The linker region of breast cancer resistance protein ABCG2 is critical for coupling of ATP-dependent drug transport.

The ATP-binding cassette (ABC) transporters of class G display a different domain organisation than P-glycoprotein/ABCB1 and bacterial homologues with a nucleotide-binding domain preceding the transmembrane domain. The linker region connecting these domains is unique and its function and structure cannot be predicted. Sequence analysis revealed that the human ABCG2 linker contains a LSGGE sequence, homologous to the canonical C-motif/ABC signature present in all ABC nucleotide-binding domains. Predictions of disorder and of secondary structures indicated that this C2-sequence was highly mobile and located between an α-helix and a loop similarly to the C-motif. Point mutations of the two first residues of the C2-sequence fully abolished the transport-coupled ATPase activity, and led to the complete loss of cell resistance to mitoxantrone. The interaction with potent, selective and non-competitive, ABCG2 inhibitors was also significantly altered upon mutation. These results suggest an important mechanistic role for the C2-sequence of the ABCG2 linker region in ATP binding and/or hydrolysis coupled to drug efflux.

Non-surgical periodontal therapy with systemic antibiotics in patients with untreated chronic periodontitis: a systematic review and meta-analysis.

OBJECTIVE: The purpose of this meta-analysis is to evaluate the effectiveness of different systemic antibiotics in combination with scaling and root planing (SRP) when compared to SRP alone in patients with untreated chronic periodontitis. BACKGROUND: Although chronic periodontitis is mostly treated without adjunctive systemic antibiotics, some recent meta-analyses have shown clinical benefit for some systemic antibiotics when used as an adjunct to SRP. However, there is a wide variety of systemic antibiotic regimens used today. It remains unclear if the selected type of systemic antibiotic influences the magnitude of clinical benefit. MATERIAL AND METHODS: The MEDLINE-PubMed database was searched from their earliest records through May 16, 2013. Several journals were hand searched and some authors were contacted for additional information. Outcome measures analysed were mean bleeding on probing change, mean clinical attachment level gain and mean probing pocket depth reduction. Extracted data were pooled using a random effect model. Weighted mean differences were calculated and heterogeneity was assessed. RESULTS: The search yielded 281 abstracts. Ultimately, 95 studies were selected, describing 43 studies meeting the eligibility criteria. Systemic antibiotics showed a significant (p < 0.05) additional pocket depth reduction for moderate (at 3 mo 0.27 mm ± 0.09, at 6 mo 0.23 mm ± 0.10 and at 12 mo 0.25 mm ± 0.27) and deep pockets (at 3 mo 0.62 mm ± 0.17, at 6 mo 0.58 mm ± 0.16 and at 12 mo 0.74 mm ± 0.30). Statistically, no specific type of antibiotic was superior over another. However, when analysing the clinical data for initially moderate pockets or deep pockets, some trends became apparent. CONCLUSION: Systemic antibiotics combined with SRP offer additional clinical improvements compared to SRP alone. Although there were no statistically significant differences, there was a trend that for initially moderate and deep pockets, metronidazole or metronidazole combined with amoxicillin, resulted in clinical improvements that were more pronounced over doxycycline or azithromycin. Additionally, there was a trend that the magnitude of the clinical benefit became smaller over time (1 year).

Non-surgical periodontal therapy with systemic antibiotics in patients with untreated aggressive periodontitis: a systematic review and meta-analysis.

OBJECTIVE: The purpose of this meta-analysis is to evaluate the effectiveness of different systemic antibiotics in combination with scaling and root planing (SRP) compared to SRP alone in patients with untreated aggressive periodontitis. BACKGROUND: In patients with aggressive periodontitis, SRP is often combined with the use of systemic antibiotics. However, the effectiveness of these antibiotics over time and differences in effectiveness between different antibiotics are hardly known. MATERIAL AND METHODS: The MEDLINE-PubMed database was searched from their earliest records until January 20, 2014. Several journals were hand searched and some authors were contacted for additional information. The following outcome measures were analysed: mean probing pocket depth reduction, mean clinical attachment level gain and mean bleeding on probing change. Extracted data were pooled using a random effect model. Weighted mean differences were calculated and heterogeneity was assessed. RESULTS: The search yielded 296 abstracts. Ultimately, 101 articles were selected of which 14 articles met the eligibility criteria. Systemic antibiotics showed a significant (p < 0.05) additional pocket depth reduction for moderate (0.36 ± 0.22 mm at 3 mo, 6 mo 0.42 ± 0.22 mm and 12 mo 0.88 ± 0.27 mm) and deep pockets (0.74 ± 0.36 mm at 3 mo, 6 mo 0.85 ± 0.55 mm and 12 mo 1.26 ± 0.81 mm) and a significant clinical attachment gain for moderate (0.26 ± 0.18 at 3 mo, 6 mo 0.52 ± 0.15 and 12 mo 0.83 ± 0.38) and deep pockets (0.59 ± 0.18 at 3 mo, 0.96 ± 0.21 at 6 mo and 1.00 ± 0.80 at 12 mo). CONCLUSION: For the treatment of patients with aggressive periodontitis, systemic antibiotics combined with non-surgical periodontal therapy resulted in a significant additional effect compared to non-surgical therapy alone. There is a visible trend that showed metronidazole + amoxicillin is the most potent antibiotic combination.

