Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate prodrugs GS-7340 and GS-9131

GS-7340 and GS-9131 {9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-propyl]adenine and 9-(R)-4'-(R)-[[[(S)-1-[(ethoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-2'-fluoro-1'-furanyladenine, respectively} are novel alkylalaninyl phenyl ester prodrugs of tenofovir {9-R-[(2-phosphonomethoxy)propyl]adenine} (TFV) and a cyclic nucleotide analog, GS-9148 (phosphonomethoxy-2'-fluoro-2', 3'-dideoxydidehydroadenosine), respectively. Both prodrugs exhibit potent antiretroviral activity against both wild-type and drug-resistant human immunodeficiency virus type 1 strains and excellent in vivo pharmacokinetic properties. In this study, the main enzymatic activity responsible for the initial step in the intracellular activation of GS-7340 and GS-9131 was isolated from human peripheral blood mononuclear cells and identified as lysosomal carboxypeptidase A (cathepsin A [CatA]; EC 3.4.16.5). Biochemical properties of the purified hydrolase (native complex and catalytic subunit molecular masses of 100 and 29 kDa, respectively; isoelectric point [pI] of 5.5) matched those of CatA. Recombinant CatA and the isolated prodrug hydrolase displayed identical susceptibilities to inhibitors and identical substrate preferences towards a panel of tenofovir phosphonoamidate prodrugs. Incubation of both enzymes with 14C-labeled GS-7340 or [3H]difluorophosphonate resulted in the covalent labeling of identical 29-kDa catalytic subunits. Finally, following a 4-h incubation with GS-7340 and GS-9131, the intracellular concentrations of prodrug metabolites detected in CatA-negative fibroblasts were approximately 7.5- and 3-fold lower, respectively, than those detected in normal control fibroblasts. Collectively, these data demonstrate the key role of CatA in the intracellular activation of nucleotide phosphonoamidate prodrugs and open new possibilities for further improvement of this important class of antiviral prodrugs.     

In vitro presentation of gliadin-derived peptides by different cell lines

BACKGROUND: Gliadin peptide presentation and T-cell activation are critical events in the pathogenesis of celiac disease. Several studies have been performed to identify the toxic gliadin peptides but the complexity of the antigenic fraction makes this analysis difficult. In this work, an in vitro model for the analysis of gliadin peptide presentation is studied. METHODS: The human cell lines U937 and THP-1 (monocytic), DUCAF and VAVY (immortalised B cells) and HT-29 and Caco-2 (intestinal epithelial cells) were incubated with biotin-labelled gliadin (bG). FITC-labelled streptavidin was used to detect biotinylated peptides at the cell surface by flow cytometry. RESULTS: All cell lines tested showed a fluorescence signal derived from bG, that was highest when cells were stimulated with IFN-gamma for 48 h. Time course experiments performed using THP-1 cells showed that after 4-h incubation, almost a maximal signal can be reached. THP-1 cells incubated at 4 degrees C or after paraformaldehyde fixation showed a substantial signal reduction, suggesting that metabolic activity was necessary for the detection of the maximal fluorescence signal at the cell surface. The presence of HLA class II-bound biotinylated peptides was observed in cell lysates of THP-1 cells incubated with bG. CONCLUSIONS: In all cell lines tested, a specific biotin-peptide-derived signal was observed. This was increased after IFN-gamma treatment and decreased after fixation or incubation at low temperature. The signal was higher in monocytic and B-cell lines than in the epithelial cell lines. The use of this procedure could be a useful tool to study the in vitro processing and presentation of naturally gliadin-derived peptides.     

Interphase FISH screening for the LCR-mediated common rearrangement of isochromosome 17q in primary myelofibrosis

Non-allelic homologous recombination (NAHR) between low-copy repeats (LCRs) has been implicated recently in somatic rearrangements including isochromosome i(17q), which is associated with hematologic malignancies as well as solid tumors. In hematological malignancies, the most common i(17q) breakpoint results from LCR-mediated NAHR, which creates a dicentric chromosome, idic(17)(p11.2). We report an elderly patient who presented with primary myelofibrosis (MF) with myeloid metaplasia (MMM), associated with idic(17)(p11.2) as the sole chromosomal abnormality, making this the first idic(17)(p11.2) myeloproliferative case reported in which the breakpoints are mapped to the breakpoint cluster region in proximal 17p. The rearrangement breakpoint maps to the previously defined LCR cluster, further suggesting that the genomic architecture of proximal 17p may be responsible for the formation of the majority of i(17q) cases. We describe our development of a rapid screening test using interphase FISH to detect idic(17)(p11.2), discuss the potential prognostic value of this molecular diagnostic test, and examine the relevance of LCR-mediated NAHR to common rearrangements in neoplasms.     

Immunohistological characterization of intraepithelial and lamina propria lymphocytes in control ileum and colon and in inflammatory bowel disease

Using monoclonal antibodies to T and B lymphocytes, to natural killer cells, and to HLA-DR antigen, we characterized the lymphocyte population within the epithelial and lamina propria regions in control intestine and colon, and in grossly involved and in grossly uninvolved intestine and colon of patients with active inflammatory bowel disease. There were significantly more intraepithelial T cells in control ileum than in control colon. In comparison to control, there was a heterogeneity of alterations in intraepithelial and lamina propria T lymphocyte subsets (T11⁺, T8⁺, T4⁺) in inflammatory bowel disease. B lymphocytes were not detected within the lamina propria, except when found in and adjacent to lymphoid aggregates. Leu 7⁺ cells were uncommon in the lamina propria of control ileum and colon and in diseased tissues. The majority of intraepithelial lymphocytes did not express HLA-DR. Epithelial cells of control colon did not express HLA-DR while epithelial cells of control ileal tissues and of diseased colonic and ileal specimens expressed HLA-DR antigen. Only small numbers of lamina propria T cells expressed HLA-DR in both control and disease tissues. There was intense expression of HLA-DR by monocytes and modest expression of HLA-DR by capillary and lymphatic endothelial cells. The induction of HLA-DR expression by diseased colonic epithelium and the observation that lymphatic endothelium expresses HLA-DR are new observations, and we established that Leu 7⁺ cells are present in very small numbers in both normal and diseased intestine and colon.Supported by funding from the National Foundation for Ileitis and Colitis and by Merit Review funds from the Veterans Administration.     

