Multi-Component Exercise with High-Intensity, Free-Weight, Functional Resistance Training in Pre-Frail Females: A Quasi-Experimental, Pilot Study.

BACKGROUND: No study has performed an exercise intervention that included high-intensity, free-weight, functional resistance training, and assessed frailty status as an inclusion criteria and outcome measure via original, standardized tools, in pre-frail females. OBJECTIVES: Determine if the intervention strategy is not only feasible and safe, but can also improve frailty status, functional task performance, and muscle strength. DESIGN: Pilot, quasi-experimental. SETTING: Community. PARTICIPANTS: 20 older-adults with pre-frailty characteristics. INTERVENTION: 12-weeks (3 days/week, 45-60 minutes/session) of multi-component exercise, inclusive of aerobic, resistance, balance and flexibility exercises. The crux of the program was balance and resistance exercises, the latter utilized high-intensity, free-weight, functional resistance training. The control group maintained their usual care. MEASUREMENTS: 1) Feasibility and safety (dropout, adherence, and adverse event); 2) Frailty (Frailty Phenotype, Clinical Frailty Scale, and gait speed); 3) Functional task performance (grip strength and sit-to-stand time); and 4) Isometric and isotonic strength of the knee extensors and elbow flexors. RESULTS: No participants dropped out of the intervention or experienced an adverse event, and adherence averaged 88.3%. The exercise group became less frail, whereas the control group became more frail. There was a significant within-group improvement in exercise participants gait speed (p ≤ 0.01, +0.24 m/sec), grip strength (p ≤ 0.01, +3.9 kg), and sit-to-stand time (p ≤ 0.01, -5.0 sec). There was a significant within-group improvement in exercise participants knee extension isometric torque (p ≤ 0.05, +7.4 Nm) and isotonic velocity (p = ≤ 0.01, +37.5 ˚/sec). Elbow flexion isotonic velocity significantly declined within the control group (p ≤ 0.01, -20.2 ˚/sec) and demonstrated a significant between-group difference (p ≤ 0.05, 40.73 ˚/sec) post-intervention. CONCLUSIONS: The intervention strategy appears to be feasible and safe, and may also improve frailty status, functional task performance, and muscle strength. These results help calculate effect size for a future randomized controlled trial.

Practical Implications for Strength and Conditioning of Older Pre-Frail Females.

Approaches to and benefits from resistance training for non-compromised older adults are well known. Less is understood about resistance training with pre-frail older adults, and even less information is available on the practical approaches to delivery. Herein, we describe an approach in pre-frail females who undertook a multi-component exercise intervention, inclusive of high-intensity, free-weight, functional resistance training. Capitalizing on the principle of overload is possible and safe for pre-frail females through constant reassurance of ability and adjustments in technique. Making exercise functionally relevant, for example, a squat is the ability to get on and off a toilet, resonates meaning. Older pre-frail females are affected by outside (clinical) influences. The exercise participant, and extraneous persons need to be educated on exercise approaches, to increase awareness, debunk myths, and enhance support for participation. Identification of individuality in a group session offers ability to navigate barriers for successful implementation.

Adherence to controller asthma medications: 6-month prevalence across a US community pharmacy chain.

WHAT IS KNOWN AND OBJECTIVE: Non-adherence to controller asthma medications is an important public health problem. It is estimated to occur in 30-70% of individuals and is a significant risk factor for asthma morbidity and mortality. The aim of this study was to determine the level of adherence, as indicated by refill rates, to controller asthma medications in a community pharmacy setting. METHODS: Secondary analyses of a community pharmacy dispensing database in 15 locations throughout Utah. RESULTS AND DISCUSSION: The dispensing records of 2193 patients who received controller medications for asthma in a 12-month period, and had a minimum of 6-month potential coverage (180 days) from the date of their first receipt of a controller medication in that period, were examined. Using standard metrics to gauge adherence, the proportion of days covered (PDC) and the medication possession ratio (MPR), the average coverage for controller asthma medications across a 6-month period (180 days) was poor, averaging less than 50% of days' availability. Standard cut-offs (≥80% medication availability) indicated that only 14-16% of patients had 'satisfactory' adherence over their 6-month follow-on period. Females and older patients had significantly greater satisfactory adherence. Medication adherence was significantly greater with inhaled corticosteroid (ICS)-long-acting ß2 -agonist (LABA) combinations than with ICS alone. WHAT IS NEW AND CONCLUSION: This study confirms the considerable scope of the asthma therapy non-adherence problem. Therefore, it is imperative to conduct survey-based research linked directly to pharmacy-based dispensing data to derive patient behavioural, attitudinal and environmental factors that may contribute to the issue, and then pilot and evaluate interventions for change.

