Infection of stromal and hemopoietic precursor cells with lentivirus vector <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

We developed a method for gene transfer into mesenchymal stromal cells. Lentivirus vector containing green fluorescent protein gene for labeling stromal and hemopoietic precursor cells was obtained using two plasmid sets from different sources. The vector was injected into the femur of mice in vivo and added into culture medium for in vitro infection of the stromal sublayer of long-term bone marrow culture. From 25 to 80% hemopoietic stem cells forming colonies in the spleen were infected with lentivirus vector in vivo and in vitro. Fibroblast colony-forming cells from the femoral bones of mice injected with the lentivirus vector carried no marker gene. The marker gene was detected in differentiated descendants from mesenchymal stem cells (bone cavity cells from the focus of ectopic hemopoiesis formed after implantation of the femoral bone marrow cylinder infected with lentivirus vector under the renal capsule of syngeneic recipient). In in vitro experiments, the marker gene was detected in sublayers of long-term bone marrow cultures infected after preliminary 28-week culturing, when hemopoiesis was completely exhausted. The efficiency of infection of stromal precursor cells depended on the source of lentivirus. The possibility of transfering the target gene into hemopoietic precursor cells in vivo is demonstrated. Stromal precursor cells can incorporate the provirus in vivo and in vitro, but conditions and infection system for effective infection should be thoroughly selected. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 1, pp. 25–28, January, 2007     

<Emphasis Type="Italic">In vivo</Emphasis> study of dihydroquercetin genotoxicity

Genotoxic properties of dihydroquercetin were in vivo studied by the method of chromosome aberrations counting and DNA-comet assay. Dihydroquercetin administered repeatedly (5 times, 0.15 and 1.5 mg/kg) or once in doses of 15, 150, and 2000 mg/kg induced no DNA damages in mouse bone marrow, blood, liver, and rectal cells. Single administration of this preparation in doses of 1.5 and 150 mg/kg and 5-fold administration in a dose of 1.5 mg/kg had no effect on the level of chromosome aberrations in mouse bone marrow cells. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 3, pp. 309–312, March, 2008     

Regulation of cytokine synthesis in cardiac surgery: Role of extracorporeal circuit and humoral mediators <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Objective and design: Cardiopulmonary bypass (CPB) impairs monocyte and neutrophil proliferation, cytokine synthesis, and antigen presentation. This study compares in vivo data with results from an extracorporeal circulation (ECC) model, distinguishing direct effects on cytokine synthesis from regulatory mechanisms. Patients and methods: Whole blood from 18 patients prior to, during and after CPB was stimulated with lipopolysaccharide (LPS). Tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 levels were measured. Additionally, blood from 4 volunteers was circulated in an ECC model. Cytokine levels were measured before and during mock ECC. Results: LPS-induced cytokine synthesis was reduced after CPB (TNF-α: 11 %; IL-6: 29 %; IL-8: 48 % of preoperative values, all p < 0.001). In mock ECC, cytokine production (except IL-8) was suppressed: TNF-α production was lowest 60 min after starting ECC, IL-6 synthesis was lowest at 90 min (33 % and 15 % vs. pre-ECC levels; both p < 0.001). Patient sera contained cytokine-inhibitory activity after CPB, an activity not found in mock ECC. Conclusions: (1) In patients, CPB induces early transient LPS hyporesponsiveness; (2) blood contact with foreign surfaces induces LPS hyporesponsiveness; (3) serum cytokineinhibitory activities are released after CPB, but not in mock ECC. Impaired leukocyte function may explain increased susceptibility to infections after CPB. Received 16 September 2006; accepted without revision by K. Visvanathan 18 October 2006     

Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> after spinal cord injury

Background  

The complement system has been suggested to affect injury or disease of the central nervous system (CNS) by regulating numerous physiological events and pathways. The activation of complement following traumatic CNS injury can also result in the formation and deposition of C5b-9 membrane attack complex (C5b-9/MAC), causing cell lysis or sublytic effects on vital CNS cells. Although complement proteins derived from serum/blood-brain barrier breakdown can contribute to injury or disease, infiltrating immune cells may represent an important local source of complement after injury. As the first immune cells to infiltrate the CNS within hours post-injury, polymorphonuclear leukocytes (PMNs) may affect injury through mechanisms associated with complement-mediated events. However, the expression/association of both early and terminal complement proteins by PMNs has not been fully characterized in vitro, and has not observed previously in vivo after traumatic spinal cord injury (SCI).     

