Phosphate-buffered Saline Mice Research Articles

Abstract 1795: Tumor-targeting Salmonella A1-R selectively delivers recombinant methioninase and inhibits syngeneic-cancer mouse models

Abstract Background: Methionine addiction, termed the Hoffman effect, is a general hallmark of cancer. Methionine restriction (MR) using an MR diet or recombinant methioninase (rMETase) is effective for all types of cancer. However, methionine restriction also inhibits the activity of CD8-positive T-lymphocytes. Therefore, we hypothesized that methionine depletion in the tumor only may be more effective for cancer therapy. We previously developed Salmonella A1-R, which selectively targets and kills tumors. In the present study, we established rMETase-producing Salmonella A1-R, by transfer of the Pseudomonas putida methioninase gene, to target and inhibit syngeneic cancer mouse models. Methods: A plasmid containing the Pseudomonas putida methioninase gene was extracted from rMETase-producing E. coli and inserted into Salmonella A1-R using electroporation. The rMETase-producing Salmonella A1-R (A1-R-rMETase) infected several cancer cell lines in vitro, including HT29, PC-3, MDA-MB-435, and Lewis Lung Carcinoma (LLC). LLC was chosen for in vivo studies as it supported extensive growth of A1-R-rMETase. We determined 108 A1-R-rMETase was a safe dosage in C57BL/6 mice. LLC cells (106) were injected in male C57BL/6 mice aged 4-6 weeks subcutaneously. After tumor growth, 18 mice were divided into three groups of 6. One group was injected with phosphate-buffered saline (PBS) via the tail vein, twice per week as a control. Another group was injected with 108 Salmonella A1-R via the tail vein, twice per week. Another group was injected with 108 A1-R-rMETase via the tail vein, twice per week for two weeks. Tumor size was measured with calipers three times per week for 3 weeks. On day 22, tumor methionine level was measured using HPLC in the PBS control and the mice injected with A1-R-rMETase. Results: The mean LLC tumor size of each group on day 22 was as follows: The PBS control: 741.5 mm3; mice injected with A1-R: 566.3 mm3; and mice injected with A1-R-rMETase: 198.8 mm3. Turkey’s multiple comparisons test showed a significant difference between the PBS control and mice injected with A1-R-rMETase (p<0.0001) and between mice injected with A1-R and mice injected with A1-R-rMETase (p=0.0036). However, the PBS control and the mice injected with A1-R did not show a significant difference (p=0.1794). The mice injected with A1-R-rMETase showed a significantly lower mean methionine level than mice injected with PBS (5.9 nM/mg protein vs. 11.1 nM/mg protein, p=0.0095, Mann Whitney test). Conclusion: Tumor-targeting Salmonella A1-R modified to express the Pseudomonas putida methioninase gene (A1-R-rMETase), inhibited LLC tumor growth in a syngeneic mouse model and reduced the methionine level in the tumor. A1-R-rMETase combines the tumor targeting and killing capability of A1-R itself and restriction methionine selectively in tumors. Citation Format: Yutaro Kubota, Yusuke Aoki, Noriyuki Masaki, Koya Obara, Sei Morinaga, Kohei Mizuta, Motokazu Sato, Ming Zhao, Qinghong Han, Bouvet Michael, Takuya Tsunoda, Robert M. Hoffman. Tumor-targeting Salmonella A1-R selectively delivers recombinant methioninase and inhibits syngeneic-cancer mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1795.

Fecal microbiota transplantation ameliorates type 2 diabetes via metabolic remodeling of the gut microbiota in db/db mice

IntroductionGut microbiome (GM) deregulation has been implicated in major conditions such as obesity and type 2 diabetes (T2DM). Our previous prospective study indicated that fecal microbiota transplantation (FMT) successfully improved...

TRAUMA-DERIVED EXTRACELLULAR VESICLES ARE SUFFICIENT TO INDUCE ENDOTHELIAL DYSFUNCTION AND COAGULOPATHY.

