U.S. Food and Drug Administration approval summary: omacetaxine mepesuccinate as treatment for chronic myeloid leukemia.

On October 26, 2012, the U.S. Food and Drug Administration (FDA) granted accelerated approval to omacetaxine mepesuccinate (Synribo; Teva Pharmaceuticals USA, Inc., North Wales, PA, http://www.tevausa.com) for the treatment of adult patients with chronic phase (CP) or accelerated phase (AP) chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs). The approval was based on the FDA review of data from 111 patients with CML in CP or in AP who had received two or more prior TKIs, including imatinib. Major cytogenetic response was achieved in 18% of patients with CP, with a median response duration of 12.5 months. Major hematologic response was achieved in 14% of patients with AP, with a median response duration of 4.7 months. The FDA safety evaluation was based on submitted data from 163 patients with CP or AP CML who had received at least one dose of omacetaxine mepesuccinate. The safety evaluation was limited by the single-arm design of the clinical trials as conducted in a small number of previously treated patients. The most common (≥20%) adverse reactions of any grade in enrolled patients included thrombocytopenia, anemia, neutropenia, diarrhea, nausea, fatigue, asthenia, injection site reaction, pyrexia, and infection. The FDA concluded that omacetaxine mepesuccinate has shown activity and a favorable benefit-to-risk profile for the studied population of adult patients with CML (CP or AP) with resistance and/or intolerance to two or more TKIs. Further evidence of response durability to verify clinical benefit is pending.

Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid.

Imbalance in the regulatory immune mechanisms that control intestinal cellular and bacterial homeostasis may lead to induction of the detrimental inflammatory signals characterized in humans as inflammatory bowel disease. Induction of proinflammatory cytokines (i.e., IL-12) induced by dendritic cells (DCs) expressing pattern recognition receptors may skew naive T cells to T helper 1 polarization, which is strongly implicated in mucosal autoimmunity. Recent studies show the ability of probiotic microbes to treat and prevent numerous intestinal disorders, including Clostridium difficile-induced colitis. To study the molecular mechanisms involved in the induction and repression of intestinal inflammation, the phosphoglycerol transferase gene that plays a key role in lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus NCFM (NCK56) was deleted. The data show that the L. acidophilus LTA-negative in LTA (NCK2025) not only down-regulated IL-12 and TNFα but also significantly enhanced IL-10 in DCs and controlled the regulation of costimulatory DC functions, resulting in their inability to induce CD4(+) T-cell activation. Moreover, treatment of mice with NCK2025 compared with NCK56 significantly mitigated dextran sulfate sodium and CD4(+)CD45RB(high)T cell-induced colitis and effectively ameliorated dextran sulfate sodium-established colitis through a mechanism that involves IL-10 and CD4(+)FoxP3(+) T regulatory cells to dampen exaggerated mucosal inflammation. Directed alteration of cell surface components of L. acidophilus NCFM establishes a potential strategy for the treatment of inflammatory intestinal disorders.

Development of a tiered multilocus sequence typing scheme for members of the Lactobacillus acidophilus complex.

Members of the Lactobacillus acidophilus complex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp60 gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of the L. acidophilus complex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection of Lactobacillus species.

Role of transporter proteins in bile tolerance of Lactobacillus acidophilus.

Lactobacillus acidophilus NCFM derivatives containing deletion mutations in the transporter genes LBA0552, LBA1429, LBA1446, and LBA1679 exhibited increased sensitivity to bile. These strains showed unique patterns of sensitivity to a variety of inhibitory compounds, as well as differential accumulations of ciprofloxacin and taurocholate.

The genomics of lactic acid bacteria.

The lactic acid bacteria (LAB) are one of the most industrially important groups of bacteria. These organisms are used in a variety of ways, including food production, health improvement and production of macromolecules, enzymes and metabolites. The genome sequencing of 20 LAB provides an expanded view of their genetic and metabolic capacities and enables researchers to perform functional and comparative genomic studies. This review highlights some of the findings from these analyses in the context of the numerous roles the LAB play.