Simultaneous real-time PCR detection of <Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>

This report describes the development of in-house real-time PCR assays using minor groove binding probes for simultaneous detection of the Bacillus anthracis pag and cap genes, the Francisella tularensis 23 KDa gene, as well as the Yersinia pestis pla gene. The sensitivities of these assays were at least 1 fg, except for the assay targeting the Bacillus anthracis cap gene, which showed a sensitivity of 10 fg when total DNA was used as a template in a serial dilution. The clinical value of the Bacillus anthracis- and Francisella tularensis-specific assays was demonstrated by successful amplification of DNA from cases of cow anthrax and hare tularemia, respectively. No cross-reactivity between these species-specific assays or with 39 other bacterial species was noted. These assays may provide a rapid tool for the simultaneous detection and identification of the three category A bacterial species listed as biological threats by the Centers for Disease Control and Prevention.     

SCC<Emphasis Type="Italic">mec</Emphasis> and <Emphasis Type="Italic">spa</Emphasis> types of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains in Israel. Detection of SCC<Emphasis Type="Italic">mec</Emphasis> type V

Molecular characterization of methicillin-resistant Staphylococcus aureus isolates from three hospitals in Israel was the aim of the study presented here. We identified 11 distinct genetic clones by pulsed-field gel electrophoresis. Molecular typing identified four different SCCmec types—I, II, IV, and V−and nine spa types. Spa type t002 was the most common. An erratum to this article can be found at     

<Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> harboring <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> increases cytopathogenicity in vitro

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. Mycoplasmas are frequently found with trichomonads but the consequences of this association are not yet known. In the present study, the effects of T. vaginalis harboring M. hominis on human vaginal epithelial cells and on MDCK cells are described. The results were analyzed by light, scanning and transmission electron microscopy, as well as using cell viability assays. There was an increase in the cytopathic effects on the epithelial cells infected with T. vaginalis associated with M. hominis compared to T. vaginalis alone. The epithelial cells exhibited an increase in the intercellular spaces, a lesser viability, and increased destruction provoked by the infected T. vaginalis. In addition, the trichomonads presented a higher amoeboid transformation rate and an intense phagocytic activity, characteristics of higher virulence behavior.     

Catalog of 86 single-nucleotide polymorphisms (SNPs) in three uridine diphosphate glycosyltransferase genes: <Emphasis Type="Italic">UGT2A1</Emphasis>, <Emphasis Type="Italic">UGT2B15</Emphasis>, and <Emphasis Type="Italic">UGT8</Emphasis>

We report here three high-density maps of variations found among 48 Japanese individuals in three uridine diphosphate glycosyltransferase (UGT) genes, UGT2A1, UGT2B15, and UGT8. A total of 86 single-nucleotide polymorphisms (SNPs) were identified through systematic screening of genomic regions containing these genes: 8 in 5′ flanking regions, 7 in coding regions, 67 in introns, 3 in 3′ untranslated regions, and 1 in a 3′ flanking region. We also discovered 14 variations of other types. Of the 86 SNPs, 63 (73%) were considered to be novel on the basis of comparison of our data with the Database of SNPs (dbSNP) of the National Center for Biotechnology Information. Among the seven SNPs identified in exonic sequences, five were non-synonymous changes that would result in amino-acid substitutions. The collection of SNPs derived from this study will serve as an additional resource for studies of complex genetic diseases and responsiveness to drug therapy. Received: June 12, 2002 / Accepted: June 13, 2002     