Although a number of studies have demonstrated increased release of extracellular vesicles (EVs) and changes in their origin differentials after trauma, the biologic significance of EVs is not well understood. We hypothesized that EVs released after trauma/hemorrhagic shock (HS) contribute to endotheliopathy and coagulopathy. To test this hypothesis, adoptive transfer experiments were performed to determine whether EVs derived from severely injured patients in shock were sufficient to induce endothelial dysfunction and coagulopathy. Methods: Total EVs were enriched from plasma of severely injured trauma/HS patients or minimally injured patients by ultracentrifugation and characterized for size and numbers. Under isoflurane anesthesia, noninjured naive C57BL/6J mice were administered EVs at varying concentrations and compared with mice receiving equal volume vehicle (phosphate-buffered saline (PBS)) or to mice receiving EVs from minimally injured patients. Thirty minutes after injection, mice were sacrificed, and blood was collected for thrombin generation (thrombin-antithrombin, thrombin-antithrombin complex [TAT] assay) and syndecan-1 by enzyme-linked immunoabsorbent assay (ELISA). Lungs were harvested for examination of histopathologic injury and costained with von Willebrand factor and fibrin to identify intravascular coagulation. Bronchial alveolar lavage fluid was aspirated from lungs for protein measurement as an indicator of the endothelial permeability. Data are presented as mean ± SD, P < 0.05 was considered significant, and t test was used. Results: An initial proof-of-concept experiment was performed in naive mice receiving EVs purified from severely injured trauma/HS patients (Injury Severity Score [ISS], 34 ± 7) at different concentrations (5 × 106 to 3.1 × 109/100 μL/mouse) and compared with PBS (control) mice. Neither TAT nor syndecan-1 levels were significantly different between groups at 30 minutes after EV infusion. However, lung vascular permeability and histopathologic injury were significantly higher in the EV group, and lung tissues demonstrated intravascular fibrin deposition. Based on these data, EVs from severely injured trauma/HS patients (ISS, 32 ± 6) or EVs from minimally injured patients (ISS, 8 ± 3) were administered to naive mice at higher concentrations (1 × 109 to 1 × 1010 EV/100 μL/mouse). Compared with mice receiving EVs from minimally injured patients, plasma TAT and syndecan-1 levels were significantly higher in the trauma/HS EV group. Similarly, bronchial alveolar lavage protein and lung histopathologic injury were higher in the trauma/HS EV group, and lung tissues demonstrated enhanced intravascular fibrin deposition. Conclusion: These data demonstrate that trauma/HS results in the systemic release of EVs, which are capable of inducing endotheliopathy as demonstrated by elevated syndecan-1 and increased permeability and coagulopathy as demonstrated by increased TAT and intravascular fibrin deposition. Targeting trauma-induced EVs may represent a novel therapeutic strategy.

Association between anti-fibrillin-2 protein induced retinal degeneration via intravitreous delivery and activated TGF-β signalling in mice.

Fibrillin-2 (FBN2) is a major component of tissue microfibrils, and the decrease of FBN2 perturbs the signalling events mediated by transforming growth factor-β (TGF-β), thereby playing a role in macular degeneration. However, the association between the retinal degeneration resulting from the abnormality of FBN2 and the activation of TGF-β signalling has not been fully addressed. In the present study, the mice were divided into a normal control group (NC group), a phosphate-buffered saline (PBS) injection group (PBS group), and an anti-FBN2 protein injection group (anti-FBN2group), and the mice in PBS and anti-FBN2groups received the relevant treatment via the intravitreal injection once a week for three consecutive weeks. One week later after injection, the retinal morphology and visual function of the fundus were detected. Further, the expression of FBN2, TGF-β1, TGF-β2 and TGF-β3 in retina was measured using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. As a result, fundus examination suggests that after intravitreous injection of anti-FBN2 protein, there were a large patchy yellow white degeneration region and numerous pigmentations in the retina in anti-FBN2-treated mice; by contrast, there was no apparent change in mice from the NC and PBS groups. The retina suffered markedly damage, and the thickness of whole retina and outer nuclear layer markedly thinned. The expression of FBN2 was decreased whereas the levels of TGF-β1, TGF-β2 and TGF-β3 were upregulated. Together, our findings indicate that the intravitreous delivery of anti-FBN2 protein could induce retina degeneration in mice, accompanied by the higher activated TGF-β. The retinal degeneration mouse model established will provide a platform for the investigation of the retinal diseases.

Colchicine prevents disease progression in viral myocarditis via modulating the NLRP3 inflammasome in the cardiosplenic axis.

