Delayed-type Hypersensitivity Response Research Articles

Immunostimulant activities of the aqueous methanolic extract of Leptadenia pyrotechnica, a plant from Cholistan desert

Ethnopharmacological relevanceLeptadenia pyrotechnica (Forssk.) Decne (Asclepiadaceae) is a well-renowned medicinal shrub, used by herbal practitioners for various ailments including allergic rhinitis, productive cough, abortion, diabetes, stomach disorders, fever, kidney disorders, stones and cancer Aim of the studyOn the basis of folkloric uses, L. pyrotechnica was selected from the wide medicinal flora of the Cholistan desert of Pakistan, for the exploration of immunomodulatory potential. Materials and methodsAqueous methanolic (30:70) extract of aerial parts of L. pyrotechnica (Lp. Cr) was prepared by 3 days thrice maceration and subsequent evaporation under reduced pressure. In-vivo experiments were performed in Wistar albino rats including neutrophil adhesion test, haemagglutinating antibody (HA) titre, delayed-type hypersensitivity response, phagocytic activity and cyclophosphamide-induced myelosuppression. ResultsLp. Cr produced a significant increase in phagocytic index in dose-dependent fashion (3.56, 4.18 and 5.42 at 30, 100 and 300mg/kg, respectively) as well as an augmented response in the delayed-type hypersensitivity reaction and HA titre induced by sheep erythrocytes. Lp. Cr also showed improved adhesion of neutrophils with nylon pellets (15.28, 27.85 and 38.42% at the doses of 30, 100 and 300mg/kg) and prevented myelosuppression in cyclophosphamide-treated rats as evidenced from the hematological parameters. The results of treatment were compared with normal and standard control groups throughout the study and the effects by Lp. Cr (300mg/kg) were found to be comparable with Levamisole. ConclusionsThe results indicated that L. pyrotechnica possesses immunostimulatory activity and justify its traditional use for the control and management of diseases in which the immune system needs to be stimulated like infectious diseases.

Comparative evaluation of organic zinc supplementation as proteinate with inorganic zinc in buffalo heifers on health and immunity

Graded Murrah buffalo heifers (18) were randomly allotted to 3 dietary groups varying in source and level of Zn supplementation in concentrate mixture to study the effect of organic (O) Zn (Zn proteinate; Zn-prot) supplementation (80 or 140 ppm) compared to inorganic Zn (I) (ZnSO4) (140 ppm) on serum biochemical parameters, antioxidant status and ovarian folliculogenesis. Mineral and biochemical constituents in serum and antioxidant enzyme activities in haemolysate were measured on 90th d of experiment. Antibody titres (log2) against Brucella abortus S19 and chicken RBC antigens was measured in serum at 7, 14, 21 and 28th d post sensitization (humoral immunity) and cell mediated immunity was assessed (120 d) by in-vivo delayed type hypersensitivity (DTH) against phytohemagglutinin-P (PHA-P). After 60 days of feeding, ovarian folliculogenesis study was made daily with ultrasound scanner in all the animals for next 60 days. Highest Zn concentration in serum without affecting the retention of other minerals (Cu, Mn and Fe) was observed with 140 ppm Zn supplementation as Zn-prot and mineral concentrations was comparable between 80 ppm Zn as Zn-prot and 140 ppm Zn as ZnSO4. Alkaline phosphatase, total protein, globulin, and glucose concentrations in serum increased with organic Zn supplementation. Organic Zn lowered lipid peroxidation (140O<80O<140I) and improved RBC catalase, glutathione peroxidase and superoxide dismutase (140O>80O>140I) activities. Antibody titres against B. abortus and chicken RBC and in vivo DTH response improved with organic Zn supplementation. Similarly, irrespective of the dose, organic Zn supplementation significantly increased the number of large follicle with greater follicular size in ovary. The study indicated that 140 ppm Zn supplementation as Zn-prot resulted in better antioxidant status, immune response and folliculogenesis in ovaries than inorganic source and the Zn supplementation could be reduced from 140 to 80 ppm as Zn-prot without any adverse effect in buffalo heifers.

Schistosoma japonicum HSP60-derived peptide SJMHE1 suppresses delayed-type hypersensitivity in a murine model.

