subtype C-infected individuals. Gag VLPs, given as a single-modality regimen, induced a predominantly CD8+ T-cell IFN-gamma response and interleukin-2 was a major cytokine within a mix of predominantly Th1 cytokines produced by a DNA-VLP prime-boost modality. The prime-boost inoculation regimen induced high serum p24 antibody titres in all baboons, which were several fold above that induced by the individual vaccines. Overall, this study demonstrated that these DNA prime/VLP boost vaccine regimens are highly immunogenic in baboons, inducing high-magnitude and broad multifunctional responses, providing support for the development of these products for clinical trials.

Detection of natural infection with Mycobacterium intracellulare in healthy wild-caught Chacma baboons (Papio ursinus) by ESAT-6 and CFP-10 IFN-gamma ELISPOT tests following a tuberculosis outbreak.

BACKGROUND: Both tuberculous and non-tuberculous mycobacteria can cause infection in nonhuman primates (NHP), indicating the existence of potential zoonotic transmission between these animals and visitors to zoos or animal handlers in primate facilities. Screening of mycobacterial infections in NHP is traditionally done by tuberculin skin test (TST), which is unable to distinguish between pathogenic and non-pathogenic mycobacterial infections. In this study, we investigated the use of ESAT-6 and CFP-10 for detection of mycobacterial infections in a wild-caught baboon colony after one baboon died of tuberculosis (TB). METHODS: Peripheral blood lymphocytes for interferon-gamma enzyme-linked immunospot assay (IFN-gamma ELISPOT) assay were obtained from TST positive baboons and those in contact with tuberculous baboons before being euthanased, autopsied and lung tissues taken for histology and mycobacterial culture. RESULTS: Both ESAT-6 and CFP-10 IFN-gamma ELISPOT assays were able to detect early M. tuberculosis but also M. intracellulare infection. Although this indicates potential cross-reactivity with M. intracellulare antigens, the method was able to distinguish M. bovis BCG vaccination from M. tuberculosis infection. This assay performed better than the TST, which failed to detect one M. tuberculosis and two early M. intracellulare infections. CONCLUSION: These results suggest that the IFN-gamma ELISPOT assay could improve the detection of M tuberculosis infections when screening NHP. There is some doubt, however, concerning specificity, as the assay scored positive three animals infected with M. intracellulare.

Effect of rapamycin on hepatic osteodystrophy in rats with portasystemic shunting.

AIM: To study if T-cell activation related to portasystemic shunting causes osteoclast-mediated bone loss through RANKL-dependent pathways. We also investigated if T-cell inhibition using rapamycin would protect against bone loss in rats. METHODS: Portasystemic shunting was performed in male Sprague-Dawley rats and rapamycin 0.1 mg/kg was administered for 15 wk by gavage. Rats received powderized chow and supplemental feeds to prevent the effects of malnutrition on bone composition. Weight gain and growth was restored after surgery in shunted animals. At termination, biochemical parameters of bone turnover and quantitative bone histology were assessed. Markers of T-cell activation, inflammatory cytokine production, and RANKL-dependent pathways were measured. In addition, the roles of IGF-1 and hypogonadism were investigated. RESULTS: Portasystemic shunting caused low turnover osteoporosis that was RANKL independent. Bone resorbing cytokine levels, including IL-1, IL-6 and TNFalpha, were not increased in serum and TNFalpha and RANKL expression were not upregulated in PBMC. Portasystemic shunting increased the circulating CD8+ T-cell population. Rapamycin decreased the circulating CD8+ T-cell population, increased CD8+ CD25+ T-regulatory cell population and improved all parameters of bone turnover. CONCLUSION: Osteoporosis caused by portasystemic shunting may be partially ameliorated by rapamycin in the rat model of hepatic osteodystrophy.

Rodent nutritional model of steatohepatitis: effects of endotoxin (lipopolysaccharide) and tumor necrosis factor alpha deficiency.

BACKGROUND AND AIMS: Intestinal endotoxin (lipopolysaccharide) is thought to contribute to liver injury in both alcoholic and nonalcoholic steatohepatitis (NASH). Tumor necrosis factor alpha (TNFalpha) is an important mediator of this process and is considered central to the inflammatory response in NASH. This study aimed to investigate the effects of lipopolysaccharide on liver injury in the methionine choline deficient (MCD) nutritional model of NASH, and to determine if TNFalpha is required for the development of steatohepatitis in this model. METHOD: Male C57/BL6 mice received a MCD diet for 4 weeks, whilst a control group received an identical diet supplemented with 0.2% choline bitartrate and 0.3% methionine. At 4 weeks, mice received either an intraperitoneal injection of lipopolysaccharide (0.5 microg/g body mass) or sterile saline, and were killed 24 h thereafter. In a separate study, TNFalpha knockout and wild type C57BL/6 mice received either MCD or control diets for 4 weeks. Serum transaminase levels, liver histology (steatosis, inflammation and apoptosis), hepatic triglyceride concentration and hepatic lipid peroxidation products (conjugated dienes, lipid hydroperoxides and thiobarbituric reactive substances, free and total) were evaluated. RESULTS: Intraperitoneal administration of lipopolysaccharide augmented serum alanine aminotransferase (ALT) levels (P<0.02), hepatic inflammation (P<0.025), apoptosis (P<0.01) and free thiobarbituric acid reactive substances (P<0.025) in MCD mice. TNFalpha knockout mice fed the MCD diet developed steatohepatitis with histological and biochemical changes similar to those seen in wild type counterparts. CONCLUSIONS: Lipopolysaccharide augments liver injury in MCD mice, and TNFalpha is not required for the development of steatohepatitis in MCD mice.

