Fermented Soy Drink (Q-CAN® PLUS) Induces Apoptosis and Reduces Viability of Cancer Cells.

This study tested the ability of a fermented soy product to induce tumor cell toxicity and to assess if this was due to fermentation of soy, and to the genistein content. Four cancer cell lines were cultured without additive, with fermented soy (Q-CAN® PLUS), nonfermented soy, or genistein, and cell viability was examined at 24 h, 48 h, and 72 h. The sensitivity of the cell lines to apoptosis by Q-CAN PLUS was tested with the Annexin V assay. All cell lines demonstrated a dose and time response reduction in tumor cell viability with exposure to Q-CAN PLUS (IC50 at 24 h 3.8 mg/mL to 9 mg/mL). Unfermented soy did not show reduction in viability of any cell line within the same concentration range. The IC50 of genistein for each of the cell lines was significantly greater than for Q-CAN PLUS. All four tumor cell lines demonstrated apoptosis in response to Q-CAN PLUS. Q-CAN PLUS reduces viability and increases apoptosis of cancer cells in a concentration- and fermentation-dependent manner. Taking into consideration the IC50 of genistein and the concentration of genistein in Q-CAN PLUS, the genistein content of Q-CAN PLUS is not responsible for the majority reduction in tumor cell viability. This suggests that fermentation of soy results in the production of metabolites that reduce cancer cell viability and induce cellular apoptosis, and play a major role in addition to any effects produced by their genistein content.

The fermented soy beverage Q-CAN® plus induces beneficial changes in the oral and intestinal microbiome.

BACKGROUND: Soy products are associated with many beneficial health consequences, but their effects on the human intestinal microbiome are poorly characterized. OBJECTIVES: To identify the changes in the oral and fecal microbiome in lean and obese participants due to consumption of Q-CAN®, and to assess the expected consequences of these changes based on the published literature. METHODS: Prospective study of lean (10) and obese (9) participants consuming Q-CAN® twice daily for 4 weeks with 8 weeks follow-up. Microbial DNA was extracted from saliva and stool samples, amplified against the V4 region of the 16S ribosomal RNA gene and data analyzed using QIIME 1.9.1 bioinformatics. Four hundred forty-four samples were collected in total, 424 of which were productive and yielded good quality data. RESULTS: STOOL. In the lean population Bifidobacteria and Blautia show a significant increase while taking Q-CAN®, and there was a trend for this in the obese population. ORAL. There were relatively fewer major changes in the oral microbiome with an increase in the family Veillonellaceae in the lean population while on Q-CAN®. CONCLUSION: Q-CAN® consumption induced a number of significant changes in the fecal and oral microbiome. Most notably an increase in the stool microbiome of Bifidobacteria and Blautia, both of which are associated with positive health benefits, and in the saliva an increase in Veillonellaceae. TRIAL REGISTRATION: This trial was registered with Clinicaltrials.gov on January 14th 2016. ClinicalTrials.gov Identifier: NCT02656056.

Fermented Soy Beverage Q-CAN Plus Consumption Improves Serum Cholesterol and Cytokines.

Soy-based beverages are well recognized for their rich nutritional contents and positive health benefits. However, there is little information regarding the composition of various commercially available soy-based beverages and uncertainty among patients regarding the utility of fermented soy products. Current study evaluates the health benefits of QCAN® Plus-an easily available fermented soy drink. This study was performed in lean (n = 10) and obese (n = 10) subjects. The subjects were observed during pre-soy (weeks -2, -1, and 0), on-soy (weeks 1, 2, 3, and 4), and post-soy (weeks 6, 8, 10, and 12) periods. The serum samples during these visits were subjected to lipid profile analysis and multiplex assay for cytokines. The results revealed that total cholesterol and low-density lipoprotein (LDL) cholesterol levels were significantly reduced in both lean and obese individuals during on-soy (P ≤ .05). Furthermore, cytokines such as platelet-derived growth factor (PDGF) AA and AB/BB were significantly lowered on-soy compared with pre-soy (P ≤ .05) in lean subjects and PDGF AA, IL-1RA, and GMCSF were significantly reduced on-soy (P ≤ .05) in obese subjects. In addition, a qualitative and quantitative analysis of the Q-CAN Plus by a third-party laboratory confirmed its chemical and microbial safety. Our preliminary study on Q-CAN Plus ensures its safety for consumption and highlights its hypolipidemic and suppressive effect on certain cytokines. These observations and relevant studies in future might guide clinicians in future to consider Q-CAN Plus as a therapeutic nutritional supplement.

