Response Of Spleen Cells Research Articles

Effect of classical conditioning over immature and mature B lymphocytes regulation.

Classical conditioning (CC) with a sweet flavored is useful for experimental models of immune response. The objective this work was analyze spleen cell response on the differentiation of B-lymphocytes associated with the CC with sweet flavored in adult rats. Twelve adult male rats were divided (n = 3) in Group 1 (control); Group 2 without conditioned stimulus (WCS) received only water; Group 3 receiving only water with salt; Group 4 (experimental) with CS of sweet flavored and salty flavored afterwards. In every work group, all animals were subjected to the unconditioned stimulus (US) treated by 10 mg/kg body weight of cyclophosphamide injected intraperitoneally (i.p.). Subsequently an immunological challenge with sheep red blood cells i.p. was applied. Spleen samples were obtained 15 days after using euthanasia with 1% sodium thiopental i.p. The evaluation B lymphocyte (immature and mature) was performed by immunohistochemistry using specific antibodies against CD20 and against CD37, and revealed with HRP/DAB. The results show in every work group significant increases of CD20+ and CD37+ cell densities in the red pulp of the spleen of male adult rats from different groups of the experimental design. We concluded that the immunomodulatory response under CC might facilitate a less aggressive and more physiological immunological response against immunosuppression. RESEARCH HIGHLIGHTS: The immune response can be reinforced by classical conditioning. The sweet taste allows to positively condition the immune response. B lymphocytes actively participate in the immune response and classical conditioning.

Abstract 708: Increase of senescent mesenchymal stromal/stem cells is predictive of unresponsiveness to the treatment with immune checkpoint inhibitors

Abstract Purposes: The therapeutic efficacy induced by immune inhibitory checkpoint inhibitors is still limited to a part pf the treated patients, and the methods effective in overcoming the treatment failures have not been established yet. A main issue under discussion is immune exhaustion as well as immune senescence with age, which is caused by persistent and chronic inflammation due to both internal and external stimuli ultimately leading to immune dysfunction in cancer patients. In literature, aging has been reported as a strong risk factor not only for causing cancers, but also for inducing hyper progression after treatment with immune checkpoint inhibitors in clinical settings. This suggests that a distinct strategy from releasing the brake on immune suppression might be needed for properly organizing anti-tumor immunity in aged hosts. In this study, we attempted to elucidate the molecular mechanisms underlying the immune exhaustion with age. Results: We used young (1 to 4-month-old) and aged (18 to 24-month-old) C57BL/6 mice, and comprehensively analyzed cell population and immune responses of spleen cells and bone marrow cells obtained from the mice. With age, CD45-CD166+ mesenchymal stromal/stem cells (MSCs) increased correlatively with increase of immunosuppressive CD4+CTLA4+Foxp3+ Tregs and CD11b+Gr1+ MDSCs, and dysfunctional CD3+PD1+Tim3+ T cells in mice. These cells were further expanded by implanting with tumor cells such as melanoma B16-F10 and lung cancer 3LL, and immune checkpoint inhibitors were ineffective in suppressing tumor progression in aged mice. As compared to the young MSCs, the aged MSCs generated much more dysfunctional T cells in vitro and in vivo, and differentiated into hypertrophic adipocytes with a large lipid droplet containing a huge amount of inflammatory adipokines. These suggest that the increasing MSCs play a key role in disturbing anti-tumor immunity both before and after differentiation in aged hosts. Utilizing cDNA microarray analysis, we identified the specific molecules those are significantly upregulated in the aged MSCs, but not young MSCs. Knockdown of the gene expressions in the aged MSCs using the specific siRNAs deprived of the morbid properties, and generated potent tumor-specific CTLs through balancing immunity in the aged mice. Conclusions: These results demonstrate that increase of the senescent and inflammatory MSCs are, at least in part, responsible for the unresponsiveness to the treatment with immune checkpoint inhibitors. Elimination of these MSCs may be a promising strategy for successfully controlling immunity against cancer. Citation Format: Chie Kudo-Saito, Takahiro Miyamoto, Mami Kawamura, Yamato Ogiwara, Kazunori Aoki. Increase of senescent mesenchymal stromal/stem cells is predictive of unresponsiveness to the treatment with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 708.

