Gastric Cancer Peritoneal Carcinomatosis Risk Score.

BACKGROUND: Gastric cancer (GC) peritoneal carcinomatosis (PC) is associated with a poor prognosis. Although grade, histology, and stage are associated with PC, the cumulative risk of PC when multiple risk factors are present is unknown. This study aimed to develop a cumulative GCPC risk score based on individual demographic/tumor characteristics. METHODS: Patient-level data (2004-2014) from the California Cancer Registry were reviewed by creating a keyword search algorithm to identify patients with gastric PC. Multivariable logistic regression was used to assess demographic/tumor characteristics associated with PC in a randomly selected testing cohort. Scores were assigned to risk factors based on beta coefficients from the logistic regression result, and these scores were applied to the remainder of the subjects (validation cohort). The summed scores of each risk factor formed the total risk score. These were grouped, showing the percentages of patients with PC. RESULTS: The study identified 4285 patients with gastric adenocarcinoma (2757 males, 64.3%). The median age of the patients was 67 years (interquartile range [IQR], 20 years). Most of the patients were non-Hispanic white (n = 1748, 40.8%), with proximal (n = 1675, 39.1%) and poorly differentiated (n = 2908, 67.9%) tumors. The characteristics most highly associated with PC were T4 (odds ratio [OR], 3.12; 95% confidence interval [CI], 2.19-4.44), overlapping location (OR 2.27; 95% CI 1.52-3.39), age of 20-40 years (OR 3.42; 95% CI 2.24-5.21), and Hispanic ethnicity (OR 1.86; 95% CI 1.36-2.54). The demographic/tumor characteristics used in the risk score included age, race/ethnicity, T stage, histology, tumor grade, and location. Increasing GCPC score was associated with increasing percentage of patients with PC. CONCLUSION: Based on demographic/tumor characteristics in GC, it is possible to distinguish groups with varying odds for PC. Understanding the risk for PC based on the cumulative effect of high-risk features can help clinicians to customize surveillance strategies and can aid in early identification of PC.

Peritoneal carcinomatosis in gastric cancer: Are Hispanics at higher risk?

BACKGROUND: A recent study from our group identified Hispanic race/ethnicity as an independent predictor of peritoneal carcinomatosis (PC) in gastric cancer. We sought to identify the tumor factors that might contribute to this strong association in Hispanics. METHODS: California Cancer Registry data were used to identify patients diagnosed with gastric adenocarcinoma from 2004 to 2014. Logistic regression analyses were performed to determine odds ratios for cancer stage, tumor location, grade, histology, and PC. RESULTS: Of 16,275 patients with gastric adenocarcinoma who met inclusion criteria, 6463 (39.7%) were non-Hispanic White (NHW), 4953 (30.4%) were Hispanic, 1020 (6.3%) were non-Hispanic Black (NHB), and 3915 (23.6%) were Asian/other. Compared to NHW, Hispanics were more likely to have a poorly differentiated grade (65.9% vs. 57.6%; p < .001), signet ring adenocarcinoma (28.1% vs. 17.6%; p < .001) and stage IV (51.9% vs. 45.0%; p < .001) gastric cancer. The proportion of stage IV patients with PC was also significantly higher in Hispanics compared to NHW, NHB, and Asian/other (28.5% vs. 16.6%, 20.5%, and 25.2%, respectively; p < .001). CONCLUSIONS: Hispanic ethnicity is an independent predictor of aggressive tumor phenotype and PC. Disproportionate incidence of signet ring adenocarcinoma and PC highlight the need to explore the genomic differences in Hispanic gastric cancer.