Why AMPK agonists not known to be stressors may surprisingly contribute to miscarriage or hinder IVF/ART.

Here we examine recent evidence suggesting that many drugs and diet supplements (DS), experimental AMP-activated protein kinase (AMPK) agonists as well as energy-depleting stress, lead to decreases in anabolism, growth or proliferation, and potency of cultured oocytes, embryos, and stem cells in an AMPK-dependent manner. Surprising data for DS and drugs that have some activity as AMPK agonists in in vitro experiments show possible toxicity. This needs to be balanced against a preponderance of evidence in vivo that these drugs and DS are beneficial for reproduction. We here discuss and analyze data that leads to two possible conclusions: First, although DS and drugs that have some of their therapeutic mechanisms mediated by AMPK activity associated with low ATP levels, some of the associated health problems in vivo and in vitro fertilization/assisted reproductive technologies (IVF/ART) may be better-treated by increasing ATP production using CoQ10 (Ben-Meir et al., Aging Cell 14:887-895, 2015). This enables high developmental trajectories simultaneous with solving stress by energy-requiring responses. In IVF/ART, it is ultimately best to maintain handling and culture of gametes and embryos in the quietest state with low metabolic activity (Leese et al., Mol Hum Reprod 14:667-672, 2008; Leese, Bioessays 24 (9):845-849, 2002) using back-to-nature or simplex algorithms to identify optima (Biggers, Reprod Biomed Online 4 Suppl 1:30-38, 2002). Stress markers, such as checkpoint proteins like TRP53 (aka p53) (Ganeshan et al., Exp Cell Res 358:227-233, 2017); Ganeshan et al., Biol Reprod 83:958-964, 2010) and a small set of kinases from the protein kinome that mediate enzymatic stress responses, can also be used to define optima. But, some gametes or embryos may have been stressed in vivo prior to IVF/ART or IVF/ART optimized for one outcome may be suboptimal for another. Increasing nutrition or adding CoQ10 to increase ATP production (Yang et al., Stem Cell Rev 13:454-464, 2017), managing stress enzyme levels with inhibitors (Xie et al., Mol Hum Reprod 12:217-224, 2006), or adding growth factors such as GM-CSF (Robertson et al., J Reprod Immunol 125:80-88, 2018); Chin et al., Hum Reprod 24:2997-3009, 2009) may increase survival and health of cultured embryos during different stress exposure contexts (Puscheck et al., Adv Exp Med Biol 843:77-128, 2015). We define "stress" as negative stimuli which decrease normal magnitude and speed of development, and these can be stress hormones, reactive oxygen species, inflammatory cytokines, or physical stimuli such as hypoxia. AMPK is normally activated by high AMP, commensurate with low ATP, but it was recently shown that if glucose is present inside the cell, AMPK activation by low ATP/high AMP is suppressed (Zhang et al., Nature 548:112-116, 2017). As we discuss in more detail below, this may also lead to greater AMPK agonist toxicity observed in two-cell embryos that do not import glucose. Stress in embryos and stem cells increases AMPK in large stimulation indexes but also direness indexes; the fastest AMPK activation occurs when stem cells are shifted from optimal oxygen to lower or high levels (Yang et al., J Reprod Dev 63:87-94, 2017). CoQ10 use may be better than risking AMPK-dependent metabolic and developmental toxicity when ATP is depleted and AMPK activated. Second, the use of AMPK agonists, DS, and drugs may best be rationalized when insulin resistance or obesity leads to aberrant hyperglycemia and hypertriglyceridemia, and obesity that negatively affect fertility. Under these conditions, beneficial effects of AMPK on increasing triglyceride and fatty acid and glucose uptake are important, as long as AMPK agonist exposures are not too high or do not occur during developmental windows of sensitivity. During these windows of sensitivity suppression of anabolism, proliferation, and stemness/potency due to AMPK activity, or overexposure may stunt or kill embryos or cause deleterious epigenetic changes.

