PubMed Health⌕ Search

Biomedical subjects

A Spada

Publications and source records attributed to A Spada.

At least 55 records · Page 3Linked to original sources
PubMed2001

Genesis of pituitary adenomas: state of the art.

In recent years, remarkable progress has been made in the understanding of the pathogenesis of pituitary tumors. Pituitary tumors originate from the uncontrolled proliferation of a single transformed cell in which an initiating event has caused a gain of proliferative function. After the initiation, promoting factors cooperate in the clonal expansion. Common oncogenes, such as ras, are only exceptionally involved. The only activating mutations identified so far are gsp mutations causing the constitutive activation of cAMP pathway. However, gsp-positive adenomas are not associated to a more aggressive tumoral phenotype. The oncogenic potential of gsp mutations is limited by a more rapid degradation of the mutant Gs(alpha) with respect to the wild-type protein, and by a faster removal of cAMP due to increased phosphodiesterase activity. Estrogen-inducible gene sequences with transforming properties (pituitary tumor-transforming gene (PTTG)) have been identified in human pituitary tumors. Human pituitary tumor-transforming gene (hPTTG) is involved both in early pituitary tumorigenesis, as it causes in vitro and in vivo transformation acting as a transcription activator, and in tumor progression, as it regulates the production of basic fibroblast growth factor (bFGF), a potent activator of angiogenesis and mitogenesis. Moreover, a role of cyclin D1 in pituitary tumorigenesis is emerging. The allelic loss of loci for unknown oncosuppressor genes are currently under investigation, while an exceedingly limited role for menin gene and RB1 has been demonstrated for sporadic pituitary tumors. Abnormal methylation that predisposing toward genetic instability may favor the allelic loss or the reduced expression of oncosuppressor genes, is also an emerging field of investigation. Several promoting factors, including the excessive action of physiological stimulators, the defective action of inhibitors, the susceptibility to respond to inappropriate stimuli and the locally produced growth factors, help in tumor progression. The study of homeobox genes that intervene in pituitary cell differentiation may help in expanding our knowledge in pituitary tumor cell genealogy.

Adenoma↗
PubMed2001

Somatostatin receptor subtype 2 and 5 in human GH-secreting pituitary adenomas: analysis of gene sequence and mRNA expression.

The role of somatostatin receptor subtypes 2 and 5 (SSTR2 and SSTR5) in determining the secretory and proliferative phenotype as well as the sensitivity to somatostatin analogue treatment is not clearly established. We quantified the expression of SSTR2 and SSTR5 mRNA using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 19 human growth hormone (GH) -secreting adenomas. Tumour characteristics and in vivo sensitivity to somatostatin analogues were assessed; tumours were screened for Gsalpha gene mutations. PCR products of SSTR2 and SSTR5 DNA from tumours resistant to somatostatin analogues were directly sequenced. All tumours expressed both SSTR2 and SSTR5 mRNA at variable levels. No significant correlation between SSTR2 and SSTR5 expression and the presence of Gsalpha mutation, GH levels, or tumour size and invasiveness was observed. A negative correlation between SSTR2 and SSTR5 mRNA levels was observed (r = 0.5; P < 0.05). No significant correlation between the levels of SSTR2 and SSTR5 expression and the in vivo responsiveness to somatostatin analogues was observed, although a tendency to a low SSTR2 expression in resistant tumours was found. No mutations in the coding or bordering regions of either SSTR2 or SSTR5 adenomatous DNA from patients totally or partially resistant to somatostatin analogues were found. The study shows that the different expression of SSTR2 and SSTR5 in GH-secreting adenomas is not significantly correlated with the secretory and proliferative phenotype, although the large, hypersecretory tumours and those with a poor sensitivity to somatostatin analogues seem to express low levels of SSTR2 mRNA. Moreover, both SSTR2 and SSTR5 DNA from tumours resistant to somatostatin analogues were found to possess intact coding sequences.

Adenoma↗
PubMed2001

Expression of cyclic adenosine 3',5'-monophosphate (cAMP)-responsive element binding protein and inducible-cAMP early repressor genes in growth hormone-secreting pituitary adenomas with or without mutations of the Gsalpha gene.

