Impaired Surfactant Production by Alveolar Epithelial Cells in a SCID-hu Lung Mouse Model of Congenital Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and life-threatening lung-associated diseases in premature infants and immunocompromised children. Although the fetal lung is a major target organ of the virus, HCMV lung pathogenesis has remained unexplored, possibly as a result of extreme host range restriction. To overcome this hurdle, we generated a SCID-hu lung mouse model that closely recapitulates the discrete stages of human lung development in utero. Human fetal lung tissue was implanted into severe combined immunodeficient (CB17-scid) mice and inoculated by direct injection with the VR1814 clinical isolate of HCMV. Virus replication in the fetal lung was assessed by the quantification of infectious virus titers and HCMV genome copies and the detection of HCMV proteins by immunohistochemistry and Western blotting. We show that HCMV efficiently replicated in the lung implants during a 2-week period, forming large viral lesions. The virus productively infected alveolar epithelial and mesenchymal cells, imitating congenital infection of the fetal lung. HCMV replication triggered apoptosis near and within the viral lesions and impaired the production of surfactant proteins in the alveolar epithelium. Our findings highlight that congenital and neonatal HCMV infection can adversely impact lung development, leading to pneumonia and acute lung injury. We have successfully developed a small-animal model that closely recapitulates fetal and neonatal lung development and provides a valuable, biologically relevant tool for an understanding of the lung pathogenesis of HCMV as well as other human respiratory viruses. Additionally, this model would greatly facilitate the development and testing of new antiviral therapies for HCMV along with select human pulmonary pathogens.     

Virulence-associated characteristics and phage lysogenicity of two morphologically distinct colonies of Vibrio cholerae O139 serogroup

Abstract The presence of a temperate phage was demonstrated in a strain of Vibrio cholerae O139 isolated from a patient. Spontaneous variants with translucent colonies had lost this phage. The loss of the phage was associated with increased hydrophobicity, indicating the loss of the capsule. These clones were sensitive to serum bactericidal activity, showed decreased expression of such presumed virulence factors as proteases, motility and mannose-sensitive pili. Furthermore, excision of the phage made the strain dependent on purines for growth.     

The Relationship between the Structure of the Tick-Borne Encephalitis Virus Strains and Their Pathogenic Properties

Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates.     

The efficacy of estrus detection and fertility following synchronization with PGF2a or synchro - mate- B in Zebu cattle

Two experiments were carried out to assess the efficacy of estrus detection and fertility in Zebu cattle after synchronization with prostaglandin F2a or a progestagen. The first experiment compared estrus detection rates and fertility following insemination in 42 cows previously synchronized with either 25 mg of PGF2a or with a 6 mg of Norgestomet implant plus 5 mg i.m. of estradiol valerate (SMB). Differences were observed in the percentage of cows detected in estrus (54 vs 95%, respectively, P < 0.05), but not in fertility at the first synchronized estrus (26 vs 15%), nor in the detection rate and fertility at the subsequent estrous period (38 v 47%). The second experiment evaluated the efficacy of estrus detection at different time intervals in 30 cows, comparing estrus synchronized with PGF2a with the subsequent estrous period. The observation periods were continuous, day and night, for 100 h both after PGF2a treatment and from Day 18 of the treatment cycle (Period 1). In addition, the animals were administered PGF2a again on Day 10 of the second cycle and observed continuously from 0600 to 1800 h, and from Day 18 of the treatment cycle (Period 2). Finally, the same treatment regimen was used except that the observation was between 0600 to 0700 h and 1800 to 1900 h (Period 3). No differences were obtained in the percentage of cows detected in estrus in the synchronized and nonsynchronized groups (average 75%); however, accuracy in the detection of estrus in Period 3 differed in the nonsynchronized and synchronized estrus groups by 40% (P < 0.05) compared with the other two, more intense observation periods.     