Antibody prophylaxis and therapy against Nipah virus infection in hamsters.

Nipah virus (NiV), a member of the Paramyxoviridae family, causes a zoonotic infection in which the reservoir, the fruit bat, may pass the infection to pigs and eventually to humans. In humans, the infection leads to encephalitis with >40 to 70% mortality. We have previously shown that polyclonal antibody directed to either one of two glycoproteins, G (attachment protein) or F (fusion protein), can protect hamsters from a lethal infection. In the present study, we have developed monoclonal antibodies (MAbs) to both glycoproteins and assessed their ability to protect animals against lethal NiV infection. We show that as little as 1.2 mug of an anti-G MAb protected animals, whereas more than 1.8 mug of anti-F MAb was required to completely protect the hamsters. High levels of either anti-G or anti-F MAbs gave a sterilizing immunity, whereas lower levels could protect against a fatal infection but resulted in an increase in anti-NiV antibodies starting 18 days after the viral challenge. Using reverse transcriptase PCR, the presence of NiV in the different organs could not be observed in MAb-protected animals. When the MAbs were given after infection, partial protection (50%) was observed with the anti-G MAbs when the animals were inoculated up to 24 h after infection, but administration of the anti-F MAbs protected some animals (25 to 50%) inoculated later during the infection. Our studies suggest that immunotherapy could be used for people who are exposed to NiV infections.

[The << king-child >>: a modern pathology? Sociopsychological thoughts and therapeutic proposals about a society symptom]. / L'<< enfant-roi >>: une pathologie moderne ? Réflexions sociopsychologiques et propositions thérapeutiques à propos d'un symptôme de société.

The symptom of << king-child >> can, in some circumstances, damage the familial relationships. It leads the childhood professionals to think about the concepts this behaviour highlights, as well on the parents'as on the child's side: what is authority today? How can we combine familiar relationships and educational structure? Do we have to set limits to the child? This article suggests to consider the evolution of mentalities within the society and to develop, based on the foundations of the main psychoaffective marks of child development, the intentions of therapeutic support. Different lines of help and care get integrated into the familial therapy being as we think, the main element in the treatment of this symptom.

Langerhans cells are susceptible to measles virus infection and actively suppress T cell proliferation.

We have previously reported that the measles virus (MV) could productively infect human dendritic cells (DC), derived in vitro from CD34+ cord blood progenitors in the presence of GM-CSF and TNF-alpha. In this study, we provide evidence that freshly isolated Langerhans cells (LC), which are immature dendritic cells located in skin and mucosal epithelia, are susceptible to MV infection in vitro as assessed by viral antigen expression by both LC syncytia and LC remaining as single cells. Moreover, MV-infected LC completely block naive allogeneic CD4+ T cell proliferation in response to uninfected LC. This active inhibitory effect is not due to virus transmission from infected LC, is independent of the maturation stage of LC at the time of infection and is antigen non-specific and MHC-unrestricted. Thus, both immature and mature LC are susceptible to measles virus infection, which turns these efficient antigen-presenting cells into active tolerogenic cells. LC infection may explain the long lasting immune suppression observed during natural measles infection.

Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells.

