Metabolic and cardiovascular profile in patients with Addison's disease under conventional glucocorticoid replacement.

OBJECTIVE: Although two studies have shown that Addison's disease (AD) is still a potentially lethal condition for cardiovascular, malignant, and infectious diseases, a recent retrospective study showed a normal overall mortality rate. Differently from secondary hypoadrenalism, scanty data exist on the role of conventional glucocorticoid replacement on metabolic and cardiovascular outcome in AD. SUBJECTS AND METHODS: In 38 AD under conventional glucocorticoid replacement (hydrocortisone 30 mg/day or cortisone 37.5 mg/day) ACTH, plasma renin activity (PRA), DHEAS, fasting glucose and insulin, 2-h glucose after oral glucose tolerance test, serum lipids, 24-h blood pressure and intima-media thickness (IMT) were evaluated and compared with 38 age-, sex- and body mass index (BMI)-matched controls (CS). RESULTS: AD had ACTH and PRA higher and DHEAS lower (p<0.0005) than CS. Mean waist was higher (p<0.05) in AD than in CS. Although no differences were found for mean gluco-lipids levels, a higher percentage of AD compared to CS were IGT (8 vs 0%), hypercholesterolemic (18 vs 8%), and hypertriglyceridemic (18 vs 8%); none of the AD and CS showed either HDL<40 mg/dl or LDL>190 mg/dl. At the multiple regression analysis, in both AD and CS, BMI was the best predictor of 2-h glucose and age of total and LDL cholesterol; in AD, no significant correlation was found between the above mentioned metabolic parameters and either hormone levels or disease duration. In both AD and CS 24-h blood pressure and IMT were normal. CONCLUSIONS: Our study shows a higher prevalence of central adiposity, impaired glucose tolerance and dyslipidemia in AD patients.

Hypothalamus-pituitary-adrenal axis evaluation in patients with hypothalamo-pituitary disorders: comparison of different provocative tests.

BACKGROUND: The insulin tolerance test (ITT) is the gold standard test to evaluate hypothalamic-pituitary-adrenal (HPA) axis in suspected ACTH insufficiency. When contraindicated, alternative tests have been proposed such as metyrapone and ACTH stimulation test. 250 microg ACTH is a supramaximal dose and unreliable in this setting. The diagnostic reliability of 1.0 microg ACTH test is controversial and very low doses have been proposed. DESIGN: In 31 patients with hypothalamo-pituitary disorders and normal basal cortisol, we compared the diagnostic sensitivity, specificity and accuracy of metyrapone [metyrapone test (MET) 30 mg/kg p.o.], high (HDT, 250 microg i.v.), low (LDT, 1.0 microg i.v.) and very-low (VLDT, 0.06 microg i.v.) dose ACTH tests. Receiver operator curve (ROC) analysis was applied with ITT as reference test. RESULTS: MET approached the best pairs of values for highest sensitivity (71.4% and 64.3%) and highest specificity (100% and 82.4%) using ACTH and 11-deoxycortisol (11-DOC) cut-off of 17.3 pmol/l and 144.3 nmol/l. Either HDT or LDT sensitivity approached 71.4% with a specificity of 82.4% or 73.3% with a specificity of 80% for cortisol cut-off of 582.1 or 477.3 nmol/l. VLDT approached the highest sensitivity (57.1%) and highest specificity (88.2%) for a cortisol cut-off of 364.2 nmol/l. CONCLUSION: Neither MET nor ACTH test can be considered completely reliable for the diagnosis of secondary hypoadrenalism, when compared with ITT that remains the best test. Either MET or ACTH stimulation test, at both high and low dose, show an overall similar reliability, provided that appropriated cut-off values were considered; testing with very low ACTH doses seems to be misleading.

[Combined dissector for laparoscopic esophageal myotomy: introduction of a prototype of dedicated instrumentation]. / Dissettore combinato per miotomia esofagea laparoscopica: presentazione di un prototipo di strumento dedicato.

The authors' aim in this article is to present the use of a combined dissector, devised, designed and patented by themselves, for laparoscopic oesophageal myomectomy in achalasic patients. The prototype was produced by Karl Storz Endoskope. This tool has a stem measuring 10 mm in diameter, with an operative push rod consisting of two upward bent jaws and an electrode that can emerge from the jaws as required by the surgeon. The authors used the dissector in two patients with a surgical achalasic mega-oesophagus. The two jaws can dissect and then divide the oesophageal muscular layer from the submucosal layer, whereas the electrode can cut the muscular fibres. The use of the combined dissector allows the surgeon to perform oesophageal myomectomy easily, with efficacy and safety, using only the right hand. The instrument requires a number of minor changes which are currently being planned.

RB18A, whose gene is localized on chromosome 17q12-q21.1, regulates in vivo p53 transactivating activity.

