No effect of salmon fish protein on 2-h glucose in adults with increased risk of type 2 diabetes: a randomised controlled trial.

The association between fish consumption and decreased risk of CVD is well documented. However, studies on health effects of fish consumption suggest that other components than n-3 PUFA have beneficial cardiometabolic effects, including effects on glucose metabolism. The aim of the present study was to investigate effects of salmon fish protein on cardiometabolic risk markers in a double-blind, randomised controlled parallel trial. We hypothesised that daily intake of a salmon fish protein supplement for 8 weeks would improve glucose tolerance in persons with increased risk of type 2 diabetes mellitus (T2DM). Our primary outcome measure was serum glucose (s-glucose) 2 h after a standardised oral glucose tolerance test. In total, eighty-eight adults with elevated s-glucose levels were randomised to 7·5 g of salmon fish protein/d or placebo, and seventy-four participants were included in the analysis. We found no significant effect of salmon fish protein supplementation on our primary outcome or other markers related to glucose tolerance, serum lipids, weight or blood pressure compared with placebo. The present study does not support the hypothesis that daily intake of a salmon fish protein supplement for 8 weeks improves glucose tolerance in persons with increased risk of T2DM.

Quantification and characterization of granulocyte macrophage colony-stimulating factor activated human peripheral blood mononuclear cells by fluorine-19 cellular MRI in an immunocompromised mouse model.

PURPOSE: The purpose of this study was to test fluorine-19 (19F) cellular magnetic resonance (MRI) as a non-invasive imaging modality to track therapeutic cell migration as a surrogate marker of immunotherapeutic effectiveness. MATERIALS AND METHODS: Human peripheral blood mononuclear cell- (PBMC)-derived antigen presenting cell (APC) were labeled with a 19F-perfluorocarbon (PFC) and/or activated with granulocyte macrophage colony-stimulating factor (GM-CSF). Viability, phenotype and cell lineage characterization preceded 19F cellular MRI of PFC+ PBMC under both pre-clinical 9.4 Tesla (T) and clinical 3T conditions in a mouse model. RESULTS: A high proportion of PBMC incorporated PFC without affecting viability, phenotype or cell lineage composition. PFC+ PBMC were in vivo migration-competent to draining and downstream lymph nodes. GM-CSF addition to culture increased PBMC migration to, and persistence within, secondary lymphoid organs. CONCLUSION: 19F cellular MRI is a non-invasive imaging technique capable of detecting and quantifying in vivo cell migration in conjunction with an established APC-based immunotherapy model. 19F cellular MRI can function as a surrogate marker for assessing and improving upon the therapeutic benefit that this immunotherapy provides.

Evaluation of iron chelating and antioxidant potential of Epilobium hirsutum for the management of iron overload disease.

The present study deals with the investigation of iron chelating and antioxidant potential of Epilobium hirsutum in iron-overloaded rats. Iron overload was induced by 6 IP injections of Iron dextran (12.5mg/100g) administered uniformly over a period of 30 days. Different fractions of E. hirsutum were given orally and deferoxamine (DFO) subcutaneously for 30 days. The extent of iron chelation and various biochemical parameters were estimated on 15th and 30th day of treatment. In-vitro study was assessed by EDTA and DFO method; the results exhibited a dose-dependant iron chelation. The methanolic fraction of methanolic extract (MFME) and methanolic fraction of aqueous extract (MFAE) of E. hirsutum showed significant (p<0.01) iron chelating and antioxidant potential as compared to disease control (DC) rats. The animals treated with MFME and MFAE of E. hirsutum showed significant (p<0.01) vital organ protection as compared to DC rats. The animals treated for longer duration (30th day) reveals better iron chelation potential than shorter ones (15th day). Superior iron chelation was seen at higher dose (300mg/kg) as compared to lower dose (150mg/kg). Taken into an account, our result reveals the reversible iron chelating and antioxidant ability of E. hirsutum and gives some evidence for its possible mechanism via excretion of iron in urine and feces.

Investigation into Iron Chelating and Antioxidant Potential of Melilotus officinalis in Iron Dextran Induced Iron Overloaded Sprague Dawley Rat Model.

