In a high-dose melphalan setting, palifermin compared with placebo had no effect on oral mucositis or related patient's burden.

This randomized-controlled trial studied the efficacy of palifermin in a chemotherapy-only, high-dose Melphalan (HDM) transplant setting, to reduce oral mucositis (OM) and its sequelae measured by patient-reported outcomes (PRO) and medical resource use. Palifermin, relative to placebo was given either pre-/post-HDM or pre-HDM in patients with multiple myeloma (MM) undergoing auto-SCT at 39 European centers. Oral cavity assessment (WHO) and PRO questionnaires (oral mucositis daily questionnaire (OMDQ) and EQ 5D) were used in 281 patients (mean age 56, ± s.d.=8 years). 57 patients received placebo. One hundred and fifteen subjects were randomized to pre-/post-HDM receiving palifermin on 3 consecutive days before HDM and after auto-SCT and 109 patients were randomized to pre-HDM, receiving palifermin (60 µg/kg/day) i.v. for 3 consecutive days before HDM. There was no statistically significant difference in maximum OM severity. Severe OM occurred in 37% (placebo), 38% (pre-/post-HDM) and 24% (pre-HDM) of patients. No significant difference was observed with respect to PRO assessments or medical resource use, but more infections and fever during neutropenia were reported in pre-/post-HDM vs placebo (for example, 51 and 26%). To conclude, palifermin was unable to reduce OM or OM-related patient's burden in MM transplant patients.

Kinetic and mechanical basis of rolling through an integrin and novel Ca2+-dependent rolling and Mg2+-dependent firm adhesion modalities for the alpha 4 beta 7-MAdCAM-1 interaction.

We studied interactions in shear flow of cells bearing integrins alpha4beta1 or alpha4beta7 with VCAM-1 and MAdCAM-1 substrates in different divalent cations. Interestingly, Ca(2+) was essential for tethering in flow and rolling interactions through both alpha4 integrins. Mg(2+) promoted firm adhesion of alpha4beta7-expressing cells on MAdCAM-1 but with much lower tethering efficiency in shear flow. The k(off) degrees of 1.28 s(-1) and resistance of the receptor-ligand bond to force (estimated as a bond interaction distance or sigma) for transient tethers on MAdCAM-1 were similar to values for E- and P-selectins. By contrast to results in Ca(2+) or Ca(2+) + Mg(2+), in Mg(2+) the alpha4beta7-MAdCAM-1 k(off) degrees decreased 20-fold to 0.046 s(-1), and the bond was weaker, providing an explanation for the finding of firm adhesion under these conditions. Shear enhanced tethering to MAdCAM-1, thereby contributing to the stability of rolling. Comparisons to selectins demonstrate that the kinetic and mechanical properties of the alpha4beta7 integrin are well suited to its intermediate position in adhesion cascades, in which it bridges rapid rolling through selectins to firm adhesion through beta2 integrins.

Solution structure of the albumin-binding GA module: a versatile bacterial protein domain.

The albumin-binding GA module is found in a family of surface proteins of different bacterial species. It comprises 45 amino acid residues and represents the first known example of contemporary module shuffling. Using 1H NMR spectroscopy we have determined the solution structure of the GA module from protein PAB, a protein of the anaerobic human commensal and pathogen Peptostreptococcus magnus. This structure, the first three-dimensional structure of an albumin-binding protein domain described, was shown to be composed of a left-handed three-helix-bundle. Sequence differences between GA modules with different affinities for albumin indicated that a conserved region in the C-terminal part of the second helix and the flexible sequence between helices 2 and 3 could contribute to the albumin-binding activity. The effect on backbone amide proton exchange rates upon binding to albumin support this assumption. The GA module has a fold that is strikingly similar to the immunoglobulin-binding domains of staphylococcal protein A but it shows no resemblance to the fold shared by the immunoglobulin-binding domains of streptococcal protein G and peptostreptococcal protein L. When the gene sequences, binding properties and thermal stability of these four domains are analysed in relation to their global folds an evolutionary pattern emerges. Thus, in the evolution of novel binding properties mutations are allowed only as long as the energetically favourable global fold is maintained.

Protein PAB, an albumin-binding bacterial surface protein promoting growth and virulence.

The anaerobic bacterium Peptostreptococcus magnus is a human commensal and pathogen. Previous work has shown that strains of P. magnus isolated from patients with gynecological disease (vaginosis) frequently express an immunoglobulin (Ig) light chain-binding protein called protein L. Here we report that strains isolated from localized suppurative infections bind human serum albumin (HSA), whereas commensal isolates bind neither Ig nor HSA. The HSA-binding protein PAB was extracted from the bacterial surface or isolated from the culture supernatant of the P. magnus strain ALB8. Protein PAB was shown to have two homologous HSA-binding domains, GA and uGA. GA is absent in the sequence of a related protein from another P. magnus strain and shows a high degree of homology to the HSA-binding domains of streptococcal protein G. Therefore GA is believed to have recently been shuffled as a module from genes of other bacterial species into the protein PAB gene. This GA module was shown to exhibit a much higher affinity for HSA than uGA and was also found to be present in all of the isolates tested from localized suppurative infections, indicating a role in virulence. Moreover, when peptostreptococci or streptococci expressing the GA module were grown in the presence of HSA, the growth rate was substantially increased. Thus, the HSA binding activity of the GA module adds selective advantages to the bacteria, which increases their virulence in the case of P. magnus strains.

