Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.

In this review the current knowledge of protein degradation during preparation, storage and release from poly(lactic-co-glycolic acid) (PLGA) microparticles is described, as well as stabilization approaches. Although we have focussed on PLGA microparticles, the degradation processes and mechanisms described here are valid for many other polymeric release systems. Optimized process conditions as well as stabilizing excipients need to be used to counteract several stress factors that compromise the integrity of protein structure during preparation, storage, and release. The use of various stabilization approaches has rendered some success in increasing protein stability, but, still, full preservation of the native protein structure remains a major challenge in the formulation of protein-loaded PLGA microparticles.

BACKGROUND

Most urticarias are induced by vasoactive mediators such as histamine released from mast cells. Although mast cells are activated by allergens through cross-linking of cell-surface--bound IgE, this mechanism does not appear to explain most cases of chronic urticaria, which, when allergic, infectious, drug-induced, or physical causes cannot be identified, are classified as idiopathic.

METHODS

We recruited 26 patients with chronic idiopathic urticaria, in whom intradermal injection of autologous serum caused a wheal-and-flare response. Serum from four patients that induced marked histamine release from basophils from a donor with very low serum IgE levels was studied with respect to the IgE dependence of the histamine release, the activity of the IgG fractions, and the neutralizing effect of a recombinant preparation of the soluble extracellular domain of the alpha subunit of the high-affinity IgE receptor (sFc epsilon RI alpha).

RESULTS

The histamine-releasing activity of the serum was abolished by passive sensitization of basophils with myeloma IgE, enhanced after dissociation of IgE by treatment with lactic acid, and induced by IgG fractions from the serum of all four patients. Preincubation of the serum and isolated IgG with sFc epsilon RI alpha resulted in almost complete neutralization.

CONCLUSIONS

Histamine-releasing IgG autoantibodies against the alpha subunit of the high-affinity IgE receptor are present in the circulation of some patients with chronic urticaria. Autoantibody-induced cross-linking of IgE receptors may be an important mechanism in the pathogenesis of chronic urticaria and other diseases mediated by mast cells.

Static muscular contraction has been firmly established to reflexly increase cardiovascular and ventilatory function. Although group III and IV fibers with endings in muscle have been shown to comprise the afferent arm of this reflex arc, little is known about the nature of the contraction-induced stimulus causing the activation of these fibers. This stimulus has often been suggested to be a metabolic product of muscular contraction. We have therefore recorded the impulse activity of group III and IV afferents with endings in the triceps surae muscles of barbiturate-anesthetized cats while we injected into the femoral artery substances believed to be metabolic products of muscular contraction. We found that lithium and sodium lactate (400 mM; 1 ml) had little or no effect on the discharge of group III and IV afferents. Likewise, monobasic sodium phosphate (20 and 400 mM; 1 ml) and 2-chloroadenosine (50-100 micrograms) had only trivial effects on the discharge of these afferents. By contrast, lactic acid (25 and 400 mM; 1 ml) and arachidonic acid (0.5-2.0 mg) caused significant increases in the activity of group III and IV afferents. Most of the excitatory effect of arachidonic acid on the discharge of the afferents was prevented by indomethacin, a cyclooxygenase inhibitor. We conclude that of the substances tested in our experiments, lactic acid and some cyclooxygenase products, such as prostaglandins and thromboxanes, are the most likely to be responsible for any metabolic stimulation of group III and IV afferents during muscular contraction.

