Delayed-type Hypersensitivity Response Research Articles

Abstract 1699: Impact of whole blood interferon gamma production for patient selection in vaccination with autologous formalin-fixed tumor vaccine

Abstract Introduction: Immune checkpoint blockade by anti-CTLA-4 antibody and anti-PD-1 antibody led to clinical breakthrough for the treatment of patients with solid tumors. Moreover, recent reports have shown that neoantigens are important functional targets mediating response to immune checkpoint inhibitors. Therefore, sequential combination of personalized cancer vaccines and immune checkpoint blockade has emerged as a potential therapeutic strategy. However, existing drawbacks is that it is difficult to identify robust biomarkers for patient selection in vaccination with cancer vaccine. In this study, we examined whether the development of delayed-type hypersensitivity (DTH) response, measured after vaccination with autologous formalin-fixed tumor vaccine (AFTV), correlates to parameters of immune function such as whole blood cytokine levels and peripheral regulatory T cells (Treg) in patients with solid tumors. Methods: Subjects comprised 52 patients with solid malignancy. AFTV treatment consisted of 3 courses of vaccination performed at intervals of 2 weeks. DTH test was performed before and after vaccination. A positive DTH response was defined as a ≥ 10mm diameter erythema/induration. We evaluated whole blood cytokine production after phytohemagglutinin (PHA) stimulation using the bioplex array system. We also assessed the number of peripheral Tregs as well as routine hematological and biochemical parameters. Results: DTH positive patients and DTH negative patients did not differ with respect to the number of peripheral Tregs, leukocytes, lymphocytes or granulocyte/lymphocyte ratio. Whole blood IFN-γ production levels after phytohemagglutinin (PHA) stimulation at baseline in DTH positive patients were significantly higher than those in DTH negative patients (p=0.0397). Conclusion: These data indicate that whole blood IFN-γ production at baseline is likely to be useful to assess immunologic competence in vaccinated patients with AFTV. Consequently, clinical utility of whole blood IFN-γ production, as immunomonitoring method for patient selection in vaccination with cancer vaccine, is required to investigate in larger clinical studies in the future. Citation Format: Naoyuki Sakamoto, Takeshi Ishikawa, Tetsuya Okayama, Tomoyo Yasuda, Toshifumi Doi, Mari Tanigawa, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. Impact of whole blood interferon gamma production for patient selection in vaccination with autologous formalin-fixed tumor vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1699. doi:10.1158/1538-7445.AM2017-1699

Protective effect of rutin on humoral and cell mediated immunity in rat model

Diet and dietary intake can persuade the development, safeguard and proper functioning of immune system. Ruin, an important bioflavonoid, is abundantly found in various foodstuffs. Rutin has been acknowledged for its protective and beneficial effects on various aspects of the biological system. The present study was aimed to examine the effect of rutin on the regulation of the immune response in experimental animal models. Effect of rutin of cellular immunity was determined by delayed-type hypersensitivity (DTH) response, carbon clearance assay, leucocyte mobilization test, and cyclophosphamide-induced myelosuppression, whereas humoral immunity was analyzed by the haemagglutinating antibody (HA) titre assay. Rutin (25, 50 and 100 mg/kg, p.o.) evoked a significant increase in antibody titre in the haemagglutination test, increased immunoglobulin levels, and enhanced the delayed type hypersensitivity reaction induced by sheep red blood cells. It also significantly restored the functioning of leucocytes in cyclophosphamide treated rats and augmented phagocytic index in the carbon clearance assay. The outcomes from the present study indicate that rutin possesses sufficient potential for increasing immune activity by cellular and humoral mediated mechanisms.

Cisplatin along with herbal drug treatment reduces the percentage of regulatory T cells and decreased the severity of experimental visceral leishmaniasis.