BackgroundParasite-derived molecules with immunomodulatory properties, which have been optimised during host-parasite co-evolution, exhibit potential applications as novel immunotherapeutics. We have previously demonstrated that Schistosoma japonicum HSP60-derived peptide SJMHE1 induces CD4+CD25+ regulatory T-cells (Tregs) and that adoptively transferred SJMHE1-induced CD4+CD25+ Tregs inhibit delayed-type hypersensitivity (DTH) in mice. However, multiple concerns regarding this method render this treatment unsuitable. To gain further insights into the potential effects of SJMHE1, we used ovalbumin (OVA)-induced DTH and evaluated the effect of SJMHE1 on DTH mice.MethodsBALB/c mice were sensitised with OVA alone or combined with SJMHE1 and then challenged with OVA to induce DTH. We first analysed the potential effects of SJMHE1 by measuring DTH responses, T-cell responses, cytokine secretion, and Treg proportions. We then evaluated the expression levels of IL-10 and TGF-β1 in CD4+CD25+ T-cells during DTH and Treg generation to identify the mechanism by which SJMHE1 suppresses DTH.ResultsSJMHE1 modulated the effector response against OVA-induced DTH and stimulated the production of the anti-inflammatory cytokines IL-10 and TGF-β1 in immunised mice through a mechanism involving CD4+CD25+ Tregs. SJMHE1-induced CD4+CD25+ Tregs expressed high levels of CTLA-4, IL-10, and TGF-β1, which substantially contributed to the suppressive activity during DTH. The administration of SJMHE1 to DTH in mice led to the expansion of CD4+CD25+ Tregs from CD4+CD25− T-cells in the periphery, which inhibited DTH responses.ConclusionsOur study proves that the parasite-driven peptide suppresses DTH in mice, which may confer a new option for inflammation treatment.

Chronic Gastrointestinal Nematode Infection Mutes Immune Responses to Mycobacterial Infection Distal to the Gut.

Helminth infections have been suggested to impair the development and outcome of Th1 responses to vaccines and intracellular microorganisms. However, there are limited data regarding the ability of intestinal nematodes to modulate Th1 responses at sites distal to the gut. In this study, we have investigated the effect of the intestinal nematode Heligmosomoides polygyrus bakeri on Th1 responses to Mycobacterium bovis bacillus Calmette-Guérin (BCG). We found that H. polygyrus infection localized to the gut can mute BCG-specific CD4(+) T cell priming in both the spleen and skin-draining lymph nodes. Furthermore, H. polygyrus infection reduced the magnitude of delayed-type hypersensitivity (DTH) to PPD in the skin. Consequently, H. polygyrus-infected mice challenged with BCG had a higher mycobacterial load in the liver compared with worm-free mice. The excretory-secretory product from H. polygyrus (HES) was found to dampen IFN-γ production by mycobacteria-specific CD4(+) T cells. This inhibition was dependent on the TGF-βR signaling activity of HES, suggesting that TGF-β signaling plays a role in the impaired Th1 responses observed coinfection with worms. Similar to results with mycobacteria, H. polygyrus-infected mice displayed an increase in skin parasite load upon secondary infection with Leishmania major as well as a reduction in DTH responses to Leishmania Ag. We show that a nematode confined to the gut can mute T cell responses to mycobacteria and impair control of secondary infections distal to the gut. The ability of intestinal helminths to reduce DTH responses may have clinical implications for the use of skin test-based diagnosis of microbial infections.

Exosomes released by granulocytic myeloid-derived suppressor cells attenuate DSS-induced colitis in mice

Myeloid-derived suppressor cells (MDSC) have been described in inflammatory bowel disease (IBD), but their role in the disease remains controversial. We sought to define the effect of granulocytic MDSC-derived exosomes (G-MDSC exo) in dextran sulphate sodium (DSS)-induced murine colitis. G-MDSC exo-treated mice showed greater resistance to colitis, as reflected by lower disease activity index, decreased inflammatory cell infiltration damage. There was a decrease in the proportion of Th1 cells and an increase in the proportion of regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) from G-MDSC exo-treated colitis mice. Moreover, lower serum levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected in G-MDSC exo-treated colitis mice. Interestingly, inhibition of arginase (Arg)-1 activity in G-MDSC exo partially abrogated the spontaneous improvement of colitis. In addition, G-MDSC exo could suppress CD4+ T cell proliferation and IFN-γ secretion in vitro and inhibit the delayed-type hypersensitivity (DTH) response, and these abilities were associated with Arg-1 activity. Moreover, G-MDSC exo promoted the expansion of Tregs in vitro. Taken together, these results suggest that G-MDSC exo attenuate DSS-induced colitis through inhibiting Th1 cells proliferation and promoting Tregs expansion.

Immunobiological effects of gemcitabine and capecitabine combination chemotherapy in advanced pancreatic ductal adenocarcinoma.