AimThe acute phase of a coxsackievirus 3 (CVB3)‐induced myocarditis involves direct toxic cardiac effects and the systemic activation of the immune system, including the cardiosplenic axis. Consequently, the nucleotide‐binding oligomerization domain‐like receptor pyrin domain‐containing‐3 (NLRP3) inflammasome pathway is activated, which plays a role in disease pathogenesis and progression. The anti‐inflammatory drug colchicine exerts its effects, in part, via reducing NLRP3 activity, and has been shown to improve several cardiac diseases, including acute coronary syndrome and pericarditis. The aim of the present study was to evaluate the potential of colchicine to improve experimental CVB3‐induced myocarditis.Methods and resultsC57BL6/j mice were intraperitoneally injected with 1 × 105 plaque forming units of CVB3. After 24 h, mice were treated with colchicine (5 μmol/kg body weight) or phosphate‐buffered saline (PBS) via oral gavage (p.o.). Seven days post infection, cardiac function was haemodynamically characterized via conductance catheter measurements. Blood, the left ventricle (LV) and spleen were harvested for subsequent analyses. In vitro experiments on LV‐derived fibroblasts (FB) and HL‐1 cells were performed to further evaluate the anti‐(fibro)inflammatory and anti‐apoptotic effects of colchicine via gene expression analysis, Sirius Red assay, and flow cytometry. CVB3 + colchicine mice displayed improved LV function compared with CVB3 + PBS mice, paralleled by a 4.7‐fold (P < 0.01) and 1.7‐fold (P < 0.001) reduction in LV CVB3 gene expression and cardiac troponin‐I levels in the serum, respectively. Evaluation of components of the NLRP3 inflammasome revealed an increased percentage of apoptosis‐associated speck‐like protein containing a CARD domain (ASC)‐expressing, caspase‐1‐expressing, and interleukin‐1β‐expressing cells in the myocardium and in the spleen of CVB3 + PBS vs. control mice, which was reduced in CVB3 + colchicine compared with CVB3 + PBS mice. This was accompanied by 1.4‐fold (P < 0.0001), 1.7‐fold (P < 0.0001), and 1.7‐fold (P < 0.0001) lower numbers of cardiac dendritic cells, natural killer cells, and macrophages, respectively, in CVB3 + colchicine compared with CVB3 + PBS mice. A 1.9‐fold (P < 0.05) and 4.6‐fold (P < 0.001) reduced cardiac gene expression of the fibrotic markers, Col1a1 and lysyl oxidase, respectively, was detected in CVB3 + colchicine mice compared with CVB3 + PBS animals, and reflected by a 2.2‐fold (P < 0.05) decreased Collagen I/III protein ratio. Colchicine further reduced Col3a1 mRNA and collagen protein expression in CVB3‐infected FB and lowered apoptosis and viral progeny release in CVB3‐infected HL‐1 cells. In both CVB3 FB and HL‐1 cells, colchicine down‐regulated the NLRP3 inflammasome‐related components ASC, caspase‐1, and IL‐1β.ConclusionsColchicine improves LV function in CVB3‐induced myocarditis, involving a decrease in cardiac and splenic NLRP3 inflammasome activity, without exacerbation of CVB3 load.

Anti-Tn Monoclonal Antibody Ameliorates Hyperoxia-Induced Kidney Injury by Suppressing Oxidative Stress and Inflammation in Neonatal Mice.

The Tn antigen, an N-acetylgalactosamine structure linked to serine or threonine, has been shown to induce high-specificity, high-affinity anti-Tn antibodies in mice. Maternal immunization with the Tn vaccine increases serum anti-Tn antibody titers and attenuates hyperoxia-induced kidney injury in neonatal rats. However, immunizing mothers to treat neonatal kidney disease is clinically impractical. This study is aimed at determining whether anti-Tn monoclonal antibody treatment ameliorates hyperoxia-induced kidney injury in neonatal mice. Newborn BALB/c mice were exposed to room air (RA) or normobaric hyperoxia (85% O2) for 1 week. On postnatal days 2, 4, and 6, the mice were injected intraperitoneally with PBS alone or with anti-Tn monoclonal antibodies at 25 μg/g body weight in 50 μL phosphate-buffered saline (PBS). The mice were divided into four study groups: RA + PBS, RA + anti-Tn monoclonal antibody, O2 + PBS, and O2 + anti-Tn monoclonal antibody. The kidneys were excised for histology, oxidative stress, cytokine, and Western blot analyses on postnatal day 7. The O2 + PBS mice exhibited significantly higher kidney injury scores, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor-κB (NF-κB) expression, and cytokine levels than did the RA + PBS mice or RA + anti-Tn mice. Anti-Tn monoclonal antibody treatment reduced kidney injury and cytokine levels to normoxic levels. The attenuation of kidney injury was accompanied by a reduction of oxidative stress and NF-κB expression. Therefore, we propose that anti-Tn monoclonal antibody treatment ameliorates hyperoxia-induced kidney injury by suppressing oxidative stress and inflammation in neonatal mice.