Cardiac phosphocreatine deficiency induced by GPA during postnatal development in rat

The effect of chronic administration of -guanidinopropionic acid (GPA) on the protein profiling, energy metabolism and right ventricular (RV) function was studied in the rat heart during the weaning and adolescence period. GPA was given in tap water (1–1.5%) using pair drink controls. The feeding of animals with GPA solution for a six week period resulted in elevation of heart to body weight ratio due to body growth retardation. GPA accumulated in the myocardium up to 67.37 ± 5.3 moles.g dry weight and the tissue content of total creatine, phosphocreatine and ATP was significantly decreased to 15%, 9% and 65% of control values respectively. Total activity of creatine kinase (CK) was not changed, but the proportion of mitochondrial (Mi) CK isoenzyme was decreased; the percentage of MB isoenzyme of CK was significantly higher. GPA treatment resulted in an elevation of the content of cardiac collagenous proteins and decrease of non-collagenous proteins in the heart; in parallel, a decrease of the collagen I to collagen III ratio was detected. The function of the RV was assessed using an isolated perfused heart with RV performing pressure-volume work. As compared to pair-drink controls, RV function was significantly impaired the GPA group: at any given right atrial filling pressure, the RV systolic pressure and the rate of pressure development were decreased by almost a factor of two. Elevation of the RV diastolic pressure with increasing pulmonary artery diastolic pressure was also significantly steeper in the GPA group which also showed decrease of cardiac output, especially at high outflow resistance. It may be assumed that chronic administration of GPA deeply influenced metabolic parameters, protein profiles and contractile function of the developing heart. On the other hand, concentrations of glucose, total lipids and triglycerides in blood plasma were not affected. All these data confirm the concept that the CK system is of central importance both for heart function and for the regulation of normal growth of cardiac myocytes.     

Matrix synthesis of chiral carboxy derivatives: Aldehyde chiral discrimination due to a two-point coordination of Mg2+

To control stereoselectivity in aldol-like reactions with chiral carbohydrate templates, we studied the interaction between completely protected dialdo compounds and magnesium enediolates of arylacetic acids. Diastereomeric mixtures of the highly functionalized acids obtained were esterified to isolate individual methyl uronates. It was found that all the diastereomeric esters exhibit Cotton effects of the same positive sign in the 220–230 nm region and so possess the same S configuration of the aryl chiral center C(6). Chiral center C(5) configurational assignments were performed using IR and ORD spectroscopy. We separated and specified four pairs of diastereomeric methyl uronates. It follows that the precursory acids have the same 5R*, 6S (major isomers) and 5S*, 6S (minor isomers) configurations. A tentative mechanism for complexation and possible models of Mg²⁺ -protected dialdose intermediate complexes has been proposed. We have concluded that a kind of orbital steering is realized, accompanied by some “tuning” of molecular assembly conditioned by two-point coordination between Mg²⁺ and potential cation-binding sites in the substrate molecules. Thus it has been demonstrated that reasonable diastereo-selectivity can be achieved even through the use of small matrix molecules using rather small functional groups, which do not impose any stringent steric requirements. © 1993 Wiley-Liss, Inc.     

Structure of the core oligosaccharide of a rough-type lipopolysaccharide of Pseudomonas syringae pv. phaseolicola.

The core structure of the lipopolysaccharide (LPS) isolated from a rough strain of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola, GSPB 711, was investigated by sugar and methylation analyses, Fourier transform ion-cyclotron resonance ESI MS, and one- and two-dimensional 1H-, 13C- and 31P-NMR spectroscopy. Strong alkaline deacylation of the LPS resulted in two core-lipid A backbone undecasaccharide pentakisphosphates in the ratio approximately 2.5 : 1, which corresponded to outer core glycoforms 1 and 2 terminated with either L-rhamnose or 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), respectively. Mild acid degradation of the LPS gave the major glycoform 1 core octasaccharide and a minor truncated glycoform 2 core heptasaccharide, which resulted from the cleavage of the terminal Kdo residues. The inner core of P. syringae is distinguished by a high degree of phosphorylation of L-glycero-D-manno-heptose residues with phosphate, diphosphate and ethanolamine diphosphate groups. The glycoform 1 core is structurally similar but not identical to one of the core glycoforms of the human pathogenic bacterium Pseudomonas aeruginosa. The outer core composition and structure may be useful as a chemotaxonomic marker for the P. syringae group of bacteria, whereas a more conserved inner core structure appears to be representative for the whole genus Pseudomonas.