Two-cell embryos are more sensitive than blastocysts to AMPK-dependent suppression of anabolism and stemness by commonly used fertility drugs, a diet supplement, and stress.

PURPOSE: This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos. METHODS: Culture mouse zygotes to two-cell embryos and test effects after 0.5-1 h AMPK agonists' (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells. RESULT(S): Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~ 20-fold, and BR-DIM caused a ~ 30-fold increase over two-cell embryos cultured for 1 h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40-85% in two-cell embryos that was ~ 60-90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30-50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease. CONCLUSION: Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

Human biofield therapy does not affect tumor size but modulates immune responses in a mouse model for breast cancer.

OBJECTIVE: To assess the effect of human biofield therapy, an integrative medicine modality, on the development of tumors and metastasis, and immune function in a mouse breast cancer model. METHODS: Mice were injected with 66cl4 mammary carcinoma cells. In study one, mice received biofield therapy after cell injection. In study two, mice were treated by the biofield practitioner only prior to cell injection. Both studies had two control groups of mock biofield treatments and phosphate-buffered saline injection. Mice were weighed and tumor volume was determined. Blood samples were collected and 32 serum cytokine/chemokine markers were measured. Spleens/popliteal lymph nodes were isolated and dissociated for fluorescent-activated cell sorting (FACS) analysis of immune cells or metastasis assays in cell culture. RESULTS: No significant differences were found in weight, tumor size or metastasis. Significant effects were found in the immune responses in study one but no additional effects were found in study two. In study one, human biofield treatment significantly reduced percentage of CD4(+)CD44loCD25(+) and percentage of CD8(+) cells, elevated by cancer in the lymph nodes, to control levels determined by FACS analysis. In the spleen, only CD11b(+) macrophages were increased with cancer, and human biofield therapy significantly reduced them. Of 11 cytokines elevated by cancer, only interferon-γ, interleukin-1, monokine induced by interfer-γ, interleukin-2 and macrophage inflammatory protein-2 were significantly reduced to control levels with human biofield therapy. CONCLUSION: Human biofield therapy had no significant effect on tumor size or metastasis but produced significant effects on immune responses apparent in the down-regulation of specific lymphocytes and serum cytokines in a mouse breast cancer model.

Poor Long-Term Efficacy of Prevnar-13 in Sickle Cell Disease Mice Is Associated with an Inability to Sustain Pneumococcal-Specific Antibody Titers.

BACKGROUND: One of the most common causes of morbidity and mortality in children with sickle cell disease (SCD) is infection with the pneumococcal bacterium (Streptococcus pneumoniae). Unfortunately, the polysaccharide-conjugate vaccine appears to be less effective in individuals with SCD when compared to the general population. We sought to better understand the relative efficacy of pneumococcal vaccination in a SCD mouse challenge model. METHODS: Transgenic control and SCD mice were monitored for mortality after intranasal pneumococcal infection or pneumococcal vaccination with Prevnar-13 and type-matched challenge. Anti-pneumococcal antibody titers were measured by ELISA and opsonophagocytosis was measured in vitro. RESULTS: Mortality after pneumococcal infection was similar between control and SCD mice. However, after three intramuscular polysaccharide-conjugate vaccinations, all control mice were protected following high-dose intranasal infection, whereas 60% of SCD mice died. Anti-pneumococcal antibody titers showed initial IgG and IgM responses in both groups, but waning titers were observed in the SCD group, even after boosting. When functionally assayed in vitro, serum from SCD mice 13 weeks after a second booster shot maintained little to no ability to opsonize pneumococci, while serum from control mice sustained a significantly higher capacity opsonization. Thus, it appears that SCD mice do not maintain antibody responses to pneumococcal polysaccharides after Prevnar-13 vaccination, thereby leaving them susceptible to mortality after type-matched infection. CONCLUSION: Our results emphasize the need to better understand the correlates of immune protection in SCD so that pneumococcal vaccines can be improved and mortality reduced in this susceptible population.