A tumorigenic anchorage-dependent cell line (H-91) was established in culture from an azo-dye-induced rat ascites hepatoma. When grown in a glucose-containing medium the cells exhibit high rates of lactic acid production characteristic of rapidly growing tumor cells. However, when glucose is replaced with galactose the cells grow equally well but exhibit only moderately elevated rates of lactic acid production. The molecular basis for this observation cannot be attributed to differences in permeability because initial rates of glucose and galactose entry into hepatoma cells are identical. Rather, the activity of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is found to be high in hepatoma cells, about 20-fold higher than that of control and regenerating rat liver. Moreover, tumor hexokinase activity is not inhibited by low concentrations (<0.6 mM) of the reaction product glucose 6-phosphate. Additionally, 50% of the hexokinase activity of hepatoma cells is found associated with the mitochondrial fraction. This fraction is 3-fold enriched in hexokinase activity relative to the homogenate and 4-fold enriched relative to the nuclear and postmitochondrial fractions. Tumor mitochondrial hexokinase appears to be coupled directly to oxidative phosphorylation, because addition of glucose to respiring hepatoma mitochondria (after a burst of ATP synthesis) results in stimulation of respiration. In contrast, glucose has no effect on the respiration of mitochondria from control and regenerating liver. These results suggest that the high glycolytic capacity of H-91 hepatoma cells is due, at least in part, to an elevated form of hexokinase concentrated in the mitochondrial fraction of the cell.

A recent demonstration of markedly reduced (-50%) activity of cytochrome oxidase (CO; complex 4), the terminal enzyme of the mitochondrial enzyme transport chain, in platelets of patients with Alzheimer's disease (AD) suggested the possibility of a systemic and etiologically fundamental CO defect in AD. To determine whether a CO deficiency occurs in AD brain, we measured the activity of CO in homogenates of autopsied brain regions of 19 patients with AD and 30 controls matched with respect to age, postmortem time, sex, and, as indices of agonal status, brain pH and lactic acid concentration. Mean CO activity in AD brain was reduced in frontal (-26%: p less than 0.01), temporal (-17%; p less than 0.05), and parietal (-16%; not significant, p = 0.055) cortices. In occipital cortex and putamen, mean CO levels were normal, whereas in hippocampus, CO activity, on average, was nonsignificantly elevated (20%). The reduction of CO activity, which is tightly coupled to neuronal metabolic activity, could be explained by hypofunction of neurons, neuronal or mitochondrial loss, or possibly by a more primary, but region-specific, defect in the enzyme itself. The absence of a CO activity reduction in all of the examined brain areas does not support the notion of a generalized brain CO abnormality. Although the functional significance of a 16-26% cerebral cortical CO deficit in human brain is not known, a deficiency of this key energy-metabolizing enzyme could reduce energy stores and thereby contribute to the brain dysfunction and neurodegenerative processes in AD.

Acute diarrhoea is a serious cause of infant morbidity and mortality, and the development of preventive measures remains an important goal. Bifidobacteria (which constitute the predominant intestinal flora of breastfed infants), as well as other lactic-acid-producing organisms such as Streptococcus thermophilus, are thought to have a protective effect against acute diarrhoeal disease. However, their efficacy has not been assessed in controlled trials. In a double-blind, placebo-controlled trial, infants aged 5-24 months who were admitted to a chronic medical care hospital were randomised to receive a standard infant formula or the same formula supplemented with Bifidobacterium bifidum and S thermophilus. Patients were evaluated daily for occurrence of diarrhoea, and faecal samples, obtained weekly, were analysed for rotavirus antigen by enzyme immunoassay. Faecal samples were also obtained during an episode of diarrhoea for virological and bacteriological analyses. 55 subjects were evaluated for a total of 4447 patient-days during 17 months. 8 (31%) of the 26 patients who received the control formula and 2 (7%) of 29 who received the supplemented formula developed diarrhoea during the course of the study (p = 0.035, Fisher's exact test, two-tailed). 10 (39%) of the subjects who received the control formula and 3 (10%) of those who received the supplemented formula shed rotavirus at some time during the study (p = 0.025). The supplementation of infant formula with B bifidum and S thermophilus can reduce the incidence of acute diarrhoea and rotavirus shedding in infants admitted to hospital.