In about 30-40% of GH-secreting adenomas, gain-of-function mutations of the Gsalpha gene, which convert this gene into an oncogene termed gsp, occur. Gsalpha mutations have been related to pituitary tumorigenesis. We focused on 2 nuclear transcription factors that are final targets of the cAMP-dependent pathway and are positively regulated by cAMP signaling, i.e. the cAMP-responsive element binding protein (CREB) and the inducible cAMP early repressor (ICER), that derives from alternative splicing of cAMP-responsive element modulator gene. We examined 21 GH-secreting adenomas, 8 with (gsp(+)) and 13 without (gsp(-)) a mutated Gsalpha. Analysis of CREB and ICER I/II messenger RNA revealed that the levels of both transcripts were higher in gsp(+) than in gsp(-) tumors (CREB/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mean optical density +/- SE, 2.34 +/- 0.36 in gsp(+) vs. 0.99 +/- 0.22 in gsp(-), P = 0.003; ICER I/GAPDH, 0.53 +/- 0.15 in gsp(+) vs. 0.14 +/- 0.07 in gsp(-), P = 0.01; ICER II/GAPDH, 1.5 +/- 0.21 in gsp(+) vs. 0.83 +/- 0.13 in gsp(-), P = 0.01), although a few cases in both groups did not display this pattern of expression. Moreover, no positive correlation between the levels of CREB and ICER transcripts was observed, suggesting the possible presence of alterations in the mechanisms by which cAMP signaling directs the expression of CREB and/or ICER genes. Our results indicate a complex pattern of expression of nuclear transcription factors that mediate cAMP action in both gsp(+) and gsp(-) tumors, suggesting that, beside Gsalpha gene mutations, different and partially unknown molecular events may contribute to the pathogenesis of these tumors.

Adenoma↗
PubMed2001

Relevant cAMP-specific phosphodiesterase isoforms in human pituitary: effect of Gs(alpha) mutations.

Both cAMP production by adenylyl cyclase and cAMP degradation by phosphodiesterases account for intracellular cAMP levels. We previously demonstrated an increased phosphodiesterase activity in GH-secreting adenomas bearing the gsp oncogene. Here we characterize both the activity and the expression of cAMP-specific phosphodiesterase genes in the human pituitary and in gsp+ and gsp- GH-secreting adenomas and analyze the impact of this intracellular feedback mechanism on the levels of cAMP-responsive element-binding protein phosphorylation. Normal pituitary and gsp- GH-secreting adenomas showed similar phosphodiesterase activities, and 7-fold higher levels were observed in gsp+ tumors. In these tumors the increased activity was mainly owing to isobutyl-methyl-xanthine-sensitive phosphodiesterase 4 and to isobutyl-methyl-xanthine-insensitive isoforms. By semiquantitative RT-PCR, all phosphodiesterase 4 transcripts were expressed in the normal and tumoral pituitary. However, the levels of phosphodiesterase 4C and 4D messenger RNAs were significantly higher in gsp+ than in gsp- GH-secreting adenomas and normal pituitary. Expression of the thyroid-specific isobutyl-methyl-xanthine-insensitive phosphodiesterase 8B was absent in the normal pituitary but detectable in almost all GH-secreting adenomas and higher in gsp+ (P < 0.02). Therefore, this study provides a characterization of phosphodiesterase expression in human pituitary and demonstrates a dramatic induction of the cAMP-specific phosphodiesterases 4C and phosphodiesterases 4D and phosphodiesterases 8B in gsp+ GH-secreting adenomas. Similar levels of cAMP-responsive element-binding protein phosphorylation were observed in gsp- and gsp+ GH-secreting adenomas; however, phosphodiesterase blockade caused an increase in cAMP-responsive element-binding protein phosphorylation that was significantly higher in gsp+ than in gsp- adenomas. Because cAMP-responsive element-binding protein represents the principal end point of the cAMP pathway, these results suggest that the enhanced phosphodiesterase activity may have a significant impact on the phenotypic expression of gsp mutations.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Mutation of somatostatin receptor type 5 in an acromegalic patient resistant to somatostatin analog treatment.

