Human Cytomegalovirus and Human Umbilical Vein Endothelial Cells: Restriction of Primary Isolation to Blood Samples and Susceptibilities of Clinical Isolates from Other Sources to Adaptation

In immunocompromised patients with disseminated infection, human cytomegalovirus (HCMV) is widespread in the microvascular endothelium of multiple organs. Human umbilical vein endothelial cells (HUVEC) were used in parallel to human embryonic lung fibroblasts (HELF) to recover HCMV from blood samples of immunocompromised patients. Using the shell vial technique, comparable median numbers of p72-positive HUVEC and HELF cells were found with the 26 HCMV-positive buffy coat samples out of 150 examined. Analysis of other clinical samples inoculated as controls revealed, in the presence of highly infected HELF monolayers, either the presence of very few infected HUVEC with urine specimens (n = 10 samples) or the lack of infected HUVEC with throat washes (n = 3) or amniotic fluid samples (n = 2). Thus, peripheral blood leukocytes (PBL) appear essential for primary isolation of HCMV in HUVEC. In this respect, HCMV strains, recovered from clinical samples other than buffy coats in HELF only, could be readily adapted to growth in HUVEC by coculturing PBL from healthy blood donors with infected HELF and then inoculating infected PBL onto HUVEC. Recently elucidated mechanisms of interaction of leukocytes and HUVEC with bidirectional transfer of virus seem to provide the basis for the restriction of HCMV primary isolation in HUVEC to blood samples. However, virus strains recovered from only HELF could be adapted to growth in HUVEC when inoculated with HELF-derived (either cell-associated or cell-free) HCMV strains upon primary isolation. In conclusion, due to the in vitro selection of virus variants provided with both PBL tropism and HUVEC tropism, HCMV recovery in HUVEC is PBL mediated and substantially restricted to blood samples. Lack of HCMV recovery in HUVEC from clinical samples other than blood leads to the assumption that epithelial cells, such as urinary, amniotic, or pharyngeal cells, do not possess adequate adhesion molecules to establish close contacts with HUVEC.     

Cloning,isolation, and IgE-binding properties of Helix aspersa (brown garden snail) tropomyosin

BACKGROUND: Gastropod consumption is quite frequent in the Mediterranean countries and cross-reactivities with crustaceans have been described, but the mechanism of this allergenic cross-reactivity has not been studied in detail. This study aimed to produce recombinant Helix aspersa (brown garden snail) tropomyosin and investigate its implication for cross-reactivity among invertebrates. METHODS: A tropomyosin-specific cDNA encoding H. aspersa tropomyosin was synthetized, and recombinant allergen was overexpressed in Escherichia coli as nonfusion protein. IgE-binding reactivity was studied by immunoblotting and immunoblot inhibition experiments with sera from snail-allergic patients. RESULTS: Cloned brown garden snail tropomyosin shares high homology with other edible mollusk tropomyosins (84-69% identity) as well as with those from arthropods (65-62%), and less homology with vertebrate ones (56% identity). Tropomyosin reacted with 18% of the sera from patients with snail allergy. Inhibition experiments, using natural and recombinant tropomyosins, showed different degrees of cross-reactivity between invertebrate tropomyosins. Sera from snail-allergic subjects recognized tropomyosins in both mollusks and crustacean extracts. CONCLUSIONS: Tropomyosin represents a minor allergen in snail extracts, but it is clearly involved in invertebrate cross-reactivity.     

Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform.     

Interleukin-6 and flow-mediated dilatation as markers of increased vascular inflammation in women receiving hormone therapy

OBJECTIVE: The lack of a beneficial long-term cardiovascular effect of hormone therapy and the early incidence of cardiovascular adverse events observed in recent randomized studies have been related to a heightened inflammatory effect of hormone therapy. DESIGN: We evaluated the effect of different postmenopause therapies on inflammatory markers and endothelial function in 205 postmenopausal women before and after therapy. RESULTS: all postmenopausal women, estrogens alone increased plasma levels of C-reactive protein (CRP) but decreased all other markers of inflammation including interleukin-6 (IL-6) (CRP: +75% +/- 11%, intracellular adhesion molecule: -21% +/- 4%, vascular cell adhesion molecule: -15% +/- 6%, E-selectin: -18% +/- 4%, s-thrombomodulin -10.5% +/- 3.7%, IL-6 -14% +/- 6%; percent changes, P < 0.01 compared with baseline). Raloxifene and tibolone did not significantly affect the overall inflammatory milieu. In a minority of patients, estrogen-progestogen associations and tibolone increased IL-6 levels and induced unfavorable changes on inflammation markers (CRP: +93% +/- 8%, intracellular adhesion molecule: -3% +/- 2%, vascular cell adhesion molecule: -5% +/- 2%, E-selectin: +6% +/- 2%, s-thrombomodulin: +5% +/- 2%, IL-6: +12% +/- 4%; percent changes compared with baseline). Patients with increased IL-6 levels were older and had a longer time since menopause. In all patients except those with increased IL-6 levels, hormone therapy improved endothelial function, whereas tibolone and raloxifene did not significantly change endothelial function compared with baseline. A worsening of endothelial function was detected in patients with increased IL-6 levels during therapy. CONCLUSIONS: Postmenopausal hormone therapy is associated with decreased vascular inflammation; however, in patients with a longer time since menopause, postmenopause hormone therapy may increase inflammation and worsen endothelial function. These unfavorable vascular effects may be detected by an elevation in IL-6 levels and by a lack of improvement in endothelial function.     