Background:Preclinical studies suggest that chemotherapy may enhance the immune response against pancreatic cancer.Methods:The levels of granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) and the associated inflammatory marker C-reactive protein (CRP) were assessed in 38 patients receiving gemcitabine and capecitabine combination chemotherapy for advanced pancreatic cancer within the TeloVac trial. Apoptosis (M30) and total immune response (delayed-type hypersensitivity and/or T-cell response) were also assessed and levels of apoptosis induction correlated with immune response. The telomerase GV1001 vaccine was given either sequentially (n=18) or concomitantly (n=24) with the combination chemotherapy.Results:There were no differences between baseline and post-treatment levels of CRP (P=0.19), IL-6 (P=0.19) and GM-CSF (P=0.71). There was a positive correlation between post-chemotherapy CRP and IL-6 levels (r=0.45, P=0.005) and between CRP with carbohydrate antigen-19-9 (CA19-9) levels at baseline (r=0.45, P=0.015) and post treatment (r=0.40, P=0.015). The change in CRP and IL-6 levels was positively correlated (r=0.40, P=0.012). Hazard ratios (95% CI) for baseline CA19-9 (1.30 (1.07–1.59), P=0.009) and CRP (1.55 (1.00–2.39), P=0.049) levels were each independently predictive of survival. The M30 mean matched differences between pre- and post-chemotherapy showed evidence of apoptosis in both the sequential (P=0.058) and concurrent (P=0.0018) chemoimmunotherapy arms. Respectively, 5 of 10 and 9 of 20 patients had a positive immune response but there was no association with apoptosis.Conclusions:Combination gemcitabine and capecitabine chemotherapy did not affect circulating levels of GM-CSF, IL-6 and CRP. Chemotherapy-induced apoptosis was not associated with the immunogenicity induced by the GV1001 vaccine in advanced pancreatic cancer.

Dietary Indoles Suppress Delayed-Type Hypersensitivity by Inducing a Switch from Proinflammatory Th17 Cells to Anti-Inflammatory Regulatory T Cells through Regulation of MicroRNA.

Aryl hydrocarbon receptor (AhR) has been shown to have profound influence on T cell differentiation, and use of distinct AhR ligands has shown that whereas some ligands induce regulatory T cells (Tregs), others induce Th17 cells. In the present study, we tested the ability of dietary AhR ligands (indole-3-carbinol [I3C] and 3,3'-diindolylmethane [DIM]) and an endogenous AhR ligand, 6-formylindolo(3,2-b)carbazole (FICZ), on the differentiation and functions of Tregs and Th17 cells. Treatment of C57BL/6 mice with indoles (I3C or DIM) attenuated delayed-type hypersensitivity (DTH) response to methylated BSA and generation of Th17 cells while promoting Tregs. In contrast, FICZ exacerbated the DTH response and promoted Th17 cells. Indoles decreased the induction of IL-17 but promoted IL-10 and Foxp3 expression. Also, indoles caused reciprocal induction of Tregs and Th17 cells only in wild-type (AhR(+/+)) but not in AhR knockout (AhR(-/-)) mice. Upon analysis of microRNA (miR) profile in draining lymph nodes of mice with DTH, treatment with I3C and DIM decreased the expression of several miRs (miR-31, miR-219, and miR-490) that targeted Foxp3, whereas it increased the expression of miR-495 and miR-1192 that were specific to IL-17. Interestingly, treatment with FICZ had precisely the opposite effects on these miRs. Transfection studies using mature miR mimics of miR-490 and miR-1192 that target Foxp3 and IL-17, respectively, or scrambled miR (mock) or inhibitors confirmed that these miRs specifically targeted Foxp3 and IL-17 genes. Our studies demonstrate, to our knowledge for the first time, that the ability of AhR ligands to regulate the differentiation of Tregs versus Th17 cells may depend on miR signature profile.

Immunomodulation Potential of Andrographis Paniculata and Tinospora Cordifolia Methanolic Extracts in Combination Forms

Objective: This work mainly focuses on the immunomodulatory effect of different drug combinations (DC-I to DC-V) of methanolic extracts of aerial parts of Andrographis paniculata (AP) and mature stems of Tinospora cordifolia (TC).Materials and Methods: The coarse powder (40-mesh) of shade dried aerial parts of AP and mature stems of TC (500g each) was subjected separately tosuccessive extraction with 1000ml each of petroleum ether (60-80oC) followed by methanol. The doses of DC-I to DC-V were selected and were administered orally at doses of 200 mg/kg body weight to Albino Wistar rats and compared with the control group and standard drug i.e. cyclophosphamide (100mg/kg) respectively.Results: Results suggest that although all the five tested combinations (DC-I to DC-V) prepared using different proportion of methanol extracts of A. paniculata and T. cordifolia (100:Zero, 75:25, 50:50, 25:75 and Zero:100) showed a significant increase in macrophase count, neutrophil adhesion(NA), haemagglutinating antibody (HA) titre and delayed type hypersensitivity (DTH) response. In rats immunized with sheep RBC, DC-I to DC-V enhanced the humoral antibody response to the antigen and significantly potentiated the cellular immunity by facilitating the footpad thickness response to sheep RBC in sensitized rats on chosen experimental models.Conclusion: The values of macrophase, NA, HA, DTH and cellular responses of combination DC-II at a 200 mg/kg body weight were statistically significant as compared to other combinations. Combination DC-II exhibited best immunostimulant potential, indicating that it has promising immunomodulatory activity, as proposed from commercial point of view.

