• Exocytotic process as a novel model for mineralization by osteoblasts in vitro and in vivo determined by electron microscopic analysis.

      Rohde, Manfred; Mayer, H; Department of Microbial Pathogenesis, Helmholtz Center for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. manfred.rohde@helmholtz-hzi.de (2007-05)
      The process of biomineralization has been examined during osteoblastic differentiation of bone marrow stroma cells (BMSCs) from embryonic chick in culture and in periosteum itself by a number of different techniques including transmission and scanning electron microscopy. In cell culture of BMSCs at days 20-25, crystals were accumulated extracellularly in the collagen matrix, resulting in large plate-like crystallites and noncollagen associated on the culture disk surface. In contrast, up to days 10-18, mainly intracellular mineralization was visible by numerous needle-like crystal structures in the cell cytoplasm and in vacuoles. After 20-30 days, the crystal content of these vacuoles is released, most probably by membrane fusion to the outside of the cells. Energy-dispersive X-ray analysis (EDX), electron spectroscopic imaging, and electron energy loss spectroscopy demonstrated that Ca, O, and P are located in the intra- and extracellular needle-like crystals. From EDX spectra a Ca/P ratio of 1.3 was estimated for the intracellular structures and a Ca/P ratio of 1.5, for the extracellular material (for comparison, the Ca/P ratio in tibiae is 1.6). X-ray diffraction and quantitative infrared spectral analyses also demonstrated an increase of crystalline bone apatite along the mineralization process. In addition to the finding in vitro, the presence of intracellular needle-like crystals in vacuoles could be demonstrated in vivo in osteoblastic cells of the periosteum in tibia of day 11. The results are in favor of a novel model for mineralization by osteoblasts, in which amorphous Ca/P material is directly secreted via an exocytotic process from vacuoles of the osteoblast, deposited extracellularly, propagated into the collagen fibril matrix, and matured to hydroxyapatite.
    • Region specific and worldwide distribution of collagen-binding M proteins with PARF motifs among human pathogenic streptococcal isolates.

      Reissmann, Silvana; Gillen, Christine M; Fulde, Marcus; Bergmann, René; Nerlich, Andreas; Rajkumari, Reena; Brahmadathan, Kootallur N; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric; Department of Microbial Pathogenesis, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2012)
      Some of the variety of Streptococcus pyogenes and Streptococcus dysgalactiae ssp. equisimilis (SDSE) M proteins act as collagen-binding adhesins that facilitate acute infection. Moreover, their potential to trigger collagen autoimmunity has been implicated in the pathogenesis of acute rheumatic fever and attributed to a collagen-binding motif called PARF (peptide associated with rheumatic fever). For the first time we determine the rate of clinical isolates with collagen-binding M proteins that use a PARF motif (A/T/E)XYLXX(L/F)N in a defined geographic region, Vellore in South India. In this region both, incidence of streptococcal infections and prevalence of acute rheumatic fever are high. M proteins with PARF motif conferred collagen-binding activity to 3.9% of 153 S. pyogenes and 10.6% of 255 SDSE clinical isolates from Vellore. The PARF motif occurred in three S. pyogenes and 22 SDSE M protein types. In one of the S. pyogenes and five of the SDSE M proteins that contained the motif, collagen-binding was impaired, due to influences of other parts of the M protein molecule. The accumulated data on the collagen binding activity of certain M protein types allowed a reanalysis of published worldwide emm-typing data with the aim to estimate the rates of isolates that bind collagen via PARF. The results indicate that M proteins, which bind collagen via a PARF motif, are epidemiologically relevant in human infections, not only in Vellore. It is imperative to include the most relevant collagen-binding M types in vaccines. But when designing M protein based vaccines it should be considered that collagen binding motifs within the vaccine antigen remain potential risk factors.