Delayed-type Hypersensitivity Response Research Articles

Immune induction by adjuvanted Leishmania donovani vaccines against the visceral leishmaniasis in BALB/c mice

Visceral leishmaniasis (VL) is a neglected tropical disease caused by Leishmania donovani or Leishmania infantum. Currently, the patients are treated with chemotherapeutic drugs; however, their toxicity limits their use. It would be desirable to develop a vaccine against this infection. In this study, we assessed the efficacy of different vaccine formulations at variable time points. Heat-killed (HK) antigen of Leishmania donovani was adjuvanted with two adjuvants (AddaVax and Montanide ISA 201) and three immunizations at a gap of 2 weeks (wk) were given to BALB/c mice. After 2 weeks of the last booster, mice were given challenge infection and sacrificed before challenge and after 4wk, 8wk, and 12 wk post-challenge. Significant protective immunity was observed in all the immunized animals and it was indicated by the notable rise in delayed-type hypersensitivity (DTH) response, remarkably declined parasite burden, a significant increase in the levels of interferon-gamma (IFN-γ), interleukin-12, interleukin-17 (Th1 cytokines), and IgG2a in contrast to infected control mice. Montanide ISA 201 with HK antigen provided maximum protection followed by AddaVax with HK and then HK alone. These findings elaborate on the importance of the tested adjuvants in the vaccine formulations against murine visceral leishmaniasis.

Capsaicin attenuates immunosuppression induced by chronic stress in BALB/C mice

Although acute stress generally exerts positive effects on the immune system, chronic stress typically causes immunosuppression via the hypothalamic–pituitary–adrenal (HPA) axis. In this study, the effects of capsaicin (1.28 mg/kg intraperitoneally [i.p.] for 7 days) on immune parameters were evaluated under conditions of chronic stress. Capsaicin treatment significantly increased the immune response as evaluated by the delayed-type hypersensitivity (DTH) reaction to dinitrofluorobenzene (DNFB) and splenocyte proliferation assays- It also is able to rescue the splenocytes of the apoptosis induced by stress. The capsaicin treatment increased the production of Th1 cytokines and decreased the production of Th2 cytokines and TGF-β1 in the plasma and culture supernatants of immunosuppressed mice, which is associated with the modulation of Th2 induced by stress cells. Moreover, the production of corticosterone significantly decreased in capsaicin-treated animals as compared to control groups. The capsaicin treatment further attenuated the immunosuppression induced by the corticosterone treatment (40 mg/kg i.p. for 7 days), albeit less potently, as exhibited in the DTH response. Intriguingly, the capsaicin treatment decreased the induction of IL-10, IL-4, and TGF-β1 through high doses of corticosterone, indicating direct cellular immunomodulation. These results show, that capsaicin is able to modulate chronic stress-induced immunosuppression, mediating corticosterone released inhibition, but also, that capsaicin significantly modulates the pharmacological action of corticosterone in vivo.

Corrective effects of original bioflavonoid complex in the cyclophosphamide-induced immunity disorders

Our aim was to evaluate immunomodulatory properties of an original bioflavonoid complex in experimental immune disturbances induced by cyclophosphamide (Cy). We have studied morphometric indexes of thymus and spleen, as well as blood leukocyte counts, cell proliferative activity in lymphoid organs, delayed hypersensitivity responses to T cell-dependent antigen, along with differentiation activity of bone marrow stem cells in experimental animals during Cy-induced immune suppression after a course of bioflavonoid treatment. Suspension of the bioflafonoid complex was introduced to the male mice (СВАхC57Bl/6)F1 aged 12- 14 weeks at a daily dose of 2 mg/animal (80 mg/kg), per os, using gastric catheter, over 14 days. Cytostatic immunosuppression was produced by a single intraperitoneal Cy injection. Proliferative activity of spleen and thymic cells was determined by standard method with Н3 -thymidine incorporation in the 72-h cell culture. Cellular immune response was assayed by the degree of delayed-type hypersensitivity development in response to sheep erythrocytes. The number of hematopoietic progenitors was evaluated by culturing bone marrow cells in methylcellulose-based medium. The experiments have shown mitigation of immunosuppressive effects induced by Cy, in the course of bioflavonoid complex treatment, with respect to absolute and relative mass of lymphoid organs and leukocyte numbers in peripheral blood. Moreover, we have demonstrated decreased effects of Cy treatment upon the spontaneous activity of spleen cells, mitogen-induced thymocyte and splenocyte proliferation, intensivity of delayed-type hypersensitivity response that reached the values of intact animals. Following the course of bioflavonoids, we have revealed an increase in early hematopoietic progenitors. Alleviation of Cy-induced suppressive effects upon cellular immune response, proliferation rates of immune cells, as well as stimulation of hematopoietic stem cell functions suggest a sufficient capacity of the original bioflavonoid complex for modulation of immunity and hematopoiesis, thus presenting experimental proofs for its potential usage as an adjuvant treatment of the patients with malignant diseases.