Abstract B063: Targeting the pathogenic mesenchymal stem cells as a new strategy for preventing immune dysfunction in cancer therapy

Abstract The immune system is exhausted by persistent and chronic inflammation due to internal and external stimuli, and ultimately loses the functions contributing to the pathogenesis of cancer. T-cell dysfunction is fatal to immunotherapy designed to elicit and intensify immune responses against cancer, and it is a crucial issue beyond immunosuppression that is recoverable by breaking immune checkpoint pathways. Although increase of exhausted T cells has been demonstrated in inflammatory cancer and aging, the reasons for the immune dysfunction and the methods for overcoming it remain undefined. In this study, we attempted to elucidate the underlying mechanisms involved in the immune exhaustion. We compared cell population and immune responses of spleen cells and bone marrow cells between young (1- to 4-month-old) and aged (18- to 24-month-old) C57BL/6 mice implanted with/without murine tumor cells such as melanoma B16-F10 and lung cancer 3LL. We found that CD45-CD166+ activated mesenchymal stromal/stem cells (MSCs) increased in the spleen and bone marrow of mice with age, correlatively with increase of CD3+PD1+Tim3+ exhausted T cells having no cytotoxic activities, and were further expanded by implantation with tumor cells. The MSCs sorted from aged mice generated the CD3+PD1+Tim3+ exhausted T cells as well as immunosuppressive Tregs and MDSCs when cocultured in vitro, and transfer with these MSCs promoted in vivo tumor growth via systemic expansion of exhausted T cells in young mice, although none or less of such properties were seen using the young MSCs. Interestingly, the aged MSCs frequently differentiated into hypertrophic adipocytes with a large lipid droplet containing a huge amount of inflammatory cytokines, implying acceleration of the immune exhaustion after adipogenic differentiation in aged mice. These suggest the key role of the increasing MSCs in deterioration of T-cell responses with age. We identified the specific molecules significantly upregulated in the aged MSCs by utilizing cDNA microarray analysis. Blocking the gene expressions using the specific siRNAs corrected unbalanced immunity through decline of immune suppression and exhaustion in the aged mice, and generated potent tumor-specific CTLs resulting in tumor regression, although immune checkpoint inhibitors are ineffective in the same settings. These results suggest that increase of the pathogenic MSCs might be a risk factor of resistance to immune checkpoint inhibitors, and targeting these MSCs may be a promising strategy for creating a suitable immune status for eliminating cancer. Citation Format: Chie Kudo-Saito, Yamato Ogiwara, Kazunori Aoki. Targeting the pathogenic mesenchymal stem cells as a new strategy for preventing immune dysfunction in cancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B063.

RGC-32 enhances experimental autoimmune encephalomyelitis by promoting Il-17 and GM-CSF production