Early-Onset Physical Inactivity and Metabolic Dysfunction in Tumor-bearing Mice Is Associated with Accelerated Cachexia.

Male C57BL/6J mice (12 wk of age) were injected with 1 × 106 LLC cells or phosphate-buffered saline (PBS) subcutaneously in the right flank, and tissue was collected 26-28 d after cell injection. Tumor volume was measured every 5 d throughout the study to calculate the tumor growth rate. Fifteen days after tumor inoculation, a subset of PBS (n = 11) and LLC (n = 16) mice were individually housed in metabolic Comprehensive Laboratory Animal Monitoring System cages for 5 d. LLC mice exhibited greater body weight loss (-5.1%), decreased muscle mass (-7%), decreased fat mass (-22%), and increased plasma interleukin-6 (212%) compared with PBS mice. Before the onset of cachexia, total cage activity was decreased in tumor-bearing mice. Cage activity was negatively associated with tumor mass and positively associated with hindlimb muscle mass. In addition, LLC mice had greater lipid oxidation than PBS mice. LLC mice exhibit early-onset physical inactivity and altered systemic lipid oxidation, which are associated with the eventual development of cachexia.

Noncovalent PEG Coating of Nanoparticle Drug Carriers Improves the Local Pharmacokinetics of Rectal Anti-HIV Microbicides.

Antiretroviral drug nanocarriers hold great promise for developing anti-human immunodeficiency virus (HIV) rectal microbicides. However, challenges remain, namely, concerning which properties are more suited for enhancing colorectal distribution and retention of microbicide compounds. In this work, we developed and assessed the in vitro and in vivo performance of poly(lactic- co-glycolic acid) (PLGA)-based nanoparticles (NPs) as carriers for the model drug efavirenz (EFV). We particularly focused on the effect of noncovalent poly(ethylene glycol) coating of PLGA NPs (PEG-PLGA NPs) conferring a mucus-diffusive behavior on the pharmacokinetics (PK) of EFV following rectal administration to mice. Drug-loaded PLGA NPs and PEG-PLGA NPs (200-225 nm) were obtained by nanoprecipitation. Both types of systems were able to retain native antiretroviral activity of EFV in vitro, while featuring lower cytotoxicity against different epithelial cell lines and HIV target cells. Also, PLGA NPs and PEG-PLGA NPs were readily taken up by colorectal cell lines and mildly reduced EFV permeation while increasing membrane retention in Caco-2 and Caco-2/HT29-MTX cell monolayer models. When administered intrarectally to CD-1 mice in phosphate-buffered saline (pH 7.4), EFV-loaded PEG-PLGA NPs consistently provided higher drug levels in colorectal tissues and lavages, as compared to free EFV or drug-loaded PLGA NPs. Mean values for the area-under-the-curve between 15 min and 12 h following administration were particularly higher for PEG-PLGA NPs in distal and middle colorectal tissues, with relative bioavailability values of 3.7 and 29, respectively, as compared to free EFV (2.2 and 6.0 over PLGA NPs, respectively). Systemic exposure to EFV was reduced for all treatments. NPs were further shown safe after once-daily administration for 14 days, as assessed by histological analysis of colorectal tissues and chemokine/cytokine assay of rectal lavages. Overall, PEG-PLGA NPs demonstrated to be safe carriers for rectal microbicide drug delivery and able to provide enhanced local PK that could be valuable in preventing rectal HIV transmission.

Harnessing the heart's resistance to malignant tumors: cardiac-derived extracellular vesicles decrease fibrosarcoma growth and leukemia-related mortality in rodents.