PAR-1 signaling on macrophages is required for effective invivo delayed-type hypersensitivity responses.

SummaryDelayed-type hypersensitivity (DTH) responses underpin chronic inflammation. Using a model of oxazolone-induced dermatitis and a combination of transgenic mice, adoptive cell transfer, and selective agonists/antagonists against protease activated receptors, we show that that PAR-1 signaling on macrophages by thrombin is required for effective granuloma formation. Using BM-derived macrophages (BMMs) in vitro, we show that thrombin signaling induced (a) downregulation of cell membrane reverse cholesterol transporter ABCA1 and (b) increased expression of IFNγ receptor and enhanced co-localization within increased areas of cholesterol-rich membrane microdomains. These two key phenotypic changes combined to make thrombin-primed BMMs sensitive to M1 polarization by 1000-fold less IFNγ, compared to resting BMMs. We confirm that changes in ABCA1 expression were directly responsible for the exquisite sensitivity to IFNγ in vitro and for the impact on granuloma formation in vivo. These data indicate that PAR-1 signaling plays a hitherto unrecognized and critical role in DTH responses.

Stimulation of humoral and cell-mediated immunities in healthy and cyclophosphamide-induced immunosuppressed rats by the lyophilized Houttuynia cordata fermented drink

ABSTRACTHouttuynia cordata fermented drinks (HCFDs) are dietary liquid supplements widely marketed and consumed for immune enhancement. In this study, the acute oral toxicity and immunostimulatory activity of the selected lyophilized HCFD in healthy and cyclophosphamide-induced immunosuppressed rats were evaluated. The lyophilized selected commercialized HCFD was safe in animal model as there was no acute oral toxic death at up to 2000 mg/kg in male Wistar rats. After 14 days of treatment, in immunosuppressed rats, lyophilized HCFD enhanced rat body weight gain and significantly increased spleen index at a dose of 100 mg/kg. Moreover, B-cell proliferation and anti-sheep red blood cell antibody production were significantly increased at doses of 100 and 200 mg/kg, respectively. The delayed-type hypersensitivity response was significantly stimulated at a dose of 200 mg/kg. These findings exhibited the immunostimulatory activity of the lyophilized HCFD by enhancing the immune responses in both humoral and cell-mediated immunities in immunosuppressed rats.

Immunopathogenesis of Dermatophytoses and Factors Leading to Recalcitrant Infections.

The pathogenesis of dermatophytic infections involves the interplay of three major factors: the dermatophyte, the inherent host defense, and the adaptive host immune response. The fungal virulence factors determine the adhesion and invasion of the skin while the immune response depends on an interaction of the pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMP) with pattern recognition receptors (PRRs) of the host, which lead to a differential Th (T helper) 1, Th2, Th17, and Treg response. While anthropophilic dermatophytes Trichophyton rubrum and now increasingly by T. interdigitale subvert the immune response via mannans, zoophilic species are eliminated due to a brisk immune response. Notably, delayed-type hypersensitivity (Th1) response of T lymphocytes causes the elimination of fungal infection, while chronic disease caused by anthropophilic species corresponds to toll-like receptor 2 mediated IL (interleukin)-10 release and generation of T-regulatory cells with immunosuppressive potential. Major steps that determine the ultimate clinical course and chronicity include genetic susceptibility factors, impaired epidermal and immunological barriers, variations in the composition of sebum and sweat, carbon dioxide tension, skin pH, and topical steroid abuse. It is important to understand these multifarious aspects to surmount the problem of recalcitrant dermatophytosis when the disorder fails conventional therapeutic agents.