Cbl-b Deficiency in Mice Results in Exacerbation of Acute and Chronic Stages of Allergic Asthma.

Mice sensitized to ovalbumin (OVA) develop allergic airway disease (AAD) with short-term daily OVA aerosol challenge; inflammation resolves with long-term OVA aerosol exposure, resulting in local inhalational tolerance (LIT). Cbl-b is an E3 ubiquitin ligase involved with CD28 signaling; Cbl-b(-/-) effector T cells are resistant to regulatory T cell-mediated suppression in vitro and in vivo. The present study utilized Cbl-b(-/-) mice to investigate the role of Cbl-b in the development of AAD and LIT. Cbl-b(-/-) mice exhibited increased airway inflammation during AAD, which failed to resolve with long-term OVA aerosol exposure. Exacerbation of inflammation in Cbl-b(-/-) mice correlated with increased proinflammatory cytokine levels and expansion of effector T cells in the BAL during AAD, but did not result in either a modulation of lymphocyte subsets in systemic tissues or in OVA-specific IgE in serum. These results implicate a role for Cbl-b in the resolution of allergic airway inflammation.

Therapeutic Touch Has Significant Effects on Mouse Breast Cancer Metastasis and Immune Responses but Not Primary Tumor Size.

Evidence-based integrative medicine therapies have been introduced to promote wellness and offset side-effects from cancer treatment. Energy medicine is an integrative medicine technique using the human biofield to promote well-being. The biofield therapy chosen for study was Therapeutic Touch (TT). Breast cancer tumors were initiated in mice by injection of metastatic 66cl4 mammary carcinoma cells. The control group received only vehicle. TT or mock treatments were performed twice a week for 10 minutes. Two experienced TT practitioners alternated treatments. At 26 days, metastasis to popliteal lymph nodes was determined by clonogenic assay. Changes in immune function were measured by analysis of serum cytokines and by fluorescent activated cells sorting (FACS) of immune cells from the spleen and lymph nodes. No significant differences were found in body weight gain or tumor size. Metastasis was significantly reduced in the TT-treated mice compared to mock-treated mice. Cancer significantly elevated eleven cytokines. TT significantly reduced IL-1-a, MIG, IL-1b, and MIP-2 to control/vehicle levels. FACS demonstrated that TT significantly reduced specific splenic lymphocyte subsets and macrophages were significantly elevated with cancer. Human biofield therapy had no significant effect on primary tumor but produced significant effects on metastasis and immune responses in a mouse breast cancer model.

The sickle cell mouse lung: proinflammatory and primed for allergic inflammation.

