Intensive monitoring of adverse drug events associated with the use of new glucose-lowering drugs: results from an inception cohort study in Portugal.

AIMS: To determine the frequency and the time-course profile of adverse drug events associated with new glucose-lowering drugs in daily practice and to explore factors potentially associated to these events. METHODS: An inception cohort study was implemented. Adults with type 2 diabetes mellitus initiating a dipeptidyl peptidase-4 inhibitor, a glucagon-like peptide-1 receptor agonist or a sodium-glucose co-transporter-2 inhibitor were eligible for inclusion. Data were collected through baseline and follow-up telephone questionnaires, administered at 2 weeks, 3 months and 6 months. Kaplan-Meier curves and log-rank were computed to compare the time to adverse drug event onset. Cox models were used to explore potential factors associated with adverse drug events. RESULTS: A total of 1328 participants were recruited to the study. In all, 1118 adverse drug events were reported (of which 36% were not listed in the summary of product characteristics) by 41% of participants. The median latency time of adverse drug events reported in ≥1% of participants ranged from 0 to 2 days. Glucagon-like peptide-1 receptor agonist and sodium-glucose co-transporter-2 inhibitor subgroups were associated with an increased likelihood of adverse drug event reporting when compared with the dipeptidyl peptidase-4 inhibitor subgroup. A total of 328 glucose-lowering drugs were withdrawn, more than half as a result of an adverse drug event. CONCLUSIONS: More than two-fifths of participants reported an adverse drug event; dipeptidyl peptidase-4 inhibitors led to the highest proportion of unlabelled adverse drug events. Adverse drug event latency time data show that counselling and adverse drug event management should be proactively addressed from treatment initiation. There should be greater focus on prevalent new users of glucose-lowering drugs, who were more complex participants in this study in terms of type 2 diabetes disease, as they were more likely to report an adverse drug event than the incident new users.

Design, synthesis and biological evaluation of multitarget hybrid molecules containing NHC-Au(I) complexes and carbazole moieties.

N-heterocyclic carbenes (NHCs) represent suitable ligands for rapid and efficient drug design, because they offer the advantage of being easily chemically modified and can bind several substituents, including transition metals as, for instance, gold derivatives. Gold-NHC complexes possess various biological activities and were demonstrated good candidates as anticancer drugs. Besides, carbazole derivatives are characterized by various pharmacological properties, such as anticancer, antibacterial, anti-inflammatory, and anti-psychotropic. Amongst the latter, N-thioalkyl carbazoles were proved to inhibit cancer cells damaging the nuclear DNA, through the inhibition of human topoisomerases. Herein, we report the design, synthesis and biological evaluation of nine new hybrid molecules in which NHC-Au(I) complexes and N-alkylthiolated carbazoles are linked together, in order to obtain novel biological multitarget agents. We demonstrated that the lead hybrid complexes possess anticancer, anti-inflammatory and antioxidant properties, with a high potential as useful tools for treating distinct aspects of several diseases, amongst them cancer.

Gene expression and phenotypic traits of Aggregatibacter actinomycetemcomitans in response to environmental changes.

BACKGROUND AND OBJECTIVE: Periodontopathogens experience several challenges in the oral cavity that may influence their transcription profile and resulting phenotype. This study evaluated the effect of environmental changes on phenotype and gene expression in a serotype b Aggregatibacter actinomycetemcomitans isolate. MATERIAL AND METHODS: Cultures in early exponential phase and at the start of stationary growth phase in microaerophilic and anaerobic atmospheres were evaluated. Cell hydrophobic properties were measured by adherence to n-hexadecane; in addition, adhesion to, and the ability to invade, KB cells was evaluated. Relative transcription of 12 virulence-associated genes was determined by real-time reverse transcritption quantitative PCR. RESULTS: The culture conditions tested in this study were found to influence the phenotypic and genotypic traits of A. actinomycetemcomitans. Cells cultured in microaerophilic conditions were the most hydrophobic, reached the highest adhesion efficiency and showed up-regulation of omp100 (which encodes an adhesion) and pga (related to polysaccharide synthesis). Cells grown anaerobically were more invasive to epithelial cells and showed up-regulation of genes involved in host-cell invasion or apoptosis induction (such as apaH, omp29, cagE and cdtB) and in adhesion to extracellular matrix protein (emaA). CONCLUSION: Environmental conditions of different oral habitats may influence the expression of factors involved in the binding of A. actinomycetemcomitans to host tissues and the damage resulting thereby, and thus should be considered in in-vitro studies assessing its pathogenic potential.

Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates.

BACKGROUND AND OBJECTIVE: Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. MATERIAL AND METHODS: Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated. RESULTS: The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes. CONCLUSION: Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.

Binding of plasma fibronectin to cell layers of human skin fibroblasts.

Human plasma fibronectin bound to confluent cell layers of cultured human-skin fibroblasts in two distinct pools. Initial binding of fibronectin occurred in a deoxycholate-soluble pool (Pool I). Binding in Pool I was reversible and reached a steady state after 3 h. After longer periods of incubation, fibronectin became bound in a deoxycholate-insoluble pool (Pool II). Binding in Pool II was irreversible and proceeded at a linear rate for 30 h. After 30 h of incubation, a significant proportion of fibronectin bound in Pool II was present as disulfide-bonded multimers. HT1080 cells, a human sarcoma cell line, did not bind fibronectin in either pool. Also, isolated cell matrices prepared by deoxycholate extraction did not bind fibronection. Binding of fibronectin in Pool I of normal fibroblasts occurred via specific, saturable receptors. There were 128,000 binding sites per cell, and KDiss was 3.6 X 10(-8) M. Fluorescence microscopic localization of fibronectin bound in Pool I and Pool II was performed using fluorescein-conjugated fibronectin. Fluorescent staining in Pool I was present in a punctate pattern and in short, fine fibrils. Pool II fluorescence was exclusively in coarse, dense fibrils. These data indicate that plasma fibronectin may become incorporated into the tissue extracellular matrix via specific cell-surface receptors.

Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts.

Plasma fibronectin binds saturably and reversibly to substrate-attached fibroblasts and is subsequently incorporated into the extracellular matrix (McKeown-Longo, P.J., and D. F. Mosher, 1983, J. Cell Biol., 97:466-472). We examined whether fragments of fibronectin are processed in a similar way. The amino-terminal 70,000-mol-wt catheptic D fragment of fibronectin bound reversibly to cell surfaces with the same affinity as intact fibronectin but did not become incorporated into extracellular matrix. The 70,000-mol-wt fragment blocked binding of intact fibronectin to cell surfaces and incorporation of intact fibronectin into extracellular matrix. Binding of the 70,000-mol-wt fragment to cells was partially abolished by cleavage into 27,000-mol-wt heparin-binding and 40,000-mol-wt gelatin-binding fragments and more completely abolished by reduction and alkylation of disulfide bonds. Binding of the 70,000-mol-wt fragment to cells was not blocked by gelatin or heparin. When coated onto plastic, the 70,000-mol-wt fragment did not mediate attachment and spreading of suspended fibroblasts. Conversely, fibronectin fragments that had attachment and spreading activity did not block binding of exogenous fibronectin to substrate-attached cells. These results indicate that there is a cell binding site in the 70,000-mol-wt fragment that is distinct from the previously described cell attachment site and is required for assembly of exogenous fibronectin into extracellular matrix.

Induction of fibronectin matrix assembly in human fibrosarcoma cells by dexamethasone.

Previous studies have suggested that the assembly of fibronectin into the extracellular matrix of cultured fibroblasts is mediated by specific matrix assembly receptors that recognize a binding site in the amino terminus of the fibronectin molecule (McKeown-Longo, P.J., and D.F. Mosher, 1985, J. Cell Biol., 100:364-374). In the presence of dexamethasone, human fibrosarcoma cells (HT-1080) acquired the ability to specifically bind exogenous plasma fibronectin and incorporate it into a detergent-insoluble extracellular matrix. Dexamethasone-induced fibronectin binding to HT-1080 cells was time dependent, dose dependent, and inhibited by cycloheximide. Saturation binding curves indicated that dexamethasone induced the appearance of 7.7 X 10(4) matrix assembly receptors per cell. The induced receptors exhibited a dissociation constant (KD) for soluble fibronectin of 5.0 X 10(-8) M. In parallel experiments, normal fibroblasts exhibited 4.1 X 10(5) receptors (KD = 5.3 X 10(-8) M) per cell. In the presence of cycloheximide, the induced fibronectin-binding activity on HT-1080 cells returned to uninduced levels within 12 h. In contrast, fibronectin-binding activity on normal fibroblasts was stable in the presence of cycloheximide for up to 54 h. The first-order rate constant (Kt = 2.07 X 10(-4) min-1) for the transfer of receptor-bound fibronectin to extracellular matrix was four- to fivefold less than that for normal fibroblasts (Kt = 1.32 X 10(-3) min-1). Lactoperoxidase-catalyzed iodination of HT-1080 monolayers indicated that a 48,000-mol-wt cell surface protein was enhanced with dexamethasone. The results from these experiments suggest that dexamethasone induces functional matrix assembly receptors on the surface of HT-1080 cells; however, the rate of incorporation of fibronectin into the matrix is much slower than that of normal fibroblasts.

