Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis. A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis. This feature allowed the expression of a number of L. lactis-derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.

1. The function of ubiquinone in Escherichia coli was studied by using whole cells and membrane preparations of normal E. coli and of a mutant lacking ubiquinone. 2. The mutant lacking ubiquinone, strain AN59 (Ubi(-)), when grown under aerobic conditions, gave an anaerobic type of growth yield and produced large quantities of lactic acid, indicating that ubiquinone plays a vital role in electron transport. 3. NADH and lactate oxidase activities in membranes from strain AN59 (Ubi(-)) were greatly impaired and activity was restored by the addition of ubiquinone (Q-1). 4. Comparison of the percentage reduction of flavin, cytochrome b(1) and cytochrome a(2) in the aerobic steady state in membranes from the normal strain (AN62) and strain AN59 (Ubi(-)) and the effect of respiratory inhibitors on these percentages in membranes from strain AN62 suggest that ubiquinone functions at more than one site in the electron-transport chain. 5. Membranes from strain AN62, in the absence of substrate, showed an electron-spin-resonance signal attributed to ubisemiquinone. The amount of reduced ubiquinone (50%) found after rapid solvent extraction is consistent with the existence of ubiquinone in membranes as a stabilized ubisemiquinone. 6. The effects of piericidin A on membranes from strain AN62 suggest that this inhibitor acts at the ubiquinone sites: thus inhibition of electron transport is reversed by ubiquinone (Q-1); the aerobic steady-state oxidation-reduction levels of flavins and cytochrome b(1) in the presence of the inhibitor are raised to values approximating those found in the membranes of strain AN59 (Ubi(-)); the inhibitor rapidly eliminates the electron-spin-resonance signal attributed to ubisemiquinone and allows slow oxidation of endogenous ubiquinol in the absence of substrate and prevents reduction of ubiquinone in the presence of substrate. It is concluded that piericidin A separates ubiquinone from the remainder of the electron-transport chain. 7. A scheme is proposed in which ubisemiquinone, complexed to an electron carrier, functions in at least two positions in the electron-transport sequence.

BACKGROUND

Culture-dependent methods have shown that meconium, the newborn's first intestinal discharge, is not sterile, but the diversity of bacteria present in this material needs to be further characterized by means of more sensitive molecular techniques.

OBJECTIVE

Our aims were to characterize molecularly the meconium microbiota in term infants, to assess whether it contributes to the future microbiota of the infants' gastrointestinal tract, and to evaluate how it relates to lifestyle variables and atopy-related conditions.

METHODS

We applied high-throughput pyrosequencing of the 16S rRNA gene to study the meconium microbiota in twenty term newborns from a Spanish birth cohort. For comparison, we characterized the microbiota in fecal samples from seven pregnant women days before delivery and in two series of infant samples spanning the first seven months of life. We also compared our data with vaginal and skin microbiota characterized in independent studies. Different types of meconium microbiota were defined based on taxonomic composition and abundance and their associations with different factors were statistically evaluated.

RESULTS

The meconium microbiota differs from those in adult feces, vagina and skin, but resembles that of fecal samples from young infants. Meconium samples clustered into two types with different bacterial diversity, richness and composition. One of the types was less diverse, dominated by enteric bacteria and associated with a history of atopic eczema in the mother (P = 0.038), whereas the second type was dominated by lactic acid bacteria and associated with respiratory problems in the infant (P = 0.040).

CONCLUSIONS

Our findings suggest that the meconium microbiota has an intrauterine origin and participates in gut colonization. Although based on a small population sample, our association analyses also suggest that the type of bacteria detected in meconium is influenced by maternal factors and may have consequences for childhood health.

Biopreservation refers to extended storage life and enhanced safety of foods using the natural microflora and (or) their antibacterial products. Lactic acid bacteria have a major potential for use in biopreservation because they are safe to consume and during storage they naturally dominate the microflora of many foods. In milk, brined vegetables, many cereal products and meats with added carbohydrate, the growth of lactic acid bacteria produces a new food product. In raw meats and fish that are chill stored under vacuum or in an environment with elevated carbon dioxide concentration, the lactic acid bacteria become the dominant population and preserve the meat with a "hidden' fermentation. The same applies to processed meats provided that the lactic acid bacteria survive the heat treatment or they are inoculated onto the product after heat treatment. This paper reviews the current status and potential for controlled biopreservation of foods.