Potent health-promoting effects of a synbiotic formulation prepared from Lactobacillus acidophilus NCDC15 fermented milk and Cichorium intybus root powder in Labrador dogs

To ascertain potent health-promoting effects of a synbiotic prepared using a combination of Lactobacillus acidophilus NCDC15 fermented milk and Cichorium intybus root powder in Labrador dogs, 15 Labrador adult female dogs were randomly distributed into three groups following a completely randomized design. Group I was fed a homemade basal diet alone and served as control (CON), while the other two groups were fed the same basal diet supplemented with low- (LSL) and high- levels (HSL) of the synbiotic. The experiment duration was of 9-weeks. The results of the study indicated that there were no variations (P > 0.05) among the groups with respect to voluntary food intake and digestibility of DM, OM, CP and EE. However, the digestibility of crude fibre tended (P = 0.051) to be higher in both the supplemented groups as compared to CON. Physical characteristics of faeces remained unaltered (P > 0.05) due to dietary treatments. However, faecal lactate was increased (P < 0.05) in both the synbiotic fed groups with a concomitant reduction in faecal ammonia compared to control. The faecal lactobacilli and bifidobacteria population exhibited higher (P < 0.05) counts in LSL and HSL groups than the CON. There was a lower population of clostridia and coliform in LSL and HSL groups than in CON. The delayed-type hypersensitivity response to PHA-P and antibody response to sheep erythrocytes showed improved (P < 0.05) immune status in response to dietary synbiotic supplementation. Overall, the findings of this study led to the conclusion that the synbiotic preparation has a beneficial impact on canine gastrointestinal health and immunity, and the response to synbiotic supplementation at lower and higher doses was similar.

Immunomodulatory effects of standardized extract of Curcuma mangga val. on cytokines, antibody and delayed-type hypersensitivity response in Wistar rats

Background and purpose:Recently, we have highlighted the immunomodulatory activity of Curcuma mangga Val. on phagocytosis ability. The current study was conducted to determine the immunomodulatory effects of the standardized extract of C. mangga rhizomes by in vitro and in vivo studies.Experimental approach:The C. mangga extract was standardized according to a guideline for herbal preparation. The extract was investigated for its immunomodulatory effects on gene expression of cytokines, cytokines and antibody production as well as delayed-type hypersensitivity (DTH) response. The gene expression of cytokines on lipopolysaccharide-induced-RAW 264.7 cells was analysed by reverse transcription-polymerase chain reaction (RT-PCR) method. The effect of the extract on DTH response was investigated by the paw edema method, meanwhile the effects of the extract on antibody and cytokine production from normal and cyclophosphamide-induced Salmonella typhimurium infected rats were determined using an enzyme-linked immunosorbent assay (ELISA).Findings/Results:The extract of C. mangga demonstrated an inhibitory effect on gene expression of interleukin-1β (IL-1β), tumor necrosis factor-α, and IL-6 as compared to lipopolysaccharide-induced cells. The extract also depicted inhibitory activity on IL-4 production as compared to the negative control. Whereas, the DTH response and production of immunoglobulin G from both groups after treatment with C. mangga extract were higher than those of negative control (P < 0.05).Conclusion and implications:The results indicated that the C. mangga extract has immunomodulatory effects, emphasizing its potential to be developed as immunotherapeutic agent.