Activation of Cellular Immunity in Herpes Simplex Virus Type 1-Infected Mice by the Oral Administration of Aqueous Extract of Moringa oleifera Lam. Leaves.

Moringa oleifera Lam. is used as a nutritive vegetable and spice. Its ethanol extract has been previously shown to be significantly effective in alleviating herpetic skin lesions in mice. In this study, we evaluated the alleviation by the aqueous extract (AqMOL) and assessed the mode of its anti-herpetic action in a murine cutaneous herpes simplex virus type 1 (HSV-1) infection model. AqMOL (300 mg/kg) was administered orally to HSV-1-infected mice three times daily on days 0 to 5 after infection. AqMOL significantly limited the development of herpetic skin lesions and reduced virus titers in the brain on day 4 without toxicity. Delayed-type hypersensitivity (DTH) reaction to inactivated HSV-1 antigen was significantly stronger in infected mice administered AqMOL and AqMOL augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice at 4 days post-infection. AqMOL administration was effective in elevating the ratio of CD11b(+) and CD49b(+) subpopulations of splenocytes in infected mice. As DTH is a major host defense mechanism for intradermal HSV infection, augmentation of the DTH response by AqMOL may contribute to their efficacies against HSV-1 infection. These results provided an important insights into the mechanism by which AqMOL activates cellular immunity. Copyright © 2016 John Wiley & Sons, Ltd.

Mixture of Alum – Naloxone and Alum – Naltrexone as a novel adjuvant elicits immune responses for Toxoplasma gondii lysate antigen in BALB /c mice

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite. Treatment of the infection induced by this parasite is not straightforward due to the toxic side effects of the available drugs. Vaccine development could be a solution to this problem. In the present study, T.gondii Lysate Antigen (TLA), as a model vaccine, in combination with the Alum-NLT (Aluminum phosphate-Naltrexone) and Alum-NLX (Aluminum phosphate-Naloxone) were evaluated for immunization BALB/c. 147female BALB/c mice which were divided into seven groups of 21, were allocated to immunization experiments. The first group was selected as the negative control group, followed by the second, third, fourth, fifth, sixth and seventh groups which were immunized with Vac, Vac-Alum, Vac-NLX, Vac-NLT, Vac-Alum-NLX, Vac-Alum-NLT, respectively. Ten days after the final immunization, mice in all groups were divided into three groups for evaluating cellular immune responses, measuring the delayed-type hypersensitivity responses (DTHs) and evaluating survival. The DTH and cellular immune responses showed that in mice immunized with the TLA vaccine combined with the Alum-NLT mixture, the efficacy improved by increasing the production of Interleukin-5(IL-5) and Interferon gamma. This consequently shifted the immune responses toward a Th1 profile by increasing the IFN-γ/IL-5 ratios. In challenge experiments, immunized mice with the Alum-NLT-Vac mixture survived for a longer period of time which indicated an improvement in protective immunity against T. gondii. Administration of the Alum-NLT mixture adjuvant in combination with TLA vaccine enhanced the cellular immunity by shifting the immune response to a Th1 pattern. This shift to the Th1 pattern plays an important role in the induction of cellular.

Study of immunomodulatory activity of aqueous extract of Carica papaya in Wistar rats

Background: Various parts of Carica papaya (CP) Linn. have been traditionally used as ethnomedicine for a number of disorders including cancer. There have been anecdotes of patients with advanced cancers achieving remission following consumption of tea extract made from CP leaves. However, the precise cellular mechanism of action of CP tea extracts remains unclear. Aims and Objectives: The aim of this study is to study the immunomodulatory activity of aqueous extract of CP in Wistar rats. Materials and Methods: The effect of test extract and standard drug on the delayed type hypersensitivity response in Wistar rats using sheep red blood cells as antigen, administration of aqueous extract of CP at the dose of 400 and 800 mg/kg and levamisole 50 mg/kg, treatments which were given orally for 14 days. Results: We observed that aqueous extract of CP showed a significant increase in paw edema compared to control group. The standard drug levamisole showed the maximum increase in paw volume compared to all groups. Conclusion: The effect of this extract was comparable to the standard drug levamisole. CP leaf extract may potentially provide the means for the treatment and prevention of selected human diseases such as cancer, various allergic disorders, and may also serve as immunoadjuvant for vaccine therapy.