Visceral leishmaniasis is the most alarming and devastating amongst the various forms of leishmaniases. It is caused by Leishmania donovani, an obligate intracellular parasite of macrophages that survives through immunosuppression. Absence of T regulatory cells provides complete clearance of the parasite. A few immunoprophylactics have been sought to battle instinctive leishmaniasis, with fluctuating achievement. Our previous studies have shown that treatment of L. donovani infected mice with cisplatin along with herbal drugs resulted in decreased parasite load with heightened delayed type hypersensitivity responses (DTH), increased levels of IgG2a, IFN-γ, IL-2, CD4+ cells, NK 1.1cells over that of IgG1, IL-4, 1L-10, CD8+ and CD19 in infected mice. Along the above lines, the present study further evaluated the percentage of CD4+ CD25+FoxP3+ T regulatory cells and ultra structural changes in kidney, liver and spleen. Cisplatin (5mg/kg b.wt. daily for 5 days, i.p.) along with Tinosporacordifolia (100mg/kg b.wt. daily for 15 days, p.o.) or Withaniasomnifera (350mg/kg b.wt. daily for 15 days, p.o.) or Asparagusracemosus (650mg/kg b.wt. daily for 15 days, p.o.) was administered to L. donovani infected BALB/c and after 30 days post treatment mice were sacrificed. The findings uncover a significant reduction in parasite load coupled with decreased percentage of Treg cells and no pathological changes at ultra structural level. In this manner, results acquired recommend that the decrease in percentage of T reg cells may further help the antileishmanial remedial impact of cisplatin alongside natural medications.

Diagnosis of Metal Hypersensitivity in Orthopedics

The pathology of early aseptic implant failure has been linked in basic science, case, and group studies to metal implant debris–induced delayed-type hypersensitivity (DTH) responses (or metal allergy). However, a precondition of metal DTH does not always result in implant failure. The methods of diagnosing metal DTH responses measure cell-mediated adaptive immunity, where conditioned lymphocytes respond to specific stimuli. Metal-sensitivity testing is a direct test of an individual’s immune response to metal challenge, which has been in use for over the past half century to measure delayed-type responses to drugs (such as antibiotics) where the results indicate levels of immune reactivity. Currently approved methods for human diagnostic testing for metal DTH include both skin testing (patch testing) and in vitro blood testing using lymphocyte transformation testing (LTT). LTT testing may be better suited for diagnosing implant-related sensitivity, given the ability of dermal exposure to metals to induce DTH responses. Metal sensitivity among the general population has been well established with a rate of 10%-15%, where hypersensitivity to nickel is the most common. However, it remains unknown about the exact percentage of these presensitized people that will go on to develop pathogenic metal-sensitivity responses to their implant. Recent studies show that when exposure to metal implant debris is minimized, the chances of metal DTH reactivity are also minimized. Further, past basic science studies, case studies, and cohort studies indicate the clinical utility of metal-sensitivity testing in specific patient populations. Continuing clinical outcome studies are needed to relate positive patch and metal-LTT test results with other clinical determinants to more definitively predict and prevent pathologic reactivity to implants.

Exosomes from B lymphocytes are a major source of Fas ligand

Abstract Exosomes are nanovesicles released by a variety of cell types, including antigen-presenting cells. The ability of exosomes to induce immunity has prompted great interest in their clinical use. Murine studies have also indicated that circulating FasL+MHCII+ exosomes can be tolerogenic in allergic and delayed-type hypersensitivity responses. B cells are capable of expressing FasL and are a major in vivo source of exosomes, but their role in producing tolerogenic exosomes is incompletely explored. In the current study, we evaluated B cells as a source of FasL+ exosomes in vivo and signals that induced B cells to release FasL+ exosomes in vitro. In B-cell deficient mice, a major loss in total splenic exosomes and exosome-associated FasL was observed, suggesting B cells are the major source of FasL+ exosomes in vivo. After sorting B cells into CD107a+, CD5+ or CD9+ cells, all subsets showed some intracellular storage of FasL, yet FasL was predominately found in the CD9+ subset. Searching for signals that induced FasL+ exosomes in vitro, we stimulated B cell signaling through Toll-like receptors (TLRs), the B-cell receptor (BCR), and/or CD40. In response to BCR ligation, B cells produced 20% more FasL compared to medium controls, which increased to 50% in the presence of any TLR ligand. Interestingly, B cells increased their intracellular FasL storage, but failed to increase their FasL+ exosome release when stimulated through CD40 alone. TLR-4, TLR-7(/8) or TLR-9 ligation with CD40 activation induced the FasL+ exosome release, which was further enhanced by BCR ligation. These data suggest that signals known to induce the differentiation of IL-10-producing B cells, also enhance FasL+ exosome production as another potential suppressive mechanism.

Effects Of Acute Eccentric Exercise On Primary Antibody Responses To Ovalbumin Vaccination In Aged Mice.