Combination therapy of phosphatidylserine liposome with cyclosporine A improves nephrotoxicity and attenuates delayed-type hypersensitivity response

This study aimed to evaluate the antioxidant capacity of phosphatidylserine liposome (PS) against oxidative stress due to cyclosporine A (CsA) and concurrent administration of PS and CsA on the attenuation of immune response. The effect of oral PS was evaluated on biochemical and oxidative renal markers and histopathology of nephrotic rats receiving CsA. The effect of co-administration of PS with CsA was also assessed on DTH (delayed-type hypersensitivity) reaction of immunized rats. The cytokines production level of IL-2 (Interleukin-2) and IFN-γ (Interferon gamma) was measured in immunized rat's splenocytes. PS treatment significantly (P < 0.05) reduced Cr and BUN of serum and MDA (malondialdehyde) in kidney tissue, and increased SOD (superoxide dismutase) and CAT (Catalase) of kidney tissue in CsA-nephrotic rats. Histopathology data indicated significantly (P < 0.05) nephrotoxicity improvement after 25-day treatment with PS. Furthermore, CsA plus PS administration significantly reduced DTH response and cytokines production of IL-2 and IFN-γ in immunized rats. In conclusion, coadministration of CsA plus PS may overcome oxidative stress and improve the performance of organ transplantation or autoimmune therapy.

Effects of combination of dihydroartemisinin and Huobahua on expression of p-p38 MAPK and ICAM-1 in mice with delayed-type hypersensitivity

The aim of this paper was to investigate the immunosuppressive effects of dihydroartemisinin and Huobahua compatibility in mice with delayed hypersensitivity and explore its possible mechanism. The delayed-type hypersensitivity(DTH) model in mice was established to observe the immunosuppressive effects of dihydroartemisinin and Huobahua compatibility in DTH mice. ELISA assay was used to detect the contents of interferon(IFN-γ); histopathological changes and degree of mononuclear infiltration of right ear tissues were examined by HE staining; the expression level of intercellular cell adhesion molecule-1(ICAM-1) in the right ear of mice was detected by immunohistochemistry; the protein expression levels of p38 phospho mitogen activated protein kinase(p-p38 MAPK) was detected by Western blot analysis. As compared with the control group, the degree of ear swelling, thymus/spleen index, serum IFN-γ as well as the number and degree of infiltration of monocytes were significantly increased in the model group. As compared with the model group, the degree of ear swelling and thymus/spleen index of the mice in the combination group were significantly reduced; the number and degree of infiltration of monocytes were significantly relieved; the serum levels of IFN-γ and the expression levels of p-p38 MAPK and ICAM-1 proteins in the right ear were also significantly reduced. The combination of dihydroartemisinin and Huobahua can significantly inhibit the DTH response, and it may regulate the production and secretion of related inflammatory factor IFN-γ by inhibiting the phosphorylation activity of p38 MAPK, thereby further reducing the expression of ICAM-1 and thus exerting the immunosuppressive effect.

A randomized controlled trial with a delayed-type hypersensitivity model using keyhole limpet haemocyanin to evaluate adaptive immune responses in man.

AimsKeyhole limpet haemocyanin (KLH) immunization is a clinical model for the evaluation of human antibody responses. The current study evaluated the anti‐KLH antibody response after KLH immunization and the delayed‐type hypersensitivity response following intradermal KLH administration, using objective imaging techniques.MethodsHealthy male subjects aged 24.5 ± 5.4 years were randomized to intramuscular immunization with 100 μg KLH (n = 12) or placebo (n = 3). Anti‐KLH antibody (Ig) M and IgG titres were determined before and every 7 days after KLH immunization for a total of 28 days. Twenty‐one days after the immunization, all subjects received 1 μg KLH intradermally. Prior to and 2 days after intradermal KLH administration, skin blood perfusion, erythema and oedema were quantified using noninvasive imaging tools. Repeated measures ANCOVAs were used to analyse data.ResultsAnti‐KLH IgM and IgG titres increased after KLH immunization compared to placebo (estimated difference [ED]: 37%, 95% confidence interval [CI]: 19–51% and ED: 68%, 95% CI: 56–76% respectively). Upon intradermal KLH administration an increase in skin blood perfusion (ED: 10.9 arbitrary units (AU), 95% CI: 1.4–20.4 AU) and erythema (ED: 0.3 AU, 95% CI: 0.1–0.5 AU) was observed in KLH‐immunized subjects compared to placebo.ConclusionKLH immunization followed by intradermal KLH administration resulted in increased anti‐KLH IgM and IgG titres and a delayed‐type hypersensitivity response quantified by an increase in skin blood perfusion and erythema. Using noninvasive imaging tools the KLH model has the potential to serve as an objective tool to study the pharmacodynamics of T‐cell‐directed immunomodulatory drugs.