MicroRNA expression profiling of PPD-B stimulated PBMC from M. bovis-challenged unvaccinated and BCG vaccinated cattle.

There is an urgent need to identify additional diagnostic biomarkers for bovine TB to complement existing read-out systems such as interferon-gamma and for predictive markers of vaccine efficacy to accelerate vaccine development. To evaluate the potential of miRNAs as such biomarkers, we have analysed their expression in bovine PPD stimulated PBMC isolated from unvaccinated and BCG vaccinated cattle before and following Mycobacterium bovis (M. bovis) infection. Using a bovine microRNA microarray, miR-155 was found to show a significant up-regulation in expression in early (week 2) and late (week 11) M. bovis post-infection samples from unvaccinated cattle, while in BCG vaccinated cattle up-regulation was observed only in late post-infection samples. No differential expression of miR-155 was observed in pre-infection samples from unvaccinated and vaccinated cattle. These observations suggest that miR-155 could be exploited as a marker distinguishing vaccinated from infected animals (DIVA). Analysis by TaqMan RT-PCR, verified the up-regulation of miR-155 in unvaccinated cattle post-infection. Significant correlation was found between the degree of pathology and miR-155 induction in the experimentally infected cattle, suggesting miR-155 is a biomarker of disease development and/or severity. Induction of miR155 expression in cattle sourced from farms with confirmed bTB that tested positive in the tuberculin skin or interferon-gamma blood test was found to be significantly higher in cattle presenting with more advanced pathology (defined by the presence of visible TB lesions) compared to infected cattle without visible pathology and thus likely to be of lower infectivity than those with more advanced disease. In conclusion, our data indicate that miR-155 has potential both as a diagnostic and prognostic biomarker that could be used to identify animals with advanced pathology and as a DIVA test read-out. Its role in the immune biology of bovine TB will also be discussed.

Reduction in inflammation in the footpad of carrageenan treated mice following the topical administration of anti-TNF molecules formulated in a micro-emulsion.

Water-in-oil micro-emulsions (ME) have been shown to have the ability to deliver water-soluble peptides and proteins into the skin. Topical administration of these formulations represents an ideal means of device-free delivery of these pharmaceutical agents. Topically administered anti-tumor necrosis factor (TNF) monoclonal antibodies formulated within a water-in-oil micro-emulsion were found to have similar pharmaceutical activity to control formulations that were administered by injection. This form of delivery presents itself as an exciting, pain-free and device-free delivery system for administration of protein pharmaceuticals and as such has enormous potential for the delivery of peptides and protein into the dermal layer of the skin.

A novel vitamin B12-nanosphere conjugate carrier system for peroral delivery of insulin.

In spite of great potential, effective oral delivery of many vitamin B(12)-peptide/protein drug conjugates does not occur due to the limited uptake capacity of the VB(12) transport system, loss of bioactivity of native protein and/or intrinsic factor affinity of VB(12) and liability to GI degradation. In order to overcome these shortcomings in a two pronged way, we have endeavoured to develop a VB(12)-Nanoparticles (NPs) system to enhance the uptake capacity of both NPs and VB(12) transport to deliver orally effective insulin. NPs were prepared using different molecular weight dextrans and epichlorohydrin as cross-linker by an emulsion method. NPs surface was modified with succinic anhydride, and conjugated with amino VB(12) derivatives of carbamate linkage. VB(12) attachment was confirmed by IR, XPS analysis, and was quantified by HPLC (4.0 to 4.4% w/w of NPs). The pre-formed NPs conjugates (Zave=160-250 nm; polydisperse) were loaded with 2, 3 and 4% w/w of insulin, and the entrapment was found to be 45-70%. NPs conjugates were found to protect 65-83% of entrapped insulin against in vitro gut proteases. In vitro release studies exhibit an initial burst followed by diffusion controlled first order kinetics with 75-95% release within 48 h. After oral administration of these carriers (20 IU/kg), a nadir of 70-75% reduction in plasma glucose was found in 5 h, reached basal levels in 8-10 h, and a prolonged second phase was found until 54 h. The % pharmacological availability (PA) of 70 K NPs conjugate containing 2, 3 and 4% w/w insulin was 1.1, 1.9 and 2.6 fold higher, respectively compared to NPs without VB(12); consistent with the hypothesis that uptake was mediated by the vitamin B(12) transport. NPs of 70 K dextran showed 1.4 fold PA compared to 10 K while negligible action was observed with 200 K. The potential utilities of VB(12)-NPs carrier as an oral delivery platform of proteins, especially insulin via dextran-coated particles necessities further elaborate investigations.

