Analgesia via blockade of NGF/TrkA signaling does not influence fracture healing in mice

Abatement of fracture‐related pain is important in patient welfare. However, the frequently used non‐steroidal anti‐inflammatory drugs are considered to impair fracture healing through blockade of cyclooxygenase‐2. An alternative for fracture‐related pain treatment may be blockade of nerve growth factor (NGF)/neurotrophic tyrosine kinase receptor type 1 (TrkA) signaling. Because the effect of blocking this signal‐pathway on bone healing has not been extensively investigated, we addressed this issue by applying neutralizing antibodies that target NGF and TrkA, respectively, in a mouse fracture model. Mice with a knock‐in for human TrkA underwent femur osteotomy and were randomly allocated to phosphate‐buffered‐saline, anti‐NGF‐antibody, or anti‐TrkA‐antibody treatment. The analgesic effect of the antibodies was determined from the activity and the ground reaction force of the operated limb. The effect of antibody administration on fracture healing was assessed by histomorphometry, micro‐computed tomography, and biomechanics. NGF/TrkA‐signaling blockade had no negative effect on fracture healing as callus formation and maturation were not altered. Mice treated with anti‐TrkA antibody displayed significantly greater activity on post‐operative day 2 compared to PBS treatment indicating effective analgesia. Our data indicate, that blockade of NGF/TrkA signaling via specific neutralizing antibodies for pain reduction during fracture healing does not influence fracture healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1235–1241, 2015.     

Structural Variations of the Cell Wall Precursor Lipid II and Their Influence on Binding and Activity of the Lipoglycopeptide Antibiotic Oritavancin

Oritavancin is a semisynthetic derivative of the glycopeptide antibiotic chloroeremomycin with activity against Gram-positive pathogens, including vancomycin-resistant staphylococci and enterococci. Compared to vancomycin, oritavancin is characterized by the presence of two additional residues, a hydrophobic 4′-chlorobiphenyl methyl moiety and a 4-epi-vancosamine substituent, which is also present in chloroeremomycin. Here, we show that oritavancin and its des-N-methylleucyl variant (des-oritavancin) effectively inhibit lipid I- and lipid II-consuming peptidoglycan biosynthesis reactions in vitro. In contrast to that for vancomycin, the binding affinity of oritavancin to the cell wall precursor lipid II appears to involve, in addition to the d-Ala-d-Ala terminus, other species-specific binding sites of the lipid II molecule, i.e., the crossbridge and d-isoglutamine in position 2 of the lipid II stem peptide, both characteristic for a number of Gram-positive pathogens, including staphylococci and enterococci. Using purified lipid II and modified lipid II variants, we studied the impact of these modifications on the binding of oritavancin and compared it to those of vancomycin, chloroeremomycin, and des-oritavancin. Analysis of the binding parameters revealed that additional intramolecular interactions of oritavancin with the peptidoglycan precursor appear to compensate for the loss of a crucial hydrogen bond in vancomycin-resistant strains, resulting in enhanced binding affinity. Augmenting previous findings, we show that amidation of the lipid II stem peptide predominantly accounts for the increased binding of oritavancin to the modified intermediates ending in d-Ala-d-Lac. Corroborating our conclusions, we further provide biochemical evidence for the phenomenon of the antagonistic effects of mecA and vanA resistance determinants in Staphylococcus aureus, thus partially explaining the low frequency of methicillin-resistant S. aureus (MRSA) acquiring high-level vancomycin resistance.     

Comparison of Two Commercial Tick-Borne Encephalitis Virus IgG Enzyme-Linked Immunosorbent Assays

Despite the availability of protective vaccines, tick-borne encephalitis virus (TBEV) infections have been increasingly reported to the European Centre for Disease Prevention and Control in the past 2 decades. Since the diagnosis of TBEV exposure relies on serological testing, we compared two commercial enzyme-linked immunosorbent assays (ELISAs), i.e., Immunozym FSME IgG assay (ELISA-1) and Euroimmun FSME Vienna IgG assay (ELISA-2). Both assays use whole TBEV antigens, but they differ in viral strains (Neudoerfl for ELISA-1 and K23 for ELISA-2) and cutoff values. In testing of samples from 398 healthy blood donors, ELISA-1 showed higher reactivity levels than ELISA-2 (P < 0.001), suggesting different assay properties. This finding was supported by Bland-Altman analysis of the optical density at 450 nm (OD₄₅₀) (mean bias, +0.32 [95% limits of agreement, −0.31 to +0.95]) and persisted after transformation into Vienna units. Concordant results were observed for 276 sera (69%) (44 positive and 232 negative results). Discordant results were observed for 122 sera (31%); 15 were fully discordant, all being ELISA-1 positive and ELISA-2 negative, and 107 were partially discordant (101 being ELISA-1 indeterminate and ELISA-2 negative and 6 having positive or indeterminate reactivity in both ELISAs). Neutralization testing at a 1:10 dilution yielded positive results for 33 of 44 concordant positive sera, 1 of 15 fully discordant sera, and 1 of 33 partially discordant sera. Indirect immunofluorescence testing revealed high antibody titers of ≥100 for yellow fever virus in 18 cases and for dengue virus in one case, suggesting that cross-reactivity contributed to the ELISA-1 results. We conclude that (i) cross-reactivity among flaviviruses remains a limitation of TBEV serological testing, (ii) ELISA-2 revealed reasonable sensitivity and specificity for anti-TBEV IgG population screening of human sera, and (iii) neutralization testing is most specific and should be reserved for selective questions.     

Lineage fate of ductular reactions in liver injury and carcinogenesis

Ductular reactions (DRs) are observed in virtually all forms of human liver disease; however, the histogenesis and function of DRs in liver injury are not entirely understood. It is widely believed that DRs contain bipotential liver progenitor cells (LPCs) that serve as an emergency cell pool to regenerate both cholangiocytes and hepatocytes and may eventually give rise to hepatocellular carcinoma (HCC). Here, we used a murine model that allows highly efficient and specific lineage labeling of the biliary compartment to analyze the histogenesis of DRs and their potential contribution to liver regeneration and carcinogenesis. In multiple experimental and genetic liver injury models, biliary cells were the predominant precursors of DRs but lacked substantial capacity to produce new hepatocytes, even when liver injuries were prolonged up to 12 months. Genetic modulation of NOTCH and/or WNT/β-catenin signaling within lineage-tagged DRs impaired DR expansion but failed to redirect DRs from biliary differentiation toward the hepatocyte lineage. Further, lineage-labeled DRs did not produce tumors in genetic and chemical HCC mouse models. In summary, we found no evidence in our system to support mouse biliary-derived DRs as an LPC pool to replenish hepatocytes in a quantitatively relevant way in injury or evidence that DRs give rise to HCCs.     

The foot in systemic disease: management of the patient with rheumatoid arthritis or diabetes mellitus

The foot is commonly affected in systemic diseases such as rheumatoid arthritis and diabetes mellitus. Treating patients who suffer with foot pathology secondary to systemic diseases requires a multidisciplinary approach, following the principles outlined within this review. There is little high level evidence in this field, such as prospective controlled clinical trials, hence much of what we know and practise is based upon the expert opinion of key individuals in specialist centres, to whom we owe a great debt.