Immunopathological effects of experimental T-2 mycotoxicosis in Wistar rats.

It is well known that T-2 toxin has cytotoxic radiomimetic like effects on the immune system. Because of scant research data demonstrating the chronic effects of low doses of the T-2 toxin on humoral and cellular responses in rats, the present experiment was undertaken. The animals were divided into four groups, namely, group I (0.5 ppm), group II (0.75 ppm) and group III (1.0 ppm) and group IV (control) were given toxin-free diet for 12 weeks and eight animals each were sacrificed at 2, 4, 6, 8, 10, and 12-week of the experimental period. The humoral immune response was evaluated based on hemagglutination test (HA), and levels of serum immunoglobulins (IgA, IgG, IgM) while the cell-mediated immune response was evaluated by delayed-type hypersensitivity (DTH) response to ovalbumin, lymphocyte stimulation index, analyses of CD4+ and CD8+ T lymphocytes and mRNA expression levels of selected cytokines like IL-2, IFN-γ, IL-4 and IL-10 by quantitative Real-time PCR in experimental groups. T-2 treatment caused suppression in both humoral and cell-mediated immune responses as evidenced by a decrease in all these parameters in toxin fed animals compared to the control in the dose and duration-dependent manner. This dose-dependent effect on the immune system has been further reflected largely by the depletion of lymphocytes from lymphoid organs as observed histopathologically in the spleen, thymus, and Peyer's patches in the present study.

In vitro and In Vivo Immunomodulatory Activity of Physalis angulata Concentrated Ethanolic Extract.

The need for new immunomodulatory drugs is due to the side effects associated with the prolonged use of the currently used immunomodulatory drugs. In this context, the present work aimed to investigate the immunomodulatory effect of an ethanolic concentrated extract from Physalis angulata. The cytotoxicity of samples was determined using peritoneal macrophages though the Alamar Blue assay. The immunomodulatory activity of the ethanolic extract from P. angulata on activated macrophages was determined by measurement of nitrite and cytokine production. The immunosuppressive effects of the ethanolic extract from P. angulata was evaluated on lymphocyte proliferation and cytokine production. The effects of the extract on cell cycle progression and cell death on lymphocytes were evaluated by flow cytometry. Lastly, the ethanolic extract from P. angulata was tested in vivo in toxicological tests and in models of peritonitis and delayed-type hypersensitivity response. The ethanolic extract from P. angulata decreased nitrite, interleukin-6, interleukin-12, and TNF-α production by activated macrophages without affecting the cell viability. In addition, the ethanolic extract from P. angulata inhibited lymphoproliferation and the secretion of interleukin-2, interleukin-6, and IFN-γ, and increased interleukin-4 secretion by activated splenocytes. Flow cytometry analysis in lymphocyte cultures showed that treatment with the ethanolic extract from P. angulata induces cell cycle arrest in the G1 phase followed by cell death by apoptosis. Moreover, mice treated with the extract from P. angulata at 100 or 200 mg/kg did not show signs of toxicity or alterations in serum components. Finally, the ethanolic extract from P. angulata significantly reduced neutrophil migration and reduced paw edema in bovine serum albumin-induced the delayed-type hypersensitivity response model. Our results demonstrate the potential of the ethanolic extract of P. angulata as an alternative for the treatment of immune-inflammatory diseases.

