Quantification of the expression level of integrin receptor alpha(v)beta3 in cell lines and MR imaging with antibody-coated iron oxide particles.

Targeted imaging requires site-specific accumulation of a contrast agent (CA), and the properties of that agent must be selected according to the abundance of the target to obtain a signal above the detection limit of the instrument. However, numerical estimates of receptors per cell are rarely found in the literature. Integrin receptors would be particularly promising targets because of their accessibility from the blood stream and expression on activated neovascular endothelial cells. We systematically estimated the number of integrin receptors of cell lines and primary cells by flow cytometry analysis. Since integrin receptors are heterodimeric molecules, and alpha(v) forms complexes with various beta subunits, the numbers of alpha(v) and beta(3) subunits are therefore dissimilar. The observed values are 3 . 10(3)-1.4 . 10(4)/cell for alpha(v), and 5.3 . 10(2)-1.1 . 10(4)/cell for beta(3). Despite the low number of exposed receptors, we show that up to single-cell MR visualization can be achieved with the use of iron oxide beads complexed with antibodies as CAs.     

Impaired modulation of motor cortex excitability by homonymous and heteronymous muscle afferents in focal hand dystonia.

A decrease of heteronymous median nerve-evoked inhibition of corticospinal projections to forearm extensor muscles was reported in a group of 10 dystonic patients by Bertolasi and colleagues in 2003. Here we tested the excitability of corticomotoneuronal connections to both wrist extensor (ECR) and flexor (FCR) muscles after conditioning stimulation of median and also radial nerve at rest in a group of 25 patients with focal hand dystonia compared to 20 healthy subjects. We also investigated the effect of the wrist dystonic posture, either in flexion or in extension, on the afferent modulation of ECR and FCR motor evolved potentials (MEPs). The heteronymous (median-induced) but also homonymous (radial-induced) inhibitions (interstimuli intervals 13-21 ms) of ECR MEP size observed in healthy subjects were decreased in patients. In addition, homonymous (median-induced) facilitation of FCR MEP size was also decreased in patients while heteronymous inhibition (radial-induced) was not. Neither the involvement of the target muscle in the dystonic posture nor the origin of the afferent volley (from a dystonic muscle) influenced the degree of impairment of afferent modulation of the MEP. These findings support the view that a global abnormal somatosensory coupling in focal hand dystonia may contribute to an inadequate motor command to wrist muscles.     

Homocysteine-Lowering Therapy and Early Progression of Transplant Vasculopathy: A Prospective, Randomized, IVUS-Based Study

Although observational studies suggest that hyperhomocysteinemia may be a risk factor for coronary allograft vasculopathy (CAV), prospective data on homocysteine-lowering interventions and CAV development are lacking. We, therefore, randomized 44 de novo heart transplant (HT) recipients to 15 mg/day of 5-methyl-tetrahydrofolate (n=22), or standard therapy (control group, n=22) to investigate the effect of homocysteine lowering on the change in coronary intimal hyperplasia during the first 12 months after transplant, as detected by intra-vascular ultrasound (IVUS). Although 12 months after HT, homocysteinemia was lower in folate-treated patients (p<0.001), coronary intimal area increased similarly in the two groups (p>0.4). Conversely, hypercholesterolemia and cytomegalovirus infection were both associated with increased intimal hyperplasia (p<0.04), independently from folate intake. Sub-group analysis revealed that folate therapy reduced intimal hyperplasia in patients with hyperhomocysteinemia before randomization (n=19; p=0.02), but increased intimal hyperplasia in patients with normal homocysteine plasma concentrations (p=0.02). This bimodal effect of folate therapy persisted significantly after adjusting for cytomegalovirus infection and hypercholesterolemia. Despite effective in prevent hyperhomocysteinemia after heart transplantation, folate therapy does not seem to affect early CAV onset. However, sub-group analysis suggests that folate therapy may delay CAV development only in patients with baseline hyperhomocysteinemia, while may favor CAV progression in recipients with normal baseline homocysteinemia.     

Prognostic relevance of histological grade and its components in node-negative breast cancer patients.

Available results highlight the lack of good level of evidence studies on the pure prognostic value of histological grade. In the present study, the prognostic relevance of histological grade and of its three components, tubule formation, nuclear pleomorphism and mitotic count, was analyzed in a series of 372 patients with node-negative breast cancer treated with locoregional therapy alone until early relapse. Histological grade was determined blindly by two observers and discordance between evaluations was resolved after joint review using a multihead microscope. No relation was observed between histological grade and any of its three components and disease-free survival. Conversely, a significant relation was observed between histological grade and distant metastasis-free survival (at 6 years, 94, 86 and 76% for grades 1, 2 and 3, respectively, P=0.013) as well as overall survival (98, 90 and 86%, P=0.001). A breakdown analysis as a function of the three components showed that neither tubule formation nor nuclear pleomorphism was associated with prognosis, and only mitotic count strongly influenced both distant metastasis-free survival (91, 82 and 74%, P=0.014) and overall survival (97, 87 and 85%, P=0.011). Histological grade suffers from a much higher subjectivity than any other microscopic evaluation of biomarkers as it is the sum of three different morphological features. Within the Italian Network for Quality Assessment of Tumor Biomarkers program we observed that histological grade is an independent prognostic variable, but also that this role is ascribable only to the number of mitotic figures. In conclusion, due to the ever smaller size of diagnosed breast cancers, resulting in less cancer tissue for biofunctional and molecular analysis, mitotic count evaluated under strict quality control conditions seems to be an accurate and feasible prognostic variable.     