Plasminogen activator inhibitor type I stabilizes vitronectin-dependent adhesions in HT-1080 cells.

Polyclonal antibodies against plasminogen activator inhibitor type-I (PAI-1) caused rapid retraction and rounding of substrate-attached HT-1080 cells. The kinetics and extent of antibody-mediated cell rounding were not dependent on either urokinase or plasmin activity. Cells adherent to vitronectin-coated substrates detached within 2 h of antibody addition. Cells adherent to fibronectin were unaffected by the antibodies. Immunoblotting of substrate-attached material indicated that HT-1080 cells deposited PAI-1 into vitronectin, but not fibronectin, dependent contacts. These data suggest that the antibody-mediated cell rounding resulted from a steric disruption of vitronectin-dependent adhesions, indicating that the binding site on vitronectin for PAI-1 is near, but does not overlap, the binding site for vitronectin receptor. The accumulation of PAI-1 into vitronectin-dependent adhesion sites correlated temporally with the preferential degradation of fibronectin from the substrate. HT-1080 cells adherent to either fibronectin or vitronectin were able to activate exogenous plasminogen to plasmin. Plasmin levels were increased 200% on cells adherent to fibronectin and 100% on cells adherent to vitronectin. In the presence of a neutralizing antibody against PAI-1, vitronectin adherent cells activated plasminogen to the same extent as fibronectin adherent cells. Plasmin levels of 200% above baseline were associated with retraction of cells from the substrate. The ability of vitronectin adherent cells to activate exogenous plasmin was completely blocked in the presence of neutralizing antibodies against urokinase. These data represent the first demonstration that vitronectin-associated PAI-1 regulates urokinase in focal contact areas.

Activation of distinct alpha5beta1-mediated signaling pathways by fibronectin's cell adhesion and matrix assembly domains.

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the alpha5beta1 integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed "matrix assembly sites," which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the alpha5beta1 integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the alpha5beta1 integrin, suggesting that these two domains of fibronectin cannot bind to the alpha5beta1 integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the alpha5beta1 integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.

Binding and degradation of platelet thrombospondin by cultured fibroblasts.

Thrombospondin was purified from human platelets and labeled with 125I, and its metabolism was quantified in cell cultures of human embryonic lung fibroblasts. 125I-Thrombospondin bound to the cell layer. The binding reached an apparent steady state within 45 min. Trichloroacetic acid-soluble radioactivity was detected in the medium after 30 min of incubation; the rate of degradation of 125I-thrombospondin was linear for several hours thereafter. Degradation of 125I-thrombospondin was saturable. The apparent Km and Vmax for degradation at 37 degrees C were 6 X 10(-8) M and 1.4 X 10(5) molecules per cell per minute, respectively. Degradation was inhibited by chloroquine or by lowering the temperature to 4 degrees C. Experiments in which cultures were incubated with thrombospondin for 45 min and then incubated in medium containing no thrombospondin revealed two fractions of bound thrombospondin. One fraction was localized by indirect immunofluorescence to punctate structures; these structures were lost coincident with the rapid degradation of 50-80% of bound 125I-thrombospondin. The second fraction was localized to a trypsin-sensitive, fibrillar, extracellular matrix. 125I-Thrombospondin in the matrix was slowly degraded over a period of hours. Binding of 125I-thrombospondin to the extracellular matrix was not saturable and indeed was enhanced at thrombospondin concentrations greater than 3 X 10(-8) M. The ability of 125I-thrombospondin to bind to extracellular matrix was diminished tenfold by limited proteolytic cleavage with trypsin. Degradation of trypsinized 125I-thrombospondin was also diminished, although to a lesser extent than matrix binding. Heparin inhibited both degradation and matrix binding. These results suggest that thrombospondin may play a transitory role in matrix formation and/or organization and that specific receptors on the cell surface are responsible for the selective removal of thrombospondin from the extracellular fluid and matrix.