Comorbid asthma in sickle cell disease (SCD) confers higher rates of vaso-occlusive pain and mortality, yet the physiological link between these two distinct diseases remains puzzling. We used a mouse model of SCD to study pulmonary immunology and physiology before and after the induction of allergic airway disease (AAD). SCD mice were sensitized with ovalbumin (OVA) and aluminum hydroxide by the intraperitoneal route followed by daily, nose-only OVA-aerosol challenge to induce AAD. The lungs of naive SCD mice showed signs of inflammatory and immune processes: (1) histologic and cytochemical evidence of airway inflammation compared with naive wild-type mice; (2) bronchoalveolar lavage (BAL) fluid contained increased total lymphocytes, %CD8+ T cells, granulocyte-colony stimulating factor, interleukin 5 (IL-5), IL-7, and chemokine (C-X-C motif) ligand (CXCL)1; and (3) lung tissue and hilar lymph node (HLN) had increased CD4+, CD8+, and regulatory T (Treg) cells. Furthermore, SCD mice at AAD demonstrated significant changes compared with the naive state: (1) BAL fluid with increased %CD4+ T cells and Treg cells, lower %CD8+ T cells, and decreased interferon gamma, CXCL10, chemokine (C-C motif) ligand 2, and IL-17; (2) serum with increased OVA-specific immunoglobulin E, IL-6, and IL-13, and decreased IL-1α and CXCL10; (3) no increase in Treg cells in the lung tissue or HLN; and (4) hyporesponsiveness to methacholine challenge. In conclusion, SCD mice have an altered immunologic pulmonary milieu and physiological responsiveness. These findings suggest that the clinical phenotype of AAD in SCD mice differs from that of wild-type mice and that individuals with SCD may also have a unique, divergent phenotype perhaps amenable to a different therapeutic approach.

Transgenic sickle cell disease mice have high mortality and dysregulated immune responses after vaccination.

BACKGROUND: Children with sickle cell disease (SCD) are susceptible to recurrent infections, which are often life threatening and necessitate frequent vaccinations. Given the altered baseline immunity and proinflammatory state associated with SCD, we sought to determine the relative safety and efficacy of vaccination in transgenic SCD mice. METHODS: Eight-week-old SCD mice were vaccinated with ovalbumin and aluminum hydroxide weekly for 3 wk by the intraperitoneal or intramuscular route. One week after the third vaccination, serum cytokines/chemokines, immunoglobulins, and bronchoalveolar lavage fluid cytokines were measured. RESULTS: Only SCD mice were prone to mortality associated with vaccination, as 40% of the animals died after the intraperitoneal vaccinations and 50% died after the intramuscular vaccinations. Serum IgG2b and IgM were significantly lower in SCD mice than in C57BL/6 mice after vaccination, but ovalbumin-specific IgE was significantly higher. Serum interleukin (IL)-1α, IL-2, IL-5, macrophage inflammatory protein 1α, and granulocyte macrophage-colony stimulating factor were significantly lower in SCD mice than in C57BL/6 mice after vaccination, whereas bronchoalveolar lavage fluid IL-1ß and IL-6 were increased. CONCLUSION: Mice with SCD appear to have a dysregulated immune response to vaccination. Thus, the relative safety and immunogenicity of vaccination should be studied in greater detail in the context of SCD.

Broncho-alveolar macrophages express chemokines associated with leukocyte migration in a mouse model of asthma.

The migration of eosinophils and lymphocytes into airways is a hallmark of allergic asthma; however, the role of broncho-alveolar macrophages (BAMs) in this inflammatory process has not been fully elucidated. Using a murine Ova model of allergic airway disease (AAD), RNA isolated from BAMs was used to assess differential gene expression via microarray and qRT-PCR. Significant increases in WBCs, eosinophilia, mucus accumulation and goblet cell hyperplasia were observed in Ova sensitized and challenged mice, which correlated with increased expression of genes associated with alternatively activated M2 macrophages (e.g. arginase 1, YM-1, YM-2, Resistin like-α, and EAR-11). Other genes associated with asthma including FcγRIIb, MMP-14, CCL-8, CCL-17, ADAM-8, LTBR1, aquaporin-9 and IL-7R were also expressed at higher levels in Ova sensitized/challenged animals when compared to BAMs isolated from control animals. Eotaxin 2 (CCL-24), which is known to influence eosinophil migration, was highly up-regulated in BAMs, but not Eotaxin-1 (CCL-11). Conversely, lung interstitial macrophages expressed high levels of CCL-11, but not CCL-24. Taken together, this study provides additional evidence to support the notion that M2 BAMs play a role in eosinophil and potentially other leukocyte migration patterns into asthmatic airways.

