The emergence and spread of multidrug-resistant gram-positive bacteria represent a serious clinical problem. Telavancin is a novel lipoglycopeptide antibiotic that possesses rapid in vitro bactericidal activity against a broad spectrum of clinically relevant gram-positive pathogens. Here we demonstrate that telavancin's antibacterial activity derives from at least two mechanisms. As observed with vancomycin, telavancin inhibited latestage peptidoglycan biosynthesis in a substrate-dependent fashion and bound the cell wall, as it did the lipid II surrogate tripeptide N,N-diacetyl-L-lysinyl-D-alanyl-D-alanine, with high affinity. Telavancin also perturbed bacterial cell membrane potential and permeability. In methicillin-resistant Staphylococcus aureus, telavancin caused rapid, concentration-dependent depolarization of the plasma membrane, increases in permeability, and leakage of cellular ATP and K ؉ . The timing of these changes correlated with rapid, concentration-dependent loss of bacterial viability, suggesting that the early bactericidal activity of telavancin results from dissipation of cell membrane potential and an increase in membrane permeability. Binding and cell fractionation studies provided direct evidence for an interaction of telavancin with the bacterial cell membrane; stronger binding interactions were observed with the bacterial cell wall and cell membrane relative to vancomycin. We suggest that this multifunctional mechanism of action confers advantageous antibacterial properties.The emergence and spread of bacterial resistance to vancomycin, an important antibiotic used to treat serious infections caused by gram-positive bacteria, has prompted active research to discover new glycopeptides and semisynthetic analogs with improved antimicrobial properties. Vancomycin and related glycopeptide antibiotics inhibit cell wall synthesis in susceptible bacteria by binding with high specificity to peptidoglycan precursors containing the C-terminal D-alanyl-D-alanine (D-Ala-DAla) motif (8). The peptide portion of glycopeptide antibiotics forms a carboxylate binding pocket that imparts, through a combination of five hydrogen bonds plus favorable hydrophobic interactions, strong affinity for the D-Ala-D-Ala-containing terminus of lipid II (8,46,54). Rational approaches toward the design of glycopeptides with improved antimicrobial activities have been described previously (for reviews, see references 35 and 36). One promising approach has been the discovery of lipoglycopeptides, analogs containing hydrophobic groups substituted at the amine position of the disaccharide moiety (20,39,40,45).Telavancin, a semisynthetic derivative of vancomycin possessing a hydrophobic (decylaminoethyl) side chain appended to the vancosamine sugar and a hydrophilic [(phosphonomethyl)aminomethyl] group on the resorcinol-like 4Ј position of amino acid 7 (33), is in late-stage clinical development for the treatment of serious gram-positive infections. Telavancin and other lipoglycopeptides exhibit superior in vitro activity compa...

Early subclinical inflammation in kidney transplants is associated with later graft fibrosis and dysfunction. Regulatory T cells (Tregs) can reverse established inflammation in animal models. We conducted a pilot safety and feasibility trial of autologous Treg cell therapy in three kidney transplant recipients with subclinical inflammation noted on 6-month surveillance biopsies. Tregs were purified from peripheral blood and polyclonally expanded ex vivo using medium containing deuterated glucose to label the cells. All patients received a single infusion of ~320 × 106 (319, 321 and 363.8 × 106) expanded Tregs. Persistence of the infused Tregs was tracked. Graft inflammation was monitored with follow-up biopsies and urinary biomarkers. Nearly 1 × 109 (0.932, 0.956, 1.565 × 109) Tregs were successfully manufactured for each patient. There were no infusion reactions or serious therapy-related adverse events. The infused cells demonstrated patterns of persistence and stability similar to those observed in non-immunosuppressed subjects receiving the same dose of Tregs. Isolation and expansion of Tregs is feasible in kidney transplant patients on immunosuppression. Infusion of these cells was safe and well tolerated. Future trials will test the efficacy of polyclonal and donor alloantigen-reactive Tregs for the treatment of inflammation in kidney transplants.

Idiopathic hypereosinophilic syndrome (HES) is a myeloproliferative disease of unknown etiology. Recently, it has been reported that imatinib mesylate (Gleevec), an inhibitor of Bcr-Abl kinase useful in the treatment of chronic myeloid leukemia, is also effective in treating HES; however, the molecular target of imatinib in HES is unknown. This report identifies a genetic rearrangement in the eosinophilic cell line EOL-1 that results in the expression of a fusion protein comprising an N-terminal region encoded by a gene of unknown function with the GenBank accession number NM 030917 and a C-terminal region derived from the intracellular domain of the platelet-derived growth factor receptor ␣ (PDGFR␣). The fusion gene was also detected in blood cells from two patients with HES. We propose naming NM 030917 Rhe for Rearranged in hypereosinophilia. Rhe-PDGFR␣ fusions result from an apparent interstitial deletion that links Rhe to exon 12 of PDGFR␣ on chromosome 4q12. The fusion kinase Rhe-PDGFR␣ is constitutively phosphorylated and supports IL-3-independent growth when expressed in BaF3 cells. Proliferation and viability of EOL-1 and BaF3 cells expressing Rhe-PDGFR␣ are ablated by the PDGFR␣ inhibitors imatinib, vatalanib, and THRX-165724.