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

Genetic diversity and toxic activity of Aggregatibacter actinomycetemcomitans isolates.

INTRODUCTION: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes. METHODS: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB. RESULTS: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells. CONCLUSION: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.

The Akt signaling pathway determines the different proliferative capacity of chronic lymphocytic leukemia B-cells from patients with progressive and stable disease.

Chronic lymphocytic leukemia (CLL) B-cells are hyporesponsive to many proliferative signals that induce activation of normal B-lymphocytes. However, a heterogeneous response has recently been observed with immunostimulatory CpG-oligodeoxynucleotides (CpG ODN). We now show that CpG ODN induce proliferation mainly in CLL B-cells from patients with progressive disease and unmutated immunoglobulin V(H) genes, whereas G(1)/S cell cycle arrest and apoptosis are induced in leukemic B-cells from stable/V(H) mutated CLL. Examination of early signaling events demonstrated that all CLL B-cells respond to CpG ODN stimulation by degradation of the NF-kappaB inhibitor IkappaB and activation of the Akt, ERK, JNK and p38 MAPK kinases, but the magnitude and duration of the signaling response was greater in the proliferating cases. Pharmacological inhibition of these pathways showed that simultaneous activation of Akt, ERK and JNK is required for cell cycle progression and proliferation. Conversely, introduction of constitutively active Akt in nonproliferating CLL B-cells resulted in induction of cyclin A following CpG ODN stimulation, indicating that increased Akt activation is sufficient to overcome the hyporesponsiveness of these cells to proliferative signals. Thus, the magnitude of Akt signaling may determine the distinct responses observed in leukemic B-cells belonging to the different prognostic subgroups.

Structure and chemistry of interfaces between ceria and yttria-stabilized zirconia studied by analytical STEM.

Epitaxial undoped and Gd2O3-doped ceria films were grown by pulsed laser deposition on (1 1 1) faced Y2O3-stabilized zirconia (YSZ). Highly localized cerium reduction at the film-substrate interfaces is revealed by atomically resolved valence EELS mapping using Cs aberration-corrected scanning transmission electron microscopy. The chemical profiles reveal interdiffusion of Ce, (Gd), Y, Zr, forming an intermixing zone at the interface 7-9 (1 1 1) lattice planes wide. In its vicinity, the fraction of Ce3+ raises gradually over 6-8 lattice planes from zero in the bulk ceria to ≈100% in one single plane at the interface. Beyond this plane the Ce3+ fraction drops sharply within the YSZ substrate. In the vicinity of the interface systematic scan deflections are observed during EELS line scans. The advancing electron probe experiences a retarding force at the ceria side, and an accelerating force at the YSZ side, irrespective of the scan direction. This behavior is suggestive of coulombic interactions between the electron probe and a charged interface. This is interpreted as an indication of the presence of a space-charge situation at the YSZ/ceria interface, resulting from an excess negative charge at the ceria side (due to Ce3+cations) and an excess positive charge at the YSZ side (due to oxygen vacancies).

Corrigendum to: Phosphonium Salt Displays Cytotoxic Effects Against Human Cancer Cell Lines.

Due to an oversight one of the author's name was published wrong in the article entitled "Phosphonium Salt Displays Cytotoxic Effects Against Human Cancer Cell Lines" in "Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 17, No. 13. pp. 1796."The correct names of all authors are given below:Dhanyalayam D, Palma G, Cappello AR, Mariconda A, Sinicropi MS, Giordano F, Del Vecchio V, Ramunno A, Arra C, Longo P, Saturnino C.

[Smoking cessation and weight gain]. / Trattamento per la disassuefazione dal fumo ed incremento ponderale.