Abstract Response Gene to Complement (RGC)-32 is an intracytoplasmatic protein expressed in a variety of cells in response to sublytic complement activation, TGF-β and several growth factors. Upon activation it translocates into the nucleus and plays an important role in cell proliferation, migration, angiogenesis and TGF-β induced epithelial mesenchymal transition and fibrosis. We have recently reported that RGC-32 promotes Th17 but not Treg, Th1 or Th2 differentiation in murine CD4 T cells in vitro. To evaluate the relevance of RGC-32 in an IL-17 dependent inflammatory condition we induced EAE in WT and RGC-32−/− mice by immunization with MOG35–55 peptide emulsified in CFA. The timing of disease onset was similar in both groups of mice. However, at peak disease, RGC-32−/− mice had significantly lower clinical scores. The attenuated EAE phenotype was confirmed by histopathologic examination of the spinal cords that showed fewer inflammatory infiltrates and demyelination foci in RGC-32−/− than in WT EAE mice. The percentage and absolute number of IL-17+ and GM-CSF+CD4+ T cells were significantly lower in the CNS of RGC-32−/− EAE mice. The percentage and number of IFNγ+ and FoxP3+ CD4+ T cells did not differ between WT and RGC-32−/− EAE mice. RGC-32 deficiency did not alter antigen specific expansion of RGC-32−/− spleen cells in response to MOG35-55 peptide. Transfer of WT CD4+ T cells into WT or RGC-32−/− mice before MOG immunization resulted in EAE of similar severity suggesting that the attenuated EAE phenotype in RGC-32−/− mice is T-cell-mediated. These data suggest that RGC-32 exerts a proinflammatory role in EAE by promoting Th17 immune responses and the generation of GM-CSF and that RGC-32 blockade might be a novel therapeutic strategy in MS.

Establishment of memory CD8+ T cells with live attenuated influenza virus across different vaccination doses.

FluMist has been used in children and adults for more than 10 years. As pre-existing CD8+ T cell memory pools can provide heterologous immunity against distinct influenza viruses, it is important to understand influenza-specific CD8+ T cell responses elicited by different live attenuated influenza virus (LAIV) regimens. In this study, we immunized mice intranasally with two different doses of live-attenuated PR8 virus (PR8 ts, H1N1), low and high, and then assessed protective efficacy by challenging animals with heterosubtypic X31-H3N2 virus at 6 weeks post-vaccination. Different LAIV doses elicited influenza-specific CD8+ T cell responses in lungs and spleen, but unexpectedly not in bronchoalveolar lavage. Interestingly, the immunodominance hierarchy at the acute phase after immunization varied depending on the LAIV dose; however, these differences disappeared at 6 weeks post-vaccination, resulting in generation of comparable CD8+ T cell memory pools. After vaccination with either dose, sufficient numbers of specific CD8+ T cells were generated for recall and protection of mice against heterosubtypic H1N1→H3N2 challenge. As a result, immunized mice displayed reduced weight loss, diminished inflammatory responses and lower viral titres in lungs, when compared to unvaccinated animals. Interestingly, the higher dose led to enhanced viral clearance on day 5 post-challenge, though this was not associated with increased CD8+ T cell responses, but with higher levels of non-neutralizing antibodies against the priming virus. Our study suggests that, while different LAIV doses result in distinct immune profiles, even a low dose produces sufficient protective CD8+ T cell memory against challenge infection, though the high dose results in more rapid viral clearance and reduced inflammation.

Immune Correlates of Resistance to Trichinella spiralis Reinfection in Mice.

The immune correlate of host resistance induced by reinfection of Trichinella spiralis remains unclear. In this study, we investigated immune correlates between the resistance and serum IgG antibody level, CD23+ IgM+ B cells, and eosinophil responses induced by T. spiralis reinfection. Mice were primarily infected with 10 or 100 T. spiralis larvae (10 TS, 100 TS), respectively, and after 4 weeks, they were challenge infected with 100 T. spiralis larvae (10–100 TS, 100-100 TS). Upon challenge infections, 10–100 TS mice induced significantly higher levels of T. spiralis-specific total IgG antibody responses in sera and antibody secreting cell responses in spleens compared to 100-100 TS mice, resulting in significantly reduced worm burdens in 10–100 TS mice (60% and 70% reductions for adult and larvae, respectively). Higher levels of eosinophils were found in mice primarily infected with 10 TS compared to those of 100 TS at week 8 upon challenge. CD23+ IgM+ B cells were found to be increased significantly in mice primarily infected with 10 TS. These results indicate that primary infection of 10 larvae of T. spiralis, rather than 100 larvae, induces significant resistance against reinfection which closely correlated with T. spiralis-specific IgG, eosinophil, and CD23+ IgM+ B cell responses.