Among the different cellular factors that regulated p53 functions, we previously identified (P. Drane et al., Oncogene, 15: 3013-3024, 1997) RB18A, a new gene whose encoded Mr 205,000 protein interacted in vitro, through its COOH-terminal domain, with p53. Therefore, we analyzed the in vivo role of RB18A by measuring its effect on the transactivating activity of p53 on physiological promoters. We herein demonstrated that RB18A, which interacted also in vivo with p53, activated Bax promoter and inhibited p21Waf1 or IGF-BP3 promoters. In addition, fluorescence in situ hybridization mapping led to localizing the RB18A gene on chromosome 17q12-q21.1, loci associated with human cancers. This is the first demonstration that in vivo RB18A, in a protein-protein interaction, regulates p53 transactivating activity.

[Cicatricial stenosis of colorectal anastomosis. Transanal treatment with circular stapler]. / Stenosi cicatriziale dell'anastomosi colorettale. Trattamento transanale con stapler circolare.

Anastomotic strictures complicating colorectal anastomoses can be difficult to treat. This condition must not be considered as an uncommon complication. In 20% of patients it may be a serious state that may require a therapy. Two patients treated successfully without complication with the transanal use of an CEEA stapler are presented. The staple cutter is safe and easy to use, and except for a conventional anoscope, no special equipment, including fluoroscope, is needed. On the basis of the successful results obtained, the procedure using staple cutter is recommended for the treatment of anastomotic stricture of the rectum.

Evidence for a new transcript of the Epstein-Barr virus/C3d receptor (CR2, CD21) which is due to alternative exon usage.

CR2 extracellular domain is constituted of 15 or 16 Short Consensus Repeats (SCR), with additional SCR 11 localized between SCRs 10 and 12. We amplified Raji cDNA library, with specific primers where SCR 11 is localized. This generated a new fragment of 643 bp (16b SCR), in addition to the two expected transcripts of 489 (15 SCR) and 667 (16a SCR) bp. Sequencing these three fragments and the corresponding genomic DNA, demonstrated the presence of a 24 bp deletion in 16b SCR, without change of open reading frame and that this 24 bp region was flanked by two splicing acceptor sites. This supported a new alternative splicing of CR2, with generation of a third distinct mRNA. This third transcript was expressed in human CR2 positive T cells, normal or transformed B cells and EBV negative B cell lines. The 24 bp deletion corresponds to a proline-rich region, which may influence CR2 conformation and more likely have consequences on CR2 extra and intracellular interactions.

Identification of RB18A, a 205 kDa new p53 regulatory protein which shares antigenic and functional properties with p53.

Immunological screening with the anti-p53 moAb, PAb1801 of a cDNA expression library, prepared from human B lymphoma cells, led us to identify a new human 205 kDa protein called RB18A for 'Recognized By PAb1801 moAntibody'. Immunoblotting or immunoprecipitation of fusion protein or in vitro translated protein, respectively, demonstrated that RB18A protein was recognized by several anti-p53 moAb reacting with the N or C-terminal domains of p53. Full length sequence of RB18A cDNA and computer analysis demonstrated that despite common antigenic determinants between RB18A and p53 proteins, nucleotide and deduced protein sequences did not reveal any significant homologies. RB18A mRNA was detected in all tissues tested except in kidney. In addition, RB18A protein shared identical functions with p53 protein: binding to DNA or to p53 and self-oligomerization. Furthermore, RB18A regulated p53 specific binding on his DNA consensus binding site. These functions were associated to the C-terminal domain of RB18A protein and more specifically to the PAb421 binding site present in this domain. The activation by RB18A of p53 binding on DNA was induced through an unstable interaction between both proteins. Altogether, our data demonstrated that RB18A protein shares antigenic and functional properties with p53 and regulated p53 functions.

Identification on melanoma cells of p39, a cysteine proteinase that cleaves C3, the third component of complement: amino-acid-sequence identities with procathepsin L.

We previously identified, on normal or tumour cells, two membrane proteinases, p57 and p65, that cleave human C3, the third component of complement, thus regulating C3's biological properties. Whereas p57 was purified from human erythrocytes, p65 was identified using polyclonal anti-p57 antibodies on a human melanoma cell line resistant to complement lysis. Analysis of cell distribution of C3-cleaving proteinases established that DSm, a murine melanoma cell line, expressed a C3-cleaving proteinase distinct from p57 and p65 proteinases. Thus we purified the C3-cleaving proteinase solubilized from membranes of DSm cells. The purified proteinase, termed 'p39' on the basis of its molecular mass of 39 kDa, was identified, using specific proteinase inhibitors, as a cysteine proteinase. Anti-p39 antibodies, prepared against highly purified p39, localized the p39 C3-cleaving proteinase mainly at the cell surface and demonstrated that p39 is also secreted. Anti-p39 antibodies inhibited solubilized C3-cleaving activity. Preincubation of DSm cells with anti-p39 F(ab')2 fragments increased up to 60% complement cell susceptibility. Amino acid analysis of N-terminal and three other regions of p39 demonstrated that this C3-cleaving proteinase carries 100% identity within four regions of procathepsin L. This is the first demonstration that a melanoma cell line expresses on its surface and secretes a p39 C3-cleaving cysteine proteinase that shares sequence identities with procathepsin L. Thus the p39 cysteine proteinase represents a new member of the C3-cleaving proteinase family associated with, and/or expressed on, the cell surface.