The enteric flora comprise approximately 95% of the total number of cells in the human body and are capable of eliciting immune responses while also protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract (GIT) may also be implicated in the pathogenesis of several chronic conditions such as inflammatory bowel disease (IBD). The University College Cork-based Probiotic Research Group has successfully isolated and identified lactic acid bacteria (LAB) which exhibit beneficial probiotic traits. These characteristics include the demonstration of bile tolerance; acid resistance; adherence to host epithelial tissue; and in vitro antagonism of potentially-pathogenic micro-organisms or those which have been implicated in promoting inflammation. The primary objective of this report is to describe the strategy adopted for the selection of potentially effective probiotic bacteria. The study further describes the evaluation of two members of the resulting panel of micro-organisms (Lactobacillus salivarius subsp. salivarius UCC118 and Bifidobacterium longum infantis 35624) under in vitro conditions and throughout in vivo murine and human feeding trials. Specifically, an initial feeding study completed in Balb/c mice focused upon (i) effective delivery of the probiotic micro-organisms to the GIT and evaluation of the ability of the introduced strains to survive transit through, and possibly colonise, the murine GIT; (ii) accepting the complexity of the hostile GIT and faecal environments, development of a method of enumerating the introduced bacterial strains using conventional microbiological techniques; and (iii) assessment of the effects of administered bacterial strains on the numbers of specific recoverable indigenous bacteria in the murine GIT and faeces. Additional research, exploiting the availability of murine models of inflammatory bowel disease, demonstrated the beneficial effects of administering probiotic combinations of Lactobacillus salivarius UCC118 and Bifidobacterium longum infantis 35624 in prevention of illness-related weight loss. A further ethically-approved feeding trial, successfully conducted in 80 healthy volunteers, demonstrated that yoghurt can be used as a vehicle for delivery of Lactobacillus salivarius strain UCC118 to the human GIT with considerable efficacy in influencing gut flora and colonisation.

Multiple sensory cues emanating from humans are thought to guide blood-feeding female mosquitoes to a host. To determine the relative contribution of carbon dioxide (CO2) detection to mosquito host-seeking behavior, we mutated the AaegGr3 gene, a subunit of the heteromeric CO2 receptor in Aedes aegypti mosquitoes. Gr3 mutants lack electrophysiological and behavioral responses to CO2. These mutants also fail to show CO2-evoked responses to heat and lactic acid, a human-derived attractant, suggesting that CO2 can gate responses to other sensory stimuli. Whereas attraction of Gr3 mutants to live humans in a large semi-field environment was only slightly impaired, responses to an animal host were greatly reduced in a spatial-scale-dependent manner. Synergistic integration of heat and odor cues likely drive host-seeking behavior in the absence of CO2 detection. We reveal a networked series of interactions by which multimodal integration of CO2, human odor, and heat orchestrates mosquito attraction to humans.

The cells of the intervertebral disc exist in an unusual environment. They are embedded in a dense matrix containing a high concentration of aggrecan whose fixed negative charges regulate the extracellular ionic composition and osmolarity; both extracellular cation concentrations and osmolarity are considerably higher than those experienced by most cell types. The disc also is avascular. Oxygen levels in the centre of the nucleus, where cells may be 6-8 mm from the blood supply, are very low. Since metabolism is mainly by glycolysis, lactic acid is produced at high rates and hence the pH is acidic. Finally, the disc is subjected to mechanical forces at all times; these vary with posture and activity. In particular, because the disc is under low loads during rest and high loads during the day's activities, it loses and regains around 25% of its fluid over a diurnal cycle with consequent changes to the concentrations of extracellular matrix macromolecules and ions and hence extracellular osmolality. Here we will briefly review these factors and discuss the influence of changes in the physicochemical environment on cellular activity, in particular on the rate at which disc cells synthesize and degrade matrix macromolecules.

