The novel CXCR4 antagonist POL5551 mobilizes hematopoietic stem and progenitor cells with greater efficiency than Plerixafor.

Mobilized blood has supplanted bone marrow (BM) as the primary source of hematopoietic stem cells for autologous and allogeneic stem cell transplantation. Pharmacologically enforced egress of hematopoietic stem cells from BM, or mobilization, has been achieved by directly or indirectly targeting the CXCL12/CXCR4 axis. Shortcomings of the standard mobilizing agent, granulocyte colony-stimulating factor (G-CSF), administered alone or in combination with the only approved CXCR4 antagonist, Plerixafor, continue to fuel the quest for new mobilizing agents. Using Protein Epitope Mimetics technology, a novel peptidic CXCR4 antagonist, POL5551, was developed. In vitro data presented herein indicate high affinity to and specificity for CXCR4. POL5551 exhibited rapid mobilization kinetics and unprecedented efficiency in C57BL/6 mice, exceeding that of Plerixafor and at higher doses also of G-CSF. POL5551-mobilized stem cells demonstrated adequate transplantation properties. In contrast to G-CSF, POL5551 did not induce major morphological changes in the BM of mice. Moreover, we provide evidence of direct POL5551 binding to hematopoietic stem and progenitor cells (HSPCs) in vivo, strengthening the hypothesis that CXCR4 antagonists mediate mobilization by direct targeting of HSPCs. In summary, POL5551 is a potent mobilizing agent for HSPCs in mice with promising therapeutic potential if these data can be corroborated in humans.

Apheresis product identification in the transplant center: development of point-of-care protocols for extended blood typing of stem cell apheresis products.

Transfusion of the 'wrong' stem cell product would almost inevitably be lethal, yet assays to confirm the contents of the product bag, except by checking labels and paperwork, are lacking. To increase the likelihood that a product mix-up would be detected in the transplant center, we developed a simple protocol for extended blood typing and hence, for confirmation of donor/product identity, on a tube segment. Apheresis samples were applied, directly or after erythrocyte enrichment, to commercially available blood typing assays, including lateral flow cards and gel agglutination cards. Without sample modification, low hematocrit and high leukocyte count obviated definitive blood typing. Using the most simple erythrocyte enrichment protocol, that is, centrifugation, reliable blood group analysis became possible with either assay. Other, more cumbersome pre-analytical protocols were also successful but provided no advantage. The preferred method was validated on 100 samples; ABD was correctly identified in 100% of cases. Of the other Rh Ags, all except two 'small e', in both cases in heterozygous individuals, were detected; there were no false positives. A simple, inexpensive point-of-care assay for extended blood typing of apheresis products is available, which can reduce the fatal risk of administering the wrong stem cell product.

Thirteen novel VKORC1 mutations associated with oral anticoagulant resistance: insights into improved patient diagnosis and treatment.

BACKGROUND: Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is the molecular target of oral anticoagulants. Mutations in VKORC1 cause partial or total coumarin resistance. OBJECTIVES: To identify new VKORC1 oral anticoagulant (OAC) resistance (OACR) mutations and compare the severity of patient phenotypes across different mutations and prescribed OAC drugs. PATIENTS/METHODS: Six hundred and twenty-six individuals exhibiting partial or complete coumarin resistance were analyzed by VKORC1 gene sequencing and CYP2C9 haplotyping. RESULTS: We identified 13 patients, each with a different, novel human VKORC1 heterozygous mutation associated with an OACR phenotype. These mutations result in amino acid substitutions: Ala26→Thr, His28→Gln, Asp36→Gly, Ser52→Trp, Ser56→Phe, Trp59→Leu, Trp59→Cys, Val66→Gly, Gly71→Ala, Asn77→Ser, Asn77→Tyr, Ile123→Asn, and Tyr139→His. Ten additional patients each had one of three previously reported VKORC1 mutations (Val29→Leu, Asp36→Tyr, and Val66→Met). Genotyping of frequent VKORC1 and CYP2C9 polymorphisms in these patients revealed a predominant association with combined non-VKORC1*2 and wild-type CYP2C9 haplotypes. Additionally, data for OAC dosage and the associated measured International Normalized Ratio (INR) demonstrate that OAC therapy is often discontinued by physicians, although stable therapeutic INR levels may be reached at higher OAC dosages. Bioinformatic analysis of VKORC1 homologous protein sequences indicated that most mutations cluster into protein sequence segments predicted to be localized in the lumenal loop or at the endoplasmic reticulum membrane-lumen interface. CONCLUSIONS: OACR mutations of VKORC1 predispose afflicted patients to high OAC dosage requirements, for which stable, therapeutic INRs can sometimes be attained.