Measles causes a profound immune suppression which is responsible for the high morbidity and mortality induced by secondary infections. Dendritic cells (DC) are professional antigen-presenting cells required for initiation of primary immune responses. To determine whether infection of DC by measles virus (MV) may play a role in virus-induced suppression of cell-mediated immunity, we examined the ability of CD1a+ DC derived from cord blood CD34+ progenitors and Langerhans cells isolated from human epidermis to support MV replication. Here we show that both cultured CD1a+ DC and epidermal Langerhans cells can be infected in vitro by both vaccine and wild type strains of MV. DC infection with MV resulted within 24-48 h in cell-cell fusion, cell surface expression of hemagglutinin, and virus budding associated with production of infectious virus. MV infection of DC completely abrogated the ability of the cells to stimulate the proliferation of naive allogeneic CD4+ T cell as early as day 2 of mixed leukocyte reaction (MLR) (i.e., on day 4 of DC infection). Mannose receptor-mediated endocytosis and viability studies indicated that the loss of DC stimulatory function could not be attributed to the death or apoptosis of DC. This total loss of DC stimulatory function required viral replication in the DC since ultraviolet (UV)-inactivated MV or UV-treated supernatant from MV-infected DC did not alter the allostimulatory capacity of DC. As few as 10 MV- infected DC could block the stimulatory function of 10(4) uninfected DC. More importantly, MV-infected DC, in which production of infectious virus was blocked by UV treatment or paraformaldehyde fixation, actively suppressed allogeneic MLR upon transfer to uninfected DC-T-cultures. Thus, the mechanisms which contribute to the loss of the allostimulatory function of DC include both virus release and active suppression mediated by MV-infected DC, independent of virus production. These data suggest that carriage of MV by DC may facilitate virus spreading to secondary lymphoid organs and that MV replication in DC may play a central role in the general immune suppression observed during measles.

Branchial HOX gene expression and human craniofacial development.

Members of the Antennapedia class of homeobox genes, known as Hox genes, are believed to be pivotal in vertebrate craniofacial development. Here we show that eight members of paralogous groups 1, 2, 3, and 4 are expressed in the human embryonic hindbrain and branchial arches at 4 weeks of development. The combinatorial patterns of expression of genes representing the first three paralogous groups parallel the patterns described for their homologues in various animal models, demonstrating a high degree of conservation of the branchial Hox code. Arch expression of group 4 genes is defined for the first time in any vertebrate. Furthermore, as development proceeds, individual paralogues of a single paralogous group (group 3), which initially share a common expression domain, are differentially down-regulated in a tissue-, organ-, or site-specific fashion.

Transcription factor GATA-1 regulates human HOXB2 gene expression in erythroid cells.

The human HOXB2 gene is a member of the vertebrate Hox gene family that contains genes coding for specific developmental stage DNA-binding proteins. Remarkably, within the hematopoietic compartment, genes of the HOXB complex are expressed specifically in erythromegakaryocytic cell lines and, for some of them, in hematopoietic progenitors. Here, we report the study of HOXB2 gene transcriptional regulation in hematopoietic cells, an initial step in understanding the lineage-specific expression of the whole HOXB complex in these cells. We have isolated the HOXB2 5'-flanking sequence and have characterized a promoter fragment extending 323 base pairs upstream from the transcriptional start site, which, in transfection experiments, was sufficient to direct the tissue-specific expression of HOXB2 in the erythroid cell line K562. In this fragment, we have identified a potential GATA-binding site that is essential to the promoter activity as demonstrated by point mutation experiments. Gel shift analysis revealed the formation of a specific complex in both erythroleukemic lines K562 and HEL that could be prevented by the addition of a specific antiserum raised against GATA-1 protein. These findings suggest a regulatory hierarchy in which GATA-1 is upstream of the HOXB2 gene in erythroid cells.

CD23/CD21 interaction is required for presentation of soluble protein antigen by lymphoblastoid B cell lines to specific CD4+ T cell clones.

Previous studies have documented a role for membrane-bound CD23 (the low affinity Fc epsilon RII) in presentation of alloantigens by B cells. The aim of the present study was to examine the involvement of cell surface CD23 in presentation of more conventional soluble protein antigens to T cells. We show that antibodies to CD23 and to its lymphocyte-associated second ligand, CD21, inhibit presentation of the cow's milk allergen casein, by autologous CD23+CD21+ B-EBV cell lines to casein-specific HLA-DP-restricted CD4+ T cell clones obtained from patients with either reaginic or enterophatic forms of cow's milk protein intolerance. Maximal inhibition was achieved when the antibodies were added at the initiation of the culture. The absence of specific inhibition by an anti-DR alpha monoclonal antibody (mAb) argues against a steric hindrance phenomenon impeding access of the T cell receptor to major histocompatibility complex class II molecules. Rather, anti-CD23 and anti-CD21 mAb-induced inhibition of antigen presentation seems to affect at least partly, heterotypic conjugate formation through CD23/CD21 interaction. Double immunofluorescence labeling of the T cell clones and antibody inhibition of T/B conjugate formation shows that functional CD23 and CD21 molecules are induced on T cells following contact with B-EBV cell lines. Taken together, these data indicate that CD23/CD21 interactions between T and B cells are required for presentation of soluble protein antigens by B-EBV cell lines to specific CD4+ T cells. The potential implications of these findings for allergen-specific T cell activation are discussed.

Immortalization of mouse intestinal epithelial cells by the SV40-large T gene. Phenotypic and immune characterization of the MODE-K cell line.