Use of targeting agents to increase uptake and localization of drugs to the intestinal epithelium.

Despite early successes in oral drug delivery of small molecules such as antibiotics, cephalosporins and vitamins, little success has been achieved with more recent pharmaceuticals such as peptides and proteins. This lack of success is primarily due to the impenetrable nature of the gastro-intestinal epithelial cell layers. Despite this, many bacteria, viruses and toxins are readily taken up from the intestine and thereby gain entry into the circulation. This review focuses on the use of various surface molecules from these organisms as well as toxin binding subunits as carriers for oral delivery of other molecules. It also describes the subversion of the natural uptake mechanisms for various vitamins and iron to allow for oral delivery of pharmaceuticals. These mechanisms provide exciting solutions to overcome the problems in oral delivery of peptides and proteins, which has been the nemesis of pharmaceutical scientists for many decades.

Comparison of biomarker and pathological responses in flounder (Platichthys flesus L.) induced by ingested polycyclic aromatic hydrocarbon (PAH) contamination.

This study investigated the response of biomarker measurements and histopathological indicators of polycyclic aromatic hydrocarbon (PAH) exposure in the flounder (Platichthys flesus L.). Flounder were fed food spiked with a mixture of four PAHs at an environmentally relevant range of concentrations for either one or six months. Ethoxyresorufin-O-deethylase (EROD) activity was elevated following 1 month exposure to PAH concentrations up to 50 mgkg(-1) in food. Bile metabolite concentrations were found to increase with PAH concentration, up to 500 mgkg(-1) PAH. By comparison, no DNA adducts were detected and there were no significant histopathological changes observed. After 6 months exposure, EROD levels were not elevated but bile metabolites showed a similar dose dependent relationship as in the 1 month experiment, while DNA adducts were only detected in the highest PAH exposure groups. No significant histopathological changes were observed. The results are discussed with respect to the implications for the use of these methods in environmental monitoring studies.

Ingestion of toxic Microcystis aeruginosa by dairy cattle and the implications for microcystin contamination of milk.

Microcystin (MCYST) toxins can be produced by the bloom-forming cyanobacterium Microcystis aeruginosa. They are chemically stable compounds and have both acute and chronic effects on the health of mammals, including cattle and humans. Cattle will drink water containing lethal cell concentrations of M. aeruginosa. When cattle consume sub-lethal doses of microcystins, the fate of those toxins is unknown. We provided drinking water containing 1 x 10(5) cells ml(-1) M. aeruginosa (strain MASH01-A19) to four lactating Holstein-Friesian dairy cattle for 21 days to determine if MCYST-LR produced by the cyanobacteria, could be detected in milk produced by the cattle. Cattle consumed up to 15 mg MCYST-LR at an ingestion rate of 1.21 microg kg (live weight) d(-1). Analysis by HPLC and ELISA indicated that no detectable amounts of microcystin from the cyanobacteria were present in the milk obtained from the treated animals. Based on the level of quantitation of the ELISA analyses, the maximum possible concentration in the milk was less than 2 ng l(-1). This is more than three orders of magnitude less that the concentration that could be considered problematic for milk of 0.86 microg l(-1) which we calculated using the World Health Organization derived tolerable daily intake for MCYST-LR and the per capita daily consumption of milk in Australia.

Comparative loss and maturation of peripheral blood dendritic cell subpopulations in African and non-African HIV-1-infected patients.

OBJECTIVES: To quantify the percentage of the two major subpopulations of blood dendritic cells (DC) in HIV-1-seropositive Ugandan individuals infected with non-clade B viruses and compare this with that seen in clade B HIV-1 infected non-African individuals. DC maturation/activation status was also investigated via the expression of CD86. METHODS: The percentage of blood DC was quantified by using flow cytometry. DC were identified as the lineage (CD3, CD14, CD16, CD19, CD20, CD56)-negative, HLA-DR-positive population and the two major subpopulations were differentiated by CD11c expression. RESULTS: The percentage of blood DC was reduced significantly in HIV-1-seropositive African individuals when compared with controls (0.21 and 0.39% respectively). A similar reduction was also seen in non-African patients residing in the UK (0.19% compared with 0.36% for controls). However, there was no selective loss in either CD11c-positive or CD11c-negative subpopulations. The percentage of blood DC expressing CD86 was significantly greater in HIV-1-seropositive individuals when compared with controls and the increased expression was largely confined to CD11c-negative DC. CONCLUSIONS: Africans infected with non-clade B HIV-1 showed similar reductions in the percentage of blood DC to non-Africans infected with clade B viruses. There was no selective loss of either DC subpopulation, suggesting that the ability of DC to acquire and present antigens or to produce interferon-alpha may both be impaired in HIV-1 infection.

Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate.

Cell quotas of microcystin (Q(MCYST); femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q(MCYST) and specific growth rate (mu), from which we propose a generalized model that enables Q(MCYST) at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q(MCYST) from mu, mu(max) (maximum specific growth rate), Q(MCYSTmax) (maximum cell quota), and Q(MCYSTmin) (minimum cell quota). Under the conditions examined in this study, we predict a Q(MCYSTmax) of 0.129 fmol cell(-1) at mu(max) and a Q(MCYSTmin) of 0.050 fmol cell(-1) at mu = 0. Net MCYST production rate (R(MCYST)) asymptotes to zero at mu = 0 and reaches a maximum of 0.155 fmol cell(-1) day(-1) at mu(max). MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with mu, whereas the MCYST/protein ratio reached a maximum at intermediate mu. In contrast, the MCYST/Chl a ratio remained constant. Cell volume correlated negatively with mu, leading to an increase in intracellular MCYST concentration at high mu. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.

Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR.

A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086-4094). We now report the cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. The heterologously expressed degradation pathway proteins are enzymatically active. Microcystinase (MlrA), the first enzyme in the degradative pathway, is a 336-residue endopeptidase, which displays only low sequence identity with a hypothetical protein from Methanobacterium thermoautotrophicum. Inhibition of microcystinase by EDTA and 1,10-phenanthroline suggests that it is a metalloenzyme. The most likely residues that could potentially chelate an active-site transition metal ion are in the sequence HXXHXE, which would be unique for a metalloproteinase. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442 residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2 family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402 residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the "penicillin-binding enzyme" family of active site serine hydrolases. The final gene in the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.

Oral absorption of peptides through the cobalamin (vitamin B12) pathway in the rat intestine.

PURPOSE: This study was aimed at examining the extent and mechanism of uptake of cobalamin (Cbl)-conjugated peptides in vitro and in vivo. METHODS: To enable acquisition of quantitative absorption data of Cbl-peptides, metabolically stable octapeptides (DP3), with (Cbl-Hex-DP3) or without a hexyl spacer (Cbl-DP3), were coupled to Cbl and radiolabeled. For comparison, LHRH coupled to Cbl was used as metabolically susceptible peptide. Biological recognition of Cbl-peptides was studied in the physiological order: binding by Intrinsic Factor (IF), recognition and transport of the IF-complexes by IF-Cbl receptors (IFCR) on Caco-2 monolayers and oral absorption of the Cbl-conjugates in the rat. RESULTS: All Cbl-peptides bound to IF and the IF-complexes were recognized by IFCR receptors on Caco-2 monolayers. Binding was saturable and could be inhibited by a 20-fold excess of IF-Cbl, but not of Non-intrinsic Factor (NIF)-Cbl. Oral administration of these ligands to rats resulted in absorption of 53%, 45%, 42%, and 23% of the applied radioactivity for Cbl, Cbl-LHRH, Cbl-Hex-DP3, and Cbl-DP3, respectively. Simultaneous administration of a >10(5)-fold excess of unlabeled Cbl reduced uptake of all compounds to <4%. Tissue distribution and elimination of the metabolically stable Cbl-conjugates were comparable to Cbl. CONCLUSIONS: The endogenous Cbl uptake pathway can be exploited for oral peptide delivery as indicated by the specific and high (40-45%) uptake of metabolically stable Cbl-coupled octapeptides.

Anti-CD4-binding domain antibodies complexed with HIV type 1 glycoprotein 120 inhibit CD4+ T cell-proliferative responses to glycoprotein 120.

HIV-specific CD4+ helper T cell responses, particularly to the envelope glycoproteins, are usually weak or absent in the majority of HIV-seropositive individuals. Since antibodies, by their capacity to alter antigen uptake and processing, are known to have modulatory effects on CD4+ T cell responses, we investigated the effect of antibodies produced by HIV-infected individuals on the CD4+ T cell response to HIV-1 gp120. Proliferative responses of gp120-specific CD4+ T cells were inhibited in the presence of either serum immunoglobulin from HIV-infected individuals or human monoclonal antibodies specific for the CD4-binding domain (CD4bd) of gp120. Human monoclonal antibodies to other gp120 epitopes did not have the same effect. The anti-CD4bd antibodies complexed with gp120 suppressed T cell lines specific for varying gp120 epitopes but did not affect T cell proliferation to non-HIV antigens. Moreover, inhibition by the anti-CD4bd/gp120 complexes was observed regardless of the types of antigen-presenting cells used to stimulate the T cells. These results indicate that the presence of anti-CD4bd antibodies complexed with gp120 can strongly suppress CD4+ helper T responses to gp120.