Mycobacterium avium infection induces the resistance of the interferon-γ response in mouse spleen cells at late stages of infection.

BackgroundBacterial infections cause an increase in the population of hematopoietic stem cells (HSCs). To investigate the downstream factors associated with hematopoietic stem cells, mice are infected with Mycobacterium avium (M. avium).Results Mycobacterium avium (M. avium) infection induces the enlargement of the spleen and changes in histopathology, including changes to the lineage populations. A dramatic expansion of Lin−c-kit+Sca-1+ (KSL) cells in mouse bone marrow cells and spleen cells was detected 4 weeks after infection with M. avium; however, there was no difference in the engraft activity between infected and un-infected mouse bone marrow cells. We tested the cytokine and cytokine-related gene expression after M. avium infection and found that IFN-γ expression increased and peaked at 4 weeks in both bone marrow and spleen cells. The expression of Sca-1 gene peaked at 4 weeks in the bone marrow but peaked at 2 weeks in spleen cells, although the Sca-1 surface marker peaked at 4 weeks after infection in both bone marrow and spleen cells. Interferon regulatory factor-2 (IRF-2) expression did not change in the bone marrow cells, whereas it decreased in spleen cells at 4 weeks and IRF-1 expression was up-regulated in both bone marrow and spleen cells after infection. However, the up-regulation of IRF-1 was not correlated with IFN-γ expression in the M. avium-infected mouse spleen cells.ConclusionsThis finding suggests that the IFN-γ production mediated by M. avium infection alters the population of KSL cells during host defense, and the down-regulation of the IFN-γ response in spleen cells occurs at the late stage after M. avium infection.

In utero exposure to gestational diabetes mellitus conditions TLR4 and TLR2 activated IL-1beta responses in spleen cells from rat offspring

Fetal exposure to gestational diabetes mellitus (GDM) is associated with a higher risk of youth-onset insulin resistance and type 2 diabetes. We have previously shown that the rat offspring of GDM dams are insulin resistant when compared to the offspring of lean dams. Since inflammation influences insulin sensitivity, we examined the impact of fetal exposure to GDM on inflammatory responses in the offspring. In rats, we compared inflammatory activity in newborn pups as well as 16week-old young-adult offspring from lean control dams with offspring from high fat and sucrose diet (HFS)-induced GDM dams. To determine whether there are additive effects of exposure to GDM and post-weaning diets, offspring of lean and GDM dams were fed either low fat or HFS diets until 16weeks of age. Plasma levels of interleukin(IL)-1β were elevated in the offspring of GDM dams. To determine whether this was related to immune reactivity, spleen cells from both the newborn and 16week-old offspring were isolated and reactivity to the toll-like receptor activators, pam3CSK4 and lipopolysaccharides were measured over a 72h timeframe. Spleen cells of GDM dams exhibited sustained stimulation of interleukin(IL)-1β and IL-10 production, whereas IL-1β and IL-10 synthesis diminished over time in spleen cells from the offspring of lean dams. Additive effects of GDM exposure and post-weaning HFS diet were not observed, suggesting the effects of GDM on cytokine production are independent of the post-weaning diet. Thus, we conclude that exposure to GDM in utero may condition the immune reactivity of spleen cells.

The immunotherapeutic GEN-003 as a prophylactic vaccine candidate for genital herpes in guinea pigs