Abstract B146: Next-generation dendritic cell vaccination in postremission therapy of AML: Results of a clinical phase I trial

Abstract Postremission therapy for acute myeloid leukemia (AML) is critical for elimination of minimal residual disease (MRD). In patients not eligible for allogeneic stem cell transplantation, alternative treatment options are needed. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with leukemia-associated antigens (LAAs) is a promising treatment strategy to induce anti-leukemic immune responses and to eradicate chemorefractory cells. We have developed a GMP-compliant 3-day protocol including a TLR7/8 agonist to differentiate monocytes of intensively pretreated AML patients into next-generation DCs. A phase I proof-of-concept study (n=6) was completed using next-generation DCs as postremission therapy of AML patients with a non-favorable genetic risk profile in CR after intensive induction therapy (NCT01734304), and we have already started enrollment into a phase II trial building on these results. DCs are loaded with ivt-RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant and surrogate antigen. Patients are vaccinated intradermally with 5x106 DCs of each antigen species up to 10 times within 26 weeks. The primary endpoint of the phase I trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control. In total, 10 patients have been enrolled into the phase I/II trial. With two screening failures, DCs of sufficient number and quality were generated from leukapheresis in 7/8 cases. DC analysis demonstrated a positive costimulatory profile, secretion of IL-12p70, migration towards a chemokine gradient, antigen processing and presentation and activation of specific T cells in vitro. 5 patients have completed the vaccination schedule; the 6th patient has received 4/10 vaccinations. We observed delayed-type hypersensitivity (DTH) responses at the vaccination site in 5/5 patients, accompanied by slight erythema and indurations at the injection site, but no grade III/IV toxicities. Multimer analysis revealed the induction of antigen-specific T cell responses in 3/3 patients tested. We detected an increase of WT1-specific T cells in one patient and strong inductions of CMVpp65-specific T cells in two CMV-seronegative patients. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. In an individual treatment attempt, we treated one patient with impending relapse with a combination of DC vaccination and 5-azacytidine, resulting in MRD conversion. From the results of our phase I trial, we conclude that vaccination with next-generation DCs in AML is feasible and safe and induces leukemia-specific immune responses in vivo. The protocol is continued to be studied in an ongoing phase II trial. Citation Format: Felix S. Lichtenegger, Frauke M. Schnorfeil, Thomas Köhnke, Torben Altmann, Veit Bücklein, Andreas Moosmann, Monika Brüggemann, Beate Wagner, Wolfgang Hiddemann, Iris Bigalke, Gunnar Kvalheim, Marion Subklewe. Next-generation dendritic cell vaccination in postremission therapy of AML: Results of a clinical phase I trial. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B146.

Bixin uptake and antioxidative effect and role in immunoregulation in domestic cats.

Bixin, a carotenoid found in the seed of the Annatto plant, , is a potent antioxidant. Carotenoids are readily absorbed from the diet; therefore, the purpose of this study was to examine uptake of bixin by plasma, lipoproteins, and leukocytes after dietary supplementation in domestic cats and to assess effects on immune response. Female domestic short hair cats (3 yr old; 4.79 ± 0.13 kg BW) were fed a single dose of 0, 1, 5, or 10 mg bixin, and blood was taken at 0, 1, 2, 4 and 8 h after administration ( = 6/treatment) to determine acute absorption rate. Then, bixin was fed daily for 14 d to examine steady-state plasma concentrations and subcellular distribution. Following these preliminary experiments, cats ( = 8/treatment) were fed diets containing 0, 1, 5, or 10 mg bixin/d for 16 wk and blood was collected on wk 0, 6, 12, and 16 for analysis of leukocyte subpopulations, cell-mediated responsiveness, and inflammatory and oxidative biomarkers. Maximal uptake in plasma occurred 1 h after a single oral dose of bixin, with a maximal concentration of 0.119 μ and elimination half-life of 1.8 to 2.2 h. Daily feeding of bixin showed a steady-state plasma concentration of 0.110 μ at the greatest doses. Bixin was primarily associated with the high-density lipoprotein fraction of blood lipoproteins and was primarily distributed in mitochondrial fractions (58-59%) of but also in microsomal and nuclear fractions (37-44%). Leukocyte subpopulations in blood were variably affected by dietary bixin, with an increase ( < 0.05) in total T cells but a concurrent decrease ( < 0.05) in CD18+ and B cell subpopulations. However, plasma IgG increased ( < 0.05) in the 10-mg treatment group by wk 6. Lymphoproliferation was stimulated ( < 0.05) in the 5-mg bixin treatment group by wk 16, and delayed-type hypersensitivity response increased after nonspecific antigenic challenge. Conversely, when a specific challenge of vaccine was assessed on wk 12 and 16, responsiveness decreased ( < 0.05) in the 10-mg bixin treatment group. Bixin supplementation surprisingly caused an increase ( < 0.05) in α-acid glycoprotein but had no effect on natural killer cell activity, other subpopulations of leukocytes, or 8-oxo-2›-deoxyguanosine, a DNA damage biomarker. This experiment demonstrated dose-dependent uptake of bixin in plasma and blood lipoproteins and distribution in leukocyte subcellular components and an impacted immune response through cell-mediated and humoral actions.