Chromosome pairing in allotetraploid hybrids of <Emphasis Type="Italic">Festuca pratensis</Emphasis> × <Emphasis Type="Italic">Lolium perenne</Emphasis> revealed by genomic <Emphasis Type="Italic">in situ</Emphasis> hybridization (GISH)

Genomic in situ hybridization (GISH) was used to make a detailed study of chromosome pairing at metaphase I (MI) of meiosis in six F(1) hybrid plants of the allotetraploid Festuca pratensis x Lolium perenne (2n = 4x = 28; genomic constitution FpFpLpLp). The mean chromosome configurations for all hybrids analysed were 1.13 univalents + 11.51 bivalents + 0.32 trivalents + 0.72 quadrivalents, and the mean chiasma frequency was 21.96 per cell. GISH showed that pairing was predominantly intragenomic, with mean numbers of L. perenne (Lp/Lp) and F. pratensis (Fp/Fp) bivalents being virtually equal at 5.41 and 5.48 per cell, respectively. Intergenomic pairing between Lolium and Festuca chromosomes was observed in 33.3% of Lp/Fp bivalents (0.62 per cell), in 79.7% of trivalents - Lp/Lp/Fp and Lp/Fp/Fp (0.25 per cell), and in 98.4% of quadrivalents - Lp/Lp/Fp/Fp and Lp/Lp/Lp/Fp (0.71 per cell). About 4.0% of the total chromosome complement analysed remained as univalents, an average 0.68 Lp and 0.45 Fp univalents per cell. It is evident that in these hybrids there is opportunity for recombination to take place between the two component genomes, albeit at a low level, and this is discussed in the context of compromising the stability of Festulolium hybrid cultivars and accounting for the drift in the balance of the genomes over generations. We speculate that genotypic differences between hybrids could permit selection for pairing control, and that preferences for homologous versus homoeologous centromeres in their spindle attachments and movement to the poles at anaphase I could form the basis of a mechanism underlying genome drift.     

Comparison of <Emphasis Type="Italic">SYBR Green I</Emphasis> and <Emphasis Type="Italic">TaqMan</Emphasis> real-time PCR formats for the analysis of <Emphasis Type="Italic">her2</Emphasis> gene dose in human breast tumors

We compared two technologies of real-time PCR (with the use of fluorescent SYBR Green I dye and specific TaqMan probe) for quantification of the dose of her2 gene in breast tumors. The maximum increase in the gene dose in TaqMan and SYBR Green I analyses was 10-and 5-fold, respectively. In was found that TaqMan and SYBR Green I technologies allow detection of the matrix in amounts corresponding to 1–100 and 2.5–40.0 ng genomic DNA, respectively. Tenfold increase in the gene dose leads to incorrect evaluation of multiplication ratio in the SYBR Green I analysis. These results suggest that TaqMan technology is more preferable for correct evaluation of her2 gene dose. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 2, pp. 201–205, February, 2008     

Detection of ion channel activity in <Emphasis Type="Italic">Xenopus laevis</Emphasis> oocytes expressing <Emphasis Type="Italic">Influenza C virus</Emphasis> CM2 protein

Summary. To demonstrate the ion channel activity of Influenza C virus CM2, we expressed this protein in oocytes of Xenopus laevis and measured whole cell currents by a two-electrode voltage-clamp method. It was found that the inward currents were induced upon hyperpolarizing the oocyte membranes. The amplitude of the currents increased slowly with time during the hyperpolarizing pulse, and the current-voltage relationship was nonlinear, having a slope that increased with the level of hyperpolarization. These results suggest strongly that CM2 forms a voltage-activated ion channel. The current amplitude was increased to a small extent by lowering the external pH. We also found that the anti-influenza A virus drug amantadine hydrochloride failed to attenuate the inward currents of CM2-expressing oocytes induced by hyperpolarization. The reversal voltage of tail currents was affected by the reduction of [Cl^–], but neither by the change of [Na⁺] nor by that of [K⁺]. Furthermore, the amplitude of the inward currents was decreased by an anion channel blocker. The data presented here suggest that CM2 protein forms a voltage-activated ion channel permeable to chloride ion.     