Effects of pyridostigmine on spontaneous and growth hormone-releasing hormone stimulated growth hormone secretion in children on daily glucocorticoid therapy after liver transplantation

OBJECTIVES: We aimed to investigate both nocturnal spontaneous and morning growth hormone (GH)-releasing hormone (GHRH)-induced GH secretion in children on daily glucocorticoid treatment after liver transplantation and to evaluate the effect of pyridostigmine (an acetylcholinesterase inhibitor thought to reduce hypothalamic somatostatin tone) on GH secretion in these patients. DESIGN: We performed a randomized, single-blind, cross-over study. PATIENTS: We studied three male and three female juvenile patients, within a year of orthotopic liver transplantation and under immunosuppressive glucocorticoid therapy (mean dose +/- SEM, 5.92 +/- 0.63 mg/day) and five normal children (four males, one female). MEASUREMENTS: Both nocturnal spontaneous and morning GHRH-induced GH secretion were evaluated after administration of placebo, 1 tablet p.o., or pyridostigmine, 2 mg/kg p.o. RESULTS: Spontaneous GH. Placebo: in liver transplanted children nocturnal GH secretion (mean GH level 10.8 +/- 2.0 mU/l) was not significantly different with respect to normal children (mean GH level 12.8 +/- 1.2 mU/l); pyridostigmine: nocturnal GH secretion was significantly increased as compared to placebo in subjects with liver transplantation but not in normal children. GHRH test. Placebo: liver transplanted patients showed a blunted GH response to GHRH with respect to normal children; pyridostigmine: the GH responses to GHRH (P less than 0.05) increased as compared to placebo and did not differ significantly in the two groups. CONCLUSIONS: Our data suggest a steroid-mediated increase in hypothalamic somatostatin tone in liver transplanted children.     

PG-M1: A New Monoclonal Antibody Directed against a Fixative-Resistant Epitope on the Macrophage-Restricted Form of the CD68 Molecule

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.     

Antibiotic resistance in exopolysaccharide-forming Staphylococcus epidermidis clinical isolates from orthopaedic implant infections

The opportunistic pathogen Staphylococcus epidermidis is able to produce biofilm and to frequently cause implant infections. In recent years, it has also exhibited an increasing antimicrobial drug resistance. Here, the resistance to a panel of 16 different antibiotics in 342 clinical strains of S. epidermidis from orthopaedic implant infections has been investigated. The isolates were pheno- and genotyped for extracellular polysaccharide production, relevant to staphylococcal biofilm formation, in order to ascertain possible associations with antibiotic resistance. Approximately 10% of the isolates were found to be sensitive to all screened antibiotics. In all, 37-38% were resistant to beta-lactams such as oxacillin and imipenem, while the resistance to penicillin, ampicillin, cefazolin, cefamandole, was consistently observed in over 80% of the strains. Erythromycin- and clindamycin- resistant strains were approximately 41% and 16%, respectively. Of the isolates, 10% was resistant to chloramphenicol, 23% to sulfamethoxazole and 26% to ciprofloxacin. Resistance to vancomycin was never observed. Interestingly, exopolysaccharide-producing strains exhibited a significantly higher prevalence in the resistance to the four aminoglycosides (gentamicin, amikacin, netilmicin, tobramycin), to sulfamethoxazole and to ciprofloxacin with respect to non-producing isolates. Moreover, multiple resistance to antibiotics was more frequent among exopolysaccharide-forming strains.     

Dpc4 is expressed in virtually all primary and metastatic pancreatic endocrine carcinomas

DPC4/Smad4 is inactivated in about 50% of pancreatic ductal cancers. It has been recently reported that this gene is also inactivated in neoplasms arising from pancreatic islet cells, a phenomenon suggested to be related to similar progressions of malignancy found in common ductal cancers. To evaluate this possibility, we analysed 20 metastases of pancreatic endocrine carcinomas and their corresponding primary lesion for inactivation of DPC4 using immunohistochemical staining. In fact, immunohistochemical labelling has been shown to correlate with DPC4 gene status with high sensitivity and specificity. The cancers included 18 nonfunctioning tumours, one gastrinoma and one ViPoma all with liver, nodal and/or adrenal metastases. Seventeen were well-differentiated and three poorly differentiated endocrine carcinomas. Dpc4 expression was absent in only one primary well-differentiated endocrine cancer and its liver metastasis, while all the remaining 19 primary tumours and their metastases stained positive for the protein. All positively staining cases showed diffuse cytoplasmic and nuclear staining in virtually all neoplastic cells. Our data suggest that DPC4 is only rarely involved in pancreatic endocrine tumourigenesis and give further weight to the hypothesis that tumours arising from pancreatic exocrine and endocrine epithelia are genetically distinct.     

Successful xenografting of cryopreserved primary pancreatic cancers

In order to assess the suitability of cryopreserved neoplastic tissues for xenografting into nude (nu/nu) mice, we compared the take rate in 28 samples of pancreatic ductal carcinoma. Eleven fresh samples were implanted in nu/nu mice, and 17 were frozen in cryopreserving solution and implanted at a later time. All samples were examined for the presence of neoplastic tissue in cryostat sections. A total of 15 tumors grew in the animals; five from the freshly implanted samples and ten from those cryopreserved. Ten xenografted tumors were characterized for alterations in p53, K-ras, and p16 genes, which were found in six, eight, and nine cases, respectively. Our results demonstrate that the take rate for xenografting is comparable between cryopreserved and fresh tissue samples. The procedure allows for the exchange of tumor material between institutions and permits the establishment of centralized facilities for the storage of an array of different primary tumor samples suitable for the production of in vivo models of cancers.