In the mature pancreas, the homeodomain transcription factor Nkx6.1 is uniquely restricted to ␤-cells. Nkx6.1 also is expressed in developing ␤-cells and plays an essential role in their differentiation. Among cell lines, both ␤-and ␣-cell lines express nkx6.1 mRNA; but no protein can be detected in the ␣-cell lines, suggesting that post-transcriptional regulation contributes to the restriction of Nkx6.1 to ␤-cells. To investigate the regulator of Nkx6.1 expression, we outlined the structure of the mouse nkx6.1 gene, and we identified regions that direct cell type-specific expression. The nkx6.1 gene has a long 5-untranslated region (5-UTR) downstream of a cluster of transcription start sites. nkx6.1 gene sequences from ؊5.6 to ؉1.0 kilobase pairs have specific promoter activity in ␤-cell lines but not in NIH3T3 cells. This activity is dependent on sequences located at about ؊800 base pairs and on the 5-UTR. Electrophoretic mobility shift assays demonstrate that homeodomain transcription factors PDX1 and Nkx2.2 can bind to the sequence element located at ؊800 base pairs. In addition, dicistronic assays establish that the 5-UTR region functions as a potent internal ribosomal entry site, providing cell type-specific regulation of translation. These data demonstrate that complex regulation of both Nkx6.1 transcription and translation provides the specificity of expression required during pancreas development.

Homeodomain transcription factor Nkx2.2 is required for the final differentiation of the ␤-cells in the pancreas and for the production of insulin. Nkx2.2 is expressed in islet cell precursors during pancreatic development and persists in a subset of mature islet cells including all ␤-cells. To understand the mechanisms regulating the expression of Nkx2.2 in these different cell populations, we outlined the structure of the mouse nkx2.2 gene and identified regions that direct cell type-specific expression. The nkx2.2 gene has two noncoding alternative first exons (exons 1a and 1b). In transgenic mice, sequences upstream from exon 1a directed expression predominantly in mature islet cells. Within this exon 1a promoter, cooperative interactions between HNF3 and basic helix-loop-helix factors neurogenin-3 or NeuroD1 binding to adjacent sites played key roles in its islet cell-specific expression. In contrast, sequences upstream from exon 1b restricted expression specifically to islet cell precursors. These studies reveal distinct mechanisms for directing the expression of a key differentiation factor in precursors versus mature islet cells.The development and differentiation of organs such as the pancreas involve sequential modifications in gene expression controlled by a cascade of transcription factors. Recently, several mouse strains with mutations in genes encoding transcription factors that are expressed in the pancreatic ␤-cells have been found to have severe abnormalities in pancreatic development (1-12). Mice homozygous for a null mutation of the homeodomain transcription factor Nkx2.2 develop severe hyperglycemia and die shortly after birth (12). The mutant embryos lack insulin-producing ␤-cells and have fewer ␣-cells and PP cells. Remarkably, in these mutants there remains a large population of islet cells that do not produce any of the four endocrine hormones. These cells express some ␤-cell markers, such as islet amyloid polypeptide and PDX-1, but lack other definitive ␤-cell markers including GLUT2, glucokinase, and the ␤-cell-specific homeodomain factor Nkx6.1. These mice demonstrate that Nkx2.2 is necessary for the final differentiation of ␤-cells.The onset of Nkx2.2 expression in mouse endoderm is coincident with the onset of dorsal pancreatic bud evagination at embryonic day 9.5. Most or all of the epithelial cells of the pancreas express Nkx2.2 from the onset of bud formation until embryonic day 12.5; thereafter, Nkx2.2 expression becomes more restricted. During the peak period for ␤-cell neogenesis, from embryonic day 13.5 to 18.5 (13), Nkx2.2 is expressed in a subset of incompletely differentiated endocrine precursor cells that coexpress the bHLH 1 proendocrine transcription factor neurogenin-3 (12, 14). Unlike neurogenin-3, which is expressed exclusively in precursor cells, Nkx2.2 is expressed also in differentiated endocrine cells. In the mature pancreas, Nkx2.2 expression is limited to the differentiated endocrine cells including ␣-, ␤-, and PP cells, but not ␦-cells. Therefore, Nkx2.2 is exp...