Human rhinoviruses enter and induce proliferation of B lymphocytes.

BACKGROUND: Human rhinoviruses (HRVs) are one of the main causes of virus-induced asthma exacerbations. Infiltration of B lymphocytes into the subepithelial tissue of the lungs has been demonstrated during rhinovirus infection in allergic individuals. However, the mechanisms through which HRVs modulate the immune responses of monocytes and lymphocytes are not yet well described. OBJECTIVE: To study the dynamics of virus uptake by monocytes and lymphocytes, and the ability of HRVs to induce the activation of in vitro-cultured human peripheral blood mononuclear cells. METHODS: Flow cytometry was used for the enumeration and characterization of lymphocytes. Proliferation was estimated using 3 H-thymidine or CFSE labeling and ICAM-1 blocking. We used bead-based multiplex assays and quantitative PCR for cytokine quantification. HRV accumulation and replication inside the B lymphocytes was detected by a combination of in situ hybridization (ISH), immunofluorescence, and PCR for positive-strand and negative-strand viral RNA. Cell images were acquired with imaging flow cytometry. RESULTS: By means of imaging flow cytometry, we demonstrate a strong and quick binding of HRV types 16 and 1B to monocytes, and slower interaction of these HRVs with CD4+ T cells, CD8+ T cells, and CD19+ B cells. Importantly, we show that HRVs induce the proliferation of B cells, while the addition of anti-ICAM-1 antibody partially reduces this proliferation for HRV16. We prove with ISH that HRVs can enter B cells, form their viral replication centers, and the newly formed virions are able to infect HeLa cells. In addition, we demonstrate that similar to epithelial cells, HRVs induce the production of pro-inflammatory cytokines in PBMCs. CONCLUSION: Our results demonstrate for the first time that HRVs enter and form viral replication centers in B lymphocytes and induce the proliferation of B cells. Newly formed virions have the capacity to infect other cells (HeLa). These findings indicate that the regulation of human rhinovirus-induced B-cell responses could be a novel approach to develop therapeutics to treat the virus-induced exacerbation of asthma.

Distinct regulation of tonsillar immune response in virus infection.

BACKGROUND: The relationships between tonsillar immune responses, and viral infection and allergy are incompletely known. OBJECTIVE: To study intratonsillar/nasopharyngeal virus detections and in vivo expressions of T-cell- and innate immune response-specific cytokines, transcription factors, and type I/II/III interferons in human tonsils. METHODS: Palatine tonsil samples were obtained from 143 elective tonsillectomy patients. Adenovirus, bocavirus-1, coronavirus, enteroviruses, influenza virus, metapneumovirus, parainfluenza virus, rhinovirus, and respiratory syncytial virus were detected using PCR. The mRNA expression levels of IFN-α, IFN-ß, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-ß, FOXP3, GATA3, RORC2, and Tbet were directly analyzed by quantitative RT-PCR. RESULTS: Fifty percentage of subjects reported allergy, 59% had ≥1 nasopharyngeal viruses, and 24% had ≥1 intratonsillar viruses. Tonsillar virus detection showed a strong negative association with age; especially rhinovirus or parainfluenza virus detection showed positive association with IFN-γ and Tbet expressions. IL-37 expression was positively associated with atopic dermatitis, whereas IFN-α, IL-13, IL-28, and Tbet expressions were negatively associated with allergic diseases. Network analyses demonstrated strongly polarized clusters of immune regulatory (IL-10, IL-17, TGF-ß, FOXP3, GATA3, RORC2, Tbet) and antiviral (IFN-α, IFN-ß, IL-28, IL-29) genes. These two clusters became more distinctive in the presence of viral infection or allergy. A negative correlation between antiviral cytokines and IL-10, IL-17, IL-37, FOXP3, and RORC2 was observed only in the presence of viruses, and interestingly, IL-13 strongly correlated with antiviral cytokines. CONCLUSIONS: Tonsillar cytokine expression is closely related to existing viral infections, age, and allergic illnesses and shows distinct clusters between antiviral and immune regulatory genes.

Suppression of B-cell activation and IgE, IgA, IgG1 and IgG4 production by mammalian telomeric oligonucleotides.