Abstract GEN‑003 is a subunit vaccine candidate containing adjuvanted gD and ICP4 antigens that has shown durable therapeutic efficacy against genital herpes in a phase 2 clinical trial. Here we describe the potential benefit of GEN-003 as a prophylactic vaccine in a guinea pig model of genital herpes. Animals were immunized three times every two weeks with GEN-003 or control and intravaginally challenged with herpes simplex virus 2 (HSV-2, MS strain) 21 days later. GEN-003 significantly reduced frequency and severity of genital lesions in both acute and early stages of recurrent infection by over 85%, and 60% of vaccinated guinea pigs did not develop any lesions during the 33 day follow-up period. Furthermore, viral genome copy numbers recovered in vaginal swabs from GEN-003-immunized animals were 100-fold lower than from controls. Recurrent shedding frequencies were reduced by 27%. GEN-003 vaccination induced antigen-specific antibody responses at least as strong as those elicited by viral infection. Driven by gD-specific antibodies, HSV-2 neutralizing antibody titers followed the same pattern. After viral challenge, peripheral antigen-specific T cell responses in spleens of GEN-003-immunized animals were lower than those in controls, as measured by interferon-γ ELISpot. This might have resulted from increased antigen-specific T cell recruitment to the genital mucosa, or decreased exposure to the virus due to fewer recurrences. Additional methods to better understand mucosal mechanisms of protection are in development. In summary, GEN-003 shows promise as a prophylactic vaccine candidate for genital herpes. Ongoing studies will determine if protection can be further enhanced by addition or exchange of antigens and/or adjuvants.

Flagellin Encoded in Gene-Based Vector Vaccines Is a Route-Dependent Immune Adjuvant.

Flagellin has been tested as a protein-based vaccine adjuvant, with the majority of studies focused on antibody responses. Here, we evaluated the adjuvant activity of flagellin for both cellular and humoral immune responses in BALB/c mice in the setting of gene-based immunization, and have made several novel observations. DNA vaccines and adenovirus (Ad) vectors were engineered to encode mycobacterial protein Ag85B, with or without flagellin of Salmonella typhimurium (FliC). DNA-encoded flagellin given IM enhanced splenic CD4+ and CD8+ T cell responses to co-expressed vaccine antigen, including memory responses. Boosting either IM or intranasally with Ad vectors expressing Ag85B without flagellin led to durable enhancement of Ag85B-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with significantly improved protection against challenge with pathogenic aerosolized M. tuberculosis. However, inclusion of flagellin in both DNA prime and Ad booster vaccines induced localized pulmonary inflammation and transient weight loss, with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in levels of mucosal CD4+ and CD8+ T cell responses following IM DNA/IN Ad mucosal prime-boosting, although antibody responses were not diminished. These findings indicate that flagellin has differential and route-dependent adjuvant activity when included as a component of systemic or mucosally-delivered gene-based prime-boost immunization. Clear adjuvant activity for both T and B cell responses was observed when flagellin was included in the DNA priming vaccine, but side effects occurred when given in an Ad boosting vector, particularly via the pulmonary route.

Effect of Maillard-type glycosylated whole buckwheat protein on immune response of spleen cells

Effect of Maillard-type glycosylated whole buckwheat protein on immune response of spleen cells

Development and validation of a SYBR Green real-time PCR assay for rapid and quantitative detection of goose interferons and proinflammatory cytokines

Real time quantitative polymerase chain reaction (RT-qPCR) based on SYBR-Green I binding is a quick, reliable, and easy method for analyzing small amounts of mRNA. Viral pathogens are recognized at the time of infection by pattern recognition receptors; thus, the inflammatory cytokines (IL1β, IL6, and IL18) and antiviral cytokines (IFNα, IFNγ) are secreted by innate immune cells and induced to respond to the pathogens. The objective of this study was to develop an effective and sensitive RT-qPCR assay for the rapid and accurate quantification of goose cytokines: IFNα, IFNγ, IL1β, IL6, and IL18. Subsequently, the established methods were employed to detect the immune response in agonist-stimulated goose spleen cells in vitro. These data indicated that the established RT-qPCR is a reliable method for determining relative gene expression. The results revealed that Imiquimod led to the significant upregulation of goose IFNα (P < 0.01), IFNγ (P < 0.01), IL1β (P < 0.01), IL6 (P < 0.01), and IL18 (P < 0.05). The established methods are important for scientific research and clinical applications, which require rapid and accurate results in a short period of time. The technique can potentially be used in the further research of goose molecular immunology, which will help us understand the interactions between hosts and pathogens.