Bromelain Inhibits Allergic Sensitization and Murine Asthma via Modulation of Dendritic Cells.

The incidence of atopic conditions has increased in industrialized countries. Persisting symptoms and concern for drug side-effects lead patients toward adjunctive treatments such as phytotherapy. Previously, we have shown that Bromelain (sBr), a mixture of cysteine proteases from pineapple, Ananas comosus, inhibits ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). However, sBr's effect on development of AAD when treatment is administered throughout OVA-alum sensitization was unknown and is the aim of the present study. C57BL/6J mice were sensitized with OVA/alum and challenged with 7 days OVA aerosol. sBr 6 mg/kg/0.5 ml or PBS vehicle were administered throughout sensitization. Lung, bronchoalveolar lavage (BAL), spleen, and lymph nodes were processed for flow cytometry and OVA-specific IgE was determined via ELISA. sBr treatment throughout OVA-alum sensitization significantly reduced the development of AAD (BAL eosinophils and lymphocytes). OVA-specific IgE and OVA TET(+) cells were decreased. sBr reduced CD11c(+) dendritic cell subsets, and in vitro treatment of DCs significantly reduced CD44, a key receptor in both cell trafficking and activation. sBr was shown to reduce allergic sensitization and the generation of AAD upon antigen challenge. These results provide additional insight into sBr's anti-inflammatory and antiallergic properties and rationale for translation into the clinical arena.

LC-MS/MS Identification of a Bromelain Peptide Biomarker from Ananas comosus Merr.

Bromelain (Br) is a cysteine peptidase (GenBank AEH26024.1) from pineapple, with over 40 years of clinical use. The constituents mediating its anti-inflammatory activity are not thoroughly characterized and no peptide biomarker exists. Our objective is to characterize Br raw material and identify peptides in the plasma of Br treated mice. After SDS-PAGE in-gel digestion, Br (VN#3507; Middletown, CT, USA) peptides were analyzed via LC/MS/MS using 95% protein probability, 95% peptide probability, and a minimum peptide number = 5. Br spiked mouse plasma (1 ug/ul) and plasma from i.p. treated mice (12 mg/kg) were assessed using SRM. In Br raw material, we identified seven proteins: four proteases, one jacalin-like lectin, and two protease inhibitors. In Br spiked mouse plasma, six proteins (ananain, bromelain inhibitor, cysteine proteinase AN11, FB1035 precursor, FBSB precursor, and jacalin-like lectin) were identified. Using LC/MS/MS, we identified the unique peptide, DYGAVNEVK, derived from FB1035, in the plasma of i.p. Br treated mice. The spectral count of this peptide peaked at 6 hrs and was undetectable by 24 hrs. In this study, a novel Br peptide was identified in the plasma of treated mice for the first time. This Br peptide could serve as a biomarker to standardize the therapeutic dose and maximize clinical utility.

Splenic morphological changes are accompanied by altered baseline immunity in a mouse model of sickle-cell disease.

Although functional asplenia from infarctions may be a major contributor to increased infectious mortality in sickle-cell disease (SCD), this relationship has not been fully defined. We used the transgenic Berkeley SCD mouse to define blood and splenic immunophenotypic differences in this model compared with C57BL/6 and hemizygous controls. In the serum of SCD mice, we found increased IgG2a and suppressed IgM, IgG2b, and IgA levels. Serum IL-6 levels in SCD mice were elevated, whereas IL-1α, CXCL10, and CCL5 levels were decreased. The blood of SCD mice had higher white blood cell counts, with an increased percentage of lymphocytes and decreases in other leukocytes. Immunophenotyping of lymphocytes revealed higher percentages of CD8(+) and T-regulatory cells and lower percentages of B cells. SCD mouse spleens exhibited histological disorganization, with reduction of defined lymphoid follicles and expansion of red pulp, a greater than fourfold increase in splenic mononuclear cells, marked expansion of the nucleated red blood cell fraction, and B-cell and CD8(+) T-cell lymphopenia. Within the splenic B-cell population, there was a significant decrease in B-1a B cells, with a corresponding decrease in IgA secreting plasma cells in the gut. Confocal microscopy of spleens demonstrated complete disruption of the normal lymphofollicular structure in the white pulp of SCD mice without distinct B, T, and marginal zones. Our findings suggest that altered SCD splenic morphological characteristics result in an impaired systemic immune response.