Evaluation of safety of modified-Danggui Buxue Tang in rodents:immunological, toxicity and hormonal aspects

Ethnopharmacological relevanceRadix Astragali (RA), Radix Angelicae Sinensis (RAS) and Folium Epimedii (FE) are three of the extensively applied herbs among traditional Chinese medicines for gynecological disorders and osteoporosis. A derivative herbal formula—RRF, consisting of the three medicines with a weight ratio of 5:1:5, is derived from a famous Chinese herbal formula—Danggui Buxue Tang (DBT). RRF has shown noteworthy perimenopause ameliorating effect in both ovariectomized rats and natural aging female rats, which might represent a promising candidate for the treatment of perimenopausal disorders. The aim of this study was to evaluate its immunological potential, chronic toxicity and reproductive effects by 26-week repeated daily administration in female rats, in order to optimize its safe use. Materials and methodsThe effect of RRF on immunological function was studied by macrophage phagocytosis, immune organ index, serum immunoglobulin level as well as delayed type hypersensitivity (DTH) in mice. For toxicity assessment, acute toxicity study was performed according to fixed dose procedure with a single oral administration of RRF to mice. In the oral chronic toxicity, 120 female rats were administrated RRF orally in 0, 1100, 4400, or 8800mg/kg/day doses for 26 weeks. Clinical signs, mortality, body weights, feed consumption, haemato-biochemical parameters, organ weights, histopathology and reproductive hormone profiles were examined at the end of the 13- and 26-week dosing period, as well as after the 4-week recovery period. ResultsOral administration of RRF at three doses (282, 564 and 1128mg/kg) significantly increased the indices of phagocytosis K, as compared with prednisone acetate (PR) group (p<0.05 or 0.01). Exposure of RRF dose-dependently boosted circulating serum IgM level (all p<0.01) in response to CRBC in PR-induced mice. Furthermore, RRF treatment elicited a significant increment (all p<0.01) in DNFB-induced DTH response and the immune organ indices in a dose-dependent manner in mice, in parellel to DNFB-induced group. In the single dose acute toxicity and repeated dose 90-day chronic toxicity investigations, no toxic signs/mortality were observed. RRF treatment did not cause any toxicologically significant changes in clinical signs, food consumption, body weight, relative organ weight, hematological parameters, clinical chemistry, gross pathology and histopathology between treatment and control groups. No treatment related gross/histopathological lesions were observed and no target organ was identified. Long-term repeated administration of RRF exerted a significant promotion on serum level of steroid hormone estradiol, progesterone and testosterone release, along with decrease of circulating pituitary follicle stimulating hormone, luteinizing hormone, and prolactin levels in female rats. The No Observed Adverse Effect Level (NOAEL) of RRF was determined to be over 8800mg/kg/day for elderly female rats, a dose that was equivalent to 50 times of human dose. ConclusionThe present investigation demonstrated that RRF possessed appreciable immunopotentiating activity and had a relatively wide margin of safety. Long-term treatment of RRF exhibited estrogenic properties, and retarded certain age-associated degenerations. RRF might have the potential for further development as a safe and effective alternative/complementary to conventional medication in relieving perimenopausal symptoms.

Tyrosine Rich Fraction as an Immunomodulatory Agent from Ficus Religiosa Bark

Ficus religiosa Linn (Moraceae) is being used in traditional medicine to improve immunity hence, petroleum ether and 70% ethanol extracts (50 and 100 mg/kg, i.p.) of F. religiosa bark were screened for immunomodulatory activity by delayed type hypersensitivity (DTH), neutrophil adhesion test and cyclophosphamide-induced neutropenia in Swiss albino mice. 70% ethanol extract showed significant immunostimulant activity hence subjected to column chromatography to find out active compounds. Tyrosine rich fraction (TRF) obtained was screened for immunomodulatory activity by above methods at the dose of 10 mg/kg, i.p. TRF showed potentiation of DTH response in terms of significant increase in the mean difference in foot-pad thickness and it significantly increased neutrophil adhesion to nylon fibres by 48.20%. Percentage reduction in total leukocyte count and neutrophil by TRF was found to be 43.85% and 18.72%, respectively. Immunostimulant activity of TRF was more pronounced and thus it has great potential as a source for natural health products.