Excess of iron leads to generates free radicals, causes organ damage. Melilotus officinalis (Fabaceae) reported to have various pharmacological activities. It contains flavonoids and phenolic compounds which have iron chelating and antioxidant property. Hence, present study was designed to investigate the beneficial effects of different fractions of M. officinalis for the management of iron overload disease and its complications. Iron overload was induced by 6 IP injections of iron dextran (12.5 mg/100 g) uniformly distributed over the period of 30 days. The different fractions of M. officinalis were given orally and Deferoxamine (DFO) subcutaneously for 30 days. The iron chelating and various biochemical parameters were estimated on 15th and 30th day. The different fractions of M. officinalis demonstrated dose dependant in-vitro iron chelating ability. There were significant (P<0.01) iron chelating potential shows in rats treated with methanolic fraction of methanolic extract (MFME) and methanolic fraction of aqueous extract (MFAE) of M. officinalis as compared to disease control (DC) rats. The rats treated with MFME and MFAE of M. officinalis shows significant (P<0.01) antioxidant and vital organ protective effect as compared to DC rats. Better iron chelation was observed on 30th day and at higher dose (300 mg/kg) as compared to 15th day and at lower dose (150 mg/kg). The present study concludes that MFME and MFAE of M. officinalis have reversible iron chelating and antioxidant potential in rats. The study also proves the possible mechanism of action, as an iron chelator by increasing the excretion of iron in urine and feces.

Nuclear magnetic resonance measurements of velocity distributions in an ultrasonically vibrated granular bed.

We report the results of nuclear magnetic resonance imaging experiments on granular beds of mustard grains fluidized by vertical vibration at ultrasonic frequencies. The variation of both granular temperature and packing fraction with height was measured within the three-dimensional cell for a range of vibration frequencies, amplitudes and numbers of grains. Small increases in vibration frequency were found--contrary to the predictions of classical 'hard-sphere' expressions for the energy flux through a vibrating boundary--to result in dramatic reductions in granular temperature. Numerical simulations of the grain-wall interactions, using experimentally determined Hertzian contact stiffness coefficients, showed that energy flux drops significantly as the vibration period approaches the grain-wall contact time. The experiments thus demonstrate the need for new models for 'soft-sphere' boundary conditions at ultrasonic frequencies.

Immunomodulatory effects of secondary hyperparathyroidism on circulating CD4+ and CD8+ T-lymphocytes in chronic renal failure patients.

The immunomodulatory effects of parathyroid hormone (PTH) in patients with end stage renal disease (ESRD) is controversial. This study was carried out to investigate the effect of PTH levels on the circulating CD4+, CD8+ T cell counts (%) in patients with chronic renal failure (CRF) on regular hemodialysis ((HD). The study included 22 patients with serum levels of PTH < 300 pg/ml (group 1), 18 patients with PTH > 300 pg/ml (group II) and 10 age and sex matched normal controls (group III). Chemiluminescence and flowcytometry assays were performed for determination of serum PTH levels and T cell subset counts respectively. The mean (%) of total lymphocyte, CD4+, CD8+ and CD4\CD8 ratio of group I were (81.68+/- 9.38), (52.00+/-6.24), (27.13+/- 6.31) and (1.99+/-0.42) respectively, as compared to (73.83+/-13.30), (46.05+/-8.59), (23.05+/-4.63) and (2.03+/-0.41) respectively in group II. Values of group I and II were significantly (P<0.001) lower than controls (88.50 +/- 6.02), (63.30 +/- 6.44), (36.80 +/- 6.44) and (1.76+/-0.36) respectively. In group II, the reduction was significantly (P<0.001) prominent in patients with high PTH levels, with significant inverse correlations (P<0.001) between PTH and % of total lymphocyte (r= -0.93), CD4+ (r= -0.74) and CD8+ % (r=-0.69). In conclusion, increased level of PTH in CRF patients on hemodialysis is associated with lymphopenia and reduction in CD4+ & CD8+ subsets of T cells. Monitoring circulating PTH levels in such patients can restore their immune competence.

CD54 is a surrogate marker of antigen presenting cell activation.