Histopathologic and electron microscopic characterization of cutaneous leishmaniasis in <Emphasis Type="Italic">Tatera indica</Emphasis> and <Emphasis Type="Italic">Gerbillus</Emphasis> spp. infected with <Emphasis Type="Italic">Leishmania major</Emphasis>

Cutaneous leishmaniasis (CL) is a zoonotic disease transmitted between rodents and canines, mainly by Phlebotomus sand flies and man. In southern Iran, the incidence of this protozoan disease has doubled over the last decade. The present study deals with histopathological features of CL in Tatera indica and Gerbillus spp. that participate in the epidemiology of CL in southern Iran. Thirty-two trapped rodents were evaluated for any Leishmania infection using enzyme electrophoresis and polymerase chain reaction and were concomitantly studied for any histopathological changes. Histopathological studies showed that bone marrow was the tissue of choice for light and electron microscopic study of Leishmania, demonstrating the macrophages infected with the amastigote form of the parasite. This is the first report of the histopathological detection of L. major in naturally infected T. indica and Gerbillus spp in the Larestan region.     

Heterogeneity of the <Emphasis Type="Italic">ospA</Emphasis> gene structure from isolates of <Emphasis Type="Italic">Borrelia garinii</Emphasis> and <Emphasis Type="Italic">Borrelia afzelii</Emphasis> from Western Siberia and Mongolia

In this work, identification and analyses of 48 full-length sequences of the ospA gene isolates of Borrelia garinii and Borrelia afzelii from Western Siberia and Mongolia has been made. It was shown that B. garinii isolates was of its high genetic heterogeneity of the ospA gene. Four genetic groups of the ospA gene from the Ixodes persulcatus tick collected in of Western Siberia and Mongolia were defined. The basic differences in the genetic variants of the ospA gene considered are seen in regions which code for antibody determinants of thhe OspA protein.     

Community-acquired <Emphasis Type="Italic">Acinetobacter</Emphasis> infections

Acinetobacter infections have been attracting increasing attention during recent years because they have become common in hospitalized patients, especially in the intensive care unit (ICU) setting. However, the available literature suggests that the pathogen has another fearful potential; it can cause community-acquired infections. We searched PubMed and the reference lists of the initially identified articles and identified six case series regarding a total of 80 patients with community-acquired Acinetobacter baumannii infections; from these, 51 had pneumonia and 29 had bacteremia. Of these 80 patients, 45 (56%) died of the infection. In addition, we identified 26 case reports regarding 43 patients with community-acquired Acinetobacter infections; from these, 38 had pneumonia, two had meningitis, one had soft-tissue infection, one had ocular infection, and one had native valve endocarditis. Comorbidity was commonly present in patients reported in the case series as well as the case reports, mainly, chronic obstructive pulmonary disease, renal disease, and diabetes mellitus; heavy smoking and excess alcohol consumption were also common. Most of the studies originated from China, Taiwan, and tropical Australia. We also identified 12 retrospective or prospective studies (seven from the Far East, two from Oceania, one from N. Guinea, one from Palestine, and one from USA/Canada) that reported the frequency of community-acquired Acinetobacter infections; the range of isolation of Acinetobacter from patients with community-acquired pneumonia in these studies was 1.3%-25.9%. In conclusion, most community-acquired Acinetobacter infections have been reported from countries with tropical or subtropical climate, and mainly affect patients with some form of comorbidity or are associated with heavy smoking and excess alcohol consumption.     