Cigarette smoking is the single most important preventable cause of death and illness. Smoking cessation is associated with substantial health benefits, but weight gain after smoking cessation is perceived to be a barrier against quitting smoking. The aim of the study was to analyse predictors of weight gain after smoking cessation. The sample included 1067 residents, aged 18-70 years, in a health district of Rome who answered to an anonymous postal questionnaire. Among them 482 were former smokers; 398 provided lifetime histories of both body weight and smoking and were considered in the analysis. 52.5% (49.3% M; 60.5% F) reported weight gain after smoking cessation; among these 25.4% reported a weight gain > or =5 kg. The multivariate logistic regression analysis showed a direct association between female gender (OR 1.9, CI 95% 1.1-3.2), age - 45 years (45-65 years: OR 2.5, CI 95% 1.4-4.4; > 64 years OR 2.1, CI 95% 1.0-4.0), number of cigarettes per day >20/day (OR 3.8, CI 95% 1.3-11.5) and weight gain after smoking cessation. The relevance of weight gain following smoking cessation suggests that health benefits associated with smoking cessation may to some extent be negated by the detrimental effects on health of associated weight gain. Smoking cessation programmes should therefore consider incorporating follow-up support to prevent weight gain; regular measurements of body weight together with dietary indications and increase of physical activity are basic factors to implement in the intervention of smoking cessation.

Transforming growth factor-ß1 functional polymorphisms in myeloablative sibling hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) with sibling donors (s.d.) is a life-saving intervention for patients with hematological malignancies. Numerous genetic factors have a role in transplant outcome. Several functional polymorphisms have been identified in TGF-ß1 gene, such as single-nucleotide polymorphism (SNP) at +29C>T within exon 1. Two hundred and forty five patient/donor pairs who underwent a s.d. HSCT in our centers were genotyped for this SNP. In the myeloablative cohort, +29CC donors were associated with an increase in severe chronic GvHD (32% vs 16%, hazard ratio (HR) 9.0, P=0.02). Regarding survival outcomes, +29CC patients developed higher non relapse mortality (NRM) (1-5 years CC 28-32% vs TC/TT 7-10%; HR 5.1, P=0.01). Recipients of +29TT donors experienced a higher relapse rate (1-5 years TT 37-51% vs TC 19-25% vs CC 13%-19%; HR 2.4, P=0.01) with a decreased overall survival (OS) (1-5 years TT 69-50% vs TC/CC 77-69%; HR 1.9, P=0.05). Similar to previous myeloablative unrelated donors HSCT results, we confirmed that +29CC patients had higher NRM. In addition we found that +29TT donors might be associated with a higher relapse rate and lower OS. These results should be confirmed in larger series. Identification of these SNPs will allow personalizing transplant conditioning and immunosuppressant regimens, as well as assisting in the choice of the most appropriate donor.

Glucocorticoid receptor determinations in leukemia patients using cytosol and whole-cell assays.

Parallel determinations of glucocorticoid receptors in the cells of patients with various forms of leukemia were made by two assay methods, one using cell-free cytosolic extracts and the other using whole-cell preparations. Both assays revealed saturable binding of triamcinolone acetonide in all cases. The mean equilibrium dissociation constant for the interaction of triamcinolone acetonide with the cytoplasmic receptor at 2 degrees was 9.45 +/- 6.33 (S.D.) nM while that for the whole-cell binding at 37 degrees was 6.13 +/- 3.25 nM, suggesting an increase in receptor affinity at physiological temperatures. Competition experiments with various unlabeled steroids revealed a higher degree of glucocorticoid specificity at 37 degrees in whole-cell suspensions than at 2 degrees in cytosol. In a comparative analysis of 41 leukemic cell specimens, it was found that determinations carried out by whole-cell assay, calculated as number of sites per cell correlated well with those performed by cytosol assay, calculated as fmol/mg protein, independent of the type of leukemia. However, for cells with low receptor content, the two assay methods were more difficult to compare. In agreement with previous reports, the cytosol assay consistently underestimated the number of receptors with respect to the whole-cell assay, particularly in cases of lymphatic leukemia. Furthermore, the underestimation decreased for increasing levels of total cellular receptor. These results suggest that, in addition to possible defects in the cytoplasm-to-nucleus translocation process, the acceptor-binding capacity of the nucleus may also represent one of the factors which determines the levels of assayable cytoplasmic receptors. Moreover, they indicate that the two assay methods furnish nonequivalent information.