Analysis of the genome sequence of Lactobacillus gasseri ATCC 33323 reveals the molecular basis of an autochthonous intestinal organism.

This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.

Functional genomics of probiotic Lactobacilli.

Lactic acid bacteria (LAB) have been used in fermentation processes for millennia. Recent applications such as the use of living cultures as probiotics have significantly increased industrial interest. Related bacterial strains can differ significantly in their genotype and phenotype, and features from one bacterial strain or species cannot necessarily be applied to a related one. These strain or family-specific differences often represent unique and applicable traits. Since 2002, the complete genomes of 13 probiotic LABs have been published. The presentation will discuss these genomes and highlight probiotic traits that are predicted, or functionally linked to genetic content. We have conducted a comparative genomic analysis of 4 completely sequenced Lactobacillus strains versus 25 lactic acid bacterial genomes present in the public database at the time of analysis. Using Differential Blast Analysis, each genome is compared with 3 other Lactobacillus and 25 other LAB genomes. Differential Blast Analysis highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Lactobacillus-specific genes include mucus-binding proteins involved in cell-adhesion and several transport systems for carbohydrates and amino acids. Comparative genomic analysis has identified gene targets in Lactobacillus acidophilus for functional analysis, including adhesion to mucin and intestinal epithelial cells, acid tolerance, bile tolerance, and quorum sensing. Whole genome transcriptional profiling of L. acidophilus, and isogenic mutants thereof, has revealed the impact of varying conditions (pH, bile, carbohydrates) and food matrices on the expression of genes important to probiotic-linked mechanisms.

The safety and regulation of natural products used as foods and food ingredients.

The use of botanicals and dietary supplements derived from natural substances as an adjunct to an improved quality of life or for their purported medical benefits has become increasingly common in the United States. This review addresses the safety assessment and regulation of food products containing these substances by the U.S. Food and Drug Administration (FDA). The issue of safety is particularly critical given how little information is available on the toxicity of some of these products. The first section uses case studies for stevia and green tea extracts as examples of how FDA evaluates the safety of botanical and herbal products submitted for consideration as Generally Recognized as Safe under the Federal Food, Drug, and Cosmetics Act. The 1994 Dietary Supplement Health Education Act (DSHEA) created a regulatory framework for dietary supplements. The article also discusses the regulation of this class of dietary supplements under DSHEA and addresses the FDA experience in analyzing the safety of natural ingredients described in pre-market safety submissions. Lastly, we discuss an ongoing interagency collaboration to conduct safety testing of nominated dietary supplements.

Characterization of a novel bile-inducible operon encoding a two-component regulatory system in Lactobacillus acidophilus.

Lactobacillus acidophilus NCFM is an industrially important strain used extensively as a probiotic culture. Tolerance of the presence of bile is an attribute important to microbial survival in the intestinal tract. A whole-genome microarray was employed to examine the effects of bile on the global transcriptional profile of this strain, with the intention of elucidating genes contributing to bile tolerance. Genes involved in carbohydrate metabolism were generally induced, while genes involved in other aspects of cellular growth were mostly repressed. A 7-kb eight-gene operon encoding a two-component regulatory system (2CRS), a transporter, an oxidoreductase, and four hypothetical proteins was significantly upregulated in the presence of bile. Deletion mutations were constructed in six genes of the operon. Transcriptional analysis of the 2CRS mutants showed that mutation of the histidine protein kinase (HPK) had no effect on the induction of the operon, whereas the mutated response regulator (RR) showed enhanced induction when the cells were exposed to bile. These results indicate that the 2CRS plays a role in bile tolerance and that the operon it resides in is negatively controlled by the RR. Mutations in the transporter, the HPK, the RR, and a hypothetical protein each resulted in loss of tolerance of bile. Mutations in genes encoding another hypothetical protein and a putative oxidoreductase resulted in significant increases in bile tolerance. This functional analysis showed that the operon encoded proteins involved in both bile tolerance and bile sensitivity.