Strukturelle Effekte in Systemen MnLi/Butadien/Styrol

The structure of the butadiene unit C₄H₆ in the hydrolysis products of the oligomers RM_nC₄H₆M_m'Li and RM_nC₄H₆Li (where R is sec-butyl, M and M' are perdeuterobutadiene or styrene) was estimated by the use of IR- and NMR-spectroscopy. The dependency of the C₄H₆-structure on the nature of both the preceding unit M and the attacking monomer M' was investigated. The variation of the concentration of the organolithium compounds during the oligomer synthesis on the one hand, and during their hydrolysis on the other hand, showed a dependency of the C₄H₆-structure on the conditions of the respective experiments.     

Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations

A study set comprised 44 Mycobacterium tuberculosis strains of the Beijing family selected for their representativeness among those previously characterized by IS6110-RFLP and spoligotyping (Northwest Russia, 1997 to 2003). In the present study, these strains were subjected to mycobacterial interspersed repetitive units (MIRU) typing to assess a discriminatory power of the 12-MIRU-loci scheme (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). The 44 Russian Beijing strains were subdivided into 12 MIRU types with identical profiles: 10 unique strains and two major types shared by 10 and 24 strains. Thus, basically, two distinct sublineages appear to shape the evolution of the Beijing strains in Russia. Most of the MIRU loci were found to be (almost) monomorphic in the Russian Beijing strains; the Hunter-Gaston discriminatory index (HGDI) for all 12 loci taken together was 0.65, whereas MIRU26 (the most variable in our study) showed a moderate level of discrimination (0.49). The results were compared against all available published MIRU profiles of Beijing strains from Russia (3 strains) and other geographic areas (51 strains in total), including South Africa (38 strains), East Asia (7 strains), and the United States (4 strains). A UPGMA (unweighted pair-group method with arithmetic averages)-based tree was constructed. Interestingly, no MIRU types were shared by Russian and South African strains (the two largest samples in this analysis), whereas both major Russian types included also isolates from other locations (United States and/or East Asia). This implies the evolution of the Beijing genotype to be generally strictly clonal, although a possibility of a convergent evolution of the MIRU loci cannot be excluded. We propose a dissemination of the prevailing local Beijing clones to have started earlier in South Africa rather than in Russia since more monomorphic loci were identified in Russian samples than in South African samples (mean HGDI scores, 0.08 versus 0.17). To conclude, we suggest to use a limited number of MIRUs for preliminary subdivision of Beijing strains in Russian (loci 26 + 31), South African (10 + 26 + 39), and global settings (10 + 26 + 39).     

Modulation of Borrelia burgdorferi stringent response and gene expression during extracellular growth with tick cells

Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.     

Setting out the frame conditions for feasible use of FFPE derived RNA

Introduction

The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.

NK cells mediate increase of phagocytic activity but not of proinflammatory cytokine (interleukin-6 [IL-6], tumor necrosis factor alpha,and IL-12) production elicited in splenic macrophages by tilorone treatment of mice during acute systemic candidiasis

The participation of NK cells in the activation of splenic macrophages or in resistance to systemic candidiasis is still a matter of debate. We had previously reported that there is a correlation between natural killer cell activation and resistance to systemic candidiasis. In those experiments we had used tilorone to boost NK cell activity in mice. Here we show a mechanism elicited by tilorone in splenic macrophages which could explain their effect on mouse survival during acute disseminated Candida albicans infection. The results demonstrate that tilorone treatment elicits, by a direct effect, the production of proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-alpha], and IL-12) by splenic macrophages. In addition, it increases the capacity of splenic macrophages to phagocytize C. albicans through activation of NK cells. We also demonstrate that the presence of NK cells is essential for maintaining a basal level of phagocytic activity, which characterizes splenic macrophages of na?ve control mice. The results demonstrate that it is possible to identify two phenotypically and functionally peculiar cell populations among splenic macrophages: (i). cells of the "stimulator/secretor phenotype," which show high levels of major histocompatibility complex (MHC) class II surface expression, are poorly phagocytic, and synthesize the proinflammatory cytokines IL-6, TNF-alpha, and IL-12, and (ii). cells of the "phagocytic phenotype," which express low levels of MHC class II molecules, are highly phagocytic, and do not secrete proinflammatory cytokines.     

Chemokine receptors that mediate B cell homing to secondary lymphoid tissues are highly expressed in B cell chronic lymphocytic leukemia and non-Hodgkin lymphomas with widespread nodular dissemination

B cell neoplasms present heterogeneous patterns of lymphoid organ involvement, which may be a result of the differential expression of chemokine receptors. We found that chemokine receptor (CCR)7, CXC chemokine receptor (CXCR)4, or CXCR5, the main chemokine receptors that mediate B cell entry into secondary lymphoid tissues and their homing to T cell and B cell zones therein, were highly expressed in B malignancies with widespread involvement of lymph nodes. Conversely, those pathologies with little or no nodular dissemination showed no expression to very low levels of CCR7 and CXCR5 and low to moderate levels of CXCR4. These findings provide evidence for the role of CCR7, CXCR4, and CXCR5 in determining the pattern of lymphoid organ involvement of B tumors. Functional studies were performed on B malignancies expressing different levels of CCR7, CXCR5, and CXCR4. Multiple myeloma (MM) cells did not express CCR7 nor CXCR5 and did not migrate in response to their ligands; a moderate expression of CXCR4 on MM cells was accompanied by a migratory response to its ligand, CXCL12. By contrast, cells from B cell chronic lymphocytic leukemia (B-CLL) expressed the highest levels of these chemokine receptors and efficiently migrated in response to all ligands of CCR7, CXCR4, and CXCR5. In addition, the migration index of B-CLL cells in response to both of the CCR7 ligands correlated with the presence of clinical lymphadenopathy, thus indicating that the high expression of functional chemokine receptors justifies the widespread character of B-CLL, representing a clinical target for the control of tumor cell dissemination.