Several studies have suggested that acute eccentric exercise can enhance vaccination responses in humans. However, the underlying mechanisms have remained understudied. We have shown that acute eccentric exercise does not improve the antibody responses to ovalbumin (OVA) vaccination in young mice. Thus, we replicated the study in an immunosenescence animal model, where there is reduced vaccine efficacy. PURPOSE: To determine the effects of acute eccentric exercise on the primary antibody response to vaccination in aged mice. METHODS: C57BL/6 male mice, aged 27 months (n=16) were randomized into either eccentric exercise (ECC, n=8) or sedentary (SED, n=8) groups. For the ECC group, mice were exercised at 17m/min at -20% grade for 45 min on a treadmill. SED mice remained in their home cages. All mice were inoculated in the gastrocnemius with 25 μg of OVA and 200 μg aluminum hydroxide (a suboptimal dosage based on titration experiments) in 50 μl sterile saline immediately after the exercise. Blood was collected prior to, and one, two and four weeks after vaccination. ELISA was performed to analyze anti-OVA IgG. At three weeks post exercise, all mice were injected with 100 μg OVA dissolved in 10 μl PBS into the dorsal side of the right ear to determine the delayed-type hypersensitivity (DTH) response. The left ear received 10 μl PBS alone as a control. Ear thickness was measured immediately before, 24 h and 48 h after intradermal injection. The measurements were performed in triplicate, where researchers were blinded to treatment. Maximum ear swelling occurred at 24 hours post injection. Results were expressed as the difference between the right and left ear thickness. RESULTS: We found a significant time main effect (p<0.001) indicating a significant increase in anti-OVA IgG at one, two and four weeks relative to pre-immunization. However, there were no significant time x treatment (p=0.652) nor treatment main effects (p=0.764). There was a significant difference between ECC and SED groups in ear DTH at 24 h post injection (p = 0.028), indicating eccentric exercise increased the DTH response. CONCLUSION: Acute eccentric exercise immediately before vaccination improved the DTH (i.e. cell-mediated immune), but not the antibody, response to vaccination in aged mice.

Induction of experimental autoimmune orchitis by immunization with xenogenic testicular germ cells in mice

We previously showed that immunization of mice with syngeneic or allogeneic testicular germ cells (TGC) alone induces autoimmune inflammation in the testes without using any adjuvant. In the present study, we examined testicular autoimmune response against xenogenic TGC antigens in mice. The mice were immunized with murine, rat or guinea pig TGC and then the histopathology, delayed type hypersensitivity (DTH) response and humoral autoimmunity were investigated. The results showed that immunization with not only murine but also rat TGC caused experimental autoimmune orchitis (EAO) with hypospermatogenesis in mice, while that with guinea pig TGC could not. The DTH response to murine TGC was significantly elevated in mice that had been immunized with murine or rat but not guinea pig TGC. Serum autoantibody to murine TGC was immunohistochemically detected in the mice immunized with either murine, rat or guinea pig TGC, however, the level of autoantibody detected by ELISA revealed significant elevation when mice were immunized with murine and rat TGC. With the immunoblotting after electrophoresis, the murine TGC proteins at molecular masses around 55kDa and 70kDa can be detected when incubated with sera from m-TGC and r-TGC groups. These results represent the cross-reactivity among TGC of the mouse, the rat and the guinea pig at the levels of humoral immunity and also demonstrate that the rat TGC could elicit significant DTH response to murine TGC with the resultant EAO. This is the first to succeed in EAO induction by the use of xenogenic TGC.

Nadroparin carries a potentially high risk of inducing cutaneous delayed-type hypersensitivity responses.

Heparins are widely used for the prophylaxis/treatment of thromboembolic events. As adverse effects, heparin-induced skin lesions occur frequently (in 7.5-39% of patients). Skin lesions may be the only clinical manifestation of life-threatening immune-mediated heparin-induced thrombocytopenia, but are commonly caused by a delayed-type hypersensitivity response [heparin-induced delayed-type hypersensitivity (HIHS)]. Risk factors have not been prospectively identified. To identify possible risk factors for heparin-induced skin lesions from three independent clinical trials in a combined analysis. A pooled analysis from prospective studies was performed, and possible risk factors were included in a multiple logistic regression analysis. Obesity (body mass index of > 25), prolonged anticoagulant therapy, prior heparin exposure and younger age (< 55 years) were confirmed as independent risk factors for HIHS. The choice of anticoagulant preparation had the greatest influence. On comparison of dalteparin, enoxaparin, fondaparinux, unfractionated heparin, and nadroparin, the latter was associated with the highest risk of eliciting HIHS (odds ratio of 30.2, 95%CI: 11.7-77.9). The high risk associated with nadroparin has been validated in controlled trials, and this emphasizes the singularity of each heparin preparation in terms of allergenicity and that individualized anticoagulation is required.

