Mesenchymal stem cells from patients to assay bone graft substitutes.

Bio-engineered scaffolds used in orthopedic clinical applications induce different tissue responses after implantation. In this study, non-stoichiometric Mg(2+) ions and stoichiometric apatites, which are used in orthopedic surgery as bone substitutes, have been assayed in vitro with human adult mesenchymal stem cells (hMSC) to evaluate cytocompatibility and osteoconductivity. hMSCs from the bone marrow aspirates of orthopedic patients were isolated and analyzed by flow cytometry for the surface markers Stro1, CD29, CD44, CD71, CD73, CD90, CD105 (positive) and CD45, CD235 (negative). The hMSC were analyzed for self-renewal capacity and for differentiation potential. The hMSC, which were grown on different biomaterials, were analyzed for (i) cytotoxicity by AlamarBlue metabolic assay, (ii) osteoconductivity by ELISA for activated focal adhesion kinase, (iii) cytoskeleton organization by fluorescence microscopy, and (iv) cell morphology which was investigated by scan electron microscopy (SEM). Results indicate that isolated cell populations agree with minimal criteria for defining hMSC cultures. Non-stoichiometric Mg(2+) and stoichiometric apatites, in granular form, represent a more favorable environment for mesenchymal stem cell adhesion and growth compared to the non-stoichiometric Mg(2+) apatite, in nano-structured paste form. This study indicates that different forms of biomaterials modulate osteoconductivity and cellular growth by differential activation focal adhesion kinase.

Osteogenic potential of cells derived from nasal septum.

BACKGROUND: The research addressed to detect new molecular targets in the development of therapeutic strategies aimed to repair bone tissues. The AIM OF THIS STUDY was to determine the potential osteogenic activity of bone cells from the nasal septum and their use to perform accurate molecular analysis from a single sample. METHODOLOGY: The cells, after nasal septum surgery, were subjected to gene silencing, Reverse Transcriptase - Polymerase Chain reactions, immunocytochemistry and chromatin immunoprecipitation. RESULTS: Cells from the nasal septum can give rise to mature osteoblasts that express osteogenic markers (ALP, Runx2, Slug) and are able to mineralize. We demonstrated that Runx2, a transcription factor critical in early osteospecific differentiation, interacts in vivo with the promoter of the SLUG gene, a marker of osteoblast maturation. CONCLUSIONS: We demonstrated that nasal septum-derived osteoblasts represent an interesting alternative source for bone forming cells, and a promising material to be utilized in bone cellular therapy.

Vitamin K-induced modification of coagulation phenotype in VKORC1 homozygous deficiency.

BACKGROUND: Combined vitamin K-dependent clotting factor (VKCF) deficiency type 2 (VKCFD2) is a rare bleeding disorder caused by mutated vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) gene. METHODS AND RESULTS: An Italian patient with moderate to severe bleeding tendency was genotyped, and found to be homozygous for the unique VKORC1 mutation (Arg98Trp) so far detected in VKCFD2. The activity levels of VKCFs were differentially reduced, and inversely related to the previously estimated affinity of procoagulant factor propeptides for the gamma-carboxylase. The normal (factor IX) or reduced antigen levels (other VKCFs) produced a gradient in specific activities. Vitamin K supplementations resulted in reproducible, fast and sustained normalization of PT and APTT. At 24 h the activity/antigen ratios of VKCFs were close to normal, and activity levels were completely (factor VII and IX), virtually (prothrombin, factor X and protein C) or partially (protein S) restored. Thrombin generation assays showed a markedly shortened lag time. The time to peak observed at low tissue factor concentration, potentially mimicking the physiological trigger and able to highlight the effect of reduced protein S levels, was shorter than that in pooled normal plasma. At 72 h the thrombin generation times were normal, and the decrease in activity of procoagulant VKCFs was inversely related to their half-life in plasma. The improved coagulation phenotype permitted the uneventful clinical course after invasive diagnostic procedures. CONCLUSIONS: Modification of coagulation phenotypes in VKCFD2 after vitamin K supplementation was clinically beneficial, and provided valuable patterns of factor specific biosynthesis, half-life and decay.

Phosphinic platinum complexes with 8-thiotheophylline derivatives: synthesis, characterization, and antiproliferative activity.