There is no single universally accepted hallmark of antigen presenting cell (APC) activation. Instead a variety of methods are used to identify APCs and assess their activation state. These activation measures include phenotypic methods [e.g., assessing the increased expression of surface markers such as major histocompatability (MHC) class II] and functional assays (e.g., evaluating the enhanced ability to take up and process antigen, or stimulate naïve T cells). Sipuleucel-T is an investigational autologous active cellular immunotherapy product designed to stimulate a T cell immune response against human prostatic acid phosphatase (PAP), an antigen highly expressed in prostate tissue. Sipuleucel-T consists of peripheral blood mononuclear cells (PBMCs), including activated APCs displaying epitopes of PAP. In order to develop a robust reproducible potency assay that is not hampered by MHC restriction we have developed a method to simply assess the biological activation of antigen presenting cells (APCs). In the course of sipuleucel-T characterization, we analyzed various phenotypic and functional parameters to define the activation state of APCs obtained from peripheral blood. Flow cytometric assays revealed that CD54+ cells are responsible for antigen uptake, and that expression of CD54 predominantly localizes to APCs. Costimulation, as measured by an allogeneic mixed lymphocytic reaction (alloMLR) assay, showed that activity was restricted to the CD54+ cell population. Similarly, CD54+ cells harbor all of the PAP-specific antigen presentation activity, as assayed using a PAP-specific HLA-DRbeta1-restricted T cell hybridoma. Finally we show that CD54 expression is substantially and consistently upregulated on APCs during culture with a GM-CSF fusion protein, and that this upregulation activity can be quantified. Thus these data support the use of CD54 upregulation as a surrogate for assessing human APC activation and validates its utility as a potency measure of sipuleucel-T.

Immunobiology of the Tomatine adjuvant.

Soluble or sub-unit protein vaccines alone are incapable of generating antigen-specific cellular immune responses. This failure can be attributed to the manner in which the immune system processes antigen; endogenous antigens are cycled through the MHC class I pathway to stimulate CD8+ restricted responses and exogenous antigens are processed through the MHC class II pathway to generate humoral immunity. Traditionally sub-unit vaccines have been formulated with adjuvants to enhance immunogenicity, however in the last decade a number of adjuvants have been developed that effectively stimulate the generation of both humoral and cellular immune responses, although the manner in which they exert their effects has not been investigated. Here we describe Tomatine, a glycoalkaloid based adjuvant, capable of stimulating potent antigen-specific humoral and cellular immune responses that contribute to protection against malaria, Francisella tularensis and regression of experimental tumors. Using in vivo models we investigated the manner in which cellular immune responses were generated by Tomatine. We established that Tomatine did not require either lymph node or splenic macrophages to generate cytotoxic T lymphocytes (CTL) and delivered soluble protein into a pathway not dependant on the machinery of the classical MHC class I pathway. We also observed that at the molecular level Tomatine required both CD80 and CD86 costimulation to engender antigen-specific cellular immunity.

Potentiation by a novel alkaloid glycoside adjuvant of a protective cytotoxic T cell immune response specific for a preerythrocytic malaria vaccine candidate antigen.

We have recently demonstrated that the novel glycoalkaloid tomatine, derived from leaves of the wild tomato Lycopersicon pimpinellifolium, can act as a powerful adjuvant for the elicitation of antigen-specific CD8+ T cell responses. Here, we have extended our previous investigation with the model antigen ovalbumin to an established malaria infection system in mice and evaluated the cellular immune response to a major preerythrocytic stage malaria vaccine candidate antigen when administered with tomatine. The defined MHC H-2kd class I-binding 9-mer peptide (amino acids 252-260) from Plasmodium berghei circumsporozoite (CS) protein was prepared with tomatine to form a molecular aggregate formulation and this used to immunise BALB/c (H-2kd) mice. Antigen-specific IFN-gamma secretion and cytotoxic T lymphocyte activity in vitro were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunised control mice. Moreover, when challenged with P. berghei sporozoites, mice immunised with the CS 9-mer-tomatine preparation had a significantly delayed onset of erythrocytic infection compared to controls. The data presented validate the use of tomatine to potentiate a cellular immune response to antigenic stimulus by testing in an important biologically relevant system. Specifically, the processing of the P. berghei CS 9-mer as an exogenous antigen and its presentation via MHC class I molecules to CD8+ T cells led to an immune response that is an in vitro correlate of protection against preerythrocytic malaria. This was confirmed by the protective capacity of the 9-mer-tomatine combination upon in vivo immunisation. These findings merit further work to optimise the use of tomatine as an adjuvant in malaria vaccine development.