Structure of SALO, a leishmaniasis vaccine candidate from the sand fly Lutzomyia longipalpis.

BackgroundImmunity to the sand fly salivary protein SALO (Salivary Anticomplement of Lutzomyia longipalpis) protected hamsters against Leishmania infantum and L. braziliensis infection and, more recently, a vaccine combination of a genetically modified Leishmania with SALO conferred strong protection against L. donovani infection. Because of the importance of SALO as a potential component of a leishmaniasis vaccine, a plan to produce this recombinant protein for future scale manufacturing as well as knowledge of its structural characteristics are needed to move SALO forward for the clinical path.Methodology/Principal findingsRecombinant SALO was expressed as a soluble secreted protein using Pichia pastoris, rSALO(P), with yields of 1g/L and >99% purity as assessed by SEC-MALS and SDS-PAGE. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-Kalpha to 1.94 Å and refined to Rfactor 17%. SALO is ~80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. SALO has three predicted human CD4+ T cell epitopes on surface exposed helices.Conclusions/SignificanceThe results indicate that SALO as expressed and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing. SALO has a novel structure, is not similar to any human proteins, is immunogenic in rodents, and does not have the anti-complement activity observed in the native salivary protein which are all important attributes to move this vaccine candidate forward to the clinical path.

Phase 1 trial of OCV-C02, a peptide vaccine for metastatic colorectal cancer patients refractory to all standard chemotherapies.

706 Background: OCV-C02 is a peptide vaccine consisting of 2 peptide epitopes derived from ring finger protein 43 and translocase of outer mitochondrial membrane 34. In colorectal cancer (CRC), they are upregulated in approximately 80% of tumor tissues. This study was conducted to assess safety, preliminary efficacy and immunological responses to OCV-C02. Methods: Key eligibility criteria for this open label, sequential cohort, dose escalation study were patients (pts) with unresectable metastatic CRC (mCRC) refractory to all standard chemotherapies and HLA-A*24:02. Pts in cohorts 1 to 4 received OCV-C02 0.3, 1, 3 and 6 mg/body respectively, subcutaneously, on days 1, 8, 15 and 22 of the 28-day treatment cycle. Cycle was repeated in pts without progressive disease, until occurrence of unacceptable toxicity or disease progression. Primary endpoint was dose limiting toxicity (DLT) that was defined as treatment-related ≥ grade 3 or 4 hematological or ≥ grade 3 non-hematological adverse events (AEs) observed from days 1 to 29 of Cycle 1. Secondary endpoints were treatment emergent AEs (TEAEs), efficacy and immunological responses. Efficacy was evaluated by overall response rate, disease control rate (DCR), time to treatment failure (TTF) and overall survival (OS). Immunological responses were evaluated by cytotoxic T lymphocytes (CTL) and delayed-type hypersensitivity (DTH). Results: A total of 24 pts were treated. No DLT occurred in cycle 1 and no major safety issues throughout the trial. All pts had ≥ 1 TEAE. Frequently reported TEAEs included injection site reaction (25%), vomiting (25%), decreased appetite (20.8%) and pyrexia (16.7%). More than 50% of TEAEs were of grade 1 or 2 in severity. None achieved complete or partial response. Six pts had stable disease and DCR were 16.7%, 50% and 33.3% for cohorts 2, 3 and 4, respectively. TTF was 1.8, 0.9, 2.3 and 1.6 months and OS was 7.9, 11.6, 8.4 and 7.4 months for cohorts 1, 2, 3 and 4, respectively. CTL and DTH responses following vaccination were observed across the 4 cohorts. Conclusions: OCV-C02 at 0.3 to 6 mg/body exhibited a safe and well tolerated profile, and showed immunological responses in mCRC pts refractory to all standard chemotherapies. Clinical trial information: NCT01801930.