Adjuvants may reduce in vivo transfection levels for DNA vaccination in mice leading to reduced antigen-specific CD8+ T cell responses

Adjuvants for DNA vaccination are designed to promote transformation of transgenes into target cells and increase inflammation in the site of injection, with resultant immune cell recruitment. Numerous studies indicated cationic liposomes as effective adjuvants for DNA vaccination due to their ability to promote in vivo transfection and innate immune system activation. Commercial reagents as Adjuplex and in vivo-JetPEI are also intended to facilitate DNA vaccination. Here, we evaluate the adjuvant properties of cationic liposomes, Adjuplex and in vivo-JetPEI compared to injection of DNA without adjuvant. In mice vaccinated with piggyBac pDNA vaccines, we assessed in vivo antigen expression, innate immune responses in draining lymph nodes, and antigen-specific T cell responses in spleens and blood. Surprisingly, vaccination with DNA in PBS emerged as the most efficient in promoting in vivo transfection and consequent antigen expression, while the addition of adjuvant reduced the amount of antigen expressed. On the other hand, we discovered higher numbers of innate immune cells and activated dendritic cells in the lymph nodes of mice injected with adjuvants than those vaccinated in PBS. The analysis of eGFP-specific immune responses revealed that all the different immunizations induced functional antigen-specific T cells in spleens, although only T cells generated by non-adjuvant vaccination and Adjuplex were identified in the blood of vaccinated mice. These results provide insight into the effects of these 3 adjuvants and may facilitate appropriate use off adjuvants by researchers using DNA vaccines in laboratory animals.

Flagellin has differential and route-dependent adjuvant effects when encoded in heterologous systemic or mucosal gene-based prime-boost vaccines (VAC4P.1107)

Abstract Flagellin has been tested as a protein-based adjuvant, with most studies focused on antibody responses. In this study, we have evaluated the adjuvant activity of flagellin for cellular and humoral immune responses in gene-based immunizations. DNA and adenovirus (Ad) vectors were constructed to encode mycobacterial Ag85B and/or flagellin (FliC) of S. typhimurium. DNA-encoded flagellin given to mice via the IM route enhanced antigen-specific splenic CD4+ and CD8+ T cell responses. Boosting either IM or intranasally (IN) with Ad vectors expressing Ag85B without flagellin enhanced antigen-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with improved protection against pulmonary challenge with Mtb. However, inclusion of flagellin in both DNA prime and Ad boosting vaccines induced localized pulmonary inflammation and transient weight loss, along with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in mucosal CD4+ and CD8+ T cell responses following IM DNA / IN Ad prime-boosting, although antibody responses were not diminished. These findings suggest that flagellin has differential and route-dependent adjuvant activity when included in systemic or mucosal gene-based prime-boost immunization. Adjuvant activity for both T and B cell responses and safety were optimal when flagellin was included only in the DNA vaccine priming phase of systemic or mucosal DNA/Ad prime-boosting.

Cytokine, antibody and proliferative cellular responses elicited by Taenia solium calreticulin upon experimental infection in hamsters.

Taenia solium causes two diseases in humans, cysticercosis and taeniosis. Tapeworm carriers are the main risk factor for neurocysticercosis. Limited information is available about the immune response elicited by the adult parasite, particularly the induction of Th2 responses, frequently associated to helminth infections. Calreticulin is a ubiquitous, multifunctional protein involved in cellular calcium homeostasis, which has been suggested to play a role in the regulation of immune responses. In this work, we assessed the effect of recombinant T. solium calreticulin (rTsCRT) on the cytokine, humoral and cellular responses upon experimental infection in Syrian Golden hamsters (Mesocricetus auratus). Animals were infected with T. solium cysticerci and euthanized at different times after infection. Specific serum antibodies, proliferative responses in mesenteric lymph nodes and spleen cells, as well as cytokines messenger RNA (mRNA) were analyzed. The results showed that one third of the infected animals elicited anti-rTsCRT IgG antibodies. Interestingly, mesenteric lymph node (MLN) cells from either infected or non-infected animals did not proliferate upon in vitro stimulation with rTsCRT. Additionally, stimulation with a tapeworm crude extract resulted in increased expression of IL-4 and IL-5 mRNA. Upon stimulation, rTsCRT increased the expression levels of IL-10 in spleen and MLN cells from uninfected and infected hamsters. The results showed that rTsCRT favors a Th2-biased immune response characterized by the induction of IL-10 in mucosal and systemic lymphoid organs. Here we provide the first data on the cytokine, antibody and cellular responses to rTsCRT upon in vitro stimulation during taeniasis.

