Comparative Proteomic Analysis of Serum from Patients with Systemic Sclerosis and Sclerodermatous GVHD. Evidence of Defective Function of Factor H

Background

Systemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD.

Methodology

Serum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein.

Principal Findings

Fourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients.

Conclusions

The comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage.     

Erythema infectiosum following generalized petechial eruption induced by human parvovirus B19

Parvovirus B19 is a DNA virus responsible for a wide spectrum of clinical illnesses. Among dermatological manifestations, the most common is erythema infectiosum, also known as the fifth disease. In 1990 Harms et al first described a papular-purpuric gloves and socks syndrome (PPGSS) due to parvovirus B19. It is an acute acral dermatosis characterized by an eruption of petechiae and small purpuric papules affecting the hands and feet in a gloves-and-socks distribution. Recently it was observed that PPGSS may be associated with involvement of the peri-oral region and the chin (acropetechial syndrome) and other sites provoking unusual presentation of the rash. We describe a patient with an acral purpura with the features of the "acropetechial syndrome" involving the buttocks, genital and axillary regions who subsequently developed a maculopapular eruption with the characteristics of the fifth disease. Parvovirus B19 DNA was detected by polymerase chain reaction (PCR) both in skin vasculitic lesions and in the serum during the petechial eruption, before the onset of antibodies. The immune response coincided with the development of the exanthem, suggesting a direct role of parvovirus B19 in the pathogenesis of endothelial cell injury.     

Lipolytic activity of Antarctic cold-adapted marine bacteria (Terra Nova Bay, Ross Sea)

AIMS: The aim of this study was to investigate the lipolytic activity of cold-adapted Antarctic marine bacteria and, furthermore, the combined effect of some environmental factors on this enzymatic process. METHODS AND RESULTS: Strains were assayed for lipolytic activity on a basal medium amended with seven individual fatty acid esters. A significant activity was observed for 148 isolates (95.5% of the total screened). The interactive effect of pH, temperature and NaCl concentration on the substrates was tested for six representative isolates, identified as Pseudoalteromonas, Psychrobacter and Vibrio. Differences between strains according to NaCl and pH tolerances were observed. Only one strain degraded the substrate more efficiently at 4 degrees C than at 15 degrees C. CONCLUSIONS: Our findings demonstrate that the lipolytic activity of Antarctic marine bacteria is rather variable, depending on culture conditions, and occurs in a wide range of salt concentration and pH. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolation and characterization of bacteria that are able to efficiently remove lipids at low temperatures will provide insight into the possibility to use cold-adapted bacteria as a source of exploitable enzymes. Moreover, research on the interactive effects of salt concentration, pH and temperature will be useful to understand the true enzyme potentialities for industrial applications.     

Feasibility and long-term results of focused radioguided parathyroidectomy using a &quot;low&quot; 37 MBq (1 mCi) <Superscript>99m</Superscript>Tc-sestamibi protocol

Aim of the present study was to investigate the feasibility and long-term results of focused radioguided parathyroidectomy using a "low" 37 MBq (1 mCi) ^99mTc-sestamibi dose protocol compared to conventional "high 740 MBq (20 mCi) ^99mTc-sestamibi dose protocol" in patients with primary hyperparathyroidism (PHPT). The data of focused radioguided surgery obtained in a group of 320 consecutive PHPT patients with high probability of the presence of a solitary parathyroid adenoma (PA) were studied. All patients underwent preoperative imaging work-up of double-tracer ^99mTc-pertechnetate/^99mTc-sestamibi subtraction parathyroid scintigraphy (Sestamibi scintigraphy) and high resolution neck ultrasound (US). In 301/320 patients (96.6%) focused minimally invasive radioguided surgery was successfully performed by administering a "low" 37 MBq (1 mCi) ^99mTc-sestamibi dose in the operating room 10 minutes before operation. No major intraoperative complications were recorded. Focused radioguided surgery required a mean time of 32 min and a mean hospital stay of 1.2 days. Local anesthesia was applied in 75 patients, 66 of whom (88%) were patients older than 65 years with comorbidities contraindicating general anesthesia. No case of persistent or recurrent PHPT was observed during post-surgical follow-up (range = 18–70 months; mean +/- SD = 15.3 +/- 9.1 months). Radiation exposure dose to the operating surgeon was 1.2 μSi/hour with the "low 37 MBq (1 mCi) ^99mTc-sestamibi dose", and less than 1.0 μSi/hour for the other operating-room personnel. Focused low dose radioguided parathyroidectomy is a safe and effective means to localize parathyroid adenomas in patients affected by solitary PA thus reducing by 20 fold the radiation exposure dose to the patients and operating room personnel.     