Functional expression in Pichia pastoris of human and rat intrinsic factor.

Intrinsic factor (IF) has been expressed previously in Baculovirus with a yield (0.1-1 mg/l) that was inadequate for structural and metabolic studies. IF cDNAs were cloned into the shuttle vector pPIC9 of P. pastoris, and the proteins were induced and purified by cobalamin (Cbl) affinity chromatography. Expression of recombinant proteins revealed a major band of 49 kDa for both human and rat IF. Expression of human IF was achieved at 1040 mg/l, but of rat IF at only 1-2 mg/l. Reaction of human IF with a photo-activatable derivative of Cbl was demonstrated by Western blotting, and detection of IF fragments by anti-Cbl monoclonal antibody and by amino-terminal sequencing revealed at least three regions (residues 129-151, 234-254, and +294) linked to Cbl. Both recombinant human and rat [125I]IF-Cbl bound to rat and guinea pig brush border membranes with similar affinity, but the binding capacity of human IF for the rat receptor was only 10% compared with rat IF. All six amino acids within the previously identified N-terminal binding region of human IF were mutated to be identical to rat IF, but the resulting chimeric IF still bound poorly to rat membranes. Mutations of residues 26/27 (Glu26 to Asp and Asn27 to Gln) and 32/34 (Ser32 to Thr and Tyr34 to Arg) showed changes in both Ka and Vmax, with great effects on Vmax. In conclusion, P. pastoris is an expression system that produces functional human IF at a higher yield than in the baculovirus system. Cbl binding was directly demonstrated at multiple sites along the linear sequence of human IF. The receptor binding function of the amino terminal sequence 25 62 has been confirmed, but it is insufficient to reproduce all the features of IF-Cbl binding.

Oral vaccine delivery.

Oral vaccination of animals and man, to provide effective mucosal and/or systemic immunity, is largely ineffective. This is due mainly to the very small quantity of antigen that survives degradation in the intestine and that crosses the intestinal wall. Over the past decade or so, a number of proteins have been identified that are effective at eliciting mucosal and systemic immune responses following oral administration. Uptake of these molecules by the gastro-intestinal tract (GIT) epithelium is dependent upon specific binding to the GIT epithelial cells. The identity of these molecules is discussed, as well as their possible application as 'carriers' for co-transporting haptens, proteins and nanoparticles across the GIT epithelium.

Synthesis and biological activity of ribose-5'-carbamate derivatives of vitamin B(12).

Twelve biologically active derivatives of vitamin B(12) (cyanocobalamin) have been synthesized in which spacers were attached to the ribose-5'-hydroxyl group of vitamin B(12). Their potential to act as oral delivery agents for proteins, nanospheres, or immunogens using the vitamin B(12) uptake system was evaluated by determining their affinity for intrinsic factor (IF) and non-IF. The ribose-5'-hydroxyl group of vitamin B(12) was activated through the use of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyldi(1,2, 4-triazole) (CDT), or di(1-benzotriazolyl) carbonate (DBTC). Subsequent addition of an aminoalkane, diaminoalkane, or alkane diacid dihydrazide gave rise to vitamin B(12) derivatives suitable for attachment to various proteins, peptides, or nanospheres to enable oral delivery utilizing the vitamin B(12) uptake system. The ribose-5'-carbamate derivatives were found to possess similar affinity for intrinsic factor as that of the e-monocarboxylic acid of vitamin B(12). The affinity for non-IF was similar to cyanocobalamin or even higher for some of the smaller derivatives. Polysciences nanoparticles derivatized with vitamin B(12) 5'-carbamate adipic dihydrazide into CaCo-2 cells showed significantly higher levels of transport of the particles, when compared to unmodified particles.

Lectin-mediated transport of nanoparticles across Caco-2 and OK cells.

Recent experiments by a number of workers have suggested that it may be possible to use various targeting molecules, which bind to the intestinal epithelium, to promote the uptake and transport of nanoparticles from the intestine to the circulation. We have used commercial nanoparticles to examine the effect of size, density and inhibitors on uptake of lectin-coated nanoparticles by epithelial cells. The degree of uptake was most influenced by the density of lectin on the particle, with size and type of lectin being less important. Uptake could be inhibited by the presence of specific sugars or free lectin. These studies should provide a good basis for the design of targetable biodegradable drug-loadable particles suitable for oral delivery.