Next-Generation Dendritic Cell Vaccination in Postremission Therapy of AML: Results of a Clinical Phase I Trial

Postremission therapy for acute myeloid leukemia (AML) is critical for elimination of minimal residual disease (MRD). In patients not eligible for allogeneic stem cell transplantation, alternative treatment options are needed. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with leukemia-associated antigens (LAAs) is a promising treatment strategy to induce anti-leukemic immune responses and to eradicate chemorefractory cells. We have developed a GMP-compliant 3-day protocol including a TLR7/8 agonist to differentiate monocytes of intensively pretreated AML patients into next-generation DCs.A phase I/II proof-of-concept study has been initiated using next-generation DCs as postremission therapy of AML patients with a non-favorable genetic risk profile in CR after intensive induction therapy (NCT01734304). DCs are loaded with in vitro transcribed RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant and surrogate antigen. Patients are vaccinated intradermally with 5x106 DCs of each antigen species up to 10 times within 26 weeks. The primary endpoint of the phase I/II trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control.Based on the safety and toxicity profile of the phase I trial (n=6), phase II has been initiated. In total, 10 patients have been enrolled into the study. DCs of sufficient number and quality were generated from leukapheresis in 8/9 cases. DCs exhibited an immune-stimulatory profile based on high surface expression of positive costimulatory molecules, the capacity to secrete IL-12p70, the migration towards a chemokine gradient and processing and presentation of antigen. 5 patients have completed the vaccination schedule; the 6th and 7th patient have received 7/10 and 4/10 vaccinations, respectively. We observed delayed-type hypersensitivity (DTH) responses at the vaccination site in 6/6 patients, accompanied by slight erythema and indurations at the injection site, but no grade III/IV toxicities. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. Limited by HLA restriction, we have so far analyzed 4 patients by multimer staining. All of them mounted DC vaccination-specific T cell responses: We detected an increase of WT1-specific T cells in one patient and strong expansion/induction of CMVpp65-specific T cells in one CMV-seropositive and two CMV-seronegative patients. In an individual treatment attempt, an enrolled patient with impending relapse was treated with a combination of DC vaccination and 5-azacytidine, resulting in MRD conversion. Long-term disease control and immunological responses are studied in the ongoing phase II trial.We conclude that vaccination with next-generation LAA-expressing DCs in AML is feasible, safe and induces anti-leukemia-specific immune responses in vivo. DisclosuresSubklewe:AMGEN Research (Munich): Research Funding.

Immunization with Single Oral Dose of Alginate‐Encapsulated BCG Elicits Effective and Long‐Lasting Mucosal Immune Responses

Effective vaccination against pathogens, which enter the body through mucosal surfaces, requires the induction of both mucosal and systemic immune responses. Here, mucosal as well as systemic immune responses in the lung and spleen of BALB/c mice which were orally vaccinated with a single dose of alginate-encapsulated bacille Calmette-Guerin (BCG) were evaluated. Twenty weeks after immunization, the vaccinated mice were challenged intranasally with BCG. Twelve weeks after immunization and 5 weeks after challenge, the immune responses were evaluated. Moreover, immune responses were compared with those of mice that were vaccinated with free BCG by subcutaneous (sc) and oral routes. Twelve weeks after the immunization, serum IgG level was higher in the sc-immunized mice, while serum IgA level was higher in the orally immunized mice with encapsulated BCG. Significant productions of both IgG and IgA were only detected in lungs of mice orally immunized with encapsulated BCG. Proliferative and delayed-type hypersensitivity responses and IFN-γ production were significantly higher in mice immunized orally with encapsulated BCG, compared to mice immunized orally with free BCG. After challenge, the levels of IFN-γ were comparable between sc-immunized mice with free BCG and orally immunized with encapsulated BCG; however, significantly less IL-4 was detected in mice which had received encapsulated BCG via oral route. Moreover, significant control of the bacilli growth in the lung of the immunized mice after intranasal challenge with BCG was documented in mice vaccinated with encapsulated BCG. These results suggest that oral immunization with alginate-encapsulated BCG is an effective mean of inducing mucosal and systemic specific immune responses.

Clinical outcome of immunotherapy with dendritic cell vaccine and cytokine-induced killer cell therapy in hepatobiliary and pancreatic cancer

The aim of this study was to determine the therapeutic effects of adoptive immunotherapy following dendritic cell (DC) vaccine and cytokine-induced killer (CIK) cell therapy and evaluate its cytotoxicity, survival benefits and quality of life (QOL) changes in patients with hepatobiliary and pancreatic cancer (HPC). We performed a retrospective analysis of 407 clinical cases, including 77 patients with HPC who received immunotherapy with DC vaccine and CIK cells (I group) and 330 patients with similar characteristics who underwent baseline treatment but did not receive immunotherapy [non-immunotherapy (NI) group)] as the control group. After a follow-up period of 294±207.5 days, the median survival time (MST) of the two groups was compared using the Kaplan-Meier method. In the I group, 61% of the patients developed a positive, delayed-type hypersensitivity response and 65% of the patients exhibited an improvement in QOL. The most notable adverse events included fever (28%), insomnia (25%), anorexia (17%), skin rash (12%) and arthralgia (31%). No severe toxicities were observed in patients in the I group; in addition, the MST was significantly longer in the I group compared with that in the NI group (P=0.014). Thus, the DC vaccine and CIK cell therapy was associated with mild adverse effects, but was able to induce an immune response and effectively eliminate tumor cells, thereby improving the QOL and prolonging the MST of the patients.