The heart is known for its resistance to cancer. Although different conjectures have been proposed to explain this phenomenon, none has been tested. We propose that the heart microenvironment may exert anti-cancer properties. So, our objective was to test the anti-oncogenic potential of cardiac-derived extracellular vesicles (EVs).For that EVs secreted by cardiosphere-derived cells (CDCs, heart progenitor cells) were tested in vitro on fibrosarcoma HT1080. In vivo models comprised the xenograft HT1080 fibrosarcoma in athymic mice (n=35), and spontaneous acute lymphocyte leukemia in old rats (n=44). CDC-EVs were compared with two control groups: EVs secreted by bone-marrow derived mesenchymal stem cells (MSC-EVs) and phosphate-buffered saline (PBS).Injection of CDC-EVs led to a 2.5-fold decrease of fibrosarcoma growth in mice (p<0.01 and p<0.05 for human and rat EVs, respectively) vs PBS group. The effect was associated with 2-fold decrease of tumor cells proliferation (p<0.001) and 1.5-fold increase of apoptosis (p<0.05) in CDC-EV vs PBS mice. Salutary changes in tumor gene and protein expression were observed in CDC-EV animals. CDC-EVs reduced tumor vascularization compared with PBS (p<0.05) and MSC-EVs (p<0.01). Moreover, CDC-EVs increased leukemia-free survival (p<0.05) in old rats vs PBS. MiR-146, highly enriched in CDC-EVs, may be implicated in part of the observed effects. In conclusion, this study presents the first evidence that ties together the long-recognized enigma of the “heart immunity to cancer” with an antioncogenic effect of heart-derived EVs. These findings open up cancer as a new therapeutic target for CDC-EVs.

Effects of Acyclovir and IVIG on Behavioral Outcomes after HSV1 CNS Infection.

Herpes simplex virus 1 (HSV) encephalitis (HSE) has serious neurological complications, involving behavioral and cognitive impairments that cause significant morbidity and a reduced quality of life. We showed that HSE results from dysregulated central nervous system (CNS) inflammatory responses. We hypothesized that CNS inflammation is casually involved in behavioral abnormalities after HSE and that treatment with ACV and pooled human immunoglobulin (IVIG), an immunomodulatory drug, would improve outcomes compared to mice treated with phosphate buffered saline (PBS) or ACV alone. Anxiety levels were high in HSV-infected PBS and ACV-treated mice compared to mice treated with ACV + IVIG, consistent with reports implicating inflammation in anxiety induced by lipopolysaccharide (LPS) or stress. Female, but not male, PBS-treated mice were cognitively impaired, and unexpectedly, ACV was protective, while the inclusion of IVIG surprisingly antagonized ACV's beneficial effects. Distinct serum proteomic profiles were observed for male and female mice, and the antagonistic effects of ACV and IVIG on behavior were paralleled by similar changes in the serum proteome of ACV- and ACV + IVIG-treated mice. We conclude that inflammation and other factors mediate HSV-induced behavioral impairments and that the effects of ACV and IVIG on behavior involve novel mechanisms.

Regulatory T Cell Depletion Abolishes the Protective Effect of Dietary Galacto-Oligosaccharides on Eosinophilic Airway Inflammation in House Dust Mite–Induced Asthma in Mice

