David H. Storms

Pyrroloquinoline Quinone Improves Growth and Reproductive Performance in Mice Fed Chemically Defined Diets

Growth, reproductive performance, and indices of collagen maturation and expression were investigated in Balb/c mice fed chemically defined, amino acid-based diets with or without the addition 6 <M pyrroloquinoline quinone (PQQ)/kg diet. The diets were fed to virgin mice for 8 weeks before breeding. At weaning, the pups from successful pregnancies were fed the same diet as their respective dams. Reproductive performance was compromised in mice fed diets devoid of PQQ, and their offspring grew at slower rates than offspring from mice fed diets supplemented with PQQ. Successful mating (confirmed vaginal plugs) was not affected by the presence or absence of PQQ; however, pup viability (number of pups at parturition/number of pups at Day 4 of lactation) was decreased in PQQ-deprived mice. Conception (percentage of females giving live births) and fertility (percentage of births) were also decreased in PQQ-deprived mice. The slower rates of growth in offspring from PQQ-deprived mice were associated with decreased steady-state mRNA levels for Type I procollagen α1-chains in skin and lungs from neonatal mice. Values for lysyl oxidase accumulation as protein in PQQ-deficient mice also tended to be lower than corresponding values from PQQ-supplemented or -replete mice. Skin collagen solubility was increased in PQQ-deprived mice. These results indicate that PQQ supplementation can improve reproductive performance, growth, and may modulate Indices of neonatal extracellular matrix production and maturation in mice fed chemically defined, but otherwise nutritionally complete diets.

Biochemistry: is pyrroloquinoline quinone a vitamin?

Arising from: T. Kasahara & T. Kato 422, 832 (2003); see also communication from Felton and Anthony; Kasahara et al. reply The announcement by Kasahara and Kato of pyrroloquinoline quinone (PQQ) as a ‘new’ vitamin has received considerable attention. We have since attempted to reproduce the findings on which their conclusion is based, namely that defects in lysine metabolism occur in PQQ-deprived rodents. However, we find that the activity of α-aminoadipic acid-δ-semialdehyde (AAS) dehydrogenase in liver and plasma levels of α-aminoadipic acid (AAA), both of which act as indicators of lysine degradation in mammals, are not affected by changes in PQQ dietary status. Our results call into question the identification of PQQ as a new vitamin.

Resveratrol Alters Proliferative Responses and Apoptosis in Human Activated B Lymphocytes in Vitro

We hypothesized that the phytochemicals resveratrol, quercetin, and kaempferol would modulate B lymphocyte proliferation, Ig synthesis, and apoptosis after activation. Peripheral blood mononuclear cells (PBMC) were isolated from 12 healthy adult human volunteers and incubated with pokeweed mitogen plus 0, 2, 5, and 10 mumol/L resveratrol, quercetin, or kaempferol. After 6 d, CD19+ B cells were analyzed for proliferation, B cell lymphoma-2 (Bcl-2) expression, and activation of caspase-3 using flow cytometry. After 8 d, cell supernatants were collected and IgM and IgG were measured by ELISA. Resveratrol at a concentration of 5 mumol/L increased the percentage of CD19+ cells compared with mitogen only-stimulated cells (P < 0.01), and a trend for increased proliferation was observed for cells treated with 0, 2, and 5 mumol/L resveratrol (P-trend = 0.01). However, 10 mumol/L resveratrol inhibited proliferation of B lymphocytes (P < 0.01). Expression of Bcl-2 and caspase-3 activation increased in B cells treated with 10 mumol/L resveratrol compared with mitogen alone (P < 0.01), and trends for dose-responsive increases in Bcl-2 expression and caspase-3 activation were observed (P-trend < 0.0001). Differences in IgM and IgG production were not observed for PBMC treated with resveratrol. Kaempferol at 10 mumol/L slightly inhibited proliferative responses (P < 0.05) but did not affect B cell function or apoptosis. Quercetin did not alter B cell proliferation, function, or apoptosis. These data show that human B lymphocyte proliferation and apoptosis are modified by physiological concentrations of resveratrol and suggest that exposure of human B cells to resveratrol may increase survival by upregulating Bcl-2.