Cytochrome P450 enzymes from the metabolically diverse bacterium Rhodopseudomonas palustris

Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically diverse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to approximately 2.0A have been obtained.     

Administration of r-VEGF-A prevents hepatic artery ligation-induced bile duct damage in bile duct ligated rats

The hepatic artery, through the peribiliary plexus, nourishes the intrahepatic biliary tree. During obstructive cholestasis, the nutritional demands of intrahepatic bile ducts are increased as a consequence of enhanced proliferation; in fact, the peribiliary plexus (PBP) displays adaptive expansion. The effects of hepatic artery ligation (HAL) on cholangiocyte functions during cholestasis are unknown, although ischemic lesions of the biliary tree complicate the course of transplanted livers and are encountered in cholangiopathies. We evaluated the effects of HAL on cholangiocyte functions in experimental cholestasis induced by bile duct ligation (BDL). By using BDL and BDL + HAL rats or BDL + HAL rats treated with recombinant-vascular endothelial growth factor-A (r-VEGF-A) for 1 wk, we evaluated liver morphology, the degree of portal inflammation and periductular fibrosis, microcirculation, cholangiocyte apoptosis, proliferation, and secretion. Microcirculation was evaluated using a scanning electron microscopy vascular corrosion cast technique. HAL induced in BDL rats 1) the disappearance of the PBP, 2) increased apoptosis and impaired cholangiocyte proliferation and secretin-stimulated ductal secretion, and 3) decreased cholangiocyte VEGF secretion. The effects of HAL on the PBP and cholangiocyte functions were prevented by r-VEGF-A, which, by maintaining the integrity of the PBP and cholangiocyte proliferation, prevents damage of bile ducts following ischemic injury.     

Sterically hindered complexes of platinum(II) with planar heterocyclic nitrogen donors. A novel complex with 1-methyl-cytosine has a spectrum of activity different from cisplatin and is able of overcoming acquired cisplatin resistance

A very interesting series of water soluble platinum compounds violating some of the classical structure-activity relationships, but still showing antitumor activity, was reported by Hollis and collaborators some 25 years ago [L.S. Hollis, A.R. Amundsenm, E.W. Stern. J. Med. Chem. 32 (1989) 128-136]. The compounds, having formula [PtClA(2)L](+) (A(2)=two monodentate or a bidentate amine, L=a secondary or tertiary amine or a N-donor heterocycle), were characterized by a positive charge and three non-labile N-donor ligands. We have extended the investigation to analogous compounds in which 2,9-dimethyl-1,10-phenanthroline has taken the place of the A(2) ligand(s) and L is 2-picoline (1), 6-amino-2-picoline (2), or 1-methyl-cytosine (3). The X-ray analysis of 2 has revealed a bow-like distortion of the phenanthroline plane, a sloping of the phenanthroline plane with respect to the coordination plane, and an overall shielding of the metallic core by the ortho substituents of the phenanthroline and pyridine ligands. In vitro grow inhibition assays have been performed on the most water soluble complex 3. The results indicate that this complex is characterized by a potent growth inhibitory activity with mean IC(50) value (in a panel of 11 human tumor cell lines) of 1.1 microM to be compared with a mean value of 3.8 microM for cisplatin. The same compound also appears to completely overcome the acquired cisplatin resistance stemming from reduced uptake or a multifocal mechanism, thus pointing to a mechanism of action distinctly different from that of cisplatin.