Background: In a murine model for house dust mite (HDM)–induced asthma, dietary galacto-oligosaccharides have been shown to suppress allergic symptoms. Previously, CD25+ regulatory T cells (Tregs) induced by nondigestible oligosaccharides were found to protect against allergy development.Objective: The aim of the current study was to examine the effect of anti–CD25-induced Treg depletion in a murine HDM-induced asthma model and to study the contribution of Tregs in the protective effect of dietary intervention with galacto-oligosaccharides.Methods: Male BALB/c mice (aged 6–8 wk) were intranasally sensitized and challenged with phosphate-buffered saline (PBS) or HDM. Two weeks before sensitization and throughout the whole experiment, mice were fed a control or 1% w/w galacto-oligosaccharide diet. Tregs were depleted by anti-mouse CD25 antibody (intraperitoneally injected). On day 14, T helper cell subtypes in lung and spleen were analyzed and cytokines were measured. Leukocyte subtypes were analyzed in the bronchoalveolar lavage fluid, and interleukin (IL)–33 and chemokines were measured in lung homogenate supernatants.Results: Anti-CD25 treatment depleted CD25+ Forkhead box P3+ Tregs in the lung and spleen of control and HDM-allergic mice (P < 0.0001) by >70% while increasing the percentage of activated T helper cells (P < 0.05) and type 2 T helper cells (P < 0.05). This was associated with increased IL-10, IL-4, and IL-13 concentrations in supernatants of ex vivo restimulated lung cells (P < 0.01). Bronchoalveolar lavage fluid leukocyte numbers and percentages of eosinophils and lymphocytes were greater in HDM-allergic mice compared with PBS mice (P < 0.01) but remained unaffected by the anti-CD25 treatment. Galacto-oligosaccharides decreased airway eosinophilia compared with HDM-allergic mice fed the control diet (from 47.8% ± 6.7% to 26.6% ± 8.5%, P < 0.01). This protective effect was lost in anti–CD25-treated mice (P < 0.05). In lung homogenates of HDM-allergic mice, IL-33 was increased compared with PBS mice (from 2.8 ± 0.3 to 5.4 ± 0.6 ng protein/mg, P < 0.01). Galacto-oligosaccharides abrogated the increase in IL-33 compared with HDM-allergic mice fed the control diet (3.0 ± 0.6 ng protein/mg, P < 0.05), which was abolished by the anti-CD25 treatment (P < 0.01).Conclusions: Treg depletion enhances pulmonary type 2 T helper cell frequency and cytokine release in HDM-induced asthma in mice. Galacto-oligosaccharides decreased airway eosinophilia and IL-33 concentrations in the lung, which was abrogated by Treg depletion. This indicates that galacto-oligosaccharides have a beneficial effect in the prevention of HDM-induced allergic asthma by supporting pulmonary Treg function.

The effect of rhinovirus on airway inflammation in a murine asthma model

PurposeThe aim of the present study was to investigate the differences in lower airway inflammatory immune responses, including cellular responses and responses in terms of inflammatory mediators in bronchoalveolar lavage fluid (BALF) and the airway, to rhinovirus (RV) infection on asthma exacerbation by comparing a control and a murine asthma model, with or without RV infection.MethodsBALB/c mice were intraperitoneally injected with a crude extract of Dermatophagoides farinae (Df) or phosphate buffered saline (PBS) and were subsequently intranasally treated with a crude extract of Df or PBS. Airway responsiveness and cell infiltration, differential cell counts in BALF, and cytokine and chemokine concentrations in BALF were measured 24 hours after intranasal RV1B infection.ResultsRV infection increased the enhanced pause (Penh) in both the Df sensitized and challenged mice (Df mice) and PBS-treated mice (PBS mice) (P<0.05). Airway eosinophil infiltration increased in Df mice after RV infection (P<0.05). The levels of interleukin (IL) 13, tumor necrosis factor alpha, and regulated on activation, normal T cells expressed and secreted (RANTES) increased in response to RV infection in Df mice, but not in PBS mice (P<0.05). The level of IL-10 significantly decreased following RV infection in Df mice (P<0.05).ConclusionOur findings suggest that the augmented induction of proinflammatory cytokines, Th2 cytokines, and chemokines that mediate an eosinophil response and the decreased induction of regulatory cytokines after RV infection may be important manifestations leading to airway inflammation with eosinophil infiltration and changes in airway responsiveness in the asthma model.

Efficacy of adipose tissue‐derived mesenchymal stem cells for fulminant hepatitis in mice induced by concanavalin A

Fulminant hepatitis is mainly caused by excessive immune response-mediated liver injury and its definitive therapy is liver transplantation. Mesenchymal stem cells, one of the adult stem cells, have an immunomodulatory effect on immune cells and reside in various tissues. The aim of this study was to investigate a therapeutic effect of adipose tissue-derived mesenchymal stem cells (ASCs) on fulminant hepatitis induced by concanavalin A (ConA). The ASCs were isolated from adipose tissues of BALB/c mice and confirmed by detection of cell surface markers and induction of multi-lineage differentiation. BALB/c mice were injected with ConA and treated with ASCs, phosphate buffered saline (PBS) or splenocytes (SPLCs). Survival rates, levels of serum liver enzymes, titers of serum cytokines, histopathology and localization of ASCs were investigated. The survival rate of ASC-injected mice significantly increased compared to PBS or SPLC-injected mice. This effect was dependent on doses and timing of ASCs injected. Improvement of liver enzyme levels, histopathological changes and suppression of inflammatory cytokine production were observed in ASC-injected mice. Fluorescent stained ASCs were detected in inflammatory liver, but not in normal liver. These results suggest that ASC treatment has a high potential to be an innovative therapy for fulminant hepatitis.