Iron-dependent transcription of the frpB gene of Helicobacter pylori is controlled by the Fur repressor protein.

We have overexpressed and purified the Helicobacter pylori Fur protein and analyzed its interaction with the intergenic regions of divergent genes involved in iron uptake (frpB and ceuE) and oxygen radical detoxification (katA and tsaA). DNase I footprint analysis showed that Fur binds specifically to a high-affinity site overlapping the P(frpB) promoter and to low-affinity sites located upstream from promoters within both the frpB-katA and ceuE-tsaA intergenic regions. Construction of an isogenic fur mutant indicated that Fur regulates transcription from the P(frpB) promoter in response to iron. In contrast, no effect by either Fur or iron was observed for the other promoters.

Regulation of transcription in Helicobacter pylori: simple systems or complex circuits?

A common strategy used by both Gram-negative and Gram-positive bacterial pathogens is based on the synchronisation of virulence gene expression using a variety of regulatory systems and networks to overcome host defence. During the last decade an exponentially growing number of studies on Helicobacter pylori, a human pathogen associated with diverse stomach diseases, have mainly focussed on the elucidation of mechanisms and functions of virulence factors. A subset of these studies were focussed on the molecular mechanisms regulating gene transcription in H. pylori with the aim of understanding the profound physiological changes that this pathogen, as well as other bacteria, undergoes during infection. Despite the limited number of putative regulatory proteins, as deduced from genome sequence analyses, evidence is accumulating for the existence of new and complex circuits regulating gene transcription and virulence of this bacterium. Here we will focus on the molecular mechanisms used by H. pylori to control gene transcription.

The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori.

The ferric uptake regulator (Fur) protein is known to act as a Fe2+-dependent transcriptional repressor of bacterial promoters. Here, we show that, in Helicobacter pylori, Fur can mediate the regulation of iron-activated genes in contrast to classical Fur regulation, in which iron acts as a co-repressor. Inactivation of the fur gene in the chromosome of H. pylori resulted in the derepression of a 19 kDa protein that was identified by N-terminal sequencing as the non-haem-containing ferritin (Pfr). Growth of the wild-type H. pylori strain on media treated with increasing concentrations of FeSO4 resulted in induction of transcription from the Ppfr promoter and, conversely, depletion of iron resulted in repression of Ppfr, indicating that this promoter is iron activated. In the fur mutant, the Ppfr promoter is constitutively highly expressed and no longer responds to iron, indicating that the Fur protein mediates this type of iron regulation. Footprinting analysis revealed that Fur binds to the Ppfr promoter region and that Fe2+ decreases the efficiency of binding. In contrast, Fe2+ increased the affinity of Fur for a classical Fur-regulated promoter, the iron-repressed frpB gene promoter. To our knowledge, this is the first evidence of direct interaction between the Fur protein and the promoter of an iron-activated (-derepressed) gene. Our results support a model in which the iron status of the Fur protein differentially alters its affinity for operators in either iron-repressed or iron-activated genes.

The autoregulatory HspR repressor protein governs chaperone gene transcription in Helicobacter pylori.

In the present study, we provide evidence that the groESL, hrcA-grpE-dnaK and cbpA-hspR-orf operons encoding the major chaperones of the human gastric pathogen Helicobacter pylori are transcribed by the vegetative sigma factor sigma80 and are regulated negatively by the transcriptional repressor HspR. In vitro studies with purified recombinant HspR protein established that the protein represses transcription by binding to large DNA regions centred around the transcription initiation site in the case of the Pcbp promoter, and around -85 and -120 in the case of the Pgro and Phrc promoters respectively. All three binding sites contain DNA motifs with some similarity to the HAIR sequence identified as a consensus for the HspR protein of Streptomyces. In contrast to the situation in Streptomyces, in which transcription of HspR-regulated genes is induced in response to heat shock, transcription of the HspR-dependent genes in H. pylori is not inducible by thermal stimuli. Transcription of the groESL and cbpA-hspR-orf operons is induced by osmotic shock, while transcription of the hrcA-grpE-dnaK operon, although HspR dependent, is not affected by salt treatment. The possibility that HspR could constitute a global transcriptional regulator for diverse cellular functions with implications for pathogenesis is discussed.