The importance of AST / ALT rate in nonalcoholic steatohepatitis diagnosis

BACKGROUND/AIMS: There is a histologic similarity between nonalcoholic steatohepatitis and alcoholic liver disease and in some cases differential diagnosis may be difficult, since some patients do not report abusive alcohol consumption. OBJECTIVE: Evaluating the usefulness of setting the rate AST/ALT for the differential diagnosis of nonalcoholic steatohepatitis and alcoholic liver disease. PATIENTS AND METHODS: Twenty nine obese patients with nonalcoholic steatohepatitis were compared with 28 patients with alcoholic liver disease. The diagnosis of nonalcoholic steatohepatitis was made after exclusion of other causes of liver disease and by histologic findings of, at least, macrovesicular steatosis and hepatocellular necrosis. RESULTS: In patients with nonalcoholic steatohepatitis the medium AST value was 52.3 +/- 21.2 U/L and ALT of 90.1 +/- 37.9 U/L, being the AST/ALT rate lower than 1 in all patients. In patients with alcoholic liver disease the medium AST value was 140 +/- 82.5 U/L and ALT was 50.6 +/- 40.3 U/L. The rate was higher than 1 in all cases and higher than 2 in 24 (85.7%), being statistically significant when compared with patients with nonalcoholic steatohepatitis. CONCLUSION: The AST/ALT rate seems to be useful in the differential diagnosis of liver diseases, while lower than 1 is highly suggestive of nonalcoholic steatohepatitis.     

Short-term exercise and platelet activity,sensitivity to agonist,and platelet-leukocyte conjugate formation

Strenuous exercise may be partially responsible for cardio-vascular events. The aim was to investigate the platelet activity, reactivity and different platelet-leukocyte-conjugate formation following maximal short-term exercises. Fifteen healthy non-smokers underwent three isokinetic maximal tests on a SRM cycle ergometry system with durations of 15, 45 and 90 s. Blood samples were taken after a 30-min rest, immediately before and after exercise, and 15 min and 1 h after completion of exercise. Platelets were detected flow-cytometrically by CD41, and activated platelets by CD62P. In addition, stimulation of the platelets in vitro with 7.5 microM TRAP-6 was initiated. For testing platelet-leukocyte-conjugates, antibodies against CD45, CD14 and CD41 were used. After the exercise tests the percent of non-stimulated CD62P-positive platelets (%PC) was unchanged. In contrast, an increase in %PC (CD62P) TRAP-6 stimulated (15-s test: 37.2+/-10.3 to 46.2+/-12.3%, P < 0.05; 90-s test: 40.6+/-9.5 to 51.7+/-10.2%, P < 0.01) and in platelet-granulocyte, platelet-lymphocyte, and platelet-monocyte conjugate formation 15 min after exercise (45- and 90-s test; P < 0.05) were observed in comparison with the changes on the control day. The changes nearly reversed 1 h after exercise. Maximal short-term exercise only leads to a moderate increase of platelet reactivity and to an increase in the different platelet-leukocyte conjugates. The implications of the changes in platelet-leukocyte conjugate formation should be investigated in future studies.     

Sequential Treatment of Biofilms with Aztreonam and Tobramycin Is a Novel Strategy for Combating Pseudomonas aeruginosa Chronic Respiratory Infections

Traditional therapeutic strategies to control chronic colonization in cystic fibrosis (CF) patients are based on the use of a single nebulized antibiotic. In this study, we evaluated the therapeutic efficacy and dynamics of antibiotic resistance in Pseudomonas aeruginosa biofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Laboratory strains PAO1, PAOMS (hypermutable), PAOMA (mucoid), and PAOMSA (mucoid and hypermutable) and two hypermutable CF strains, 146-HSE (Liverpool epidemic strain [LES-1]) and 1089-HSE (ST1089), were used. Biofilms were developed using the flow cell system. Mature biofilms were challenged with peak and 1/10-peak concentrations of ATM (700 mg/liter and 70 mg/liter), TOB (1,000 mg/liter and 100 mg/liter), and their alternations (ATM/TOB/ATM and TOB/ATM/TOB) for 2 (t = 2), 4 (t = 4), and 6 days (t = 6). The numbers of viable cells (CFU) and resistant mutants were determined. Biofilm structural dynamics were monitored by confocal laser scanning microscopy and processed with COMSTAT and IMARIS software programs. TOB monotherapy produced an intense decrease in CFU that was not always correlated with a reduction in biomass and/or a bactericidal effect on biofilms, particularly for the CF strains. The ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to that with the individual regimens, potentiating the bactericidal effect and/or the reduction in biomass, particularly at peak concentrations. Resistant mutants were not documented in any of the regimens at the peak concentrations and only anecdotally at the 1/10-peak concentrations. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to the current maintenance treatments with just one antibiotic in monotherapy.