The metabolic environment of disc cells is governed by the avascular nature of the tissue. Because cellular energy metabolism occurs mainly through glycolysis, the disc cells require glucose for survival and produce lactic acid at high rates. Oxygen is also necessary for cellular activity, although not for survival; its pathway of utilization is unclear. Because the tissues are avascular, disc cells depend on the blood supply at the margins of the discs for their nutrients. The nucleus and inner anulus of the disc are supplied by capillaries that arise in the vertebral bodies, penetrate the subchondral bone, and terminate at the bone-disc junction. Small molecules such as glucose and oxygen then reach the cells by diffusion under gradients established by the balance between the rate of transport through the tissue to the cells and the rate of cellular demand. Metabolites such as lactic acid are removed by the reverse pathway. The concentrations of nutrients farthest from the source of supply can thus be low; oxygen concentrations as low as 1% have been measured in the discs of healthy animals. Although gradients cannot be measured easily in humans, they can be calculated. Measured concentrations in surgical patients are in agreement with calculated values.

OBJECTIVE

We examined the antimicrobial activity and composition of vaginal fluid.

METHODS

Vaginal fluid from preweighed tampons was assayed for pH, lactic acid, and antimicrobial polypeptides. The fluid was also fractionated by molecular filtration. Antimicrobial activity of whole fluid was determined against representative resident and exogenous microbes, and its fractions were tested against Escherichia coli.

RESULTS

Vaginal fluids (5/5 donors) were permissive for Lactobacillus crispatus and vaginalis and Candida albicans, but not for Escherichia coli, Streptococcus group B, and Lactobacillus jensenii in three of five donors. The antimicrobial activity against E coli was predominantly in a <3-kd fraction and correlated with both low pH and high lactic acid content. Compared with a matched pH buffer, lactic acid markedly suppressed the growth of E coli. Concentrated 2- or 5-fold, the protein-rich fraction was active against E coli.

CONCLUSIONS

Vaginal fluid exerts selective antimicrobial activity against nonresident bacterial species. The activity is mediated by lactic acid, low pH, and antimicrobial polypeptides.

The gut microbiota is vital in the maintenance of homeostasis in the gut immune system. Its diversity and composition play major roles in relation to allergies and inflammatory bowel diseases, and administration of lactic acid bacteria (LAB), such as lactobacilli and bifidobacteria, has positive effects on these pathologies. However, the mechanisms behind the beneficial effects are largely unknown. Here we reveal divergent roles played by Toll-like receptor-2 (TLR2) and nucleotide-binding oligomerization domain-2 (NOD2) in dendritic cell (DC) recognition of LAB. Murine bone-marrow-derived DC lacking NOD2 produce higher levels of interleukin-10 (IL-10) and reduced levels of IL-12 and tumour necrosis factor-alpha (TNF-alpha) in response to LAB. This indicates that peptidoglycan is partly responsible for the T helper type 1 skewing effect of certain LAB. Dendritic cells that are TLR2-/- produce less IL-12 and TNF-alpha and more IL-10 in response to some strains of lactobacilli, while they produce more IL-12 and less IL-10 in response to bifidobacteria. The same tendency was found in human monocyte-derived DC. We have previously reported that the weak IL-12-inducing and TNF-alpha-inducing bifidobacteria inhibit the T helper type 1 skewing effect induced by strong immunostimulatory lactobacilli. Here we show that this immunoinhibitory effect of bifidobacteria is dependent on TLR2 and independent of NOD2. Moreover, independently of the cytokine pattern induced by intact LAB, cell wall fractions of all LAB, as well as synthetic lipoproteins possess immunoinhibitory capacities in both human and murine DC. These novel findings suggest that LAB act as immunoregulators through interaction of lipoprotein with TLR2 and as immunostimulators through interaction of peptidoglycan with NOD2.

METHODS

In vitro measurements of metabolic rates of isolated bovine nucleus pulposus cells at varying levels of oxygen, glucose, and pH.

OBJECTIVE

To obtain quantitative information on the interactions between oxygen and glucose concentrations and pH, and the rates of oxygen and glucose consumption and lactic acid production, for disc nucleus cells.