Predicting methotrexate resistance in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is an incurable, systemic autoimmune disease that decreases quality of life and can lead to severe disability. While there are many medications available to treat RA, the first-line of therapy is low-dose methotrexate (MTX), a small molecule disease-modifying anti-rheumatic drug (DMARD). MTX is the recommended therapy due to its affordability and efficacy in reducing symptoms in most RA patients. Unfortunately, there is great person-to-person variability in the physiological response to MTX, with up to 50% of patients showing little response to the medication. Thus, many RA patients initially placed on MTX do not experience an adequate reduction of symptoms, and could have benefited more in both the short and long terms if initially prescribed a different drug that was more effective for them. To combat this problem and better guide treatment decisions, many research groups have attempted to develop predictive tools for MTX response. Currently, there is no reliable, clinical-grade method to predict an individual's response to MTX treatment. In this review, we describe progress made in the area of MTX non-response/resistance in RA patients. We specifically focus on application of the following elements as predictive markers: proteins related to MTX transport and function, intracellular MTX concentration, immune cell frequencies, cytokines, and clinical factors.

Mechanism of chimeric vaccine stimulation of indoleamine 2,3-dioxygenase biosynthesis in human dendritic cells is independent of TGF-ß signaling.

Cholera toxin B subunit fusion to autoantigens such as proinsulin (CTB-INS) down regulate dendritic cell (DC) activation and stimulate synthesis of DC immunosuppressive cytokines. Recent studies of CTB-INS induction of immune tolerance in human DCs indicate that increased biosynthesis of indoleamine 2,3-dioxygenase (IDO1) may play an important role in CTB-INS vaccine suppression of DC activation. Studies in murine models suggest a role for transforming growth factor beta (TGF-ß) in the stimulation of IDO1 biosynthesis, for the induction of tolerance in DCs. Here, we investigated the contribution of TGF-ß superfamily proteins to CTB-INS induction of IDO1 biosynthesis in human monocyte-derived DCs (moDCs). We show that CTB-INS upregulates the level of TGF-ß1, activin-A and the TGF-ß activator, integrin αvß8 in human DCs. However, inhibition of endogenous TGF-ß, activin-A or addition of biologically active TGF-ß1, and activin-A, did not inhibit or stimulate IDO1 biosynthesis in human DCs treated with CTB-INS. While inhibition with the kinase inhibitor, RepSox, blocked SMAD2/3 phosphorylation and diminished IDO1 biosynthesis in a concentration dependent manner. Specific blocking of the TGF-ß type 1 kinase receptor with SB-431542 did not arrest IDO1 biosynthesis, suggesting the involvement of a different kinase pathway other than TGF-ß type 1 receptor kinase in CTB-INS induction of IDO1 in human moDCs. Together, our experimental findings identify additional immunoregulatory proteins induced by the CTB-INS fusion protein, suggesting CTB-INS may utilize multiple mechanisms in the induction of tolerance in human moDCs.

The function of myeloid dendritic cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease that causes joint pain, inflammation, and loss of function. Disease pathogenesis involves activation and proliferation of autoreactive pro-inflammatory effector T cells. While the details of RA onset and progression remain controversial, dendritic cell (DC) numbers dramatically increase in the synovial joint tissues of RA patients. Based on their key functions as antigen-presenting cells and inducers of T cell differentiation, DCs may play an important role in the initiation of joint inflammation. Myeloid DC contributions are likely central to the development of RA, as they are more efficient at antigen presentation in comparison with their closely related cousins, plasmacytoid DCs. Synovial fluid in the joints of RA patients is enriched with pro-inflammatory cytokines and chemokines, which may stimulate or result from DC activation. Epidemiological evidence indicates that smoking and periodontal infection are major environmental risk factors for RA development. In this review, factors in the synovial environment that contribute to altered myeloid DC functions in RA and the effects of environmental risk factors on myeloid DCs are described.

Identification of Anti-Long Chain Saturated Fatty Acid IgG Antibodies in Serum of Patients with Type 2 Diabetes.