Bromelain limits airway inflammation in an ovalbumin-induced murine model of established asthma.

CONTEXT: Allergic asthma continues to increase despite new pharmacological advances for both acute treatment and chronic-disease management. Asthma is a multifactorial disease process with genetic, allergic, infectious, environmental, and dietary origins. Researchers are investigating the benefits of lifestyle changes and alternative asthma treatments, including the ability of bromelain to inhibit inflammation. Bromelain is a commonly used, proteolytically active pineapple extract. OBJECTIVE: The present study intended to determine the ability of bromelain to reduce the inflammation of preexisting asthma via an ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). DESIGN: The research team designed a study examining the effects of bromelain in a control group of mice that received phosphate buffered saline (PBS) only and in an intervention group that received bromelain in PBS. Setting The study took place in the Department of Immunology at the University of Connecticut's School of Medicine, Farmington. Intervention The research team sensitized female C57BL/6J mice with intraperitoneal OVA/alum and then challenged them with OVA aerosolization for 10 consecutive days. On day 4, the team began administering daily doses of PBS to the control group (n = 10) and bromelain (6mg/kg) in PBS to the bromelain (intervention) group (n = 10). OUTCOME MEASURES: The primary measures included bronchoalveolar lavage (BAL) cellular differential, cellular phenotype via flow cytometry, and lung histology. Additional outcomes included testing for serum cytokines and immunoglobulin. RESULTS: Bromelain treatment of AAD mice (bromelain group) resulted in significant anti-inflammatory activity as indicated by reduced BAL total leukocytes (P < .05), eosinophils (P < .05), and cellular infiltrates via lung pathology (P < .005), as compared to the control group. In addition, bromelain significantly reduced BAL CD4+ and CD8+ T cells without affecting cell numbers in the spleen or hilar lymph node. The study found decreased interleukins IL-4, IL-12, IL-17, as well as IFN-α in the serum of bromelain-treated animals. CONCLUSIONS: The results suggest that bromelain has a therapeutic effect in established AAD, which may translate into an effective adjunctive therapy in patients with similar conditions, such as allergic asthma, who have chosen to initiate treatment after the onset of symptoms.

Phenotypic changes to the endogenous antigen-specific CD8+ T cell response correlates with the development and resolution of allergic airway disease.

The role of CD8(+) T cells in the pathogenesis of asthma remains controversial, as both pro- and anti-inflammatory functions have been suggested. This study was designed to examine the endogenous CD8(+) T cell response in a biphasic ovalbumin (OVA)-induced model of allergic airway disease (AAD) and its subsequent resolution with the development of local inhalational tolerance (LIT). We observed increases in OVA-specific CD8(+) T cell numbers in the local lung compartments (bronchoalveolar lavage, lung tissue, hilar lymph node) at AAD and LIT; systemic compartments (spleen, inguinal lymph node) displayed no such increases in CD8(+) T cell numbers. OVA-specific CD8(+) T cells appeared to exhibit plasticity both phenotypically and functionally. They possessed pro-inflammatory characteristics at AAD, with high phenotypic expression of CD11a and increased functional expression of granzyme B and interferon-γ. In contrast, at LIT they showed increased phenotypic expression of the inhibitory marker NKG2A and functionally did not produce granzyme B or interferon-γ. In addition, in a discontinuous model the OVA-specific CD8(+) T cells could be recalled on re-exposure to OVA, demonstrating memory. Finally, confocal microscopy results showed that OVA-specific CD8(+) T cells at AAD are associated with B cell aggregates in lung tissue. These B cell aggregates resembled tertiary ectopic lymphoid tissue and may thus provide a local environment for the salient cellular interactions that contribute to the development of LIT.