Identification of interdomain sequences promoting the intronless evolution of a bacterial protein family.

In the evolution of eukaryotic genes, introns are believed to have played a major role in increasing the probability of favorable duplication events, chance recombinations, and exon shuffling resulting in functional hybrid proteins. As a rule, prokaryotic genes lack introns, and the examples of prokaryotic introns described do not seem to have contributed to gene evolution by exon shuffling. Still, certain protein families in modern bacteria evolve rapidly by recombination of genes, duplication of functional domains, and as shown for protein PAB of the anaerobic bacterial species Peptostreptococcus magnus, by the shuffling of an albumin-binding protein module from group C and G streptococci. Characterization of a protein PAB-related gene in a P. magnus strain with less albumin-binding activity revealed that the shuffled module was missing. Based on this fact and observations made when comparing gene sequences of this family of bacterial surface proteins interacting with albumin and/or immunoglobulin, a model is presented that can explain how this rapid intronless evolution takes place. A new kind of genetic element is introduced: the recer sequence promoting interdomain, in frame recombination and acting as a structure-less flexibility-promoting spacer in the corresponding protein. The data presented also suggest that antibiotics could represent the selective pressure behind the shuffling of protein modules in P. magnus, a member of the indigenous bacterial flora.

The GA module, a mobile albumin-binding bacterial domain, adopts a three-helix-bundle structure.

We present the first study of the secondary structure and global fold of an albumin-binding domain. Our data show that the GA module from protein PAB, an albumin-binding protein from the anaerobic bacterial species Peptostreptococcus magnus, is composed of a left-handed three-helix bundle. The helical regions were identified by sequential and medium range NOEs, values of NH-C alpha H coupling constants, chemical shift indices, and the presence of slowly exchanging amide protons, as determined by NMR spectroscopy. In addition, circular dichroism studies show that the module is remarkably stable with respect to both pH and temperature.

Protein PAB, a mosaic albumin-binding bacterial protein representing the first contemporary example of module shuffling.

Some strains of the anaerobic human commensal and pathogen Peptostreptococcus magnus bind human serum albumin (HSA), whereas other strains of this species express protein L, an immunoglobulin light chain-binding surface protein. A novel HSA-binding protein called protein PAB was purified in one step from the culture supernatant of an HSA-binding strain of P. magnus by affinity chromatography on HSA-Sepharose. The apparent size of the molecular was 47 kDa on SDS-polyacrylamide gel electrophoresis. Amino acid sequence analysis of protein PAB demonstrated that the 4 NH2-terminal residues were identical to the corresponding sequence in protein L. In a polymerase chain reaction, oligonucleotides based on extragenic 5'- and 3'-end sequences of the protein L gene generated a product of the expected size: 1.3 kilobase pairs. A recombinant protein with retained albumin binding capacity was expressed in Escherichia coli, and the nucleotide sequence of the protein PAB gene was determined. The structural gene is 1161 nucleotides long, corresponding to a preprotein of 387 amino acids and a molecular mass of 43,043 Da. Unlike most other Gram-positive bacterial surface proteins described, protein PAB contains no internal homologies. However, substantial homologies were found to both proteins L and G (the IgG- and HSA-binding surface protein of group C and G streptococci). The derived amino acid sequence of the 135-base pair-long region homologous to protein G corresponds to the HSA-binding domain of that protein, and in protein PAB, this region is inserted between sequences showing extensive homology to COOH-terminal regions of peptostreptococcal protein L. This mosaic organization of protein PAB demonstrates that the molecule is a product of intergenic interspecies recombination of a functional domain into a common framework for peptostreptococcal surface proteins. Such an interspecies exchange of a functional protein module has previously not been described in prokaryotic cells.

On the interaction between protein L and immunoglobulins of various mammalian species.

Protein L, a cell wall molecule of certain strains of the anaerobic bacterial species Peptostreptococcus magnus, shows high affinity for human immunoglobulin (Ig) light chains. In the present study protein L was tested against a panel of human myeloma proteins of the IgG, IgM, IgA and IgE classes, and strong binding was seen with antibodies carrying kappa light chains. A high degree of specificity for Ig was demonstrated in binding experiments with human plasma proteins. Apart from human Ig, strong protein L-binding activity was also detected in the serum of 12 out of 23 tested additional mammalian species, including other primates and rodents. Subsequent analysis with purified Ig samples demonstrated the binding of protein L to Ig of important laboratory animal species such as the mouse, the rat and the rabbit. The affinity constants for the interactions between protein L and polyclonal IgG of these species were 2.6 x 10(9), 3.9 x 10(8) and 7.4 x 10(7), respectively. In non-human species, the binding of protein L was also found to be mediated through Ig light chains, and the results demonstrate the potential value of protein L as an immunochemical tool.