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.     

Genomic structure and evolution of the ancestral chromosome fusion site in 2q13-2q14.1 and paralogous regions on other human chromosomes

Human chromosome 2 was formed by the head-to-head fusion of two ancestral chromosomes that remained separate in other primates. Sequences that once resided near the ends of the ancestral chromosomes are now interstitially located in 2q13-2q14.1. Portions of these sequences had duplicated to other locations prior to the fusion. Here we present analyses of the genomic structure and evolutionary history of >600 kb surrounding the fusion site and closely related sequences on other human chromosomes. Sequence blocks that closely flank the inverted arrays of degenerate telomere repeats marking the fusion site are duplicated at many, primarily subtelomeric, locations. In addition, large portions of a 168-kb centromere-proximal block are duplicated at 9pter, 9p11.2, and 9q13, with 98%-99% average sequence identity. A 67-kb block on the distal side of the fusion site is highly homologous to sequences at 22qter. A third ~100-kb segment is 96% identical to a region in 2q11.2. By integrating data on the extent and similarity of these paralogous blocks, including the presence of phylogenetically informative repetitive elements, with observations of their chromosomal distribution in nonhuman primates, we infer the order of the duplications that led to their current arrangement. Several of these duplicated blocks may be associated with breakpoints of inversions that occurred during primate evolution and of recurrent chromosome rearrangements in humans.     

Cytotoxic molecule expression and epithelial cell apoptosis in oral and cutaneous lichen planus

We evaluated the expression of T cell-restricted intracellular antigen (Tia-1), granzyme B, and perforin by lymphocytes and the degree of epithelial apoptosis in oral and cutaneous lichen planus (LP) in 51 untreated cases, including 27 oral LP (OLP) and 24 cutaneous LP (CLP) cases. The number of total dermal-positive lymphocytes in OLP and CLP was similar, indicating similar activity of the inflammatory process. Intraepithelial Tia-1-positive, perforin-positive, and granzyme B-positive lymphoid cells were more numerous in OLP than in CLP (P < .05). The epithelial cell apoptotic index (AI) was increased significantly in OLP (P < .05), particularly in erosive-atrophic variants. A linear correlation between AI and the mean +/- SEM number of intraepithelial and dermal perforin+ cells (6.85 +/- 2.44 and 27.48 +/- 10.19, respectively), per 10 high-power fields for OLP and for CLP (1.17 +/- 0.88 and 10.42 +/- 5.74, respectively), was found (intraepithelial, r = 0.50; dermal, r = 0.51; P < .01). These data suggest a pivotal role for perforin in triggering epithelial cell apoptosis. The differences of infiltrating cytotoxic cells and related AI observed in OLP and CLP are in keeping with the clinical behaviors that distinguish these LP variants.     

Cutaneous papillary squamous cell carcinoma. Report of three new cases and review of the literature

Squamous cell carcinoma is one of the most common malignant cutaneous tumors. Several histological variants have been described; the papillary subtype is one of the most infrequent, with only four cases having being reported previously. We report three new cases of this unusual variant of cutaneous squamous cell carcinoma, review the literature and consider the main differential diagnoses. Polymerase chain reaction performed in our three cases did not demonstrate human papilloma virus infection.     

Breakpoints for predicting Pseudomonas aeruginosa susceptibility to inhaled tobramycin in cystic fibrosis patients: use of high-range Etest strips

Inhaled administration of tobramycin assures high concentrations in cystic fibrotic lungs, improving the therapeutic ratio over that of parenteral tobramycin levels, particularly against Pseudomonas aeruginosa. Conventional Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) breakpoints only consider parenteral levels and do not take into account these high antimicrobial concentrations. The Spanish Antibiogram Committee (The MENSURA Group) has tentatively defined specific breakpoint values for inhaled tobramycin when testing P. aeruginosa isolates from cystic fibrosis (CF) patients (susceptible, < or =64 microg/ml; resistant, > or =128 microg/ml). The antimicrobial susceptibilities of 206 prospectively collected CF P. aeruginosa isolates were determined by the reference agar dilution method. For tobramycin, the performance of high range tobramycin Etest strips (AB Biodisk, Solna, Sweden) and conventional tobramycin disks were assessed with the same collection. Applying MENSURA proposed breakpoints, 95.1% of the strains were categorized as susceptible to tobramycin, either using agar dilution or Etest high-range strips (99% categorical agreement between both methods). With CLSI breakpoints, susceptibility rates decreased to 79.1 and 81.1% for agar dilution and Etest strips, respectively (83.5% categorical agreement). Minor, major, and very major errors for Etest strips (CLSI criteria) were 13.6, 1.2, and 14.8%, respectively. Upon applying the new proposed criteria for inhaled tobramycin, only one major and one very major error were observed with Etest strips. Whenever inhaled tobramycin is considered for therapy, we suggest that P. aeruginosa strains from CF patients categorized as intermediate or resistant to tobramycin according to the CLSI criteria should be retested with high-range Etest strips and recategorized using MENSURA interpretive criteria. CLSI breakpoints should still be followed when intravenous tobramycin is used in CF patients, particularly during the course of exacerbations.     

Processing of influenza HA protein in MDCK cells: Components with different mobilities in polyacrylamide gel electrophoresis and their precursor-product relationships

Summary In influenza virus-infected MDCK cells labelled with¹⁴C-chlorella hydrolysate or³⁵S-methionine a virus-specific protein component is revealed migrating slightly faster than HA protein in polyacrylamide gel electrophoresis. Under chase conditions the component disappears either completely or partially, with a concomitant intensification of the HA band. The rate and extent of this transition are strain-dependent. Both the HA band and the faster moving component are not revealed if the cells are labelled in the presence of 20mM of D-glucosamine. In primary cell cultures of chick embryos a single HA band with a mobility similar to that of the faster moving component in MDCK cells has been observed. It is suggested that the transition of the label from the faster moving component to the HA band reflects the final step of HA processing specific for MDCK cells.With 7 Figures     

Antibiotic resistance in exopolysaccharide-forming Staphylococcus epidermidis clinical isolates from orthopaedic implant infections

The opportunistic pathogen Staphylococcus epidermidis is able to produce biofilm and to frequently cause implant infections. In recent years, it has also exhibited an increasing antimicrobial drug resistance. Here, the resistance to a panel of 16 different antibiotics in 342 clinical strains of S. epidermidis from orthopaedic implant infections has been investigated. The isolates were pheno- and genotyped for extracellular polysaccharide production, relevant to staphylococcal biofilm formation, in order to ascertain possible associations with antibiotic resistance. Approximately 10% of the isolates were found to be sensitive to all screened antibiotics. In all, 37-38% were resistant to beta-lactams such as oxacillin and imipenem, while the resistance to penicillin, ampicillin, cefazolin, cefamandole, was consistently observed in over 80% of the strains. Erythromycin- and clindamycin- resistant strains were approximately 41% and 16%, respectively. Of the isolates, 10% was resistant to chloramphenicol, 23% to sulfamethoxazole and 26% to ciprofloxacin. Resistance to vancomycin was never observed. Interestingly, exopolysaccharide-producing strains exhibited a significantly higher prevalence in the resistance to the four aminoglycosides (gentamicin, amikacin, netilmicin, tobramycin), to sulfamethoxazole and to ciprofloxacin with respect to non-producing isolates. Moreover, multiple resistance to antibiotics was more frequent among exopolysaccharide-forming strains.