Short day length enhances physiological resilience of the immune system against 2-deoxy-d-glucose-induced metabolic stress in a tropical seasonal breeder Funambulus pennanti

Studies demonstrate the importance of metabolic resources in the regulation of reproduction and immune functions in seasonal breeders. In this regard, the restricted energy availability can be considered as an environmental variable that may act as a seasonal stressor and can lead to compromised immune functions. The present study explored the effect of photoperiodic variation in the regulation of immune function under metabolic stress condition. The T-cell-dependent immune response in a tropical seasonal breeder Funambulus pennanti was studied following the inhibition of cellular glucose utilization with 2-deoxy-d-glucose (2-DG). 2-DG treatment resulted in the suppression of general (e.g., proliferative response of lymphocytes) and antigen-specific [anti-keyhole limpet hemocyanin IgG titer and delayed-type hypersensitivity response] T-cell responses with an activation of the hypothalamic–pituitary–adrenal axis, which was evident from the increased levels of plasma corticosterone. 2-DG administration increased the production of inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α] and decreased the autocrine T-cell growth factor IL-2. The immunocompromising effect of 2-DG administration was retarded in animals exposed to short photoperiods compared with the control and long photoperiod-exposed groups. This finding suggested that short photoperiodic conditions enhanced the resilience of the immune system, possibly by diverting metabolic resources from the reproductive organs toward the immune system. In addition, melatonin may have facilitated the energy “trade-off” between reproductive and immune mechanisms, thereby providing an advantage to the seasonal breeders for their survival during stressful environmental conditions.

St20, a new venomous animal derived natural peptide with immunosuppressive and anti-inflammatory activities

Peptide toxins from venomous animals are natural resources with diverse biological functions and therapeutic potential towards human diseases. For venomous scorpions, many valuable peptide toxins have been discovered from Buthidae scorpions, but few works were done about non-buthidae scorpions. Here, we cloned and characterized the first disulfide-bridged toxin peptide St20 from the non-buthidae scorpion Scorpiops tibetanus. St20 has a putative 23-residue signal peptide, followed by a presumed 34-residue mature peptide including 8 cysteines. Sequence alignments and structural analysis suggested that St20 is a new member of α-KTx23 scorpion toxin subfamily with a conserved CSα/β structural fold. Functional studies showed that St20 inhibited human T lymphocyte surface marker CD69 expression and cytokine IL-2 secretion. Beside this, St20 inhibited two important pro-inflammatory factors TNF-α and IFN-γ secretion in the activated human T lymphocyte. Animal experiments showed that the delayed-type hypersensitivity response in rat autoimmune disease model was ameliorated in the present of peptide toxin St20. Together, our results showed that St20 is the first disulfide-bridged toxin peptide from the non-buthidae scorpion Scorpiops tibetanus with immunosuppressive and anti-inflammatory activities, suggesting that toxins from non-buthidae scorpions might be a new source of peptide drug discovery towards human diseases.

Validation of the Immunomodultory Potential of Amarkand Species

Amarkand plants are routinely used by Indian tribes for health and longevity. Taxonomically, Amarkand includes species from genera Eulophia and Dioscorea. Comparative immunomodulatory potential of Amarkand extracts were examined through biochemical, hematological, serological and histopathological analysis. A significant effect on the modulation of immune reactivity was observed in bulbils of Dioscorea bulbifera and tubers of Eulophia ochreata. A significant increase was also observed in relative organ weights, hematological parameters and total protein by E. ochreata (500 mg/kg) treatment compared to the negative control group. While aminotransferase enzyme levels have shown a significant decrease by the treatment of E. ochreata and D. bulbifera extracts, D. bulbifera (300 mg/kg) showed greater (61.7±3.62%) neutrophil adhesion and a significant increase in delayed type hypersensitivity response. E. ochreata treatment showed increased haemagglutination titre compared to negative control, thereby providing evidence that D. bulbifera and E. ochreata possessed immunomodulatory activity. Histopathology of rat organs treated with D. bulbifera and E. ochreata, showed normal cytoarchitecture and less neutrophil infiltration as compared to the negative control group, suggesting no evidence of pathological signs. Our findings demonstrated that E. ochreata and D. bulbifera have immunomodulatory potential and provide the basis for an alternative approach to investigate new traditional remedies.