BACKGROUND

Disc cells depend on diffusion from blood vessels at the disc margins for supply of nutrients. Loss of supply is thought to lead to disc degeneration, but how loss of supply affects nutrient concentrations in the disc is not known; nutrient concentrations within discs can normally only be calculated, because concentration measurements are invasive. However, realistic predictions cannot be made until there are data from measurements of metabolic rates at conditions found in the disc in vivo, i.e., at low levels of oxygen, glucose, and pH.

METHODS

A metabolism chamber was designed to allow simultaneous recording of oxygen and glucose concentrations and of pH. These concentrations were measured electrochemically with custom-built glucose and oxygen sensors; lactic acid was measured biochemically. Bovine nucleus pulposus cells were isolated and inserted into the chamber, and simultaneous rates of oxygen and glucose consumption and of lactic acid production were measured over a range of glucose, oxygen, and pH levels.

RESULTS

There were strong interactions between rates of metabolism and oxygen consumption and pH. At atmospheric oxygen levels, oxygen consumption rate at pH 6.2 was 32% of that at pH 7.4. The rate fell by 60% as oxygen concentration was decreased from 21 to 5% at pH 7.4, but only by 20% at pH 6.2. Similar interactions were seen for lactic acid production and glucose consumption rates; we found that glycolysis rates fell at low oxygen and glucose concentrations and low pH. Equations were derived that satisfactorily predict the effect of nutrient and metabolite concentrations on rates of lactic acid production rate and oxygen consumption.

CONCLUSIONS

Disc cell metabolism in air and at pH 7.4 differs markedly from that found in the disc nucleus in vivo, where low levels of oxygen, glucose, and pH all coexist.

Enterococci are lactic acid bacteria of importance in food, public health and medical microbiology. Many strains produce bacteriocins, some of which have been well characterized. This review describes the structural and genetic characteristics of enterocins, the bacteriocins produced by enterococci. Some of these can be grouped with typical bacteriocins produced by lactic acid bacteria according to traditional classification, whereas others are atypical and structurally distinct from the general classes of bacteriocins. These atypical enterocins recently played an important role in and prompted reclassification of the class II bacteriocins into a new scheme. In this review, a more simplified classification scheme for enterocins based on amino acid sequence homologies is proposed. Enterocins are of interest for their diversity and potential for use as food biopreservatives. The emergence of multiple antibiotic-resistant enterococci among agents of nosocomial disease and the presence of virulence factors among food isolates requires a careful safety evaluation of isolates intended for potential biotechnical use. Nevertheless, enterococcal bacteriocins produced by heterologous hosts or added as cell-free preparations may still be attractive for application in food preservation.

This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.

As an alternative to cell specific cancer targeting strategies (which are often afflicted with the heterogeneity of cancer cells as with most biological systems), a novel polymeric micelle constitute of two block copolymers of poly(L-lactic acid)-b-poly(ethylene glycol)-b-poly(L-histidine)-TAT (transactivator of transcription) and poly(L-histidine)-b-poly(ethylene glycol) was developed. The micelle formed via the dialysis method was approximately 95 nm in diameter and contained 15 wt.% of doxorubicin (DOX) by weight. The micelle surface hides TAT during circulation, which has the strong capability to translocate the micelle into cells, and exposes TAT at a slightly acidic tumor extracellular pH to facilitate the internalization process. The micelle core was engineered for disintegration in early endosomal pH of tumor cells, quickly releasing DOX. The ionization process of the block copolymers and ionized polymers assisted in disrupting the endosomal membrane. This processes permitted high DOX concentrations in the cytosol and its target site of the nucleus, thus increasing DOX potency in various wild and multidrug resistant (MDR) cell lines (3.8-8.8 times lower IC50 than free DOX, depending on cell line). When tested with the xenografted tumors of human ovarian tumor drug-resistant A2780/AD, human breast tumor drug-sensitive MCF-7, human lung tumor A549 and human epidermoid tumor KB in a nude mice model, all tumors significantly regressed in size by three bolus injections at a dose of DOX 10 mg equivalent/kg body per injection of DOX-loaded micelle at three day interval, while minimum weight loss was observed. This approach may replace the need for cell-specific antibodies or targeting ligands, thereby providing a general strategy for solid tumor targeting.