Introduction of somatostatin analogs has greatly contributed to improving the prognosis of acromegaly. Although the majority of patients are effectively treated by these agents, resistance occurs in a subset of patients. So far, resistance to somatostatin has never been associated with mutations of the somatostatin receptor subtypes (sst2 and sst5) that inhibit GH secretion. Molecular analysis of genomic DNA from pituitary tumor and peripheral blood obtained from an acromegalic resistant to octreotide showed a somatic activating mutation of Gsalpha (Arg201Cys), no mutation in sst2, and one polymorphism (Pro109Ser) and one germ line mutation (Arg240Trp) in sst5. Wild-type (WT) and mutant sst5 PCR products were cloned and transfected into Chinese hamster ovary K1 cells. In Chinese hamster ovary K1 cells stably expressing mutant sst5, somatostatin-28 was less potent in inhibiting cyclic AMP levels than in WT cells. Proliferation of mutant cells exceeded that of WT by 50%. Moreover, somatostatin reduced cell growth and MAPK activity in WT but not in mutant cells in which the peptide even increased MAPK activity. We suggest that this mutation that abrogates the antiproliferative action of somatostatin and activates mitogenic pathways may be involved in the resistance to somatostatin treatment.

Acromegaly↗
PubMed2001

G protein mutations in endocrine diseases.

This review summarizes the pathogenetic role of naturally occurring mutations of G protein genes in endocrine diseases. Although in vitro mutagenesis and transfection assays indicate that several G proteins have mitogenic potential, to date only two G proteins have been identified which harbor naturally occurring mutations, Gsalpha, the activator of adenylyl cyclase and Gi2alpha, which is involved in several functions, including adenylyl cyclase inhibition and ion channel modulation. The gene encoding Gsalpha (GNAS1) may be altered by loss or gain of function mutations. Indeed, heterozygous inactivating germ line mutations in this gene cause pseudohypoparathyroidism type Ia, in which physical features of Albright hereditary osteodystrophy (AHO) are associated with resistance to several hormones, i.e. PTH, TSH and gonadotropins, that activate Gs-coupled receptors or pseudopseudohypoparathyroidism in which AHO is the only clinical manifestation. Evidence suggests that the variable and tissue-specific hormone resistance observed in PHP Ia may result from tissue-specific imprinting of the GNAS1 gene, although the Gsalpha knockout model only in part reproduces the human AHO phenotype. Activating somatic Gsalpha mutations leading to cell proliferation have been identified in endocrine tumors constituted by cells in which cAMP is a mitogenic signal, i.e. GH-secreting pituitary adenomas, hyperfunctioning thyroid adenomas and Leydig cell tumors. When the same mutations occur very early in embryogenesis they cause McCune-Albright syndrome. Although these mutations would in principle confer growth advantage, studies failed to detect differences in the clinical and hormonal phenotypes, suggesting the existence of mechanisms able to counteract the activation of the cAMP pathway. Activating mutations of Gi2alpha have been identified in a subset of ovarian, adrenal and pituitary tumors, but their prevalence and significance are still controversial. Finally, although Galpha subunits are the only components of the heterotrimeric GTP binding proteins which harbor known mutations, beta/gamma subunits should be considered possible targets of genetic alterations as suggested by the frequent presence of beta3 subunit variants in patients with essential hypertension.

Animals↗
PubMed2001

Genetic aspects of pituitary tumors.

Pituitary adenomas arise from the replication of a single mutated cell in which growth advantage may result from either activation of protooncogenes or inactivation of antioncogenes. The search for oncogenes in the genesis of these tumors has yielded negative results except for the gsp oncogene, that has been identified in about 30-40% of GH-secreting adenomas. gsp mutations cause constitutive activation of the Gs alpha subunit, leading to elevated cAMP formation and growth hormone hypersecretion. In the remaining tumor types several lines of evidence suggest that genes implicated in cell proliferation, such as early immediate genes, growth factors and growth factor receptors, are overexpressed. As far as the loss of antioncogenes in pituitary adenomas is concerned, no inactivating mutations of these genes have yet been identified in pituitary adenomas. However, despite the lack of mutations, several antioncogene proteins have been detected at extremely low levels in pituitary adenomas, consistent with a possible role of these tumor suppressors in pituitary tumor formation.

Animals↗
PubMed2000

A case of central nervous system vasculitis related to an episode of Guillain-Barrè syndrome.