The platinum mixed-phosphine complexes (SP-4,2)-[PtCl(8-MTT)(PPh3)(PTA)] (2) and cis-[Pt(8-MTT)2(PPh3)(PTA)] (3) (MTTH2 = 8-(methylthio)theophylline, PTA = 1,3,5-triaza-7-phosphaadamantane) have been prepared from the precursor cis-[PtCl2(PPh3)(PTA)] (1), which has been fully characterized by X-ray diffraction determination. Antiproliferative activity tests indicated that the presence of one lipophilic PPh3 and one hydrophilic PTA makes 1-3 more active than the analogues bearing two PPh3 or two PTA. The reactivity of cis-[PtCl2(PPh3)2], cis-[PtCl2(PTA)2], and cis-[PtCl2(PPh3)(PTA)] with the bis(thiopurines) bis(S-8-thiotheophylline)methane (MBTTH2), 1,2-bis(S-8-thiotheophylline)ethane (EBTTH2), and 1,3-bis(S-8-thiotheophylline)propane (PBTTH2) has also been investigated. New binuclear complexes have been prepared and identified by spectroscopic techniques and their antiproliferative activities on T2 and SKOV3 cell lines evaluated.

Biologically active platinum complexes containing 8-thiotheophylline and 8-(methylthio)theophylline.

Complexes [Pt(mu-N,S-8-TT)(PPh(3))(2)](2) (1), [Pt(mu-S,N-8-TT)(PTA)(2)](2) (2), [Pt(8-TTH)(terpy)]BF(4) (3), cis-[PtCl(8-MTT)(PPh(3))(2)] (4), cis-[Pt(8-MTT)(2)(PPh(3))(2)] (5), cis-[Pt(8-MTT)(8-TTH)(PPh(3))(2)] (6), cis-[PtCl(8-MTT)(PTA)(2)] (7), cis-[Pt(8-MTT)(2)(PTA)(2)] (8), and trans-[Pt(8-MTT)(2)(py)(2)] (9) (8-TTH(2) = 8-thiotheophylline; 8-MTTH = 8-(methylthio)theophylline; PTA = 1,3,5-triaza-7-phosphaadamantane) are presented and studied by IR and multinuclear ((1)H, (31)P[(1)H]) NMR spectroscopy. The solid-state structure of 4 and 9 has been authenticated by X-ray crystallography. Growth inhibition of the cancer cells T2 and SKOV3 induced by the above new thiopurine platinum complexes has been investigated. The activity shown by complexes 4 and 9 was comparable with cisplatin on T2. Remarkably, 4 and 9 displayed also a valuable activity on cisplatin-resistant SKOV3 cancer cells.

Supra-agonist peptides enhance the reactivation of memory CTL responses.

Single amino acid substitutions at TCR contacts may transform a natural peptide Ag in CTL ligands with partial agonist, antagonist, or null activity. We obtained peptide variants by changing nonanchor amino acid residues involved in MHC class I binding. These peptides were derived from a subdominant HLA-A2-presented, latent membrane protein 2-derived epitope expressed in EBV-infected cells and in EBV-associated tumors. We found that small structural changes produced ligands with vastly different activities. In particular, the variants that associated more stably to HLA-A2/molecules did not activate any CTL function, behaving as null ligands. Interestingly, T cell stimulations performed with the combination of null ligands and the natural epitope produced significantly higher specific CTL reactivation than reactivation of CTLs induced by the wild-type epitope alone. In addition, these particular variants activated memory CTL responses in the presence of concentrations of natural epitope that per se did not induce T cell responses. We show here that null ligands increased ZAP-70 tyrosine kinase activation induced by the natural epitope. Our results demonstrate for the first time that particular peptide variants, apparently behaving as null ligands, interact with the TCR, showing a supra-agonist activity. These variant peptides did not affect the effector T cell functions activated by the natural epitope. Supra-agonist peptides represent the counterpart of antagonists and may have important applications in the development of therapeutic peptides.

Selective amino acid substitutions of a subdominant Epstein-Barr virus LMP2-derived epitope increase HLA/peptide complex stability and immunogenicity: implications for immunotherapy of Epstein-Barr virus-associated malignancies.