Damage to peripheral nerves often cannot be repaired by the juxtaposition of the severed nerve ends. Surgeons have typically used autologous nerve grafts, which have several drawbacks including the need for multiple surgical procedures and loss of function at the donor site. As an alternative, the use of nerve guidance channels to bridge the gap between severed nerve ends is being explored. In this paper, the electrically conductive polymer--oxidized polypyrrole (PP)--has been evaluated for use as a substrate to enhance nerve cell interactions in culture as a first step toward potentially using such polymers to stimulate in vivo nerve regeneration. Image analysis demonstrates that PC-12 cells and primary chicken sciatic nerve explants attached and extended neurites equally well on both PP films and tissue culture polystyrene in the absence of electrical stimulation. In contrast, PC-12 cells interacted poorly with indium tin oxide (ITO), poly(L-lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) surfaces. However, PC-12 cells cultured on PP films and subjected to an electrical stimulus through the film showed a significant increase in neurite lengths compared with ones that were not subjected to electrical stimulation through the film and tissue culture polystyrene controls. The median neurite length for PC-12 cells grown on PP and subjected to an electrical stimulus was 18.14 micron (n = 5643) compared with 9.5 micron (n = 4440) for controls. Furthermore, animal implantation studies reveal that PP invokes little adverse tissue response compared with poly(lactic acid-co-glycolic acid).

Stimulating the effector functions of tumor-infiltrating T lymphocytes (TIL) in primary and metastatic tumors could improve active and adoptive T-cell therapies for cancer. Abnormal glycolysis, high lactic acid production, proton accumulation, and a reversed intra-extracellular pH gradient are thought to help render tumor microenvironments hostile to roving immune cells. However, there is little knowledge about how acidic microenvironments affect T-cell immunity. Here, we report that lowering the environmental pH to values that characterize tumor masses (pH 6-6.5) was sufficient to establish an anergic state in human and mouse tumor-specific CD8(+) T lymphocytes. This state was characterized by impairment of cytolytic activity and cytokine secretion, reduced expression of IL-2Rα (CD25) and T-cell receptors (TCR), and diminished activation of STAT5 and extracellular signal-regulated kinase (ERK) after TCR activation. In contrast, buffering pH at physiologic values completely restored all these metrics of T-cell function. Systemic treatment of B16-OVA-bearing mice with proton pump inhibitors (PPI) significantly increased the therapeutic efficacy of both active and adoptive immunotherapy. Our findings show that acidification of the tumor microenvironment acts as mechanism of immune escape. Furthermore, they illustrate the potential of PPIs to safely correct T-cell dysfunction and improve the efficacy of T-cell-based cancer treatments.

Poly(lactic-co-glycolic) acid (PLGA) has attracted considerable interest as a base material for biomedical applications due to its: (i) biocompatibility; (ii) tailored biodegradation rate (depending on the molecular weight and copolymer ratio); (iii) approval for clinical use in humans by the U.S. Food and Drug Administration (FDA); (iv) potential to modify surface properties to provide better interaction with biological materials; and (v) suitability for export to countries and cultures where implantation of animal-derived products is unpopular. This paper critically reviews the scientific challenge of manufacturing PLGA-based materials with suitable properties and shapes for specific biomedical applications, with special emphasis on bone tissue engineering. The analysis of the state of the art in the field reveals the presence of current innovative techniques for scaffolds and material manufacturing that are currently opening the way to prepare biomimetic PLGA substrates able to modulate cell interaction for improved substitution, restoration, or enhancement of bone tissue function.