Pep34, a synthetic peptide whose sequence corresponds to the intracytoplasmic domain of the Epstein-Barr virus receptor (CR2, CD21), regulates human B lymphocyte proliferation triggered through CR2.

CR2 is involved in regulation of human B lymphocyte proliferation by interacting, through distinct domains, with extracellular, cell surface or intracellular components. Contribution of CR2 intracytoplasmic domain in CR2 regulatory functions remains unclear. Thus, we used pep34, a 34 amino acid synthetic peptide whose sequence corresponds to CR2 intracytoplasmic domain. Pep34 was incorporated into B lymphocytes which were then activated by EBV or C3d through CR2. Our data demonstrate that pep34 inhibits 100% B lymphocyte proliferation triggered by EBV or C3d. Irrelevant peptide had no effect. When B lymphocyte proliferation was triggered by a multipotent B cell activator as SAC, pep34 did not exert any inhibitory effect. Our data demonstrate that pep34 inhibits B lymphocyte proliferation only when lymphocytes are triggered through CR2. Thus, this strongly supports that despite its short length. CR2 intracytoplasmic domain participates to regulatory functions of this receptor.

Epstein-Barr virus/C3d receptor (CR2, CD21) activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways.

We previously demonstrated that human C3d or pep16, a 16-amino acid synthetic peptide derived from human C3d, induced in vivo and in vitro tyrosine phosphorylation of pp105, an intracellular component found only in human cells that express CR2 at their surface. To determine the contribution of CR2 molecules to this enzymatic regulation, we first analyzed whether activation of CR2 by other extracellular CR2 ligands could trigger such regulation in cell extracts. Subsequently, we used cell extracts of either CR2-positive cells depleted in CR2 molecules by absorption with anti-CR2 antibodies or CR2-negative cells transfected with CR2 cDNA. We demonstrate here that pp105 phosphorylation was induced when CR2 was activated by C3d and pep16 as well as by gp350, the Epstein-Barr virus capsid protein or OKB7, an anti-CR2 monoclonal antibody (mAb). HB5, another anti-CR2 mAb, which did not activate B lymphocytes through CR2, did not induce pp105 phosphorylation. Thus, C3d, pep16, gp350, and OKB7 presented similar properties in activating CR2 to trigger pp105 phosphorylation and in regulating B lymphocyte proliferation, while HB-5 had no effect on either assays. Furthermore, our data demonstrate that the presence of CR2 activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways: one which also requires the presence of non-activated CD19, and one which is independent of CD19. The involvement of CD19 in the first pathway was not due to the formation of putative CR2-CD19 complexes. Both pathways were TAPA-1 independent. This is the first demonstration that activated CR2 molecules can play a regulatory role in enzymatic function, such as phosphorylation, despite the absence of CD19 and TAPA-1.

Binding sites of the Epstein-Barr virus and C3d receptor (CR2, CD21) for its three intracellular ligands, the p53 anti-oncoprotein, the p68 calcium binding protein and the nuclear p120 ribonucleoprotein.

Epstein-Barr virus/C3d receptor (CR2, CD21) interacts with three intracellular proteins: the p53 anti-oncoprotein expressed in human B lymphoma cells, the p68 calcium binding protein expressed in normal B lymphocytes and the nuclear p120 ribonucleoprotein (RNP). We previously demonstrated that p53 and p68 interacted with the intracytoplasmic carboxy-terminal domain of CR2. To analyse the amino acid sequence of CR2 binding sites for p53 and p68, we synthesized different peptides whose sequences were derived from this carboxy-terminal domain. Thus, CR2 bound to p53 and p68 through two distinct binding sites localized on the N-terminal and on the central part of its carboxy-terminal domain, characterized by the amino acid sequences of KHRERNYYTD and KEAFHLEARE, respectively. CR2 site reacting with the nuclear p120RNP was determined using either anti-CR2 mAb directed against its extracellular domain or pep34, pep14/SCR3 and pep14/SCR4, synthetic peptides whose sequences corresponded to the intracellular 34 amino acid domain or to sites of the extracellular domain of CR2, respectively. Data support that CR2 interacts with p120RNP through the DEGYRLQGPPSSRC amino acid sequence of its extracellular SCR4 domain. Furthermore, phosphorylation of CR2 inhibits its interaction with the nuclear p120RNP. Binding of CR2, through its intracellular and extracellular domains, with the p53 oncoprotein and p120RNP, respectively, and the co-localization of these three proteins on nuclear interchromatin fibrils, suggest that CR2 could act as a crosslinker between these two nuclear proteins to regulate their functions.

Occlusal splint therapy in MPD and internal derangements of the TMJ.

Although occlusal splints are useful in the treatment of MPD and internal derangements, they must be used in a rational manner. Two basic appliances are employed in Phase I palliative therapy: a maxillary full coverage (MAR) and a mandibular orthopedic repositioning appliance (MORA). The MAR is used in MPD patients primarily to disengage the occlusion and reduce parafunctional activity. In many cases of internal derangement, protrusive mandibular repositioning is indicated. By using the MORA during the day and the MAR at night, the disadvantages of each appliance are minimized.