Short Communication: Stimulatory effect of Curcuma mangga on immune response against Staphylococcus aureus

Abstract. Yuandani, Nugraha SE, Laila L, Silaban SD, Ramadhani F. 2020. Short Communication: Stimulatory effect of Curcuma mangga on immune response against Staphylococcus aureus. Nusantara Bioscience 12: 109-113. Recently, the ethanol extract of Curcuma mangga Val. rhizomes were found to have immunomodulatory activity by enhancing phagocytic ability. This study was carried out to evaluate the effect of C. mangga rhizomes on antibody titer and delayed-type hypersensitivity response in normal and doxorubicin-induced rats against Staphylococcus aureus. The extract was administered orally at doses of 100, 200, and 400 mg/kg body weight (bw) to rats for 72 hours prior to sensitization of Staphylococcus aureus, then continued for 14 days. Doxorubicin (4.67 mg/kg bw) was administered on days 8 and 11 to suppress the immune system. The antibody titer of normal and immune-suppressed rats after the treatment with C. mangga ethanolic extract was significantly higher than the rats with negative control (P&lt;0.05). Ethanol extract of C. mangga also demonstrated stimulation on delayed-type hypersensitivity response which was indicated by the increase of paw volume. The extract at dose of 400 mg/kg bw showed higher immunostimulatory activity than Levamisole as a positive control. The results indicated that the ethanol extract of C. mangga shows immunostimulatory activity, confirming its potential to be developed as a therapeutic agent.

Phlebotomus perniciosus Recombinant Salivary Proteins Polarize Murine Macrophages Toward the Anti-Inflammatory Phenotype.

Phlebotomus perniciosus (Diptera: Phlebotominae) is a medically and veterinary important insect vector. It transmits the unicellular parasite Leishmania infantum that multiplies intracellularly in macrophages causing life-threatening visceral diseases. Leishmania establishment in the vertebrate host is substantially influenced by immunomodulatory properties of vector saliva that are obligatorily co-injected into the feeding site. The repertoire of P. perniciosus salivary molecules has already been revealed and, subsequently, several salivary proteins have been expressed. However, their immunogenic properties have never been studied. In our study, we tested three P. perniciosus recombinant salivary proteins—an apyrase rSP01 and yellow-related proteins rSP03 and rSP03B—and showed their anti-inflammatory nature on the murine bone-marrow derived macrophages. Even in the presence of pro-inflammatory stimuli (IFN-γ and bacterial lipopolysaccharide, LPS), all three recombinant proteins inhibited nitric oxide production. Moreover, rSP03 seems to have a very strong anti-inflammatory effect since it enhanced arginase activity, increased the production of IL-10, and inhibited the production of TNF-α even in macrophages stimulated with IFN-γ and LPS. These results suggest that P. perniciosus apyrase and yellow-related proteins may serve as enhancing factors in sand fly saliva, facilitating the development of Leishmania infection along with their anti-haemostatic properties. Additionally, rSP03 and rSP03B did not elicit the delayed-type hypersensitivity response in mice pre-exposed to P. perniciosus bites (measured as visible skin reaction). The results of our study may help to understand the potential function of recombinant's native counterparts and their role in Leishmania transmission and establishment within the host.

Evaluation of Adjuvant Effectiveness of Alum-Propranolol Mixture on the Immunogenicity of Excreted/Secreted Antigens of Toxoplasma gondii RH Strain.

Purpose: The introduction of novel adjuvants is an important step in attempts to develop a safe and more efficient vaccine. The present study was performed to determine whether the use of a mixed beta-adrenergic receptor antagonist propranolol (PRP) and aluminum (alum), as an adjuvant, have efficacy for Toxoplasma gondii vaccine to induce protective immunity in a mouse model.Methods: Female BALB/c mice divided into five groups were immunized with excretorys-ecretory antigens (ESA) vaccine, alum-ESA vaccine, PRP-ESA vaccine, and alum-PRP ESA vaccine, as well as with phosphate buffered saline (PBS), as a negative control group. The immune responses were evaluated by lymphocyte proliferation assay for measuring delayedtype hypersensitivity (DTH) response and by cytokine assay for evaluating IFN-γ and IL-5 levels. The survival rate of mice in all groups was assessed during a three-week monitoring period after an intraperitoneal challenge with T. gondii tachyzoites. Results: The results showed that mice immunized with PRP, as an adjuvant, could secret a higher level of IFN-γ, which was significant in comparison to other groups. However, mice vaccinated with alum-precipitated ESA antigen had ability to produce an elevated level of IL-5 compared to other mouse groups (P ≤ 0.05). Moreover, alum-PRP co-administration together with ESA vaccine resulted in the longer survival of mice.Conclusion: The findings of this study revealed that the combination of alum-PRP adjuvants and ESA vaccine of T. gondii elicits both humoral and cellular immune responses, which are comparable to either alum or PRP alone.