Lactobacillus and bifidobacterial cultures are increasingly used as probiotics in pharmaceuticals and in foods. The selection of strains is performed often for technological rather than for microecological reasons. Detailed reports about species and strains composition of these microorganisms in the intestinal microflora of man are rare. Our investigations were performed with samples originating from infants and adults, taken from faeces and from upper sections of the intestinal tract including mouth and stomach, and from caecum and colon. Post mortem cases as well as test subjects under physiological conditions were analyzed using an automatic capsule system sampling at defined times in different parts of the intestinal tract. The fate of selected strains after oral intake was studied, too. Furthermore, influences of the microflora originating from food were considered. The identification of autochthonous (indigenous) and allochthonous (transient) species could be achieved with descriptions of new species in the genera Lactobacillus and Bifidobacterium. L. gasseri and L. reuteri proved to be predominant autochthonous Lactobacillus species in infants as well as in adults. Both species were occasionally present even in the stomach. This was also the case with an anaerobic lactic acid bacterium, previously named Catenabacterium catenaforme, later classified as L. ruminis, a non-motile variant of this species. The bifidobacterial microflora differed in composition between infants and adults and in different stages of the host's life. Up to 5 species or special strains of bifidobacteria could be present in different, individually fixed, combinations. Species typical for infants were B. bifidum, B. infantis, B. breve, and B. parvulorum. Typical for adults were 4 different variants of B. adolescentis. B. bifidum and B. longum could often be found in both groups, but in lower numbers. B. longum showed some oxygen tolerance whereas B. bifidum and B. adolescentis required strict anaerobic and fastidious conditions for cultivation. The autochthonous Lactobacillus and Bifidobacterium microflora in man will remain stable life-long. With lactobacilli, however, some successions may be caused by transient species derived from food or from the oral cavity, thus giving the impression of an altered microflora. Nevertheless L. gasseri, L. reuteri, L. ruminis, and to some degree, L. salivarius, may be present as autochthonous species all of the time. With bifidobacteria, a decreasing tendency in counts and in multiple composition in elderly people exists. Furthermore, this microflora is also influenced by consumption habits, which are probably caused by geographical circumstances.

Probiotics are live microorganisms that provide health benefits to the host when ingested in adequate amounts. The strains most frequently used as probiotics include lactic acid bacteria and bifidobacteria. Probiotics have demonstrated significant potential as therapeutic options for a variety of diseases, but the mechanisms responsible for these effects have not been fully elucidated yet. Several important mechanisms underlying the antagonistic effects of probiotics on various microorganisms include the following: modification of the gut microbiota, competitive adherence to the mucosa and epithelium, strengthening of the gut epithelial barrier and modulation of the immune system to convey an advantage to the host. Accumulating evidence demonstrates that probiotics communicate with the host by pattern recognition receptors, such as toll-like receptors and nucleotide-binding oligomerization domain-containing protein-like receptors, which modulate key signaling pathways, such as nuclear factor-ĸB and mitogen-activated protein kinase, to enhance or suppress activation and influence downstream pathways. This recognition is crucial for eliciting measured antimicrobial responses with minimal inflammatory tissue damage. A clear understanding of these mechanisms will allow for appropriate probiotic strain selection for specific applications and may uncover novel probiotic functions. The goal of this systematic review was to explore probiotic modes of action focusing on how gut microbes influence the host.

A technique is described for the quantitative recovery of monocytes from horse blood by means of flotation on dense albumin solutions. Monocytes are concentrated in a surface pellicle along with a few lymphocytes which are then removed when the monocytes adhere to a glass surface. The in vitro cultivation of homogeneous populations of monocytes results in an increase in (a) cell size, (b) number of mitochondria, and (c) phase-dense granules of the centrosphere. The phase-dense granules are osmiophilic and acid phosphatase positive. Quantitative biochemical analysis during cultivation have revealed increased levels of cytochrome oxidase, acid phosphatase, arylsulfatase, and BPN hydrolase. In addition, glucose utilization and lactic acid production are stimulated under the same conditions. The uptake of both bacteria and colloidal gold is stimulated during in vitro cultivation. The phagocytic activity of cultured monocytes may be enhanced by a purified bacterial lipopolysaccharide. These data are consistant with the in vitro maturation of monocytes to macrophages, a cell with greater metabolic and functional potentional.

The enteric flora comprises approximately 95% of the total number of cells in the human body and can elicit immune responses while protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract may also be implicated in the pathogenesis of diseases such as inflammatory bowel disease (ulcerative colitis and Crohn disease). The objectives of the Probiotic Research Group based at University College Cork were to isolate and identify lactic acid bacteria exhibiting beneficial probiotic traits, such as bile tolerance in the absence of deconjugation activity, acid resistance, adherence to host epithelial tissue, and in vitro antagonism of pathogenic microorganisms or those suspected of promoting inflammation. To isolate potentially effective probiotic bacteria, we screened the microbial population adhering to surgically resected segments of the gastrointestinal tract (the environment in which they may subsequently be reintroduced and required to function). In total, 1500 bacterial strains from resected human terminal ilea were assessed. From among these organisms, Lactobacillus salivarius subsp. salivarius strain UCC118 was selected for further study. In mouse feeding trials, milk-borne L. salivarius strain UCC118 could successfully colonize the murine gastrointestinal tract. A human feeding study conducted in 80 healthy volunteers showed that yogurt can be used as a vehicle for delivery of strain UCC118 to the human gastrointestinal tract with considerable efficacy in influencing gut flora and colonization. In summary, we developed criteria for in vitro selection of probiotic bacteria that may reflect certain in vivo effects on the host such as modulation of gastrointestinal tract microflora.