Bromelain treatment reduces CD25 expression on activated CD4+ T cells in vitro.

Bromelain (Br), an extract from pineapple stem with cysteine protease activity, exerts anti-inflammatory effects in a number of inflammatory models. We have previously shown that Br treatment decreased activated CD4(+) T cells and has a therapeutic role in an ovalbumin-induced murine model of allergic airway disease. The current study was designed to determine the effect of Br on CD4(+) T cell activation, specifically the expression of CD25 in vitro. CD25 is up regulated upon T cell activation, found as a soluble fraction (sCD25) and is a therapeutic target in inflammation, autoimmunity and allergy. Br treatment of anti-CD3 stimulated CD4(+) T cells reduced CD25 expression in a dose and time dependent manner. This reduction of CD25 was dependent on the proteolytic action of Br as the addition of E64 (a cysteine protease inhibitor) abrogated this response. The concentration of sCD25 was increased in supernatants of Br treated activated CD4(+) T cells as compared to control cells, suggesting that Br proteolytically cleaved cell-surface CD25. This novel mechanism of action identifies how Br may exert its therapeutic benefits in inflammatory conditions.

Regulatory role of B cells in a murine model of allergic airway disease.

Mice sensitized to OVA and subjected to acute OVA aerosol exposures develop allergic airway disease (AAD). However, chronic continuous Ag exposure results in resolution of AAD and the development of local inhalational tolerance (LIT). Because we have previously observed the persistence of B cells in the bronchoalveolar lavage (BAL) and hilar lymph nodes (HLN) at the resolution stage of this model, we investigated the role of B cells in the modulation of AAD. Although B cell-deficient mice developed LIT, adoptive transfer of HLN B cells from LIT mice to OVA-sensitized recipients resulted in attenuated AAD following subsequent OVA aerosol exposure, as determined by reduced BAL leukocytosis and eosinophilia, decreased tissue inflammation, and absent methacholine hyper-responsiveness. In similar adoptive transfer studies, HLN B cells from AAD mice were without effect. The protection transferred by LIT HLN B cells was Ag specific and was associated with accumulation of Foxp3(+) T regulatory cells regionally in BAL and HLN, but not systemically in the spleen. Fluorescent labeling of LIT HLN B cells before adoptive transfer demonstrated that these cells had the capacity to migrate to local inflammatory sites. In vitro assessment demonstrated that the LIT HLN B cells exerted this regulatory effect via TGF-beta induced conversion of CD4(+)CD25(-) T effector cells into functionally suppressive CD4(+)CD25(+)Foxp3(+) T regulatory cells. These findings illustrated a novel regulatory role for regional B cells in AAD and suggested a possible contributory role of B cells, along with other cell types, in the establishment of LIT.

Subcutaneous late phase responses are augmented during local inhalational tolerance in a murine asthma model.

Acute exposure of sensitized mice to antigen elicits allergic airway disease (AAD) characterized by Th2 cytokine-dependent pulmonary eosinophilia, methacholine hyperresponsiveness and antigen-specific IgE elevation. However, chronic exposure induces a local inhalational tolerance (LIT), with resolution of the airway responses but persistent systemic IgE production. To further determine if systemic immunologic responses were maintained during LIT, we assessed subcutaneous late phase responses to ovalbumin in this model. Sensitized and AAD mice developed small subcutaneous responses to ovalbumin, with footpad thickness increasing to 113.7 and 113.6% of baseline, respectively. In comparison, LIT mice developed marked foot swelling (141.6%). Histologic examination confirmed increased inflammation in the chronic animals, with a significant contribution by eosinophils. Thus, the resolution of airway inflammatory responses with chronic antigen inhalation is a localized response, not associated with loss of systemic responses to antigen.