Balanced TH1 and TH2 immunopotentiating effects of silicates partly containing nanoparticles present in calcined serpentine

Calcined Serpentine (CS) is used in various formulations of alternative systems of medicine as a tonic to vital organs and as an anti-inflammatory agent. The process of calcination or incineration is believed to render non-toxic, gently absorbable, adaptable and digestible properties to the mineral compounds. The present study characterized CS and also evaluated its immunostimulatory potential. CS was characterized by using transmission electron microscopy (TEM), X-ray powder diffraction, atomic absorption spectroscopy and CHNS analysis. The characterized CS was further evaluated for its immunomodulatory potential in Swiss mice. X-Ray diffraction analysis revealed that the CS contained silicates of magnesium, calcium and iron as major minerals. Elemental composition and heavy metal analyses showed a presence of various inorganic elements/heavy metals, albeit at levels well below daily permissive intake values. TEM analysis of the test CS revealed a presence of nano particles with an average size of 10–20 nm (≈ 26% of total material). Oral administration of CS to mice at 50, 75, 100 or 200 μg/kg body weight for 10 days led to enhanced levels of total IgG, IgG1, IgG2a and IgG2b in ovalbumin-immunized mice as well as ex vivo lymphocyte proliferation and levels of TH1 (IL-2, IFNγ) and TH2 (IL-4, IL-10) cytokines produced by their cultured splenocytes. Similarly, CS treatment resulted in enhanced delayed-type hypersensitivity responses in GRBC-primed hosts. CS also activated host peritoneal macrophages, as indicated by increases in phagocytic activity and in TLR-2, CD80 and CD86 expression. The CS did not affect liver, kidney and spleen histology. Taken together, the results indicated that absorbed CS was stimulatory of host cell-mediated immune responses. It is hypothesized for now that the immunomodulatory effect of CS may have been due, in part, to a presence of nanoparticles on the CS; further study is required to validate this viewpoint.

CORR Insights(®): do patients with a failed metal-on-metal hip implant with a pseudotumor present differences in their peripheral blood lymphocyte subpopulations?

T he study by Catelas and colleagues adds to the current scientific consensus that adaptive immune responses to metals occurs in a subset of all people with metal implants. The study authors also found that the incidence of pseudotumors correlates with systemic evidence of metal-related delayed-type hypersensitivity (DTH) responses and elevated metal ion levels. The current study identifies alterations in circulating adaptive immune cells in people with elevated levels of metal debris and pseudotumors. These findings support past histological and T-cell function studies, indicating pseudotumors are more prevalent in people with DTH-type immune reactivity. This highlights the growing recognition of these phenomena as central to implant performance in some individuals/implant designs. Case and group studies [4, 5] indicate that once locally activated, adaptive immune inflammation to metal contributes to (if not, mediates) the pathogenesis of poor implant performance. Metal DTH reactivity is a lymphocyte mediated response, similar to a Type IV DTH reaction. Metal-on-metal (MoM) hip failures have been associated with classic DTH responses including periimplant activated lymphocyte accumulations (aseptic lymphocytic vasculitisassociated lesions, [ALVAL]), and dermal and functional lymphocyte hypersensitivity responses to metals [2, 7, 11]. Elevated metal exposure can also cause different forms of toxicity responses in tissue, bone, and immune cells, including cobalt-induced hypoxia-like responses, postulated as causing pseudotumors [10]. Lymphocyte reactivity (metal DTH responses/ALVAL) and pseudotumors can be caused separately [6, 8]. Currently, there are no accepted thresholds for systemic metal ion levels that necessarily indicate for pseudotumor formation or immune reactions [1, 7]; it remains an increased risk association. This CORR Insights is a commentary on the article ‘‘Do Patients With a Failed Metal-onmetal Hip Implant With a Pseudotumor Present Differences in Their Peripheral Blood Lymphocyte Subpopulations?’’ by Catelas and colleagues available at: DOI: 10.1007/ s11999-015-4466-8. The author certifies that she, or any members of her immediate family, has no funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research editors and board members are on file with the publication and can be viewed on request. The opinions expressed are those of the writers and do not reflect the opinion or policy of CORR or The Association of Bone and Joint Surgeons. This CORR Insights comment refers to the article available at DOI: 10.1007/s11999015-4466-8. N. J. Hallab PhD (&) Department of Orthopedic Surgery, Rush University Medical Center, 1735 W Harrison, Cohn Research Bldg, #722, Chicago, IL 60612, USA e-mail: nhallab@rush.edu CORR Insights Published online: 13 October 2015 The Association of Bone and Joint Surgeons1 2015