OBJECTIVE

To investigate the correlation between in vitro and in vivo immunomodulation potential of the probiotic strain and its ability to prevent experimental colitis in mice.

METHODS

In vitro immunomodulation was assessed by measuring interleukin (IL)-12p70, IL-10, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) release by human peripheral blood mononuclear cells (PBMCs) after 24 h stimulation with 13 live bacterial strains. A murine model of acute TNBS-colitis was next used to evaluate the prophylactic protective capacity of the same set of strains.

RESULTS

A strain-specific in vivo protection was observed. The strains displaying an in vitro potential to induce higher levels of the anti-inflammatory cytokine IL-10 and lower levels of the inflammatory cytokine IL-12, offered the best protection in the in vivo colitis model. In contrast, strains leading to a low IL-10/IL-12 cytokine ratio could not significantly attenuate colitis symptoms.

CONCLUSIONS

These results show that we could predict the in vivo protective capacity of the studied lactic acid bacteria (LAB) based on the cytokine profile we established in vitro. The PBMC-based assay we used may thus serve as a useful primary indicator to narrow down the number of candidate strains to be tested in murine models for their anti-inflammatory potential.

Poly(D,L-lactic-co-glycolic acid) (PLGA) is the most frequently used biodegradable polymer in the controlled release of encapsulated drugs. Understanding the release mechanisms, as well as which factors that affect drug release, is important in order to be able to modify drug release. Drug release from PLGA-based drug delivery systems is however complex. This review focuses on release mechanisms, and provides a survey and analysis of the processes determining the release rate, which may be helpful in elucidating this complex picture. The term release mechanism and the various techniques that have been used to study release mechanisms are discussed. The physico-chemical processes that influence the rate of drug release and the various mechanisms of drug release that have been reported in the literature are analyzed in this review, and practical examples are given. The complexity of drug release from PLGA-based drug delivery systems can make the generalization of results and predictions of drug release difficult. However, this complexity also provides many possible ways of solving problems and modifying drug release. Basic, generally applicable and mechanistic research provides pieces of the puzzle, which is useful in the development of controlled-release pharmaceuticals.

Controlled release from biodegradable polymers is a novel approach to replace daily painful injections of protein drugs. A major obstacle to development of these polymers is the need to retain the structure and biological activity of encapsulated proteins during months of incubation under physiological conditions. We encapsulated bovine serum albumin (BSA) in injectable poly(DL-lactide- co-glycolide) (PLGA) 50/50 cylindrical implants and determined the mechanism of BSA instability. Simulations of the polymer microclimate revealed that moisture and acidic pH (<3) triggered unfolding of encapsulated BSA, resulting in peptide bond hydrolysis and noncovalent aggregation. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polyester, we coincorporated into the polymer an antacid, Mg(OH)2, which increased microclimate pH and prevented BSA structural losses and aggregation for over one month. We successfully applied this stabilization approach in both cylinder- and microsphere-injectable configurations and for delivery of angiogenic basic fibroblast growth factor and bone-regenerating bone morphogenetic protein-2.