Early nonspecific immune responses and immunity to blood-stage nonlethal Plasmodium yoelii malaria.

The early role of natural killer cells and gamma delta T cells in the development of protective immunity to the blood stage of nonlethal Plasmodium yoelii infection was studied. Splenic cytokine levels were measured 24 h after infection of natural killer cell-depleted immunodeficient and littermate mice or transiently T-cell-depleted normal mice. Splenic gamma interferon levels were significantly increased above background in immunodeficient and littermate mice 24 h after infection. Depletion of natural killer cells resulted in markedly depressed gamma interferon levels and poor control of parasitemia, particularly in severe combined immunodeficient mice. In the littermates, gamma interferon levels were partially reduced, but parasitemias were resolved normally. However, in athymic mice, natural killer cell depletion had no effect on gamma interferon production. Levels of tumor necrosis factor alpha were increased in all animals 24 h after infection, and responses were not affected by natural killer cell depletion. However, in T-cell-depleted animals, both gamma interferon and tumor necrosis factor alpha levels were decreased 24 h after infection, and depleted mice were unable to control their parasitemia. These results suggest that the early production of both cytokines is important in the early control of parasitemia and that both natural killer and gamma delta T cells contribute equally towards their production. The data also suggest that the subsequent resolution of infection requires early production of gamma interferon, which might act by switching on the appropriate T-helper-cell subsets and other essential parasitotoxic effector mechanisms.

Delivery systems for molecular vaccination.

Vaccination is one of the medical success stories of the 20th century, however, there are many diseases for which no prophylactic regimes are available. A major hindrance that has prevented the development of effective mass immunization programs is the inability to induce an appropriate, protective, immune response. For example, for vaccines against intracellular pathogens there is a requirement for cell-mediated immunity as characterized by cytolytic T-lymphocyte activity. However, such a response can be extremely difficult to elicit, especially those employing recombinant, soluble protein subunits. This deficiency is due to the inability of these antigens to access the machinery of the appropriate antigen-processing pathway. Following an improved understanding of the mechanisms underlying such processing, as well as the realization that delivery systems can affect, quantitatively and qualitatively, the resulting immune response, the last decade has witnessed an intense research effort in this field. In this article we will review the major developments in the area of antigen delivery as related to vaccination.

Novel aggregate structure adjuvants modulate lymphocyte proliferation and Th1 and Th2 cytokine profiles in ovalbumin immunized mice.

Cytokines are important mediators of effector lymphoid cell function during an immune response. The principal cytokine producers are the T helper (Th) cells and macrophages. Vaccine strategies need to take into account the balance of Th (Th1/Th2) cytokines they induce. Adjuvants are compounds that, when combined with an antigen, potentiate an immune response in an immunized species. The use of adjuvants has been shown to activate differentially Th1 and Th2 subsets. In this study we describe the immunopotentiating properties of three novel molecular aggregate formulations based on tomatine (RAM1), a glycosylamide lipid (RAM2) and a fifth generation dendrimeric polymer (RAM3) respectively. These formulations were evaluated for their ability to augment Th1 or Th2 cytokine responses when administered with a soluble protein antigen. Of the three formulations, RAM1 was found to induce predominantly Th1 cytokines; the levels of which were substantially higher than those induced by reference control adjuvants. It was also found that at a late post-vaccinated period, RAM1 can stimulate Th2 responses. In contrast, RAM2 and RAM3 induced cytokine profiles typically associated with Th2 responses.

Generation of antigen specific CD8+ cytotoxic T cells following immunization with soluble protein formulated with novel glycoside adjuvants.