This study investigates formulation and process modifications to improve the versatility of the nanoprecipitation technique, particularly with respect to the encapsulation of hydrophilic drugs (e.g. proteins). More specifically, the principal objective was to explore the influence of such modifications on nanoparticle size. Selected parameters of the nanoprecipitation method, such as the solvent and the non-solvent nature, the solvent/non-solvent volume ratio and the polymer concentration, were varied so as to obtain polymeric nano-carriers. The feasibility of such a modified method was assessed and resulting unloaded nanoparticles were characterized with respect to their size and shape. It was shown that the mean particle size was closely dependent on the type of non-solvent selected. When alcohols were used, the final mean size increased in the sequence: methanol<ethanol<propanol. Surfactants added to the dispersing medium were usually unnecessary for final suspension stabilization. Changing the solvent/non-solvent volume ratio was also not a determinant factor for nanoparticle formation and their final characteristics, provided that the final mixture itself did not become a solvent for the polymer. A too high polymer concentration in the solvent, however, prevented nanoparticle formation. Both poly(<em>lactic</em> <em>acid</em>) (PLA) and poly(d,l-<em>lactic</em>-co-glycolic <em>acid</em>) (PLGA) could be used by accurately choosing the polymer solvent and in this respect, some non-toxic solvents with different dielectric constants were selected. The nanoparticles obtained ranged from about 85-560 nm in size. The nanoparticle recovery step however needs further improvements, since bridges between particles which cause flocculation could be observed. Finally, the presented results demonstrate that the nanoprecipitation technique is more versatile and flexible than previously thought and that a wide range of parameters can be modified.

Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication. Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH. In order to characterize the genes involved in acid tolerance responses, we selected acid-resistant insertional mutants of the L. lactis strain MG1363. Twenty-one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism. None of these genes was identified previously as involved in lactococcal acid tolerance. The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress-resistant strains. Our results reveal that L. lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance. In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction. Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.

In order to study the effect of glucose on the differentiation of cultured human colon cancer cells, a subpopulation of HT-29 cells was selected for its capacity to grow in the total absence of sugar. These cells (Glc-cells) exhibit, after confluency, an enterocytic differentiation, in contrast to cells grown with glucose (Glc+ cells), which always remain undifferentiated. The differentiation is characterized by a polarization of the cell layer with apical brush borders and tight junctions, and by the presence of sucrase-isomaltase. The differentiation of Glc- cells is reversible: the addition of glucose to postconfluent cultures of Glc- cells results in an inhibiting effect on the expression of sucrase-isomaltase; switching growing cultures of Glc- cells to the Glc+ medium for several passages results in a progressive reversion to the undifferentiated state, which is completed after seven passages. The dedifferentiation process is associated with a parallel, passage-related, increase in the rates of glucose consumption and lactic acid production, and decreases of intracellular glycogen content, which return to the values of the undifferentiated original Glc+ cells. The values of these metabolic parameters are correlated, at each passage, with the degree of dedifferentiation of the cells. When these dedifferentiated cells, after having been cultured in Glc+ medium for 20 passages, are switched back to the Glc- medium, they readily grow without mortality, and reexpress the same enterocytic differentiation as the parent Glc- cells. These results show that the capacity of this subpopulation to grow and differentiate in the absence of sugar is a stable characteristic. They further suggest that glucose metabolism interferes with the program of differentiation of HT-29 cells.

OBJECTIVE

To investigate the antimicrobial properties of phenolic compounds present in Finnish berries against probiotic bacteria and other intestinal bacteria, including pathogenic species.

RESULTS

Antimicrobial activity of pure phenolic compounds representing flavonoids and phenolic acids, and eight extracts from common Finnish berries, was measured against selected Gram-positive and Gram-negative bacterial species, including probiotic bacteria and the intestinal pathogen Salmonella. Antimicrobial activity was screened by an agar diffusion method and bacterial growth was measured in liquid culture as a more accurate assay. Myricetin inhibited the growth of all lactic acid bacteria derived from the human gastrointestinal tract flora but it did not affect the Salmonella strain. In general, berry extracts inhibited the growth of Gram-negative but not Gram-positive bacteria. These variations may reflect differences in cell surface structures between Gram-negative and Gram-positive bacteria. Cloudberry, raspberry and strawberry extracts were strong inhibitors of Salmonella. Sea buckthorn berry and blackcurrant showed the least activity against Gram-negative bacteria.

