Citations

ABSTRACT Recent molecular studies have shown Mycobacterium porcinum , recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the M. fortuitum third biovariant complex. We identified 67 clinical and two environmental isolates of the M. fortuitum third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the hsp65 gene of M. porcinum (ATCC 33776 T ) and were studied in more detail. Most U.S. patient isolates were from Texas (44%), Florida (19%), or other southern coastal states (15%). Clinical infections included wound infections (62%), central catheter infections and/or bacteremia (16%), and possible pneumonitis (18%). Sequencing of the 16S rRNA gene (1,463 bp) showed 100% identity with M. porcinum ATCC 33776 T . Sequencing of 441 bp of the hsp65 gene showed four sequevars that differed by 2 to 3 bp from the porcine strains. Clinical isolates were positive for arylsulfatase activity at 3 days, nitrate, iron uptake, d -mannitol, i- myo -inositol, and catalase at 68°C. They were negative for l -rhamnose and d -glucitol (sorbitol). Clinical isolates were susceptible to ciprofloxacin, sulfamethoxazole, and linezolid and susceptible or intermediate to cefoxitin, clarithromycin, imipenem, and amikacin. M. porcinum ATCC 33776 T gave similar results except for being nitrate negative. These studies showed almost complete phenotypic and molecular identity between clinical isolates of the M. fortuitum third biovariant d -sorbitol-negative group and porcine strains of M. porcinum and confirmed that they belong to the same species. Identification of M. porcinum presently requires hsp65 gene PRA or 16S rRNA or hsp65 gene sequencing.

AbstractPrevious studies have demonstrated the in vitro and in vivo activity of CC-5013 (Revlimid), an immunomodulatory analog (IMiD) of thalidomide, in multiple myeloma (MM). In the present study, we have examined the anti-MM activity of rapamycin (Rapamune), a specific mTOR inhibitor, combined with CC-5013. Based on the Chou-Talalay method, combination indices of less than 1 were obtained for all dose ranges of CC-5013 when combined with rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or adherence to bone marrow stromal cells (BMSCs). Combining rapamycin and CC-5013 induced apoptosis of MM cells. Differential signaling cascades, including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3′-kinase/Akt kinase (PI3K/Akt) pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies, therefore, provide the framework for clinical evaluation of mTOR inhibitors combined with IMiDs to improve patient outcome in MM.

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.

ABSTRACTPreviously, we identified a naturally processed and presented measles virus (MV) 19-amino-acid peptide, ASDVETAEGGEIHELLRLQ (MV-P), derived from the phosphoprotein and eluted from the human leukocyte antigen (HLA) class II molecule by using mass spectrometry. We report here the identification of a 14-amino-acid peptide, SAGKVSSTLASELG, derived from the MV nucleoprotein (MV-N) bound to HLA-DRB1*0301. Peripheral blood mononuclear cells (PBMC) from 281 previously vaccinated measles-mumps-rubella II (MMR-II) subjects (HLA discordant) were studied for peptide recognition by T cells. Significant gamma interferon (IFN-γ) responses to MV-P and MV-N peptides were observed in 55.9 and 15.3% of subjects, respectively. MV-P- and MV-N-specific interleukin-4 (IL-4) responses were detected in 19.2 and 23.1%, respectively, of PBMC samples. Peptide-specific cytokine responses and HLA-DRB1 allele associations revealed that, for the MV-P peptide, the allele with the strongest association with both IFN-γ (P= 0.02) and IL-4 (P= 0.03) secretion was DRB1*0301. For MV-N, the allele with the strongest association with IFN-γ secretion was DRB1*1501 (P= 0.04), and the alleles with the strongest associations with IL-4 secretion were DRB1*1103 and DRB1*1303 (P= 0.01). These results indicate that HLA class II MV proteins can be processed, presented, and identified, and the ability to generate cell-mediated immune responses can be demonstrated. This information is promising for new vaccine design strategies with peptide-based vaccines.

AbstractGenetic heterogeneity between individuals confounds the comparison of gene profiling of multiple myeloma (MM) cells versus normal plasma cells (PCs). To overcome this barrier, we compared the gene expression profile of CD138+ MM cells from a patient bone marrow (BM) sample with CD138+ PCs from a genetically identical twin BM sample using microarray profiling. Two hundred and ninety-six genes were up-regulated and 103 genes were down-regulated at least 2-fold in MM cells versus normal twin PCs. Highly expressed genes in MM cells included cell survival pathway genes such as mcl-1, dad-1, caspase 8, and FADD-like apoptosis regulator (FLIP); oncogenes/transcriptional factors such as Jun-D, Xbp-1, calmodulin, Calnexin, and FGFR-3; stress response and ubiquitin/proteasome pathway–related genes and various ribosomal genes reflecting increased metabolic and translational activity. Genes that were down-regulated in MM cells versus healthy twin PCs included RAD51, killer cell immunoglobulin-like receptor protein, and apoptotic protease activating factor. Microarray results were further confirmed by Western blot analyses, immunohistochemistry, fluorescent in situ hybridization (FISH), and functional assays of telomerase activity and bone marrow angiogenesis. This molecular profiling provides potential insights into mechanisms of malignant transformation in MM. For example, FGFR3, xbp-1, and both mcl-1 and dad-1 may mediate transformation, differentiation, and survival, respectively, and may have clinical implications. By identifying genes uniquely altered in MM cells compared with normal PCs in an identical genotypic background, the current study provides the framework to identify novel therapeutic targets.

CD28 is the quintessential costimulatory molecule expressed on CD4+ and CD8+ T cells. During chronic infections and the normal aging process, CD28 expression is lost, compromising the functional activity of T cells. CD28 loss is promoted by replicative stress, particularly in the presence of tumor necrosis factor–α, owing to an inoperative CD28 initiator element. It is currently unknown whether CD28 loss is irreversible. The present study examined cytokines for their ability to reinduce CD28 expression. CD4+CD28null T cells constitutively expressed interleukin-12 (IL-12) α and β receptors, which were functional and allowed for the up-regulation of the signal transducer and activator of transcription–4 (STAT-4)–dependent gene CD161. Costimulation of the T-cell and IL-12 receptors induced the transcription of CD28 in approximately 50% of CD4+CD28null T-cell clones and lines. IL-12 by itself did not restore CD28 expression. Up-regulation of CD28 after IL-12 exposure correlated with the reassembly of the CD28–initiator protein complex. The re-expressed CD28 was functional and restored the ability of CD4+CD28null T cells to express CD25 and CD40 ligand. Our data suggest that IL-12 may, in part, functionally rescue senescent CD4+ T cells.