In vitro Studies of the Antibody Response: Antibodies of Different Specificity are Made in Different Populations of Cells.

In vitro Studies of the Antibody Response: Antibodies of Different Specificity are Made in Different Populations of Cells.

Effect of Acanthopanax senticosus on the Accumulation of Cadmium and on the Immune Response of Spleen Cells

Exposure to cadmium (Cd) is known to alter immune responses. Acanthopanax senticosus (Rupr. et Maxim.) Harms (AS) extract, an antioxidant-containing complex of phenolic compounds, tetracyclic triterpenoids/steroids, and polysaccharides, is known to produce Cd mobilization and excretion in vivo. Building upon earlier findings, the aim of the study was to evaluate the effects of an AS extract on Cd accumulation and changes in the presence of splenic immune cells in hosts during a chronic metal exposure. Chronic Cd exposure of BALB/c mice was induced by providing them solutions containing different levels of CdCl2 (25 or 250 mg/L) in double-distilled water, with/without a concurrent presence of AS root extract (approximately 151 g material/L), for 8 wk. At the study end, Cd levels in spleen were measured. Levels of key splenic immune cells, including macrophages, T-lymphocytes, and B-lymphocytes, were determined by immunohistochemistry using, respectively, CD68, CD3, and CD20 antibodies. The results indicated that chronic consumption of AS extract in the presence of the high dose of CdCl2 led to a significant decrease in Cd levels in mouse spleen. The effects of AS on the lower CdCl2 dose were less apparent. In addition, the presence of AS and Cd increased the amount of macrophages and both B and T lymphocytes in mouse spleen relative to concentrations that were lowered as a result of chronic metal only intake.

Optimization of Mucosal Responses after Intramuscular Immunization with Integrase Defective Lentiviral Vector

Many infectious agents infiltrate the host at the mucosal surfaces and then spread systemically. This implies that an ideal vaccine should induce protective immune responses both at systemic and mucosal sites to counteract invasive mucosal pathogens. We evaluated the in vivo systemic and mucosal antigen-specific immune response induced in mice by intramuscular administration of an integrase defective lentiviral vector (IDLV) carrying the ovalbumin (OVA) transgene as a model antigen (IDLV-OVA), either alone or in combination with sublingual adjuvanted OVA protein. Mice immunized intramuscularly with OVA and adjuvant were compared with IDLV-OVA immunization. Mice sublingually immunized only with OVA and adjuvant were used as a positive control of mucosal responses. A single intramuscular dose of IDLV-OVA induced functional antigen-specific CD8+ T cell responses in spleen, draining and distal lymph nodes and, importantly, in the lamina propria of the large intestine. These results were similar to those obtained in a prime-boost regimen including one IDLV immunization and two mucosal boosts with adjuvanted OVA or vice versa. Remarkably, only in groups vaccinated with IDLV-OVA, either alone or in prime-boost regimens, the mucosal CD8+ T cell response persisted up to several months from immunization. Importantly, following IDLV-OVA immunization, the mucosal boost with protein greatly increased the plasma IgG response and induced mucosal antigen-specific IgA in saliva and vaginal washes. Overall, intramuscular administration of IDLV followed by protein boosts using the sublingual route induced strong, persistent and complementary systemic and mucosal immune responses, and represents an appealing prime-boost strategy for immunization including IDLV as a delivery system.