Optimization of intracerebral tumour protection by active-specific immunization against murine melanoma B16/G3.12.

Development of brain metastases despite extracerebral response to systemic immunotherapy is a common problem in melanoma patients. We have previously described a murine melanoma vaccine of interferon-gamma (IFNgamma)-treated, irradiated syngeneic B16/G3.12 and allogeneic (Cloudman) melanoma cells, plus the adjuvant DETOX, that is protective against subcutaneous (93%) or intracerebral (69%) syngeneic challenge. This study aimed to optimize this vaccine. Groups of nine or 10 mice were immunized five times in 5 weeks with: (i) complete vaccine +/- IFNgamma (VAC+, VAC-); (ii) syngeneic 2 x 106 G3.12 cells plus DETOX (Syn+D), (iii) 2 x 106 allogeneic Cloudman cells plus DETOX (Allo+D); (iv) VAC+ without DETOX (no DETOX); (v) DETOX alone (DETOX); or (vi) phosphate buffered saline (PBS). Mice were challenged subcutaneously with 104 viable G3.12 (or Cloudman cells) and after 35 days intracerebrally with 104 G3.12 cells. Expression of H-2 antigens (measured using fluorescence-activated cell sorting), splenocyte cytotoxicity (measured using 51Cr release) and median overall survival (OAS) were analysed using the log-rank test. VAC+, VAC- and G3.12 mice were equally protected from subcutaneous (s.c.) and intracerebral (i.c.) melanoma challenge (OAS 65 days for s.c., 30 days for i.c.). Protection was less (P < 0.05) in DETOX mice (48 days for s.c.), PBS mice (47 days for s.c., 21 days for i.c.) or no DETOX mice (51 days for s.c.). Allo+D mice showed s.c. (59 days) but not i.c. protection (20 days). IFNgamma incubation did not increase the effect in either the challenge cells or the vaccine cells (P > 0.05). Specific cytotoxicity was seen with G3.12 targets in VAC+ (27%) but not PBS (2%; P < 0.05) mice with equal NK (YAC-1) lysis (10% versus 7%; P< 0.05). Optimal protection against s.c./i.c. experimental murine melanoma was yielded by irradiated syngeneic cells plus DETOX. DETOX alone was not active. Upregulation of H-2 antigens with IFNgamma under these conditions does not augment protection.

Congenital toxoplasmosis in the Balb/c mouse: prevention of vertical disease transmission and fetal death by vaccination

Vertical disease transmission only occurs in Balb/c mice infected with Toxoplasma gondii for the first time during pregnancy. This is similar to the situation in humans, where a previous infection with T. gondii tends to give life-long immunity against reinfection and fetal disease transmission. The Balb/c mouse therefore provides a suitable model to study the effectiveness of T. gondii vaccine candidates. A soluble tachyzoite antigen (STAg) preparation was used to vaccinate female Balb/c mice. STAg was inoculated subcutaneously into Balb/c mice in phosphate-buffered saline (PBS), emulsified in Freund's complete adjuvant (FCA), or entrapped within non-ionic surfactant vesicles (NISV). While all inocula induced cellular immunity as measured by parasite-specific spleen cell proliferation in vitro, the highest mean proliferative values were observed in spleens from mice where NISV had been used as the adjuvant and the lowest values were observed where FCA had been used. More importantly, cultures from the NISV/STAg vaccinated mice produced significantly more γ-interferon than the other experimental groups. This vaccine formulation was therefore identified as that most likely to induce protective immunity against toxoplasmosis. Mice were inoculated subcutaneously with either NISV/STAg or STAg in PBS 4 and 2 weeks before mating and infected orally with 20 tissue cysts of T. gondii on day 12 of pregnancy. The incidence of fetal infection and death in these mice and non-vaccinated infected dams was compared. Of 84 pups born to 14 non-vaccinated dams 45 were viable, of which 18 were found to be infected on reaching maturity. All 67 of the pups born to the 11 dams vaccinated with STAg/NISV survived into maturity, of which a mere nine were infected. These studies emphasize that, given appropriate antigen and adjuvant preparations for vaccination, sufficient immunity can be generated in Balb/c dams to severely limit the likelihood of T. gondii-induced abortion and congenital disease.