The effect of intraperitoneally administered microcystin-LR on the gastrointestinal tract of Balb/c mice.

Microcystin-LR (MCLR) has been associated with the development of gastrointestinal complaints in people ingesting cyanobacterial bloom contaminated water. The symptoms usually present a day or two after exposure raising questions as to the toxic effects of MCLR on the gastrointestinal tract. This study investigated the apoptotic effect of ip administered MCLR over time on the duodenum, jejenum and ileum of mice receiving a single 75% LD(50) dose. The apoptotic index was significantly raised in all sections at 8 h post exposure (pe) and continued to rise for the 16, 24 and 32 h pe groups, while the glycogen levels were normal at 24 h pe. The duodenum exhibited the most significant increase in apoptotic index overall, followed by the jejenum and ileum. Immunohistochemistry indicated the presence of MCLR in the lamina propria suggesting a role for MCLR in the induction of apoptosis in the GIT of mice exposed to a single sublethal dose of MCLR.

The role of microcystin-LR in the induction of apoptosis and oxidative stress in CaCo2 cells.

The increasing presence of toxic cyanobacteria in drinking and recreational water bodies, and their potential to impact on human and animal health is cause for concern. Recent work suggests that apoptosis plays a major role in the toxic effects induced by microcystin-LR (MCLR) in the gastrointestinal tract; however, the biochemical pathway remains elusive. Exposure of CaCo2, a human colon carcinoma cell line, and MCF-7, a cell line deficient in pro-caspase-3, cells to 50 microM MCLR induced similar reductions in cell viability as measured by MTT and LDH leakage. The role of MCLR induced oxidative stress in the initiation of apoptosis was investigated over a 2-hr period, and it was found that there was an increase in the release of H(2)O(2) in the first 30 min of exposure for both cell lines. Both cell lines exhibited a dose-dependent increase in both micro- and millicalpain after 24 h exposure to MCLR suggesting a role for protease activation in MCLR-induced apoptosis in non-hepatic human derived cell lines.

An investigation into the detoxification of microcystin-LR by the glutathione pathway in Balb/c mice.

Toxin-producing cyanobacteria pose a world-wide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. The predominant cyanotoxin, microcystin-LR (MCLR), targets the liver and its toxicity depends on the uptake and removal rates in the liver. The role of the glutathione detoxification pathway in protecting the liver from the effects of MCLR was investigated. Mice exposed to a single 75% LD(50) dose of pure MCLR were sacrificed at 8, 16, 24 and 32 h post-exposure (pe). Toxin induced liver damage was observed 8 and 16 h pe as evidenced by raised serum ALT and LDH levels, reduced glycogen levels and liver histology. A significant increase in lipid peroxidation was seen at 16 h pe that decreased after 24 and 32 h pe, the time-points which showed significant increases in GPX activity. An increase in soluble GST activity was noted between 8 and 16 h pe, levels of total GSH increased at 24 h while oxidised glutathione increased throughout the investigation. The increase in activity of both GPX and GST corresponded with increased transcription of these enzymes, as well as the rate-limiting enzyme in GSH synthesis, gamma-glutamyl transferase. In conclusion, this study confirms that an increase in GST activity is critical for the detoxification of MCLR, that this is regulated at the transcriptional level, and that exposure to MCLR induces the de novo synthesis of GSH. Finally, we report the involvement of GPX in the removal of MCLR-induced lipid hydroperoxides.

Fumonisin B1-induced hepatocellular and cholangiocellular tumors in male Fischer 344 rats: potentiating effects of 2-acetylaminofluorene on oval cell proliferation and neoplastic development in a discontinued feeding study.