Antithrombocytopenic and immunomodulatory potential of metabolically characterized aqueous extract of Carica papaya leaves

Context: Carica papaya Linn. (Caricaceae) leaf (CPL) juice has long been traditionally used in ethnomedicine for dengue fever. Objective: The study examines the effects of standardized CPL aqueous extract (SCPLE) on platelet count, extramedullary haematopoiesis (EMH), and immunomodulation in cyclophosphamide (CP)-induced animal model of thrombocytopenia. Materials and methods: The extract was analyzed for myricetin, caffeic acid, trans-ferulic acid, and kaempferol using HPTLC for standardization followed by UPLC-qTOF/MS fingerprinting for metabolite signature. The effects of SCPLE (50 and 150 mg/kg p.o.) on proliferative response of platelet count and total leucocyte count (TLC) were observed up to 14 days in Wistar rat. However, delayed-type hypersensitivity (DTH), haemagglutination titre (HT), and in vivo carbon clearance were examined as immunomodulatory parameters in albino mice at 150 mg/kg p.o. against CP. Results: The quantitative HPTLC estimation of SCPLE showed the presence of myricetin, caffeic acid, trans-ferulic acid, and kaempferol up to 280.16 ± 5.99, 370.18 ± 6.27, 1110.86 ± 2.97, and 160.53 ± 2.48 (μg/g), respectively. Twenty-four metabolites were identified using UPLC-qTOF/MS. Oral administration of SCPLE (150 mg/kg) in thrombocytopenic rats exhibited significant (p < 0.01) increase in thrombocytes (1014.83 × 103 cells/mm3), DTH response (0.16 ± 0.004), and phagocytic index (63.15% increase) as compared to CP-induced thrombocytopenia group. Histopathological studies showed minimal fibrosis in spleen histology. Discussion and conclusions: Results suggest CPL can mediate the release of platelets providing the means for the treatment and prevention of dengue.

Toxicological Evaluation of the Aqueous Extract of Caralluma europaea and Its Immunomodulatory and Inflammatory Activities.

Background:Caralluma europaea (CE) has been studied for its chemical constituents, and no information is available on its toxicity or its pharmacological activities.Objective:To determine the toxicity of an aqueous extract of CE stems in vitro and in vivo after acute and subchronic oral gavages in Swiss albino's mice and its immunomodulatory and inflammatory activities.Materials and Methods:The extract was administrated in single oral dose at 5 g/kg body weight for the acute toxicity test and by gavages daily at doses of 1, 2.5, or 5 g/kg for 30 consecutive days for the subchronic toxicity test. The immunomodulatory activities and inflammatory activities were tested by the evaluation of hemagglutination antibodies (HAs) titers and delayed-type hypersensitivity (DTH) response.Results:For the dose of 1 g/kg, no visible toxic effects were observed. However, for the higher doses, clinical observations of toxicity were noted after 1 week of treatment. This was confirmed by the biochemical parameters values and the histology analyses of the spleen, liver, and kidney tissues. The high cellular mortality rate in vitro when treated with CE extract confirmed their toxicity potential. There was also increase of “HA titer” and “DTH” response in mice treated with nontoxic dose of CE (1 g/kg) compared to control group. This immune activity was confirmed by the high number of lymphocytes infiltrates noted in the different organs.Conclusion:We conclude that CE at the dose up of 1 g/kg produced toxic effect in mice that induced an immune inflammatory reaction.SUMMARY Caralluma europaea (CE) has been studied for its chemical constituents, and no information is available on its toxicity or its pharmacological activities. The objective is to determine the toxicity of an aqueous extract of CE stems in vitro and in vivo after acute and subchronic oral gavages in Swiss albino's mice and its immunomodulatory and inflammatory activities. For the dose of 1 g/kg, no visible toxic effects were observed. However, for the higher doses, clinical observations of toxicity were noted after 1 week of treatment. This was confirmed by the biochemical parameters values and the histology analyses of the spleen, liver, and kidney tissues. The high cellular mortality rate in vitro confirmed their toxicity potential. There was also increase of “hemagglutination antibody titer” and “delayed-type hypersensitivity” response in mice treated with nontoxic dose of CE (1 g/kg) compared to control group. This immune activity was confirmed by the high number of lymphocytes infiltrates noted in the different organs. We conclude that CE at the dose up of 1 g/kg produced toxic effect in mice that induced an immune inflammatory reaction. Abbreviations Used: CE: Caralluma europaea, ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, RRBCs: Rat red blood cells, DTH: Delayed-type hypersensitivity response, PBS: Phosphate buffer solution.