There are 3 distinct yet closely integrated processes that operate together to satisfy the energy requirements of muscle. The anaerobic energy system is divided into alactic and lactic components, referring to the processes involved in the splitting of the stored phosphagens, ATP and phosphocreatine (PCr), and the nonaerobic breakdown of carbohydrate to lactic acid through glycolysis. The aerobic energy system refers to the combustion of carbohydrates and fats in the presence of oxygen. The anaerobic pathways are capable of regenerating ATP at high rates yet are limited by the amount of energy that can be released in a single bout of intense exercise. In contrast, the aerobic system has an enormous capacity yet is somewhat hampered in its ability to delivery energy quickly. The focus of this review is on the interaction and relative contribution of the energy systems during single bouts of maximal exercise. A particular emphasis has been placed on the role of the aerobic energy system during high intensity exercise. Attempts to depict the interaction and relative contribution of the energy systems during maximal exercise first appeared in the 1960s and 1970s. While insightful at the time, these representations were based on calculations of anaerobic energy release that now appear questionable. Given repeated reproduction over the years, these early attempts have lead to 2 common misconceptions in the exercise science and coaching professions. First, that the energy systems respond to the demands of intense exercise in an almost sequential manner, and secondly, that the aerobic system responds slowly to these energy demands, thereby playing little role in determining performance over short durations. More recent research suggests that energy is derived from each of the energy-producing pathways during almost all exercise activities. The duration of maximal exercise at which equal contributions are derived from the anaerobic and aerobic energy systems appears to occur between 1 to 2 minutes and most probably around 75 seconds, a time that is considerably earlier than has traditionally been suggested.

About half of all cancer patients show a syndrome of cachexia, characterized by loss of adipose tissue and skeletal muscle mass. Such patients have a decreased survival time, compared with the survival time among patients without weight loss, and loss of total body protein leads to substantial impairment of respiratory muscle function. These changes cannot be fully explained by the accompanying anorexia, and nutritional supplementation alone is unable to reverse the wasting process. Despite a falling caloric intake, patients with cachexia frequently show an elevated resting energy expenditure as a result of increases in Cori cycle (i.e., catalytic conversion of lactic acid to glucose) activity, glucose and triglyceride-fatty acid cycling, and gluconeogenesis. A number of cytokines, including tumor necrosis factor-apha, interleukins 1 and 6, interferon gamma, and leukemia-inhibitory factor, have been proposed as mediators of the cachectic process. However, the results of a number of clinical and laboratory studies suggest that the action of the cytokines alone is unable to explain the complex mechanism of wasting in cancer cachexia. In addition, cachexia has been observed in some xenograft models even without a cytokine involvement, suggesting that other factors may be involved. These probably include catabolic factors, which act directly on skeletal muscle and adipose tissue and the presence of which has been associated with the clinical development of cachexia. A polyunsaturated fatty acid, eicosapentaenoic acid, attenuates the action of such catabolic factors and has been shown to stabilize the process of wasting and resting energy expenditure in patients with pancreatic cancer. Such a pharmacologic approach may provide new insights into the treatment of cachexia.

BACKGROUND

The origin of the lactic acid that acidifies the vagina is not well established. It is widely accepted that during times of high oestrogen (during the neonatal period and again during a woman's reproductive years) large amounts of glycogen are deposited in the vaginal epithelium and that the glycogen is anaerobically metabolized to lactic acid. What is not established is whether lactic acid is primarily produced by vaginal bacteria or by vaginal epithelial cells. Human cells can make only L-lactate, while bacteria can produce both D- and L-, thus the D- to L-lactate ratio can indicate the relative contribution of bacterially derived lactic acid.

METHODS

In this study, we used chiral HPLC to examine the percentages of D- and L-lactate in vaginal secretions, in primary cultures of bacteria from these vaginal secretions, and in cultures of lactobacillus isolates of vaginal origin.

RESULTS

We found that in most vaginal secretion samples, >50% of the lactic acid was the D-isoform (mean 55%, range 6-75%, n = 14).

CONCLUSIONS

Our results thus support the hypothesis that vaginal bacteria, not epithelial cells, are the primary source of lactic acid in the vagina.

BACKGROUND

Recognition of myocardial ischemia is critical both for the diagnosis of coronary artery disease and the selection and evaluation of therapy. Recent advances in proteomic and metabolic profiling technologies may offer the possibility of identifying novel biomarkers and pathways activated in myocardial ischemia.