CONCLUSIONS

Different bacterial species exhibit different sensitivities towards phenolics.

CONCLUSIONS

These properties can be utilized in functional food development and in food preservative purposes.

Antimicrobial peptides, bacteriocins, produced by lactic acid bacteria were adsorbed on the cells of producing strains and other gram-positive bacteria. pH was a crucial factor in determining the degree of adsorption of these peptides onto cell surfaces. In general, between 93 and 100% of the bacteriocin molecules were adsorbed at pHs near 6.0, and the lowest (< or = 5%) adsorption took place at pH 1.5 to 2.0. On the basis of this property, a novel isolation method was developed for bacteriocins from four genera of lactic acid bacteria. By using this method we made preparations of pediocin AcH, nisin, sakacin A, and leuconocin Lcm1 that were potent and concentrated. This method produced a higher yield than isolation procedures, which rely on precipitation of the bacteriocins from the cell-free culture liquor. It is simple and can be used to produce large quantities of bacteriocins from lactic acid bacteria to be used as food biopreservatives.

To analyze the relationship between immunophenotyping profile and main clinicopathological features and outcome in diffuse large B-cell lymphoma (DLBCL), we studied 128 patients (59 men, 69 women; median age 65 years) consecutively diagnosed with de novo DLBCL in a single institution. Cells from each patient were immunostained with CD20, CD79a, CD5, CD10, bcl-6, MUM1, CD138, bcl-2, p53, p27, and Ki-67 antibodies. Four immunophenotyping profiles were distinguished according to the pattern of differentiation: germinal center-CD10(+) (GC-CD10(+); CD10(+)/Bcl-6(+)/MUM1(-)/CD138(-)), germinal center-CD10(-) (GC-CD10(-); CD10(-)/Bcl-6(+)/ MUM1(-)/CD138(-)), post-germinal center (pGC; CD10(-)/bcl-6(+/-)/ MUM1(+)/CD138(-)), and plasmablastic (CD10(-)/bcl-6(-)/MUM1(+)/CD138(+)). Rearrangement of bcl-2 was studied by polymerase chain reaction (PCR) in 57 patients. Single-antigen expression was as follows: CD5, 2%; CD10, 21%; bcl-6, 72%; MUM1, 54%; CD138, 2%; bcl-2, 59%; p53, 28%; p27, 40%. Distribution according to differentiation profiles was as follows: GC-CD10(+), 24 patients, GC-CD10-, 30 patients; pGC, 60 patients; plasmablastic, 2 patients; other patterns, 12 patients. The pGC profile was associated with primary nodal presentation and immunoblastic morphology, whereas GC-CD10(+) tumors showed disseminated disease, centroblastic morphology, bcl-2 rearrangement, and lower Ki-67 proliferative index. GC-CD10(-) patients more often presented with primary extranodal origin, early stage, normal lactic acid dehydrogenase (LDH) levels, and low or low/intermediate International Prognostic Index (IPI) scores than the others. However, no significant difference was found in terms of response or overall survival (OS) according to these profiles. Expression of bcl-2 was associated with advanced stage, high or high-intermediate IPI, and poor OS. Expression of bcl-2 maintained predictive value in multivariate analysis, with stage and LDH. In conclusion, differentiation profile was associated with particular clinicopathological features but was not essential to predicting outcome in DLBCL patients.

As molecular, cellular, and tissue-level treatments for spinal cord injury are discovered, it is likely that combinations of such treatments will be necessary to elicit functional recovery in animal models or patients. We describe multiple-channel, biodegradable scaffolds that serve as the basis for a model to investigate simultaneously the effects on axon regeneration of scaffold architecture, transplanted cells, and locally delivered molecular agents. Poly(lactic-co-glycolic acid) (PLGA) with copolymer ratio 85:15 was used for these initial experiments. Injection molding with rapid solvent evaporation resulted in scaffolds with a plurality of distinct channels running parallel along the length of the scaffolds. The feasibility of creating scaffolds with various channel sizes and geometries was demonstrated. Walls separating open channels were found to possess void fractions as high as 89%, with accessible void fractions as high as 90% through connections 220 microm or larger. Scaffolds degraded in vitro over a period of 30 weeks, over which time-sustained delivery of a surrogate drug was observed for 12 weeks. Primary neonatal Schwann cells were distributed in the channels of the scaffold and remained viable in tissue culture for at least 48 h. Schwann-cell containing scaffolds implanted into transected adult rat spinal cords contained regenerating axons at one month post-operation. Axon regeneration was demonstrated by three-dimensional reconstruction of serial histological sections.