Presentation of peptide on MHC class I molecules is essential to elicit cytolytic T cell (CTL) activity. Such peptides are a result of the cytosolic, or class I, antigen processing pathway. Due to the segregation of the class I and the exogenous processing pathway, soluble protein cannot enter the class I pathway and is thus incapable of inducing CTL. However careful formulation with adjuvants can overcome this obstacle. In this study we evaluated the capacity of two novel amphiphilic adjuvants, better termed delivery vehicles, to elicit CTL activity in a C57Bl/6 murine model with ovalbumin (OVA) as an antigen. Incomplete Freund's adjuvant and aluminium hydroxide (Alhydrogel) were used as reference adjuvants. In addition the oil-in-water emulsion Provax was used throughout as a positive control adjuvant. Both amphiphile preparations were capable of eliciting potent CTL activity after administration of one immunizing dose of ovalbumin. CTL were CD8+ restricted as assessed by in vitro depletion of CD8+ and CD4+ T cells. CTL activity was also MHC-restricted as well as specific for the H-2Kb OVA motif SIINFEKL.

Evaluation of novel aggregate structures as adjuvants: composition, toxicity studies and humoral responses.

Adjuvants are compounds that, when combined with an antigen, potentiate an immune response in an immunized species. There are numerous pathogens for which there are no protective vaccines and since alum is the only adjuvant licensed for use in humans, there is a clear need for more effective adjuvant preparations. In this study we describe the immunopotentiating properties of three novel molecular aggregate formulations based on tomatine (RAM1), a glycosylamide lipid (RAM2) and a fifth generation dendrimeric polymer (RAM3) respectively. These formulations were evaluated for their ability to augment antigen-specific antibody responses when administered with a soluble protein antigen. All three adjuvants were shown to be nontoxic to mice and elicited antigen-specific antibody responses. Of the three formulations, RAM1 was found to induce the highest titers of antibody; these were substantially higher than those induced by reference control adjuvants. RAM1 elicited antibodies of the IgG1 and IgG2a subclasses indicating, indirectly, that this adjuvant can stimulate Th2 and Th1 type immunity.

Structural similarities among malaria toxins insulin second messengers, and bacterial endotoxin.

Malaria toxin causes hypoglycemia and induction of tumor necrosis factor. Extracts of parasitized erythrocytes which were coeluted and copurified with one of the two subtypes of mammalian insulin-mimetic inositolphosphoglycans similarly induced fibroblast proliferation in the absence of serum. In addition, induction of tumor necrosis factor in macrophages by malaria toxin and by lipopolysaccharide from Escherichia coli was enhanced by pretreatment of these toxins with alpha-galactosidase. Thus, parasitized erythrocytes contain both soluble inositolphosphoglycan-like insulin second messengers and endotoxin-like lipidic molecules.

Malaria: a tumour necrosis factor inhibitor from parasitized erythrocytes.

The excessive production of tumour necrosis factor (TNF) is associated with the pathology of blood-stage malaria and phosphatidylinositol-containing phospholipid antigens from parasitized erythrocytes stimulate its secretion by macrophages, thus acting as toxins. This brief report describes some properties of an inhibitor present in lysates from erythrocytes infected with malarial parasites that blocked the detection of recombinant TNF in an enzyme-linked immunosorbent assay and diminished or abolished the cytotoxicity of TNF. It was not found in control lysates of normal erythrocytes. Its addition to macrophage cultures stimulated by toxic malarial preparations or by bacterial lipopolysaccharide also blocked the detection of TNF. These findings may explain the contradictory results obtained from different assays for TNF, and emphasize the need for caution when interpreting the results of a single assay system. If released when parasitized erythrocytes rupture in vivo, the inhibitor could help protect both parasite and host from the damaging effects of TNF.

A multi-centre study of sulindac versus naproxen in the treatment of elderly osteoarthritic patients.

Ninety-five geriatric patients with osteoarthritis of the hip and/or knee were entered into an open parallel group study of sulindac (400 mg/day) and naproxen (500 mg/day), both given in twice-daily regimens. Patients received the drugs for 12 weeks. Both drugs produced improvements in the patients' overall condition. There were no statistically significant differences between the effects of the two drugs. Overall, both drugs proved beneficial and well tolerated.