Motility of Helicobacter pylori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog.

sigma54 is the subunit of bacterial RNA polymerase that transcribes from promoters with enhancer elements bound by enhancer-binding proteins. By computer searches of Helicobacter pylori genomic sequences, chromosomal gene disruption, and RNA analyses, we have identified sigma54-recognized promoters that regulate transcription of flagellar basal body and hook genes, as well as the enhancer-binding protein FlgR (flagellum regulator), a transactivating protein of the NtrC family. We demonstrate that FlgR is required for bacterial motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR acts as a repressor of transcription of the sigma28-regulated flaA flagellin gene promoter, while changes in DNA topology repress transcription of the sigma54-regulated flaB flagellin gene promoter. Our data indicate that regulation of flagellar gene expression in H. pylori shows similarities with that in enterobacteriaceae and Caulobacter.

Functional analysis of the Helicobacter pylori principal sigma subunit of RNA polymerase reveals that the spacer region is important for efficient transcription.

We have cloned the rpoD gene encoding the principal sigma (sigma) factor of Helicobacter pylori. The deduced amino acid sequence reveals a predicted polypeptide of 676 residues that has amino acid homology with the principal sigma factors of a number of divergent prokaryotes. We have designated this factor sigma80. Amino acid sequence analysis suggests that region 1.1 is missing in sigma80 and that a region with homology to a regulatory protein from Bacillus subtilis phage SPO1 is present. Genetic studies have indicated that sigma80 is not compatible with the transcriptional machinery of Escherichia coli. However, in vitro sigma80 could be assembled into the E. coli RNA polymerase and could bind to E. coli and H. pylori promoters, suggesting that the sigma80-containing RNA polymerase has the same stoichiometry as the native complex. By exchanging protein domains between E. coli and H. pylori sigma factors, we demonstrate that the sigma80 domain inhibiting transcription from E. coli promoters is confined within the non-conserved spacer region, implying that the spacer region of prokaryotic primary sigma factors plays an important role in the process of transcription. Consistent with its restricted niche and with the availability of a very restricted number of transcriptional regulators, H. pylori may have evolved a spacer region of the sigma factor to modulate total transcription and to quickly respond to microenvironmental changes.

Identification and characterization of an operon of Helicobacter pylori that is involved in motility and stress adaptation.

We identified a novel stress-responsive operon (sro) of Helicobacter pylori that contains seven genes which are likely to be involved in cellular functions as diverse as chemotaxis, heat shock response, ion transport, and posttranslational protein modification. The products of three of these genes show amino acid homologies to known proteins, such as the flagellar motor switch protein CheY, a class of heat shock proteins, and the ribosomal protein L11 methyltransferase, and to a phosphatidyltransferase. In addition to containing an open reading frame of unknown function, the product of which is predicted to be membrane associated, the sro locus contains three open reading frames that have previously been described as constituting two separate loci, the ftsH gene and the copAP operon of H. pylori. Knockout mutants showed that CheY is essential for bacterial motility and that CopA, but not CopP, relieves copper toxicity. Transcriptional analyses indicated that this locus is regulated by a single promoter and that a positive effect on transcription is exerted by the addition of copper to the medium and by temperature upshift from 37 to 45 degrees C. The possible role of this locus in H. pylori virulence is discussed.

Transcriptional analysis of the divergent cagAB genes encoded by the pathogenicity island of Helicobacter pylori.

Helicobacter pylori strains isolated from most patients with peptic ulcer disease and adenocarcinoma express the vacuolating toxin VacA and contain a pathogenicity island named cag. The cag pathogenicity island codes for more than 40 putative proteins with features similar to bacterial secretion systems. One of these proteins, CagA, is an immunodominant antigen with unknown function encoded by the cagA gene. In the present study, we have analysed the functional promoter elements of the H. pylori cagA gene as well as of the divergently transcribed cagB gene. Primer extension analyses identified a single 5' end of the cagA mRNA, while two initiation sites were mapped in the case of the cagB mRNA. The promoters deduced upstream of these start points of transcription contained conserved -10 regions but no -35 regions with respect to the Escherichia coli sigma70 consensus sequence. Nevertheless, they could be activated in E. coli and in vitro by purified E. coli RNA polymerase. Deletion analyses indicated that the cagA and cagB genes are transcribed by overlapping promoters and that full activation requires sequences up to -70 and -96 respectively. Instead, basal transcription is likely to be mediated by -10 extended promoter-like sequences. RNA polymerase is able to bind the -40 to -60 region of the cagA promoter, and its binding is mediated by the alpha-subunit. This region resembles the UP elements of prokaryotic promoters in location, sequence and mechanism of interaction with the RNA polymerase. We discuss the features of these promoters and propose that they could represent a class of minimum promoters, which ensures a basic level of transcription, while full activation requires regulatory elements or a defined promoter context.