C-<Emphasis Type="Italic">erb</Emphasis>B-2 oncogene and <Emphasis Type="Italic">P21</Emphasis><Superscript><Emphasis Type="Italic">WAF/CIP1</Emphasis></Superscript> tumor suppressor gene expression as prognostic factors in canine mammary adenocarcinomas

To evaluate the use of c-erbB-2 oncogene and p21 ^WAF1/CIP1 suppressor gene products as the prognosis markers for canine mammary tumors, expression of these gene products were examined immunohistochemically using tumor tissues and clinical data from 96 dogs with malignant mammary tumors. Semiquantitative data was compared with histopathological grades, proliferating cell nuclear antigen (PCNA)-positive index, and clinicopathological matters. The expression c-erbB-2 protein was found in the cellular membrane and cytoplasm of neoplastic epithelial cells, and the positive index had no significant relation to the histopathological features and PCNA-positive index, except for the individual age of affected dogs (P < 0.05). The product of p21 ^WAF1/CIP1 was mostly found in cytoplasm and occasionally in the nucleus of neoplastic cells. The quantitative data had significant association to the malignancy grade and size of tumors (P < 0.05). However, that had no significant relationship to the PCNA-positive index. The present study concluded that both gene products could not apply as the direct markers to evaluate the prognosis of canine mammary tumors. The detection of c-erbB-2 product may be partly beneficial to the differential diagnosis of epithelial type of mammary cancer. The use of p21 ^WAF1/CIP1 product in prognosis of canine mammary cancer needs further investigation.     

Structural and functional analysis of <Emphasis Type="Italic">araC</Emphasis> gene in <Emphasis Type="Italic">Yersinia pestis</Emphasis> of various origins

Structural and functional analyses of the araC gene, which is involved in regulation of expression of diagnostically important characteristics, such as arabinose fermentation in the plague microbe strain, were carried out. It was established that the reason for the lack of arabinose fermentation in the altaica and hissarica subspecies and strains of talassica groups is the presence of the insertion of a single nucleotide in the araC gene at position 773 bp. The strains of the basic, caucasia, and ulegeica subspecies do not contain this mutation in the araC gene, which correlates to their ability to ferment arabinose.     

Complicating infectious foci in patients with <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> or <Emphasis Type="Italic">Streptococcus</Emphasis> species bacteraemia

Complicating infectious foci resulting from haematogenous or local spread of microorganisms are observed frequently in patients with Staphylococcus aureus bacteraemia (SAB) or Streptococcus species bacteraemia (SSB). The aim of this study was to compare the epidemiology of complicating infectious foci during SAB and SSB in a university hospital in The Netherlands. The charts of all adult patients diagnosed with SAB or SSB (except for Streptococcus pneumoniae bacteraemia) from July 2002 until December 2004 were reviewed retrospectively. Overall, 180 immunocompetent patients were identified, 127 with SAB and 53 with SSB. The percentage of patients with complicating infectious foci (39% of SAB patients, 25% of SSB patients) did not differ significantly between the groups. Endocarditis and cerebral involvement, however, were significantly more common in the SSB group. Of all complicating infectious foci, 32% lacked guiding signs or symptoms and 10% were detected only at autopsy. Factors associated with the development of complicating infectious foci were a delay in treatment for more than 48 h after the onset of symptoms, community acquisition, persistently positive blood cultures, congenital heart disease, and the presence of foreign bodies or prosthetic valves. Infection-related mortality was 18% in SAB patients and 11% in SSB patients and was significantly higher in patients with complicating infectious foci (29 vs. 9%). In conclusion, complicating infectious foci develop in approximately one-third of all patients with SAB and SSB. An active approach that entails searching for the complicating infectious foci is warranted in these patients, because only two-thirds of complicated infectious foci have guiding symptoms or signs, and infection-related mortality is significantly increased in patients with complicating infectious foci compared to patients without these infections. Authors M.L.H. Cuijpers and F.J. Vos contributed equally to this work.