The latent membrane protein 2 is an immunogenic antigen expressed in Epstein-Barr virus (EBV)-associated tumors and consequently it may represent a target for specific cytotoxic T lymphocyte (CTL)-based immunotherapies. However, the efficacy of such a therapy is limited by the poor immunogenicity of the protein that induces weak CTL responses directed to the CLGGLLTMV (CLG) epitope only in the minority of EBV-seropositive donors. We have now demonstrated that selective peptide stimulation of peripheral blood lymphocytes induced CLG-specific CTL in all donors, suggesting that this epitope can be a suitable target for specific immunotherapies. We found that the CLG peptide has a low affinity for HLA-A*0201 and does not produce stable complexes, both factors that are likely to determine the strength of CTL responses to this epitope. Therefore, we synthesized and tested CLG analogues carrying single or combined amino acid substitutions to increase HLA/peptide stability. Among the analogues tested we identified two peptides which, compared to the natural epitope, showed higher affinity for HLA-A*0201 molecules, and produced stable complexes. These peptides demonstrated a potent, specific stimulatory capacity and could be used for selective CTL-based therapies.

The lifespan of major histocompatibility complex class I/peptide complexes determines the efficiency of cytotoxic T-lymphocyte responses.

Major histocompatibility complex (MHC)/peptide association and stability are determined by specific amino acid interactions between peptide antigens and the MHC groove, and are regarded as a critical feature in ensuring efficient monitoring by T cells. In this investigation we examined the relationship between MHC/peptide stability and the immunostimulatory capacity of MHC/peptide complexes. For this purpose we compared synthetic peptide analogues derived from the immunodominant HLA-A11-presented IVTDFSVIK (IVT) epitope, for their capacity to reactivate IVT-specific memory cytotoxic T-lymphocyte (CTL) responses. The analogues differentiated from the wild-type epitope by single amino acid substitution at position 2. All peptides showed similar affinity for HLA-A11 molecules and were recognized by IVT-specific CTL clones, but induced HLA-A11 complexes at the cell surface with different lifespan. This model offered the possibility of comparing the capacity of an immunogenic epitope to stimulate a unique population of T-cell precursors depending on the lifespan of its presentation at the cell surface. We demonstrated that stable HLA-A11/peptide complexes efficiently stimulate IVT-specific CTL responses, while HLA-A11/peptide complexes with short lifespan do not. The precise identification of the role of amino acid residues in the formation of stable MHC/peptide complexes may be relevant for the design of wild-type-derived epitopes with high immunogenicity. These analogues may have important applications in the immunotherapy of infectious diseases and immunogenic tumours.

[The role of supraduodenal choledocho-duodenostomy in non-neoplastic obstructive pathology of the principal bile duct]. / Il ruolo della coledocoduodenostomia sopraduodenale nella patologia ostruttiva non neoplastica della via biliare principale.

The authors review the indications of their 146 cases of supraduodenal choledochoduodenostomy (SDC) from 1970 to 1977. The conditions for which the procedure was carried out were dilatation of the choledochus (100%), multiple calculosis (83%), and primitive or secondary stenosing odditis (17%). After a detailed discussion of these indications, the authors report their immediate and long-term results, which were good in 96% of the patients. In the matter of surgical technic the authors give preference to laterolateral SDC, stressing that effective anastomosis requires a choledochal diameter of at least 10 or 12 mm. In good accord with data published from other sources, the authors' data suggest further restriction of direct procedures (in view of the associated risk of acute pancreatitis) in favor of SDC. The authors also review the objections to, and complications of, this surgical procedure, and conclude that these are never so important as to discourage the procedure when definitely indicated.

[Agenesis of the gallbladder. Presentation of a case]. / Sull'agenesia della colecisti. Presentazione di un caso.

The authors describe one case of agenesis of the gallbladder associated with papillary stenosis, dyskinesia of the extrahepatic bile ducts, and chronic appendicitis. They devote particular attention to the composition of hepatic bile and choledochal bile, which was investigated in detail after surgical exploration of the extrahepatic bile passages, transduodenal papillosphincterotomy, and appendectomy. They conclude that this rare congenital failure of organ development, in the long run, even a slight alteration of bile composition in the hepatic and/or choledochal segment of the biliary tract (the latter somewhat serving the purpose of the missing gallbladder) may invite calculosis of the common bile duct.

[Benzogammapirone in the treatment of digestive hemorrhages]. / Il "benzogammapirone" nel trattamento di emorragie digestive

Given the difficulties of treating digestive haemorhages with normal coagulant methods, and considering that most haemorrhages are due to vascular factors, a vasoprotective substance was associated with common coagulant therapies. Benzogammapirone was used orally and by selective arterial infusion. The series is presented and the results discussed.