High levels of serum long chain saturated fatty acids (LCSFAs) have been associated with inflammation in type 2 diabetes. Dietary SFAs can promote inflammation, the secretion of IgG antibodies, and secretion of the proinflammatory cytokine IL-1ß. This study characterizes anti-LCSFA IgG antibodies from patients with type 2 diabetes. Serum samples from several cohorts with type 2 diabetes were analyzed for the presence of anti-LCSFA IgG, the cytokine IL-1ß, and nonesterified fatty acids. Anti-LCSFA IgG was isolated from patient samples and used for in vitro characterization of avidity and specificity. A cohort participating in En Balance, a diabetes health education program that improved diabetes management, tested positive for anti-LCSFA IgG. Following the 3-month program, the cohort showed a significant reduction in anti-LCSFA IgG levels. Anti-LCSFA antibodies isolated from these patients demonstrated high avidity, were specific for long chain SFAs, and correlated with serum fatty acids in patients with managed type 2 diabetes. Interestingly, anti-LCSFA IgG neutralized PA-induced IL-1ß secretion by dendritic cells. Our data shows that nonesterified SFAs are recognized by IgG antibodies present in human blood. The identification of anti-LCSFA IgG antibodies in human sera establishes a basis for further exploration of lipid induced immune responses in diabetic patients.

Persistent suppression of type 1 diabetes by a multicomponent vaccine containing a cholera toxin B subunit-autoantigen fusion protein and complete Freund's adjuvant.

Data presented here demonstrate multifunctional vaccination strategies that harness vaccinia virus mediated delivery of a gene encoding an immunoenhanced diabetes autoantigen in combination with complete Freund's adjuvant (CFA) that can maintain safe and durable immunologic homeostasis in NOD mice. Systemic coinoculation of prediabetic mice with recombinant vaccinia virus rVV-CTB::GAD and undiluted or 10-fold diluted CFA demonstrated a significant decrease in hyperglycemia and pancreatic islet inflammation in comparison with control animals during 17-61 and 17-105 weeks of age, respectively. Synergy in these beneficial effects was observed during 43-61 and 61-105 wks of age, respectively. Inflammatory cytokine and chemokine levels in GAD-stimulated splenocytes isolated from vaccinated mice were generally lower than those detected in unvaccinated mice. The overall health and humoral immune responses of the vaccinated animals remained normal throughout the duration of the experiments.

A CTB-SARS-CoV-2-ACE-2 RBD Mucosal Vaccine Protects Against Coronavirus Infection.

Mucosal vaccines protect against respiratory virus infection by stimulating the production of IgA antibodies that protect against virus invasion of the mucosal epithelium. In this study, a novel protein subunit mucosal vaccine was constructed for protection against infection by the beta coronavirus SARS-CoV-2. The vaccine was assembled by linking a gene encoding the SARS-CoV-2 virus S1 angiotensin converting enzyme receptor binding domain (ACE-2-RBD) downstream from a DNA fragment encoding the cholera toxin B subunit (CTB), a mucosal adjuvant known to stimulate vaccine immunogenicity. A 42 kDa vaccine fusion protein was identified in homogenates of transformed E. coli BL-21 cells by acrylamide gel electrophoresis and by immunoblotting against anti-CTB and anti-ACE-2-RBD primary antibodies. The chimeric CTB-SARS-CoV-2-ACE-2-RBD vaccine fusion protein was partially purified from clarified bacterial homogenates by nickel affinity column chromatography. Further vaccine purification was accomplished by polyacrylamide gel electrophoresis and electro-elution of the 42 kDa chimeric vaccine protein. Vaccine protection against SARS-CoV-2 infection was assessed by oral, nasal, and parenteral immunization of BALB/c mice with the CTB-SARS-CoV-2-ACE-2-RBD protein. Vaccine-induced SARS-CoV-2 specific antibodies were quantified in immunized mouse serum by ELISA analysis. Serum from immunized mice contained IgG and IgA antibodies that neutralized SARS-CoV-2 infection in Vero E6 cell cultures. In contrast to unimmunized mice, cytological examination of cell necrosis in lung tissues excised from immunized mice revealed no detectable cellular abnormalities. Mouse behavior following vaccine immunization remained normal throughout the duration of the experiments. Together, our data show that a CTB-adjuvant-stimulated CTB-SARS-CoV-2-ACE-2-RBD chimeric mucosal vaccine protein synthesized in bacteria can produce durable and persistent IgA antibodies in mice that neutralize the SARS-CoV-2 subvariant Omicron BA.1.1.

En Balance: The Contribution of Physical Activity to the Efficacy of Spanish Diabetes Education of Hispanic Americans with Type 2 Diabetes.