Porphyromonas gingivalis infection enhances Th17 responses for development of atherosclerosis

ObjectivesPorphyromonas gingivalis has been shown to associate with the development of atherosclerosis. Recent studies indicate that IL-17-producing T helper 17 (Th17) cells have been correlated with the emergence of atherosclerosis. Therefore, we investigated whether the Th17 cell response and expression of Th17-related molecules, in contrast with Th1- and Treg cells, are enhanced by P. gingivalis-challenge in Apolipoprotein E knockout (ApoE KO) mice. DesignFive mice were intravenously injected with P. gingivalis three times a week for 3 weeks and killed at 15 weeks of age. The proximal aorta lesion area, flow cytometry analysis and IL-17, IL-10, IFN-γ, and IL-1β levels in splenic cultures, and expression of Th17-related molecules in spleen and hearts were examined. ResultsP. gingivalis-challenge showed notable accumulation of atherosclerotic plaques by Oil Red O-staining in ApoE KO mice. Intracellular cytokine staining revealed that significantly elevated CD4+ interleukin (IL)-17A+ T cells and slightly increased CD4+ Foxp3+ T cells was recognized in spleen cells of P. gingivalis-challenged mice compared with those from non-infected mice. P. gingivalis-challenge significantly increased IL-17 and IL-1β production and RORγt expression in splenic cells. Furthermore, the expression of Th17-related genes such as IL-6, TGF-β, RORγt and STAT3 were elevated in splenic cells as well as heart tissue of P. gingivalis-challenged mice. ConclusionThese results suggest that P. gingivalis infection may enhance pro-inflammatory Th17 cell responses in lesion areas and spleen, thereby accelerating atherosclerosis.

Dietary Bovine Lactoferrin Alters Mucosal and Systemic Immune Cell Responses in Neonatal Piglets1–3

Lactoferrin (LF) is a multifunctional immune protein found at high concentrations in human milk. Herein, the effect of dietary bovine LF (bLF) on mucosal and systemic immune development was investigated. Colostrum-deprived piglets were fed formula containing 130 [control (Ctrl)], 367 (LF1), or 1300 (LF3) mg of bLF/(kg body weight · d). To provide passive immunity, sow serum was provided orally during the first 36 h of life. Blood, spleen, mesenteric lymph node (MLN), and ascending colon (Asc) contents were collected on day 7 (n = 10–14/group) and day 14 (n = 10–12/group). Immune cell populations were quantified by flow cytometry and immunoglobulins (Igs) were measured by ELISA. Additionally, immune cells were isolated from spleen and MLNs (n = 7/group) on day 7 and stimulated ex vivo with phytohemagglutinin or lipopolysaccharide (LPS) ± LF for 72 h. Secreted cytokine concentrations were quantified by multiplex assay. Lymphocyte populations [cluster determinant (CD)4, CD8, and natural killer cells] developed normally and were unaffected by dietary bLF. LF3 piglets tended to have 1.4 to 2 times more serum IgG than Ctrl piglets (P = 0.07) or LF1 piglets (P = 0.03), but IgA in Asc contents was unaffected by bLF. Asc IgA was 4 times higher on day 14 than day 7. Spleen cells from LF3 piglets produced 2 times more interleukin (IL)-10 and tumor necrosis factor (TNF)-α ex vivo than those from Ctrl or LF1 piglets. MLN cells from LF1 and LF3 piglets produced 40% more IL-10 and tended to produce 40% more IL-6 (P = 0.05) than those from Ctrl piglets. However, ex vivo bLF did not affect the cytokine response of spleen or MLN cells to LPS. In summary, dietary bLF alters the capacity of MLN and spleen immune cells to respond to stimulation, supporting a role for LF in the initiation of protective immune responses in these immunologically challenged neonates.