Given that miR-124 is preferentially expressed in differentiating and mature neurons and external granule cells of cerebellum are thought to be cells-of-origins of medulloblastomas, we investigated if miR-124 played a role in the development of medulloblastomas. Quantitative expression analysis of 29 medulloblastomas demonstrated significant down-regulation of miR-124 in 21 (72%) tumors by at least 2-fold, with 11 of them exhibiting greater than 10-fold reduced level compared to normal cerebella (P < .01). Ectopic expression of miR-124 in medulloblastoma cell lines, ONS-76 and DAOY, inhibited cell proliferation. Using computational and expression analyses, solute carrier family 16, member 1 (SLC16A1) was identified as a candidate target of miR-124. Transfection of miR-124 resulted in down-regulation of SLC16A1 at both transcript and protein levels. Reporter assay with 3' untranslated region of SLC16A1 cloned downstream of the luciferase gene showed reduced luciferase activity in the presence of miR-124, providing strong evidence that miR-124 is a direct regulator of SLC16A1. Expression analysis further revealed that SLC16A1 transcript was elevated in 26 (90%) of 29 tumors examined. Knockdown of SLC16A1 by siRNA induced cell death in medulloblastoma cells. SLC16A1 functions to efflux lactic acid during aerobic glycolysis. We speculated that inhibition of SLC16A1 function resulted in a decrease of intracellular pH to a lethal level. In conclusion, our study demonstrates that miR-124 deregulation is common in medulloblastomas, and restoration of its function inhibits cell proliferation, suggesting that miR-124 may act as a growth suppressor. Our findings also raise the possibility that the miR-124/SLC16A1 pathway may represent a novel therapeutic target for treatment of malignant medulloblastomas.

Ninety patients with progressive recurrent lymphoma were treated with a combination of cisplatin 100 mg/m2 intravenously (IV) by continuous infusion over 24 hours, followed by cytosine arabinoside in two pulses each at a dose of 2 g/m2 given 12 hours apart. Dexamethasone, 40 mg orally or IV, was given on days 1 through 4. Vigorous hydration was reinforced by routine use of mannitol. Treatments were repeated at 3- to 4-week intervals for six to ten courses. Most patients had not achieved complete remission (CR) with prior therapies, which included Adriamycin (all patients) and methotrexate and VP-16 (58 patients). Median patient age was 55 years. Intermediate-grade lymphoma was the most frequent pathologic diagnosis. Seven patients died within two weeks of therapy; of the remaining 83 patients, 28 (34%) or 31% if all patients are considered, achieved CR, and 22 (26.5%) achieved partial remission (PR). Response was evident after the first two cycles of chemotherapy and appeared to be independent of the histopathologic type of lymphoma. To date, only eight of the complete responders have relapsed at a median follow-up of 11 months. The overall 2-year survival in 25%. Further analysis showed that patients with low tumor burden and normal lactic acid dehydrogenase (LDH) had a high CR response rate (67%) and a survival rate of 61% at 2 years. In contrast, patients with both high tumor burden and elevated serum LDH levels had a negligible CR rate, and only 5% are surviving at 1 year. Patients with either high tumor burden with normal LDH or low tumor burden with elevated LDH had an intermediate survival. Myelosuppression-related infection was the most frequent serious complication of this regimen (31%) and the cause of death of ten patients. Acute lysis syndrome was also observed in five patients with high tumor burden and was the cause of death in three of these patients. DHAP has proven to be an effective non-crossresistant regimen for patients with relapsing or refractory lymphoma, particularly for patients who have favorable prognostic characteristics.