PURPOSE: En Balance, a culturally sensitive diabetes education program, improves glycemic control in Hispanics with type 2 diabetes. The program emphasized diet, physical activity, and other factors important for glycemic control. However, the individual contributions of these education factors are unclear. The purpose of this study is to assess the contribution of physical activity to the success of En Balance in improving the health of Mexican Americans with type 2 diabetes. METHODS: A retrospective study was conducted with plasma samples collected pre- and post-3-month study. Samples from 58 (18 males and 40 females) Hispanic subjects with type 2 diabetes were analyzed for the concentration of kynurenines, known to decrease in response to exercise. After three months, health outcomes for the active group (decreased kynurenines) and the rest of the cohort were evaluated by paired Wilcoxon signed-rank test. RESULTS: Half of the subjects had increased kynurenine levels at the end of the educational program. We found that the subjects in the active group with decreased kynurenine concentrations displayed statistically greater improvements in fasting blood glucose, A1C, cholesterol, and triglycerides despite weight loss being higher in the group with increased kynurenine concentrations. CONCLUSIONS: En Balance participants with decreased kynurenine levels had significantly improved glycemic control. These data suggest that physical activity significantly contributes to the success of the En Balance education program. This analysis indicates that diabetes public health educators should emphasize the benefit of physical activity on glycemic control even in the absence of major weight loss.

Synthesis and assembly of Porphyromonas gingivalis fimbrial protein in potato tissues.

Periodontal disease caused by the gram-negative oral anaerobic bacterium Porphyromonas gingivalis is thought to be initiated by the binding of P. gingivalis fimbrial protein to saliva-coated oral surfaces. To assess whether biologically active fimbrial antigen can be synthesized in edible plants, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein, fimA (amino acids 266-337), was cloned behind the mannopine synthase promoter in plant expression vector pPCV701. The plasmid was transferred into potato (Solanum tuberosum) leaf cells by Agrobacterium tumefaciens in vivo transformation methods. The fimA cDNA fragment was detected in transformed potato leaf genomic DNA by PCR amplification methods. Further, a novel immunoreactive protein band of ~6.5 kDa was detected in boiled transformed potato tuber extracts by acrylamide gel electrophoresis and immunoblot analysis methods using primary antibodies to fimbrillin, a monomeric P. gingivalis fimbrial subunit. Antibodies generated against native P. gingivalis fimbriae detected a dimeric form of bacterial-synthesized recombinant FimA(266-337) protein. Further, a protein band of ~160 kDa was recognized by anti-FimA antibodies in undenatured transformed tuber extracts, suggesting that oligomeric assembly of plant-synthesized FimA may occur in transformed plant cells. Based on immunoblot analysis, the maximum amount of FimA protein synthesized in transformed potato tuber tissues was approximately 0.03% of total soluble tuber protein. Biosynthesis of immunologically detectable FimA protein and assembly of fimbrial antigen subunits into oligomers in transformed potato tuber tissues demonstrate the feasibility of producing native FimA protein in edible plant cells for construction of plant-based oral subunit vaccines against periodontal disease caused by P. gingivalis.

Immunogenicity of a cholera toxin B subunit Porphyromonas gingivalis fimbrial antigen fusion protein expressed in E. coli.

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.

Extracellular vimentin modulates human dendritic cell activation.