The authors report their knowledge about an uncommon case of isolated vasculitis, restricted to the left sylvian artery during an auto-immune Guillain-Barrè syndrome (GBS), sustained by cytomegalovirus (CMV). An acute cardiopulmonary failure requiring a ventilator and vasopressor support manifested, notwithstanding plasma exchanging and immune-modulating therapy. An IgM-enriched formula administration coincided with a rapid amelioration of GBS and vasculitis to a complete recovery the next month after her discharge to a rehabilitation centre.

Adjuvants, Immunologic↗
PubMed2000

Oxidative diphosphonylation of 1,4-dihydropyridines and pyridinium salts

An oxidative double phosphonylation of dihydropyridines 1 and pyridinium salts 2 is achieved through the use of dialkyl phosphites, DDQ, and triethylamine. Acceptable to good yields of 2, 6-diphosphonylated-1,2-dihydropyridines 3 are obtained in a one-pot reaction involving tandem nucleophilic addition/oxidation processes. Isomerization of 3 to the more stable 2,4-diphosphonylated-1, 4-dihydropyridine 4 was observed in some cases.

Journal Article↗
PubMed2000

Introduction of heteroatom-based substituents into 1,4-dihydropyridines by means of a halogen-mediated, oxidative protocol: diamination, sulfonylation, sulfinylation, bis-sulfanylation, and halo-phosphonylation processes

The natural tendency of 1,4-dihydropyridines to undergo "biomimetic" oxidation to afford pyridinium salts can be switched off and, through the use of reagents that interact electrophilically with the enamine moiety present in the heterocyclic system, it is possible to promote alternative oxidations. In this way, efficient regio- and stereocontrolled synthetic methods have been developed that lead to diversely substituted di- and tetrahydropyridines. These include iodoazidation, diamination, bis-sulfonamidation, sulfonylation, sulfinylation, thiocyanation, sulfanylation, bis-sulfanylation, and halo-phosphonylation processes.

Journal Article↗
PubMed2000

Calcium-sensing receptor expression and signalling in human parathyroid adenomas and primary hyperplasia.

OBJECTIVE: Both in vivo and in vitro evidence indicates that primary hyperparathyroidism is characterized by a reduced sensitivity to extracellular calcium ([Ca2+]o). The existence of alterations in the expression and signalling of calcium sensing receptor (CaSR) in parathyroid neoplasia is still uncertain. In order to clarify the role of CaSR in the reduced [Ca2+]o sensing of parathyroid neoplasia we investigated PTH secretion and intracellular effectors triggered by CaSR activation as well as the levels of expression of CaSR and CaSR coupled G proteins (Gq/G11) in parathyroid adenomas and primary hyperplasia. MATERIALS AND METHODS: The study included 27 parathyroid adenomas, 4 cases of primary hyperplasia and pools of normal parathyroid biopsies. Tissues were either snap frozen in liquid nitrogen or placed in sterile medium for cell dispersion. The effects of increasing [Ca2+]o on in vitro PTH release, intracellular cAMP levels and intracellular calcium ([Ca2+]i) in cells loaded with the Ca2 + indicator fura-2 were evaluated. CaSR mRNA levels were assessed by semiquantitative RT-PCR analysis, using GAPDH as internal standard, while CaSR protein was detected by western blot analysis using a specific polyclonal antibody. Purified antisera selective for G11alpha and Gqalpha were used to detect this class of proteins. RESULTS: In basal conditions (at 0.5 mM [Ca2+]o) in vitro PTH released ranged from 29.4 to 1186 pg/well/60 minutes. Increasing [Ca2+]o from 0.5 to 1, 2.5 and 5 mM caused a variable effect. One group (n = 7) showed a significant but partial reduction of PTH release (of 17 to 60% of basal levels) that occurred at physiological [Ca2+]o concentrations (1 mM) while the remainder showed either inhibition detectable only at 2.5 mM (n = 15) or total (n = 9) resistance to [Ca2+]o. In the responsive cells, [Ca2+]o (1-5 mM) caused a pertussis toxin-insensitive [Ca2+]i rise (ranging from 10% to 260%), due to Ca2+ release from intracellular stores, and an inhibition of forskolin-stimulated cAMP levels. By RT-PCR almost all tumours tested showed a substantial reduction in CaSR mRNA levels when compared to the normal tissue (CaSR/GAPDH ratio: 3.1 +/- 0.5 vs. 15.5 +/- 3.1; P < 0.001), which was confirmed by immunoblotting analysis demonstrating low levels of CaSR protein in tumour tissues. Moreover, low amounts of G11alpha and Gqalpha, the G proteins involved in CaSR coupling, were observed in the majority of pathological tissues. CONCLUSIONS: The study shows that the activation of the calcium sensing receptors expressed in adenomatous parathyroid glands modulates intracellular effectors in a similar way to those operating in the normal parathyroid. Although a reduction of calcium sensing receptor expression is probably involved in the poor inhibition of PTH release induced by [Ca2+]o, this is not the only factor altering [Ca2+]o sensing in parathyroid adenomas, since tumours characterized by different in vitro sensitivity to [Ca2+]o showed similar CaSR levels. The low content of G proteins of the Gq subfamily might represent an additional alteration leading to a defective [Ca2+]o sensing.