BACKGROUND: The fine balance of immunoglobulins (Ig) E, IgG1, IgG4 and IgA in healthy production is maintained by the interaction of B cells with adaptive and innate immune response. The regulation of toll-like receptors (TLRs)-driven innate and adaptive immune effector B-cell response and the role of mammalian telomeric TTAGGG repeat elements represent an important research area. METHODS: Human PBMC and purified naive and memory B cells were stimulated with specific ligands for TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 and TLR9 in the presence or absence of telomeric oligonucleotides. B-cell proliferation, differentiation and antibody production were determined. RESULTS: TLR9 ligand directly activates naive and memory B cells, whereas TLR7 can stimulate them in the presence of plasmacytoid dendritic cells. Human B cells proliferate and turn into antibody-secreting cells in response to TLR3, TLR7 and TLR9, but not to TLR2, TLR4, TLR5 and TLR8 ligands. Stimulation of B cells with intracellular TLR3, TLR7 and TLR9 induced an activation cascade leading to memory B-cell generation and particularly IgG1, but also IgA, IgG4 and very low levels of IgE production. Mammalian telomeric oligodeoxynucleotide (ODN) significantly inhibited all features of TLR ligand-induced events in B cells including B-cell proliferation, IgE, IgG1, IgG4, IgA production, class switch recombination, plasma cell differentiation induced by TLR3, TLR7 and TLR9 ligands. CONCLUSION: B cells require specific TLR stimulation, T-cell and plasmacytoid dendritic cell help for distinct activation and Ig production profiles. Host-derived telomeric ODN suppress B-cell activation and antibody production demonstrating a natural mechanism for the control of overexuberant B-cell activation, antibody production and generation of memory.

Complexes at the replication origin of Bacillus subtilis with homologous and heterologous DnaA protein.

The initial steps in the formation of the initiation complex at oriC of Bacillus subtilis were analyzed with special emphasis on the exchangeability of B. subtilis DnaA protein by DnaA of Escherichia coli. The DNA binding domain of B. subtilis DnaA protein was localized in the 93 C-terminal amino acids. Formation of the "initial complex", as analyzed by electron microscopy, was indistinguishable with B. subtilis DnaA protein or with E. coli DnaA. Similarly, both proteins were able to form loops by interaction of DnaA proteins bound to the DnaA box regions upstream and downstream of the dnaA gene in B. subtilis oriC. The region of local unwinding in the "open complex" was precisely defined. It is located at one side of a region of helical instability, a DNA unwinding element (DUE). Unwinding in oriC could only be catalyzed by the homologous DnaA protein.

Sequential headful packaging and fate of the cleaved DNA ends in bacteriophage SPP1.

The virulent Bacillus subtilis bacteriophage SPP1 packages its DNA from a precursor concatemer by a headful mechanism. Following disruption of mature virions with chelating agents the chromosome end produced by the headful cut remains stably bound to the phage tail. Cleavage of this tail-chromosome complex with restriction endonucleases that recognize single asymmetric positions within the SPP1 genome yields several distinct classes of DNA molecules whose size reflects the packaging cycle they were generated from. A continuous decrease in the number of molecules within each class derived from successive encapsidation rounds indicates that there are several packaging series which end after each headful packaging cycle. The frequency of molecules in each packaging class follows the distribution expected for a sequential mechanism initiated unidirectionally at a defined position in the genome (pac). The heterogeneity of the DNA fragment sizes within each class reveals an imprecision in headful cleavage of approximately 2.5 kb (5.6% of the genome size). The number of encapsidation events in a packaging series (processivity) was observed to increase with time during the infection process. DNA ejection through the tail can be induced in vitro by a variety of mild denaturing conditions. The first DNA extremity to exit the virion is invariably the same that was observed to be bound to the tail, implying that the viral chromosome is ejected with a specific polarity to penetrate the host. In mature virions a short segment of this chromosome end (55 to 67 bp equivalent to 187 to 288 A) is fixed to the tail area proximal to the head (connector). Upon ejection this extremity is the first to move along the tail tube to exit from the virion through the region where the tail spike was attached.

Distribution of molecularly imprinted polymer layers on macroporous silica gel particles by STEM and EDX.

Using transmission electron microscopy (TEM) in parallel and scanning mode (STEM) combined with energy dispersive X-ray spectroscopy (EDX) the distribution of molecularly imprinted polymer layers covalently grafted on the inner and outer surface of macroporous silica gel particles was observed. The grafting was achieved using a special initiator system (iniferter) and applied on a well known model system producing molecularly imprinted layers with L-phenyl alanine analide (L-PA) as template. By staining the sample with RuO(4) before its embedment in epoxy resin and cutting ultra thin slices it was possible to identify the stained grafted polymer on the silica matrix by EDX. Based on this, complete particle slices could be investigated by visual means according to the presence or absence of grafted polymer and its layer homogeneity.

Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors.

A broad-host-range vector, pKT240, containing the structural gene (aph) for aminoglycoside phosphotransferase (APH), without promoter, has been constructed. Insertion of DNA fragments carrying promoters upstream of aph gene into the unique EcoRI site of this vector results in the expression of the aph gene and consequently the resistance of the host cells to streptomycin. The new vector has been used to show that the hybrid trp-lac (tac) promoter and the promoter of the lacIQ gene of Escherichia coli are active in Pseudomonas putida. Derivatives of pKT240 containing tac and lacIQ sequences may be used as wide-host-range expression vectors. Regulated overproduction of APH and catechol 2,3-oxygenase can be obtained with the aid of the new vectors in both E. coli and P. putida.

Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas.

Host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. They comprise restriction-negative host strains of Pseudomonas aeruginosa and P. putida and new cloning vectors derived from the high-copy-number, broad-host-range plasmid RSF1010, which are stably maintained in a wide range of Gram-negative bacteria. These plasmids contain EcoRI, SstI, HindIII, XmaI, XhoI, SalI, BamHI, and ClaI insertion sites. All cloning sites, except for BamHI and ClaI, are located within antibiotic-resistance genes' insertional inactivation of these genes during hybrid plasmid formation provides a readily scored phenotypic change for the rapid identification of bacterial clones carrying such hybrids. One of the new vector plasmids is a cosmid that may be used for the selective cloning of large DNA fragments by in vitro lambda packaging. An analogous series of vectors that are defective in their plasmid-mobilization function, and that exhibit a degree of biological containment comparable to that of current Escherichia coli vector plasmids, are also described.

Enhancement of ColE1-type replication promoted by the Gp alpha initiator protein of bacteriophage P4.

Gp alpha, the phage P4 specific replication protein, increases in vitro replication of pCN51, a pBR322 based replicon, by a factor of two. This effect is dependent on DNA polymerase I and requires transcription by host RNA polymerase. Electron microscopic analysis of replicating intermediates indicates that pCN51 replication occurred from the same origin and with the same directionality in the presence and in the absence of Gp alpha. These results reveal that Gp alpha can influence the replication of an heterologous replicon and show that this effect occurs in ColE1-type replicons without altering the normal pattern of initiation. Further analysis of replicating intermediates shows an increase in the average size of the ColE1-type replication 'bubble' obtained in the presence of Gp alpha. It is proposed that Gp alpha interacts with the ColE1 replisome complex at an early replication stage.

Distinct leucocyte redistribution after glucocorticoid treatment among difficult-to-treat asthmatic patients.

Difficult-to-treat asthma (DTA) represents a heterogeneous subgroup of asthma. Up to now, the lack of specific diagnosis not only complicates appropriate specification and control of asthma, but also makes targeted research difficult. The aim of this study is to categorize this heterogeneous group of DTA patients (n=27; referring to the GINA guidelines) based on the distinct leucocyte redistribution (LR) after glucocorticoid (GC) treatment. Furthermore, the effect of adjuvant therapies was investigated for its impact on LR. The frequency of CD3+, CD4+, CD8+, CD14+, CD19+ and NK cells was analysed in peripheral blood before and 3 h after systemic GC treatment, along with the markers of activation HLA-DR and CD25. Within 3 h of GC administration, a significant average decrease of 16% in CD3+CD4+ (P < or = 0.001) and a 12% increase in NK-cell frequency (P < or = 0.001) clearly distinguished two groups of patients: LR-responsive and LR-unresponsive patients. The CD3+CD8+ T-cell number and activation marker remained unchanged. Patients who received adjuvant therapy, such as methotrexate or interferon-alpha, because of poor clinical response to GC showed an LR similar to that showed by responsive patients. DTA patients comprise at least two immunologically distinct groups: patients showing an immediate decrease in CD3+CD4+ T cells and an increase in NK cells following GC administration and patients lacking an immediate change. Analysis of LR not only may allow the identification of immunologic steroid resistance, but also may be of value for immunologic determination of effective steroid doses.

Induction of the IL-10 gene via the fas receptor in monocytes--an anti-inflammatory mechanism in the absence of apoptosis.

Death receptors play an important role in controlling cell numbers and immune responses. In contrast to TNF receptors, little is known about non-apoptosis functions of the Fas receptor (CD95, APO-1). Here we demonstrate that Fas receptor engagement results in the induction of the IL-10 gene in monocytes, but not in lymphocytes or dendritic cells. In contrast, TNF-alpha stimulated IL-10 production in dendritic cells but not monocytes. Fas receptor-mediated transcriptional activation of the IL-10 gene was followed by the release of large amounts of the cytokine in cell cultures and occurred in the absence of apoptosis induction. Since caspase activation did not occur in monocytes following Fas receptor engagement, it is unlikely that caspases are involved in IL-10 gene activation. Monocyte-derived IL-10 suppressed T cell proliferation induced by anti-CD3 monoclonal antibody without affecting CD3-mediated transmembrane signal transduction. In conclusion, we report about a novel pathway initiated via the Fas receptor leading to transcriptional activation of at least one cytokine gene. Fas ligand-induced IL-10 production in monocytes might represent an important anti-inflammatory mechanism in secondary immune responses.