Fumonisin B1 (FB1) is a naturally occurring mycotoxin produced by Fusarium verticillioides. Dietary exposure to FB1 has been linked to human cancer in certain parts of the world, and treatment with FB1 causes oval cell proliferation and liver tumors in rats. To study the potential role of oval (liver progenitor) cells in the cellular pathogenesis of FB1-induced liver tumors, we gave male F344 rats prolonged treatment with FB1 for 25 weeks, followed by return to control diet until 50 weeks ('stop study'). The time course of FB1-induced liver lesions was followed by examination of serial liver biopsies at set time intervals and post-mortem liver tissue at the end of the study. The effects of different FB1 treatment regimens (5 versus 25 weeks), as well as the modulating effect of 2-acetylaminofluorene (2-AAF), on the kinetics of oval cell proliferation and development of liver tumors were compared. Prolonged treatment with FB1 in normal diet caused persistent oval cell proliferation and generation of both hepatic adenomas and cholangiofibromas (CFs). These liver lesions occurred in the setting of chronic toxic hepatitis and liver fibrosis/cirrhosis, similar to that seen in human hepatocarcinogenesis. Some adenomas and CFs were dysplastic, and one post-mortem liver contained a hepatocellular carcinoma. OV-6+ oval cells were noted in close relation to proliferative neoplastic liver lesions, and some of these lesions expressed OV-6, suggesting that all these cell types were derived from a common progenitor cell. 2-AAF enhanced the size of FB1-induced glutathione S-transferase pi+ hepatocellular lesions and the incidence of CFs in post-mortem liver specimens, but this was not statistically significant. In conclusion, this study supports the involvement of dietary FB1 in liver carcinogenesis in male F344 rats. Oval cells may be the source of both the hepatocellular and cholangiocellular tumors induced by prolonged treatment with FB1. 2-AAF appears to have an enhancing effect on FB1-induced liver tumors, presumably due to its potent inhibitory effects on hepatocyte regeneration.

Rodent nutritional model of non-alcoholic steatohepatitis: species, strain and sex difference studies.

BACKGROUND AND AIM: The methionine choline-deficient (MCD) diet leads to steatohepatitis in rodents. The aim of the present study was to investigate species, strain and sex differences in this nutritional model of non-alcoholic steatohepatitis (NASH). METHODS: Male and female Wistar, Long-Evans and Sprague-Dawley rats, and C57/BL6 mice (n = 6 per group) were fed a MCD diet for 4 weeks. Control groups received an identical diet supplemented with choline bitartrate (0.2% w/w) and methionine (0.3% w/w). Liver pathology (steatosis and inflammation) and ultrastructure, liver lipid profile (total lipids, triglycerides, lipid peroxidation products), liver : body mass ratios and serum alanine aminotransferase (ALT) levels were compared between these groups. RESULTS: The MCD diet-fed male rats developed greater steatosis (P < 0.001), had higher liver lipid content (P < 0.05) and had higher serum ALT levels (P < 0.005) than did female rats. Wistar rats (both sexes) had higher liver lipid levels (P < 0.05), serum ALT levels (P < 0.05), and liver mass : body mass ratios (P < 0.025) than did Long-Evans and Sprague-Dawley rats. In female groups, Wistar rats showed greater fatty change than did the other two strains (P < 0.05). All rats fed the MCD diet developed hepatic steatosis, but necrosis and inflammation were minor features and fibrosis was absent. Compared with Wistar rats, male C57/BL6 mice showed a marked increase in inflammatory foci (P < 0.001), end products of lipid peroxidation (free thiobarbituric acid reactive substances) (P < 0.005), and mitochondrial injury, while showing less steatosis (P < 0.005), lower hepatic triglyceride levels, (P < 0.005) and lower early lipid peroxidation products (conjugated dienes and lipid hydroperoxides; P < 0.005 and P < 0.01, respectively). CONCLUSIONS: The Wistar strain and the male sex are associated with the greatest degree of steatosis in rats subjected to the MCD diet. Of the groups studied, male C57/BL6 mice develop the most inflammation and necrosis, lipid peroxidation, and ultrastructural injury, and best approximate the histological features of NASH.

An investigation into the effect of selenium supplementation on microcystin hepatotoxicity.

Toxin-producing cyanobacteria pose a worldwide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. Little work has, however, been done on a preventative therapy for anyone at risk of exposure to cyanobacterial toxins. The potential benefits of dietary supplementation of selenium, an antioxidant, to protect against the mouse liver injury induced by the toxin, microcystin-LR, has been investigated. BALB/c mice were pretreated for two weeks with sodium selenite (1.5 microg/mouse/day) before an intraperitoneal injection of microcystin-LR. Selenium-supplementation was found to provide some protection to the action of the toxin. In addition selenium pretreatment reduced the liver damage caused by lethal and sub-lethal toxin doses as reflected in liver pathology, decreased serum ALT and lipid peroxidation levels as well as prevention of glycogen loss compared to non-selenium supplemented toxin treated mice. The increased level of liver glutathione peroxidase activity following selenium-supplementation may indicate the possible route of selenium protection in the mice.