Effect of levamisole administration on immunogenic and protective capacity of Brucella abortus RB51

Background: Brucella abortus strain RB51 is a rough strain that was derived after multiple passages of virulent strain 2308. Levamisole, a potent anthelmintic that is widely used in veterinary medicine, is also known as an immune stimulant. Aim and Objectives: Determine the capability of levamisole to enhance the humoral antibody response and cell-mediated immunity as well as protective capacity of RB51 vaccine in mice against challenge with virulent Brucella melitensis strain. Materials and Methods: Six groups of 7–8-week-old female Bulb/C mice were used. Group I was vaccinated I/P with 5 × 108 colony-forming units (CFU) of RB51 strain. Group II was vaccinated I/P with 5 × 108 CFU of RB51 strain simultaneously injected (at 0 day) s/cu with 12.5 mg/Kg levamisole. Group III was vaccinated I/P with 5 × 108 CFU of RB51 strain and injected at 7-day postvaccination with 12.5 mg/Kg levamisole. Group IV was vaccinated I/P with 5 × 108 CFU of RB51 strain and injected at the day of vaccination (0 day) and 7-day postvaccination with 12.5 mg/Kg levamisole. Group V was injected with levamisole alone. Group VI was kept as control group. 8-week postvaccination; all vaccinated as well as control animals groups received I/P challenge of 2 ml dose of 2 × 104 CFU/ml of virulent strain of B. melitensis biovar 3. Results: (Groups III and IV) At the day of vaccination (0 day) and 7-day postvaccination showed the highest significant serum antibody titer measured by ELISA all over the experimental period as well as increase in delayed type hypersensitivity (DTH) response 48 h after elicitation by intradermal inoculation of B. abortus soluble antigen compared to vaccinated control group (Group I). While RB51 vaccinated and simultaneously treated with levamisole (Group II) showed mild elevation in antibody titer as well as the lowest significant increase in DTH response compared to vaccinated control group. Bioassay test of challenged mice groups showed significant improvement in the resistance of vaccinated groups treated with levamisole either 7-day post-vaccination (Group III) or 0 and 7 days postvaccination (Group IV) which showed 10% bacteriologically positive mice, whereas only 30% of the vaccinated control mice (Group I) and vaccinated simultaneously treated with levamisole (Group II), were bacteriologically positive.

Dinitrophenyl hapten with laser immunotherapy for advanced malignant melanoma: A clinical study.

The present study aimed to evaluate the efficacy and safety of in situ immunotherapy with dinitrophenyl (DNP) hapten in combination with laser therapy for patients with malignant melanoma (MM). Between February 2008 and March 2012, 72 patients with stage III or IV MM were enrolled. Patients received in situ DNP alone (n=32) or in combination with laser therapy (n=32), and each group received dacarbazine chemotherapy. The levels of peripheral cluster of differentiation (CD)4+CD25+ regulatory T cells (Tregs), interleukin (IL)-10 and tumor growth factor (TGF)-β were detected by ELISA. The association between delayed-type hypersensitivity (DTH) and survival time was evaluated. Although peripheral Treg levels significantly decreased over time in the two groups (P<0.001), there was no significant difference between the treatment groups (P=0.098). Patients receiving the combination treatment exhibited significantly higher interferon-γ production by CD8+ and CD4+ T cells (both P<0.001), as well as significantly reduced levels of IL-10, TGF-β1 and TGF-β2. In addition, patients in the combination treatment group experienced significantly longer overall survival (OS; P=0.024) and disease-free survival (DFS; P=0.007) times; a DTH response of ≥15 mm was also associated with increased OS time and DFS time (P≤0.001). Finally, no severe adverse events were observed in either treatment group. Overall, in situ immunization with DNP in combination with laser immunotherapy may activate focal T cells, producing a regional antitumor immune response that increases cell-mediated immunity and improves survival in MM patients. Thus, this may represent a novel therapeutic strategy for patients with unresectable, advanced MM.

Electro-acupuncture at Acupoint ST36 Ameliorates Inflammation and Regulates Th1/Th2 Balance in Delayed-Type Hypersensitivity.