Pathogenesis of adenocarcinoma in Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant condition characterized by intestinal hamartomatous polyps, mucocutaneous melanin deposition, and increased risk of cancer. Families with PJS from the Johns Hopkins Polyposis Registry were studied to identify the molecular basis of this syndrome and to characterize the pathogenesis of gastrointestinal hamartomas and adenocarcinomas in PJS patients. Linkage analysis in the family originally described by Jeghers in 1949 and five other families confirmed linkage to 19p13.3 near a recently identified gene responsible for PJS. Germ-line mutations in this gene, STK11, were identified in all six families by sequencing genomic DNA. Analysis of hamartomas and adenocarcinomas from patients with PJS identified loss of heterozygosity (LOH) of 19p markers near STK11 in 70% of tumors. Haplotype analysis indicated that the retained allele carried a germ-line mutation, confirming that STK11 is a tumor suppressor gene. LOH of 17p and 18q was identified in an adenocarcinoma but not in hamartomas, implying that allelic loss of these two regions corresponds to late molecular events in the pathogenesis of cancer in PJS. The adenocarcinomas showing 17p LOH also demonstrated altered p53 by immunohistochemistry. None of the 18 PJS tumors showed microsatellite instability, LOH on 5q near APC, or mutations in codons 12 or 13 of the K-ras proto-oncogene. These data provide evidence that STK11 is a tumor suppressor gene that acts as an early gatekeeper regulating the development of hamartomas in PJS and suggest that hamartomas may be pathogenetic precursors of adenocarcinoma. Additional somatic mutational events underlie the progression of hamartomas to adenocarcinomas, and some of these somatic mutations are common to the later stages of tumor progression seen in the majority of colorectal carcinomas.

Degradation of distinct forms of multimeric vitronectin by human fibroblasts.

The plasma protein vitronectin is thought to be an important regulator of extravascular plasminogen activation. In previous studies we have shown that a disulfide stabilized multimeric form of vitronectin is endocytosed and degraded by fibroblast cells (T.S. Panetti, P.J. McKeown-Longo, J. Biol. Chem. 268 (1993) 11988-11993; P.J. McKeown-Longo, T.S. Panetti, in: K.T. Preissner, S. Rosenblatt, C. Kost, J. Wegerhoff, D.F. Mosher (Eds.), Biology of Vitronectins and their Receptors, Elsevier Science Publishers, Amsterdam, 1993, pp. 111-118). The preparation of multimeric vitronectin used in these earlier studies was in the form of high molecular weight disulfide-bonded aggregates which were stable in sodium dodecyl sulfate (SDS). To address the question of whether vitronectin needed to be in the form of disulfide stabilized multimers in order to be endocytosed, a multimeric vitronectin, which was not disulfide stabilized, was prepared from vitronectin that had been treated with reducing agent and alkylated with iodoacetamide. The resulting protein migrated as a 65/75 kDa protein on SDS gels in the absence of reducing agent, confirming that this form of vitronectin was no longer stabilized into disulfide-bonded aggregates. However, the protein was still multimeric when analyzed by native gels and could be converted to SDS stable multimers by cross-linking agents. This result demonstrated that reduced and alkylated vitronectin aggregates into multimeric forms which are not stable in SDS. Similar to disulfide stabilized multimers, alkylated multimers of vitronectin bound to sulfated proteoglycans in the extracellular matrix and were endocytosed and degraded. Degradation of both forms of vitronectin was inhibited with arginine-glycine-aspartic acid peptides, an anti-alphavbeta5 antibody and heparin. Chloroquine and wortmannin were also able to inhibit degradation of both forms of vitronectin, indicating that both multimeric forms were following the same endocytic and degradative pathway. These results suggest that the organization of vitronectin into a multimeric form which will be recognized for endocytosis does not require disulfide bond stabilization. This study further suggests that recognition of vitronectin for endocytosis is dependent upon its conversion from a monomeric to a multivalent form (C.E. Wilkins-Port, P.J. McKeown-Longo, Mol. Biol. Cell 8:S:64A (1997).