Adenoma↗
PubMed2000

Mutational analysis of GNAS1 in patients with pseudohypoparathyroidism: identification of two novel mutations.

Pseudohypoparathyroidism (PHP) refers to two major variants that generally coexist in the same family, PHP type Ia (PHP Ia), in which both PTH resistance and a constellation of physical features, termed Albright's hereditary osteodystrophy (AHO), are present, and pseudopseudohypoparathyroidism (PPHP), in which AHO occurs without PTH resistance. Most patients with PHP Ia show a partial deficiency (50%) of Gs activity, due to loss of function mutations in Gsalpha gene (GNAS1). The present study reports clinical, biochemical, and molecular data of 8 unrelated families with PHP Ia and PPHP. The 13 exons of GNAS1 were screened for mutations by PCR and direct sequencing of the amplified products. We detected heterozygous mutations in the affected members of the 4 families in which PHP Ia was present. In 2 families 2 previously reported deletions in exons 5 and 7 were found, whereas in the other 2 families, 2 novel frameshift deletions were identified in exons 1 and 11, causing a premature stop codon in the mutant allele. No mutation was detected in the families in which PPHP was the only clinical manifestation. In conclusion, we report the first mutational analysis of Italian patients with PHP Ia and PPHP, and we describe two novel deletions in GNAS1. Furthermore, we confirm that these mutations cannot be detected in families with isolated PPHP, suggesting that these forms of AHO are genetically distinct from PHP Ia.

Adolescent↗
PubMed2000

Induction of specific phosphodiesterase isoforms by constitutive activation of the cAMP pathway in autonomous thyroid adenomas.

Thyrocytes largely depend on cAMP signaling for replication and differentiation. This pathway may be constitutively activated by mutations of the TSH receptor (TSHR) and Gsalpha in autonomous thyroid adenomas (ATAs). Because steady state cAMP results from production by adenylyl cyclase and degradation by phosphodiesterases (PDEs), we evaluated PDE activity and expression in ATAs with wild-type and mutant TSHR and Gsalpha. Activating mutations of TSHR and Gsalpha were identified in 7 and 1 of 18 ATAs, respectively. No difference was observed in the cAMP content in ATAs with or without activating mutants. In the surrounding normal thyroid tissue (NTs), PDE activity was 80% isobutylmethylxanthine sensitive, with the major contribution by PDE1 and a minor contribution by PDE4. No differences were observed in PDE activities between NTs and ATAs with wild-type TSHR and Gsalpha. In contrast, in the presence of mutant TSHRs or Gsalpha, total PDE activity was higher. This increase was primarily due to PDE4 induction (917 +/- 116% over NTs), associated with a minor PDE1 increase only in ATAs with mutant TSHR. By RT-PCR, increments of PDE4D and 4C messenger ribonucleic acids were found in the ATAs with mutant TSHR or Gsalpha, whereas messenger ribonucleic acids encoding other cAMP-specific PDEs were not significantly increased. This study provides a characterization of the PDEs expressed in human thyroid and demonstrates a dramatic PDE4 induction in the ATAs bearing mutant TSHR or Gsalpha genes. The increase in cAMP-degrading activity may represent a marker of constitutive adenylyl cyclase activation and constitutes an intracellular feedback mechanism with significant impact on the phenotypic expression of the activating mutations.

Adenoma↗
PubMed2000

Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells.

This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by delta12PGJ2 was first observed at 10 microM, rose to 4000-5000 ng/mL within one log unit and a maximum response was not observed; concentrations of delta12PGJ2 higher than 30 microM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 microM and maintained to 1 microM without toxicity. Both, delta12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 microM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with delta12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of delta12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3-4 hours of exposure to delta12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or delta12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.