A Novel Murine Model of Early Abdominal Aortic Aneurysm Reflects Immunopathology in Human Abdominal Aortic Aneurysms

The pathogenesis of abdominal aortic aneurysm (AAA) remains poorly understood but there is mounting evidence that AAA may be an immune mediated condition. Current mouse models of AAA were developed primarily to reproduce mature aneurysms and may poorly reflect the pathology found in early AAA. We propose a novel murine model of AAA incorporating sensitization to aortic antigens, which Results in more robust AAA and duplicates the proposed autoimmune mechanisms in humans. C57B/L6 mice were randomized to sensitization to fragmented elastin (fELA) or control. One week later AAA was induced in all mice with the topical elastase method. Prior to sacrifice, development of a delayed-type hypersensitivity (DTH) reaction was assessed by cutaneous challenge in the ear pinna with fELA. Blood and periaortic lymph nodes (LN) were collected for quantification of type 1 regulatory T-cells (Tr1) and T-helper 17 (Th17) cells by flow cytometry. The mice were perfused with a silicone injection compound to fill and opacify their vasculature and microcomputed tomography scans were obtained for three-dimensional reconstruction of the infrarenal aorta (Fig 1) to evaluate volume and surface area of the aneurysms. Mice sensitized to fELA had larger aneurysms: volume 5.31 mm3 versus 4.16 mm3 (P = .034) and surface area 34.55 mm2 versus 30.87 mm2 (P = .038). They had positive DTH reactions to fELA represented by a greater weight difference between challenged and unchallenged ears: 4.13 mg vs 0.21 mg (P = .0004). The degree of DTH response was found to positively correlate with both volume (Fig 2) (R = 0.79; P = .00025) and surface area of aneurysm (R = 0.71; P = .0019). Flow cytometry Results were noTable for a significantly decreased Tr1/Th17 ratio in the sensitized group both in the blood (P = .0005) and LNs (P = .031) and in periaortic LNs this Tr1/Th17 ratio negatively correlated with AAA volume (R = –0.52; P = .021) with a trend toward a similar correlation in the blood (R = –0.49; P = .056). The addition of sensitization to fELA to a surgical topical elastase model of AAA Results in the development of larger aneurysms whose size correlates both with the DTH response to the antigen and with a decreasing Tr1/Th17 ratio in blood and periaortic LNs. The decreased Tr1/Th17 ratio is of particular interest as this was noted to be highly predictive of the presence of AAA in a study of Veterans Affairs patients presenting for screening ultrasound examination (0.5287 in AAA vs 2.6570 in non-AAA; P < .00001; receiver operating characteristic curve with an area under the curve of 0.925; 58 patients).Fig 2The strength of the delayed type-hypersensitivity (DTH) reaction to cutaneous challenge with elastin fragments correlates with aneurysm volume as calculated from three-dimensional reconstructions of the infrarenal aorta on microcomputed tomography scans (MicroCT). Cutaneous challenge was performed on the pinna of the ear and reaction quantified by weight difference compared to contralateral ear which was injected with vehicle.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Therapeutic and Prophylactic Antitumor Activity of an Oral Inhibitor of Fucosylation in Spontaneous Mammary Cancers.