Two unrelated cell-derived sequences in the genome of avian leukemia and carcinoma inducing retrovirus MH2.

Molecularly cloned proviral DNA of avian replication-defective retrovirus Mill Hill No. 2 (MH2) was analyzed. The MH2 provirus measures 5.5 kb including two long terminal repeats (LTR), and contains a partial complement of the structural gene gag, 1.5 kb in size, near the 5' terminus, and a 1.3-kb segment of the v-myc transforming gene near the 3' terminus. These v-myc sequences are closely related to the v-myc transforming gene of avian acute leukemia virus MC29, and to the cellular chicken gene c-myc. The gag and myc domains on the MH2 provirus are separated by unique sequences, 1.3 kb in size and termed v-mil, which are unrelated to v-myc, or to other oncogenes or structural genes of the avian leukemia-sarcoma group of retroviruses. Normal chicken DNA contains sequences closely related to v-mil, termed c-mil. Analyses of chicken c-mil clones isolated from a recombinant DNA library of the chicken genome reveal that c-mil is a single genetic locus with a complex split gene structure. In the MH2 genome, v-mil is expressed via genome-sized mRNA as a gag-related hybrid protein, p100gag-mil, while v-myc is apparently expressed via subgenomic mRNA independently from major coding regions of structural genes. The presence in the MH2 genome of two unrelated cell-derived sequences and their independent expression may be significant for the oncogenic specificities of this virus.

Plasmid replication functions. VII. Electron microscopic localization of RNA polymerase binding sites in the replication control region of plasmid R6-5.

RNA polymerase binding sites on the R6-5 miniplasmid derivative, plasmid pKT401, were mapped by electron microscopy of DNA:RNA polymerase complexes formed with both circular-supercoiled and restriction endonuclease-linearized plasmid DNA molecules. Of eight specific binding sites on pKT401 that were identified, three were found to be in the P-6 fragment of the plasmid replication region, three in the Tn3 element, and two in other parts of the plasmid molecule. Binding sites 1 and 3 in the P-6 fragment are most probably the promoters of the copB and copA/incA plasmid replication control genes, respectively, whereas site 2 in this fragment appears to be the promoter of the essential replication gene, repA. The location of these promoters in relation to the site of action of the plasmid replication control elements, copT, and the origin of replication, oriV, suggests that replication control may be effected by regulation of transcription events initiated at site 2, or of the activity of transcripts initiated from this site, i.e., by regulation of the expression of the repA gene or another function dependent upon these events.

DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.

The lytic replicon of bacteriophage P1 is controlled by an antisense RNA.

The lytic replicon of phage P1 is used for DNA replication during the lytic cycle. It comprises about 2% of the P1 genome and contains the P1 C1 repressor-controlled operator-promoter element Op53.P53 and the kilA and the repL genes, in that order. Transcription of the lytic replicon of P53 and synthesis of the product of repL, but not kilA, are required for replicon function. We have identified an additional promoter, termed P53as (antisense), at the 5'-end of the kilA gene from which a 180 base transcript is constitutively synthesized and in the opposite direction to the P53 transcript. By using a promoter probe plasmid we show that transcription from P53 is strongly repressed by the C1 repressor, whereas that of P53as remains unaffected. Accordingly, the C1 repressor inhibits binding of Escherichia coli RNA polymerase to P53, but not to P53as, as shown by electron microscopy. Under non-repressed conditions transcription from P53 appears to be inhibited by P53as activity and vice versa. An inhibitory effect of P53as on the P1 lytic replicon was revealed by the construction and characterization of a P53as promoter-down mutant. Under non-repressed conditions transcription of repL and, as a consequence, replication of the plasmid is strongly enhanced when P53as is inactive. The results suggest a regulatory role for P53as on the P1 lytic replicon.

Replication of the broad host range plasmid RSF1010: requirement for three plasmid-encoded proteins.

Cloning of specific regions of plasmid RSF1010, in conjunction with in vitro replication studies, has revealed three novel genes: repA, repB, and repC. They are clustered in one region of the plasmid, separated from the origin of replication by regions that are not essential for plasmid viability in an Escherichia coli host. In vivo, a 2.1-kilobase segment of the plasmid, bearing the replication origin, can establish itself as an autonomous replicon if the DNA region carrying the three rep genes is present in the same cell on an independent plasmid. In vitro, RSF1010 DNA is efficiently replicated by an ammonium sulfate fraction from the E. coli extract, provided the extracts are prepared from cells that can supply the required rep gene products. Using cells containing the cloned rep gene region as a source of elevated levels of the rep proteins, we have partially purified these proteins in functional form. When added to an enzyme fraction derived from plasmid-free cells, they specifically promote the replication of plasmid DNA bearing the RSF1010 origin.