Dietary strawberry powder reduces blood glucose concentrations in obese and lean C57BL/6 mice, and selectively lowers plasma C-reactive protein in lean mice

The purpose of the present study was to test the anti-inflammatory and blood glucose (BG)-regulating capacity of strawberries in a mouse model of diet-induced obesity. A total of thirty-six male C57BL/6J mice were randomly divided into four groups (nine mice per group). Mice were fed a low-fat diet (LF, 13 % fat), the LF supplemented with 2·6 % freeze-dried strawberry powder (LFSB), a high-fat diet (HF, 44 % fat) or the HF supplemented with 2·6 % strawberry powder (HFSB). Blood samples were collected to measure BG, inflammation and systemic markers for endocrine function of pancreas and adipose tissue. Splenocytes were harvested at the end of the study and activated with either anti-cluster of differentiation (CD) 3/anti-CD28 antibodies or lipopolysaccharide to test immune responsiveness. The HF increased non-fasted BG, insulin, soluble intracellular adhesion molecule-1, E-selectin, leptin, resistin and plasminogen activator protein-1 (P < 0·05). High dietary fat decreased IL-4 production from activated splenocytes (P < 0·05). BG concentrations were lower in the mice supplemented with SB (10·64 mmol/l) compared to the non-supplemented mice (11·37 mmol/l; P = 0·0022). BG values were approximately 6·5 % lower in the supplemented mice. Additionally, SB lowered plasma C-reactive protein in the LFSB group compared to the other three groups (P < 0·05). The dietary intake of SB approximated one human serving of strawberries. These results, although modest, support a promising role for dietary strawberries in reducing the risks associated with obesity and diabetes, and regulating the levels of inflammatory markers in non-obese individuals.

Dietary grape powder increases IL-1β and IL-6 production by lipopolysaccharide-activated monocytes and reduces plasma concentrations of large LDL and large LDL-cholesterol particles in obese humans

Obese individuals are at an increased risk of developing CVD, hypertension, type 2 diabetes, and bacterial and viral infections when compared with the normal-weight population. In a 9-week randomised, double-blind, cross-over study, twenty-four obese subjects aged between 20 and 60 years and with a BMI between 30 and 45 kg/m2 were fed grape or placebo powder for 3-week intervals to determine the effects of dietary grapes on blood lipid profiles, plasma inflammatory marker concentrations and immune cell function. Blood samples were collected on days 1 and 8 for obtaining baseline information and at weeks 3, 4, 8 and 9. Comprehensive chemistry panels, lipid profile analyses by NMR, measurement of plasma inflammatory marker concentrations, and analyses of cytokine production by activated T lymphocytes and monocytes were performed for each blood draw. Dietary grape powder reduced the plasma concentrations of large LDL-cholesterol and large LDL particles compared with the placebo powder (P< 0·05). The concentrations of interferon-γ, TNF-α, IL-4 and IL-10 were measured in supernatants from peripheral blood mononuclear cells (PBMC) activated with anti-CD3/CD28 antibodies and those of TNF-α, IL-1β, IL-6 and IL-8 were measured in supernatants from PBMC activated with lipopolysaccharide (LPS). No difference in the production of T-cell cytokines was observed between the two intervention groups. The production of IL-1β and IL-6 was increased in supernatants from LPS-activated PBMC in the grape powder group compared with the placebo powder group (P< 0·05). These data suggest that dietary grapes may decrease atherogenic lipid fractions in obese individuals and increase the sensitivity of monocytes in a population at a greater risk of developing infections.

Carnosol Delays Chemotherapy-Induced DNA Fragmentation and Morphological Changes Associated With Apoptosis in Leukemic Cells

Carnosol, from the herb rosemary, has been shown to induce apoptotic cell death in high-risk pre-B acute lymphoblastic leukemia (ALL). In the present study, carnosol was tested for its ability to sensitize leukemia cells to chemotherapeutic agents. Carnosol reduced the percentage of cell death in the pre-B ALL lines SEM, RS4;11, and REH when combined with cytarabine, methotrexate, or vincristine compared to the chemotherapeutic agents alone. Analysis of DNA strand breaks by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that carnosol delayed DNA cleavage in the cells when combined with chemotherapeutic drugs. Co-treatment of the cells with carnosol and chemotherapeutic drugs did not reduce mitochondrial membrane depolarization compared to the drug treatment alone. Time course analysis of caspase-3 activation by flow cytometry showed co-treatment with carnosol and drugs increased the activation of caspase-3 above that observed for the chemotherapeutic drugs alone. A lower percentage of caspase-3 positive cells progressed to an apoptotic phenotype when co-treated with carnosol and the chemotherapeutic drugs compared to drugs alone. These data show that carnosol blocks the terminal apoptotic events induced by chemotherapeutic drugs and suggest that increased dietary intake of carnosol may potentially decrease the effectiveness of some standard chemotherapy treatments used for leukemia.