Vimentin is an intermediate filament protein traditionally considered to be an intracellular protein with a structural role. However, recent evidence suggests that vimentin can also be found outside the cell in disease conditions such as cancer, traumatic tissue injury, and inflammation. Extracellular vimentin was previously found to stimulate innate immunity by increasing monocyte and macrophage ability to kill bacteria. However, vimentin has also been previously found to decrease neutrophil infiltration into inflamed tissue. How extracellular vimentin affects the initiation of adaptive immune responses is unknown. Initiation of adaptive immunity involves priming of naïve T cells by antigen-presenting cells, the most effective of which are dendritic cells (DCs). In this study, we demonstrate how extracellular vimentin modulates lipopolysaccharide (LPS) - induced activation of human DCs. Using cytometric bead arrays, we show that extracellular vimentin decreases LPS-activated DC secretion of pro-inflammatory cytokines IL-6 and IL-12 while increasing secretion of the anti-inflammatory cytokine IL-10. Using flow cytometry, we show that extracellular vimentin does not significantly affect LPS-induced DC surface expression of MHC I (HLA-ABC) or MHC II (HLA-DR) presentation molecules, costimulatory factors (CD80, CD86), or the DC maturation marker (CD83). Further, LPS-stimulated DCs co-cultured with allogeneic naïve CD4 + T cells (Th0) induced less secretion of the pro-inflammatory Th1 effector cytokine IFN-γ in the presence of vimentin than in the presence of LPS alone. This result suggests that vimentin reduces Th1 differentiation. Taken together, our data suggest that extracellular vimentin may inhibit pro-inflammatory adaptive immune responses, by blocking DC secretion of pro-inflammatory cytokines. Thus, extracellular vimentin may play an important role in cancer or trauma-complications by inducing suppression of the adaptive immune response. In a positive sense, the presence of extracellular vimentin may prevent tissue-damage from contributing to the development of autoimmunity. Consequently, extracellular vimentin may become a novel drug target for treatment of a variety of pro- and anti-inflammatory disease conditions.

The role of TGF-beta signaling in dendritic cell tolerance.

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine present in vertebrate and invertebrate organisms that functions in numerous physiological and pathological processes. TGF-ß impacts all the cells of the immune system, and of the three known TGF-ß isoforms, TGF-ß1 is the predominant isoform expressed in immune cells. TGF-ß1 is known to play a pivotal role in the function of all immune cells especially in the regulation of T cell development and in the induction of immunological tolerance in dendritic cells (DCs). Based on the importance of DCs in regulation of the innate and adaptive arms of the immune system, in this review we explore the regulatory functions of TGF-ß required for establishment and maintenance of DC-mediated immune tolerance.

The Role of Dendritic Cell Maturation in the Induction of Insulin-Dependent Diabetes Mellitus.

Dendritic cells (DCs) are the dominant class of antigen-presenting cells in humans and are largely responsible for the initiation and guidance of innate and adaptive immune responses involved in maintenance of immunological homeostasis. Immature dendritic cells (iDCs) phagocytize pathogens and toxic proteins and in endosomal vesicles degrade them into small fragments for presentation on major histocompatibility complex (MHC) II receptor molecules to naïve cognate T cells (Th0). In addition to their role in stimulation of immunity, DCs are involved in the induction and maintenance of immune tolerance toward self-antigens. During activation, the iDCs become mature. Maturation begins when the DCs cease taking up antigens and begin to migrate from their location in peripheral tissues to adjacent lymph nodes or the spleen where during their continued maturation the DCs present stored antigens on surface MHCII receptor molecules to naive Th0 cells. During antigen presentation, the DCs upregulate the biosynthesis of costimulatory receptor molecules CD86, CD80, CD83, and CD40 on their plasma membrane. These activated DC receptor molecules bind cognate CD28 receptors presented on the Th0 cell membrane, which triggers DC secretion of IL-12 or IL-10 cytokines resulting in T cell differentiation into pro- or anti-inflammatory T cell subsets. Although basic concepts involved in the process of iDC activation and guidance of Th0 cell differentiation have been previously documented, they are poorly defined. In this review, we detail what is known about the process of DC maturation and its role in the induction of insulin-dependent diabetes mellitus autoimmunity.

Palmitic acid is a toll-like receptor 4 ligand that induces human dendritic cell secretion of IL-1ß.