2-fluorofucose (2FF) inhibits protein and cellular fucosylation. Afucosylation of IgG antibodies enhances antibody-dependent cell-mediated cytotoxicity by modulating antibody affinity for FcγRIIIa, which can impact secondary T-cell activation. Immune responses toward most common solid tumors are dominated by a humoral immune response rather than the presence of tumor-infiltrating cytotoxic T cells. IgG antibodies directed against numerous tumor-associated proteins are found in the sera of both patients with breast cancer and transgenic mice bearing mammary cancer. We questioned whether 2FF would have antitumor activity in two genetically distinct transgenic models; TgMMTV-neu (luminal B) and C3(1)-Tag (basal) mammary cancer. 2FF treatment significantly improved overall survival. The TgMMTV-neu doubled survival time compared with controls [P < 0.0001; HR, 7.04; 95% confidence interval (CI), 3.31-15.0], and survival was significantly improved in C3(1)-Tag (P = 0.0013; HR, 3.36; 95% CI, 1.58-7.14). 2FF treated mice, not controls, developed delayed-type hypersensitivity and T-cell responses specific for syngeneic tumor lysates (P < 0.0001). Serum IgG from 2FF-treated mice enhanced tumor lysis more efficiently than control sera (P = 0.004). Administration of 2FF for prophylaxis, at two different doses, significantly delayed tumor onset in both TgMMTV-neu; 20 mmol/L (P = 0.0004; HR, 3.55; 95% CI, 1.60-7.88) and 50 mmol/L (P = 0.0002; HR: 3.89; 95% CI, 1.71-8.86) and C3(1)-Tag; 20 mmol/L (P = 0.0020; HR, 2.51; 95% CI, 1.22-5.18), and 50 mmol/L (P = 0.0012; HR, 3.36; 95% CI, 1.57-7.18). Mammary cancer was prevented in 33% of TgMMTV-neu and 26% of C3(1)-Tag. 2FF has potent antitumor effects in mammary cancer models. The agent shows preclinical efficacy for both cancer treatment and prevention.

EVALUATION OF IN VIVO IMMUNOMODULATORY ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACT OF CAPPARIS MOONII

Objective: The present study was intended to evaluate the in vivo immunomodulatory activity of aqueous and ethanolic extract of dried fruits of Capparis moonii Wight. &#x0D; Methods: The effect of both the extracts was evaluated by carbon clearance assay in mice, and they showed a significant increase in the phagocytic index at 50, 100, and 200 mg/kg dose. The effect of the extracts on delayed-type hypersensitivity (DTH) and antibody titer assay was evaluated in ovalbumin immunized mice.&#x0D; Results: Both extracts showed a significant effect on DTH response and stimulation of antibody titer at 200 mg/kg dose. The effects of the extracts in cyclophosphamide-induced myelosuppression mice were also statistically significant.&#x0D; Conclusion: The studies demonstrated specific and non-specific immunostimulating properties of both the ethanol and aqueous extract of C. moonii fruits in various in vivo experimental methods suggesting its therapeutic usefulness in immunocompromised conditions.

Identification of Secreted O-Mannosylated Proteins From BCG and Characterization of Immunodominant Antigens BCG_0470 and BCG_0980.

Bacterial glycoproteins have been investigated as vaccine candidates as well as diagnostic biomarkers. However, they are poorly understood in Mycobacterium bovis strain bacille Calmette-Guérin (BCG), a non-pathogenic model of Mycobacterium tuberculosis. To understand the roles of secreted O-mannosylated glycoproteins in BCG, we conducted a ConA lectin-affinity chromatography and mass spectra analysis to identify O-mannosylated proteins in BCG culture filtrate. Subsequent screening of antigens was performed using polyclonal antibodies obtained from a BCG-immunized mouse, with 15 endogenous O-mannosylated proteins eventually identified. Of these, BCG_0470 and BCG_0980 (PstS3) were revealed as the immunodominant antigens. To examine the protective effects of the antigens, recombinant antigens proteins were first expressed in Mycobacterium smegmatis and Escherichia coli, with the purified proteins then used to boost BCG primed-mice. Overall, the treated mice showed a greater delayed-type hypersensitivity response in vivo, as well as stronger Th1 responses, including higher level of IFN-γ, TNF-α, and specific-IgG. Therefore, mannosylated proteins BCG_0470 and BCG_0980 effectively amplified the immune responses induced by BCG in mice. Together, our results suggest that the oligosaccharide chains containing mannose are the antigenic determinants of glycoproteins, providing key insight for future vaccine optimization and design.