Dietary resveratrol does not delay engraftment, sensitize to vincristine or inhibit growth of high-risk acute lymphoblastic leukemia cells in NOD/SCID mice

Acute lymphoblastic leukemia (ALL) with translocation t(4;11) is a high-risk leukemia found in 60–85% of infants with ALL and is often refractory to conventional chemotherapeutics after relapse. To evaluate the efficacy of dietary resveratrol in vivo, 5-week-old NOD.CB17-Prkdcscid/J mice were fed a control diet or a diet containing 0.2% w/w resveratrol. After 3 weeks of dietary treatment, mice were engrafted with the human t(4;11) ALL line SEM by tail vein injection. Engraftment was monitored by evaluating the presence of human CD19+ cells in peripheral blood using flow cytometry. Relative to control diet, dietary resveratrol did not delay the engraftment of the leukemia cells. To determine if dietary resveratrol could increase efficacy of a chemotherapeutic agent, vincristine was injected intraperitoneally into leukemic mice fed the control or supplemented diet. Survival curves and monitoring the percentage of human leukemia cells in peripheral blood showed that resveratrol did not inhibit leukemia cell growth or influence the activity of vincristine. Mass spectrometric analysis of mouse serum revealed that the majority of resveratrol was present as glucuronidated and sulfated metabolites. These data do not support the concept that dietary resveratrol has potential as a preventative agent against the growth of high-risk t(4;11) ALL.

Dietary strawberries increase the proliferative response of CD3/CD28-activated CD8+ T cells and the production of TNF-α in lipopolysaccharide-stimulated monocytes from obese human subjects

Obesity increases the risk of developing bacterial and viral infections compared with normal weight. In a 7-week double-blind, randomised, cross-over trial, twenty obese volunteers (BMI between 30 and 40 kg/m²) were fed freeze-dried strawberry powder or strawberry-flavoured placebo preparations to determine the effects of dietary strawberries on immune function. Blood was collected at six time points during the study and peripheral blood mononuclear cells (PBMC) were isolated at each time point and activated with CD3 plus CD28 antibodies (T-lymphocyte activation) or lipopolysaccharide (LPS, monocyte activation). Interferon-γ, TNF-α, IL-4 and IL-10 were measured in supernatants from the activated T cells. Supernatants from the activated monocytes were analysed for the production of TNF-α, IL-1β, IL-6 and IL-8. PBMC were pre-stained with PKH (Paul Karl Horan) dye and activated with CD3 plus CD28 antibodies to determine the proliferative responses of CD4⁺ and CD8⁺ T-lymphocytes by flow cytometry. To detect global changes in gene expression, microarray analysis was performed on LPS- and vehicle-treated PBMC from two subjects before and after the strawberry intervention. No difference was observed for the production of T-cell cytokines between the intervention groups. The production of TNF-α was increased in the supernatants from LPS-activated PBMC in the group consuming strawberries compared with the placebo. A modest increase in the proliferation of the CD8⁺ T-lymphocyte population was observed at 24 h post-activation. These data suggest that dietary strawberries may increase the immunological response of T-lymphocytes and monocytes in obese people who are at greater risk for developing infections.

Resveratrol given intraperitoneally does not inhibit the growth of high-risk t(4;11) acute lymphoblastic leukemia cells in a NOD/SCID mouse model

The efficacy of resveratrol as a preventive agent against the growth of t(4;11) acute lymphoblastic leukemia (ALL) was evaluated in NOD.CB17-Prkdcscid/J mice engrafted with the human t(4;11) ALL SEM cell line. SEM cells were injected into the tail vein and engraftment was monitored by flow cytometry. Once engraftment was observed, mice were injected intraperitoneally with resveratrol (10 mg/kg body weight) dissolved in dimethylsulfoxide (DMSO) or DMSO alone (control) every other day, or vincristine (0.5 mg/kg body weight) 3 times per week for 4 weeks (n=16 per group). Comparisons of the percent of human leukemia cells in blood and survival curves showed resveratrol did not inhibit progression of the disease. Liquid chromatography-tandem mass spectrometry analyses of mouse sera showed resveratrol was rapidly metabolized to glucuronidated and sulfated forms 1 h post-injection, with low to no resveratrol or metabolites observed in sera by 24–48 h. These data indicate that in contrast to findings in in vitro models, parenterally administered resveratrol does not have potential as a preventive agent against high risk t(4;11) ALL.

Novel in vivo model of inducible multi-drug resistance in acute lymphoblastic leukemia with chromosomal translocation t(4;11)

Acute lymphoblastic leukemia (ALL) with translocation t(4;11) is found in 60-85% of infants with ALL and is often refractory to conventional chemotherapeutics after relapse. Using the t(4;11) ALL line SEM, we evaluated chemotherapy resistance in NOD.CB17-Prkdcscid/J mice. SEM cells were injected into the tail vein and engraftment was monitored by flow cytometry. Once engraftment was observed, mice were injected intraperitoneally with phosphate-buffered saline (PBS), or vincristine (0.5mg/kg body weight) three times per week for 4weeks (n=8 per group). The level of P-glycoprotein in SEM cells was increased 3-fold by vincristine treatment compared to PBS-treated mice. Survival curves showed that leukemia cell growth was initially delayed by vincristine treatment, but the mice eventually succumbed to disease. These data describe a novel inducible model for investigating multi-drug resistance mechanisms in high-risk t(4;11) ALL.