Palmitic acid (PA) and other saturated fatty acids are known to stimulate pro-inflammatory responses in human immune cells via Toll-like receptor 4 (TLR4). However, the molecular mechanism responsible for fatty acid stimulation of TLR4 remains unknown. Here, we demonstrate that PA functions as a ligand for TLR4 on human monocyte derived dendritic cells (MoDCs). Hydrophobicity protein modeling indicated PA can associate with the hydrophobic binding pocket of TLR4 adaptor protein MD-2. Isothermal titration calorimetry quantified heat absorption that occurred during PA titration into TLR4/MD2, indicating that PA binds to TLR4/MD2. Treatment of human MoDCs with PA resulted in endocytosis of TLR4, further supporting the function of PA as a TLR4 agonist. In addition, PA stimulated DC maturation and activation based on the upregulation of DC costimulatory factors CD86 and CD83. Further experiments showed that PA induced TLR4 dependent secretion of the pro-inflammatory cytokine IL-1ß. Lastly, our experimental data show that PA stimulation of NF-κB canonical pathway activation is regulated by TLR4 signaling and that reactive oxygen species may be important in upregulating this pro-inflammatory response. Our experiments demonstrate for the first time that PA activation of TLR4 occurs in response to direct molecular interactions between PA and MD-2. In summary, our findings suggest a likely molecular mechanism for PA induction of pro-inflammatory immune responses in human dendritic cells expressing TLR4.

Ricin toxin B subunit enhancement of rotavirus NSP4 immunogenicity in mice.

A 90-amino acid peptide from the simian rotavirus SA-11 nonstructural protein, NSP4 was linked to the N-terminus of the Ricinus communis A-B toxin B subunit protein (RTB) and synthesized in Escherichia coli. Recombinant RTB and the NSP4(90)::RTB fusion protein bound artificial receptor glycoprotein asialofetuin in an in vitro enzyme-linked immunosorbent assay (ELISA), demonstrating biological activity of the recombinant protein. Mice co-inoculated with purified recombinant RTB plus NSP4(90) peptide proteins or heat denatured NSP4(90)::RTB fusion protein generated higher titers of serum anti-NSP4(90) IgG antibodies than mice immunized with NSP4(90) peptide alone, indicating the presence of adjuvant functions for N-terminal linked RTB. Serum anti-NSP4(90) IgG titers were highest in mice immunized with native recombinant NSP4(90)::RTB fusion protein, confirming the immunostimulatory function of RTB. Results of experiments described here demonstrate the feasibility of using RTB mediated adjuvant functions for stimulation of the antigenicity of a rotavirus nonstructural protein. The ability of recombinant NSP4(90)::RTB fusion protein synthesized in E. coli to bind glycoprotein receptor molecules effectively indicates that protein linkage to the RTB N-terminus and synthesis of the recombinant NSP4(90)::RTB fusion protein in bacteria do not interfere with the immunostimulatory properties of the RTB subunit.

Synthesis of a ricin toxin B subunit-rotavirus VP7 fusion protein in potato.

A gene encoding the outer capsid glycoprotein (VP7) of simian rotavirus SA11, was genetically linked to the amino terminus of the ricin toxin B subunit (RTB) isolated from castor-oil plant (Ricinus communis) seeds. To assess fusion protein expression in plant cells, the VP7::RTB fusion gene was transferred into potato (Solanum tuberosum) cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The fusion gene was detected in transformed potato genomic DNA by polymerase chain reaction DNA amplification methods. Immunoblot analysis with anti-SA11 antiserum as the primary antibody verified the presence of VP7::RTB fusion protein in transformed potato tuber tissues. The plant-synthesized fusion protein bound RTB membrane receptors as measured by asialofetuin-enzyme-linked immunosorbent assay (ELISA). The ELISA results indicated that the VP7::RTB fusion protein was biologically active and made up approx 0.03% of total soluble transformed tuber protein. The biosynthesis of receptor binding VP7::RTB fusion protein in potato tissues demonstrates the feasibility of producing monomeric ricin toxin B subunit adjuvant-virus antigen fusion proteins in crop plants for enhanced immunity.

Suppression of hyperglycemia in NOD mice after inoculation with recombinant vaccinia viruses.