Intestinal epithelial cells from the mouse small intestine were immortalized by SV40 large T gene transfer through a murine ecotropic virus. The resulting cell lines expressed the SV40 large T mRNA and exhibited morphological and phenotypic characteristics of normal enterocytes, including intercellular junctions, and expression of cytokeratin, villin, poly-Ig receptor (i.e., secretory component) and vasoactive intestinal peptide receptors. All expressed cell surface major histocompatibility complex class I molecules, but cell surface class II antigens were undetectable. Functional studies on antigen presentation were carried out using the MODE-K cell line established from the mouse duodenum. Interferon-gamma treatment of MODE-K cells resulted in a high level of class II molecule expression, and the ability to process and present native protein antigens to specific CD4+ T-cell hybridomas, via functional class II molecules. These data suggest that the MODE-K cell line is a suitable model for the analysis of intestinal epithelial cell function in mucosal immunity.

Mouse intestinal epithelial cells express the self superantigen Mls1a.

In previous studies, we demonstrated that intestinal epithelial cells of the mouse small intestine could present exogenous antigen to specific CD4+ T cell hybridomas. We now report on the ability of normal enterocytes to present the self superantigen Mls1a. Enterocytes from Mls1a but not from Mls1b strains stimulated interleukin-2 production through a V beta 6+ T cell hybridoma specific for Mls1a determinants. Antibody inhibition experiments showed that enterocytes presented Mls determinants via a major histocompatibility complex class II-dependent mechanism. Furthermore, the ability of enterocytes to activate V beta 6+ Mls1a-specific T cells was inhibited by monoclonal antibodies against the Orf protein encoded by an Mtv-7 provirus which is associated with Mls1a expression. These findings provide evidence for the first time that Mls determinants are expressed on normal enterocytes and support the theory of a possible role of these cells in extrathymic selection of T cell receptor V beta repertoire of intraepithelial T lymphocytes.

Intestinal epithelial cells express the CD23/Fc epsilon RII molecule: enhanced expression in enteropathies.

Immunohistochemical analysis of normal human intestine revealed that two anti-CD23 monoclonal antibodies (mAb), EBVCS 1 and EBVCS 2, reacted with human intestinal epithelial cells. Both mAb exhibited an exclusive reactivity with epithelial cells of the small and large bowels. Staining with both EBVCS 1 and EBVCS 2 was localized on the apical and basal sides of enterocytes. Enhanced expression of CD23 on gut epithelial cells was found in inflammatory bowel diseases, in children with food intolerance to cows' milk proteins and in a young infant with severe autoimmune enteropathy. Western blot analysis of anti-CD23 mAb reactivity with gut epithelial cell extracts showed the presence of a non-reducible 42,000-45,000 M(r) polypeptide compatible with the membrane form of the intact CD23 molecule. These data show that CD23 is constitutively expressed by intestinal epithelial cells and that its expression is enhanced in enteropathies.

[Homeoproteins: participation in hematopoietic processes?]. / Les homéoprotéines: participation aux processus hématopoïétiques?

The molecular basis of commitment and differentiation of hematopoietic cells remain poorly understood at the genetic level. Among putative candidates involved in these processes are homeoproteins, a large family of transcription factors which play a major role during development. Using a strategy based on the polymerase chain reaction (PCR) we have isolated nine different Antennapedia-like homeobox (HOX) genes from purified human hematopoietic precursors. Their expression patterns, analyzed with a panel of leukemia-derived cell lines representing various blood cells phenotypes, appears to be lineage-restricted. Extending our study to all the known members of the HOX 1 and HOX 2 clusters, we found that HOX 1 genes are predominantly detected within cell of myelomonocytic origin whereas HOX 2 genes transcripts are principally expressed in erythro-megakaryocytic cell lines. Furthermore, we have observed that the expression of three HOX 1 genes within B lymphoid lineages is stage-related and that the expression of several of them is switched off during TPA-induced differentiation of KG1 and U937 cells. These observations support the idea that homeoproteins could be regulators of lineage determination during hematopoiesis.

Identification of homeobox-containing genes expressed in hematopoietic blast cells.

Among putative candidates involved in commitment and differentiation of hematopoietic cells are homeoproteins, a large family of transcription factors playing a major role during development. Using a polymerase chain reaction (PCR)-derived protocol, we have investigated for Antennapedia-like homeobox-containing (HOX) gene expression in an enriched population of human hematopoietic progenitors. Nine members of HOX 1 and HOX 2 loci were isolated. Together with recent studies using established cell lines, this indicates a large representation of HOX genes in the hematopoietic compartment and suggests a participation of this class of nuclear proteins to early steps of hematopoiesis.