Increasing evidence indicates anti-allergic and anti-inflammatory effects of electro-acupuncture (EA) therapy. However, its underlying mechanism on delayed-type hypersensitivity (DTH), a classic allergic inflammatory disease, still remains unclear. In this study, we aimed to explore the immunomodulatory mechanism of EA intervention in a mouse model of ovalbumin (OVA)-induced DTH. Mice were randomly divided into four groups: Control, OVA-DTH, DTH + EA, DTH + Sham. "Zusanli" acupoint (ST36) was used for DTH + EA, whereas a non-acupoint (localized 5mm below the "Zusanli" acupoint) was selected for DTH + Sham. Footpad thickness was checked, and the infiltration of inflammatory cells was estimated by hematoxylin and eosin staining. Levels of IgG and IgE in serum of different groups and inflammatory cytokines in the supernatants from homogenized footpads, including IFN-γ, TNF-α, IL-4, and IL-5, were determined by ELISA. Cell proliferation of spleen lymphocytes was assayed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). The frequency of CD4+IFN-γ+ and CD4+IL-4+ T cells was analyzed with flow cytometry. In addition, the mRNA and protein expression of T-bet and GATA-3 were evaluated by real-time PCR and Western blotting, respectively. Our data showed EA treatment at acupoint ST36 relieved the pathological progression of DTH responses via reduction in footpad swelling, infiltration of inflammatory cells, levels of IgG and IgE as well as decreased production of IFN-γ and TNF-α in homogenized footpad tissue. Moreover, detailed studies were performed revealing that EA attenuated the percentage of CD4+IFN-γ+ T cells and prevented Th cells differentiation into Th1 cells, and this results from inhibiting secretion of IFN-γ and suppressing expression of T-bet, an IFN-γ transcription factor. The results indicated that EA treatment improved Th1-mediated allergic skin inflammation via restoring Th1/Th2 balance by curbing Th1 differentiation. These findings suggested that EA at acupoint ST36 might be a useful and promising therapeutic for allergic inflammatory as well as Th1-mediated inflammation response.

A Key Role for Inhibins in Dendritic Cell Maturation and Function.

Inhibins are members of the TGFβ superfamily, which regulate many cellular processes including differentiation, proliferation, survival and apoptosis. Although initially described as hormones regulating the hypothalamus-pituitary-gonadal axis, based on their ability to antagonize Activins, our group has recently reported that they play a role in thymocyte differentiation and survival, as well as in thymic stromal cell maturation and nTreg generation. Here, we used Inhibin knock out mice (Inhα-/-) to investigate the role of Inhibins in peripheral dendritic cell maturation and function. We first demonstrated that LPS treated Inhα+/+ bone marrow derived dendritic cells (BMDC) were capable to produce significant levels of Inhibin A. Interestingly, Inhα-/- BMDC showed reduced MHCII and CD86 upregulation and increased PD-L1 expression in response to LPS compared to Inhα+/+, which correlated with reduced ability to induce proliferation of allogeneic T cells. The “semi-mature” phenotype displayed by Inhα-/- mBMDC correlated with increased levels of IL-10 and slightly decreased IL-6 production after LPS stimulation. In addition, Inhα-/- mBMDC showed impaired migration towards CCL19 and CCL21, assessed by in vitro chemotaxis and in vivo competitive homing experiments, despite their normal CCR7 expression. Furthermore, in vivo LPS-induced DC maturation was also diminished in Inhα-/- mice, specially within the LC (CD207+ CD11b+ CD103-) subpopulation. Finally, analysis of delayed type hypersensitivity responses in Inhα-/- mice, showed reduced ear swelling as a result of reduced cellular infiltration in the skin, correlating with impaired homing of CD207+ DCs to the draining lymph nodes. In summary, our data demonstrate for the first time that Inhibins play a key role in peripheral DC maturation and function, regulating the balance between immunity and tolerance.

Chickadees Faced with Unpredictable Food Increase Fat Reserves but Certain Components of Their Immune Function Decline

In winter, temperate resident birds are often faced with periodic low natural food availability. This reduction or unpredictability in resource availability might then have a negative impact on immune function, given that immune system support is highly resource dependent. We investigated the balance between energetic and immune management in captive black-capped chickadees (Poecile atricapilus) by manipulating the predictability of resources. The control group received food ad lib. every day, while the experimental group received a reduced amount of food on random days and food ad lib. on all other days. We measured two key metrics of energetic management (body and fat mass) as well as a suite of immune system components. Compared with control birds, experimental birds maintained significantly higher total body and fat mass, had lower acute phase protein concentrations, and had decreased body temperature and lost more body mass during the fever response following injection with lipopolysaccharides. Interestingly, birds in both groups had similar levels of complement lysis, delayed-type hypersensitivity response (phytohemagglutinin), and primary antibody production (keyhole limpet hemocyanin). This experiment demonstrates that black-capped chickadees strategically increase their fat mass in response to decreased food availability and that this might allow the birds to maintain most of the immune system unaltered, except some of the most costly immune components.