In autoimmune (type 1) diabetes, autoreactive lymphocytes destroy pancreatic beta-cells responsible for insulin synthesis. To assess the feasibility of gene therapy for type 1 diabetes, recombinant vaccinia virus (rVV) vectors were constructed expressing pancreatic islet autoantigens proinsulin (INS) and a 55-kDa immunogenic peptide from glutamic acid decarboxylase (GAD), and the immunomodulatory cytokine interleukin (IL)-10. To augment the beneficial effects of recombinant virus therapy, the INS and GAD genes were fused to the C terminus of the cholera toxin B subunit (CTB). Five-week-old non-obese diabetic (NOD) mice were injected once with rVV. Humoral antibody immune responses and hyperglycemia in the infected mice were analyzed. Only 20% of the mice inoculated with rVV expressing the CTB::INS fusion protein developed hyperglycemia, in comparison to 70% of the mice in the uninoculated animal group. Islets from pancreatic tissues isolated from euglycemic mice from this animal group showed no sign of inflammatory lymphocyte invasion. Inoculation with rVV producing CTB::GAD or IL-10 was somewhat less effective in reducing diabetes. Humoral antibody isotypes of hyperglycemic and euglycemic mice from all treated groups possessed similar IgG1/IgG2c antibody titer ratios from 19 to 32 wk after virus inoculation. In comparison with uninoculated mice, 11-wk-old NOD mice injected with virus expressing CTB::INS were delayed in diabetes onset by more than 4 wk. The experimental results demonstrate the feasibility of using rVV expressing CTB::INS fusion protein to generate significant protection and therapy against type 1 diabetes onset and progression.

Bacterial and plant enterotoxin B subunit-autoantigen fusion proteins suppress diabetes insulitis.

Several bacterial and plant enterotoxin B subunit-islet autoantigen fusion proteins were compared for their ability to serve as islet autoantigen carriers and adjuvants for reduction of pancreatic islet inflammation associated with type 1 diabetes. The cholera toxin B subunit (CTB), the heat-labile toxin B subunit from enterotoxigenic Escherichia coli (LTB), the Shigella toxin B subunit (STB), and the plant toxin ricin B subunit (RTB) were genetically linked to the islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The adjuvant-autoantigen gene fusions were transferred to a bacterial expression vector and the corresponding fusion proteins synthesized in E. coli. The purified adjuvant-autoantigen proteins were fed to 5-wk-old nonobese diabetic (NOD) mice once a week for 4 wk. Histological examination of pancreatic islets isolated from inoculated mice showed significant levels of insulitis reduction in comparison with uninoculated mice. The ratio of serum anti-INS and anti-GAD IgG2c to IgG1 antibody isotype titers increased in all ligand-autoantigen inoculated animal groups, suggesting an increase in effector Th2 lymphocytes in B subunit-mediated insulitis suppression. The results of these experiments indicate that bacterial and plant enterotoxin B subunit ligand-autoantigens enhance insulitis reduction in NOD mice. This research prompts further exploration of a multiadjuvant/autoantigen co-delivery strategy that may facilitate type 1 diabetes prevention and suppression in animals and humans.

Mucosal immunization with a ricin toxin B subunit-rotavirus NSP4 fusion protein stimulates a Th1 lymphocyte response.

The castor-oil plant Ricinus communis A-B dimeric toxin B subunit (RTB) was genetically linked at its N-terminus with a 90 amino acid peptide from simian rotavirus SA-11 non-structural protein NSP4(90) and produced in Escherichia coli BL21 cells. Biologically active recombinant NSP4(90)-RTB fusion protein was shown to bind glycoprotein asialofetuin receptor molecules in an in vitro enzyme-linked immunosorbent assay (ELISA). Oral inoculation of the purified NSP4(90)-RTB ligand-antigen fusion protein delivered the chimeric protein to intestinal epidermal cells for mucosal immunization against rotavirus infection. Mice fed the NSP4(90)-RTB fusion protein generated higher humoral and intestinal antibody titers than mice inoculated with NSP4(90) alone. Titers of serum IgG2a antibodies were significantly higher than IgG1 titers suggesting a dominant Th1 lymphocyte immune response. ELISA measurement of cytokines secreted from splenocyte isolated from immunized mice confirmed NSP4(90)-RTB fusion protein stimulates a strong Th1 cell-mediated immune response. The experimental results demonstrate that the ricin toxin B subunit N-terminus can be used as a site for delivery of virus antigens to the gut associated lymphoid tissues for RTB-mediated immune stimulation of antiviral mucosal immune responses.