RESULTS

Blood samples were obtained before and after exercise stress testing from 36 patients, 18 of whom demonstrated inducible ischemia (cases) and 18 of whom did not (controls). Plasma was fractionated by liquid chromatography, and profiling of analytes was performed with a high-sensitivity electrospray triple-quadrupole mass spectrometer under selected reaction monitoring conditions. Lactic acid and metabolites involved in skeletal muscle AMP catabolism increased after exercise in both cases and controls. In contrast, there was significant discordant regulation of multiple metabolites that either increased or decreased in cases but remained unchanged in controls. Functional pathway trend analysis with the use of novel software revealed that 6 members of the citric acid pathway were among the 23 most changed metabolites in cases (adjusted P=0.04). Furthermore, changes in 6 metabolites, including citric acid, differentiated cases from controls with a high degree of accuracy (P<0.0001; cross-validated c-statistic=0.83).

CONCLUSIONS

We report the novel application of metabolomics to acute myocardial ischemia, in which we identified novel biomarkers of ischemia, and from pathway trend analysis, coordinate changes in groups of functionally related metabolites.

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides >> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.

In order to overcome multidrug resistance in solid tumors, doxorubicin (DOX) loaded pH-sensitive micelles of which surface was decorated with folate (PHSM/f) were evaluated both in vitro and in vivo experiments. PHSM/f were fabricated from a mixture of two block copolymers of poly(L-histidine) (M(n): 5K)-b-PEG (M(n): 2K)-folate (polyHis/PEG-folate) (75 wt.%) and poly(L-lactic acid) (M(n): 3K)-b-PEG (M(n): 2K)-folate (PLLA/PEG-folate) (25 wt.%). The PHSM/f showed more than 90% cytotoxicity of DOX resistant MCF-7 (MCF-7/DOX(R)) when cultured with PHSM/f at a concentration of 10 microg/ml DOX. The result was interpreted by a sequential event of active internalization of PHSM/f via folate-receptor mediated endocytosis and ionization of His residues which result in micelle destabilization and probably disturbance of endosomal membranes. This potential mechanism may endow the drug carriers to bypass Pgp efflux pump and sequestration of DOX in acidic intracellular compartments, yielding high cytotyoxicity. Experimental evaluation of tumor regression was carried out in a small animal model bearing s.c. MCF-7 or MCF-7/DOX(R) xenografts. The tumor (MCF-7/DOX) volumes of mice treated with PHSM/f were significantly less than control groups treated with free DOX or similar micelles but without folate (PHSM). In the MCF-7/DOX(R) xenograft model, the accumulated DOX level of PHSM/f in solid tumors was 20 times higher than free DOX group, and 3 times higher than PHSM group. The results demonstrate that PHSM/f is a viable means for treating drug resistant tumors.

Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone formation and remodeling. Since vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are key regulators of angiogenesis and osteogenesis during bone regeneration, the aim of this study was to investigate if their sequential release could enhance BMP-2-induced bone formation. A composite consisting of poly(lactic-co-glycolic acid) microspheres loaded with BMP-2 embedded in a poly(propylene) scaffold surrounded by a gelatin hydrogel loaded with VEGF was used for the sequential release of the growth factors. Empty composites or composites loaded with VEGF and/or BMP-2 were implanted ectopically and orthotopically in Sprague-Dawley rats (n=9). Following implantation, the local release profiles were determined by measuring the activity of (125)I-labeled growth factors using scintillation probes. After 8 weeks blood vessel and bone formation were analyzed using microangiography, microCT and histology. The scaffolds exhibited a large initial burst release of VEGF within the first 3 days and a sustained release of BMP-2 over the full 56-day implantation period. Although VEGF did not induce bone formation, it did increase the formation of the supportive vascular network (p=0.03) in ectopic implants. In combination with local sustained BMP-2 release, VEGF significantly enhanced ectopic bone formation compared to BMP-2 alone (p=0.008). In the orthotopic defects, no effect of VEGF on vascularisation was found, nor was bone formation higher by the combination of growth factors, compared to BMP-2 alone. This study demonstrates that a sequential angiogenic and osteogenic growth factor release may be beneficial for the enhancement of bone regeneration.