Anti-Bacterial Agents↗
PubMed2000

Effect of aging on serum gonadotropin levels in healthy subjects and patients with nonfunctioning pituitary adenomas.

OBJECTIVE: Nonfunctioning pituitary adenomas (NFPA), which represent about one-quarter of human pituitary tumors, occur in middle or old age. Determination of gonadotropin levels, which are not expected to be high during the early postmenopause in normal women and which are low in women with NFPA, is important to distinguishing hypogonadal status due to the normal decline of gonadal function from that due to hypothlalamic-pituitary dysfunction. The aim of the study was to verify whether this difference still persists in old subjects, despite the physiological decline of gonadotropins in the last decades of life. DESIGN AND METHODS: The study included 154 healthy subjects (aged 50-104 years) and 47 patients with NFPA (aged 50-80 years). Blood samples were collected after an overnight fast and hormone levels were measured by two immunofluorimetric assays. RESULTS: In healthy women the highest serum levels of gonadotropins were present in the 50-60 year age group, with a slight but progressive age-associated decrease in serum FSH and LH being observed thereafter. In healthy men serum gonadotropin levels were stable up to 70 years, increased up to 75-85 years and thereafter gradually decreasing. Female patients with NFPA showed levels of gonadotropins which were far lower than controls. Only three patients had levels of both FSH and LH above the 2.5 centile for normal subjects. A high sensitivity and specificity of gonadotropin measurements (about 90%) for the diagnosis of NFPA was observed in female patients aged 50-80 years. In male subjects, a large overlap of gonadotropin values in NFPA and controls, namely over the 50-70 years age range, was observed. CONCLUSIONS: Our study demonstrates that despite the gradual decline of gonadotropin levels in healthy postmenopsausal women, the reduction of both FSH and LH persists in old patients with NFPA, suggesting that measurement of gonadotropin levels could prove useful in the evaluation of pituitary lesions even in old women. More subtle differences seem to occur in male subjects.

Adenoma↗
PubMed2000

Peripheral blood neutrophils from hepatitis C virus-infected patients are replication sites of the virus.

BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) is able to cause not only acute and chronic liver disease, but also immunologic and hematologic disorders. In order to clarify the extra-hepatic tropism of HCV, and to understand the pathogenetic mechanisms of HCV infection, we evaluated viral replication in peripheral blood mononuclear cells. DESIGN AND METHODS: The presence of genomic and antigenomic (replicative) forms of HCV in B- and T-lymphocytes, monocytes, and polymorphonuclear leukocytes (PML) was determined by reverse transcriptase-polymerase chain reaction in 54 HCV-RNA positive patients and, as control groups, in 10 patients who had recovered from HCV infection without evidence of serum HCV-RNA, and in 10 HCV-negative subjects. RESULTS: In HCV-RNA positive patients, the genomic RNA was found in 94% of B-cells, in 14% of T-cells, in 40% of monocytes and in 77% of PML, while only 1 of the HCV-RNA negative subjects showed positivity in B-cells. The anti-genomic form of HCV-RNA was found in 52% of B-cells, in 3% of monocytes, and in 31% of PML. By contrast, it was never detected in T-cells and in HCV-RNA negative subjects. Neither genomic nor anti-genomic forms were found in HCV-negative cases. INTERPRETATION AND CONCLUSIONS: These data suggest that PML are replication sites of HCV. Whether the infection occurs at the level of the stem cells or subsequently during myeloid cell differentiation is, as yet, unknown. The absence of correlation between the presence of replicative forms and any clinical and/or laboratory data opens the question of the role of HCV replication in extra-hepatic sites.

Adult↗
PubMed1999

Aminoisoquinolines: design and synthesis of an orally active benzamidine isostere for the inhibition of factor XA.

The design, synthesis and SAR of sulfonamidopyrrolidinone fXa inhibitors incorporating a new benzamidine isostere, namely aminoisoquinolines, is described. These inhibitors have higher Caco-2 cell permeability than comparable benzamidines and attain higher levels of exposure upon oral dosing. The most potent member 14b (fXa Ki=6 nM) is selective against other serine proteases of interest (>600 fold).

Administration, Oral↗