An emerging player in knee osteoarthritis: the infrapatellar fat pad

The role of inflammation in the development, progression, and clinical features of osteoarthritis has become an area of intense research in recent years. This led to the recognition of synovitis as an important source of inflammation in the joint and indicated that synovitis is intimately associated with pain and osteoarthritis progression. In this review, we discuss another emerging source of inflammation that could play a role in disease development/progression: the infrapatellar fat pad (IFP). The aim of this review is to offer a comprehensive view of the pathology of IFP as obtained from magnetic resonance studies, along with its characterization at both the cellular and the molecular level. Furthermore, we discuss the possible function of this organ in the pathological processes in the knee by summarizing the knowledge regarding the interactions between IFP and other joint tissues and discussing the pro- versus anti-inflammatory functions this tissue could have. We hope that this review will offer an overview of all published data regarding the IFP and will indicate novel directions for future research.     

TLR Accessory Molecule RP105 (CD180) Is Involved in Post-Interventional Vascular Remodeling and Soluble RP105 Modulates Neointima Formation

Background

RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown.

Methods and Results

TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105^−/− mice resulting in a higher proliferation rate. In RP105^−/− mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982±974 µm² vs.1947±278 µm²,p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105^−/− mice this effect was more pronounced (10316±1243 µm² vs.4208±555 µm²,p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500±573 vs.6581±1894 µm²,p<0.05), whereas expression of the single factors RP105 or MD1 had no effect.

Conclusion

RP105 is a potent inhibitor of post-interventional neointima formation.     

PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P₂ is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P₂-containing liposomes of the PtdIns(4,5)P₂ binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P₂ on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P₂ mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P₂ may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis.     

High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics

The observation of biological processes at the molecular scale in real time requires high spatial and temporal resolution. Magnetic tweezers are straightforward to implement, free of radiation or photodamage, and provide ample multiplexing capability, but their spatiotemporal resolution has lagged behind that of other single-molecule manipulation techniques, notably optical tweezers and AFM. Here, we present, to our knowledge, a new high-resolution magnetic tweezers apparatus. We systematically characterize the achievable spatiotemporal resolution for both incoherent and coherent light sources, different types and sizes of beads, and different types and lengths of tethered molecules. Using a bright coherent laser source for illumination and tracking at 6 kHz, we resolve 3 Å steps with a 1 s period for surface-melted beads and 5 Å steps with a 0.5 s period for double-stranded-dsDNA-tethered beads, in good agreement with a model of stochastic bead motion in the magnetic tweezers. We demonstrate how this instrument can be used to monitor the opening and closing of a DNA hairpin on millisecond timescales in real time, together with attendant changes in the hairpin dynamics upon the addition of deoxythymidine triphosphate. Our approach opens up the possibility of observing biological events at submillisecond timescales with subnanometer resolution using camera-based detection.     

Comparison of the accuracy of disk diffusion zone diameters obtained by manual zone measurements to that by automated zone measurements to determine antimicrobial susceptibility

Although a variety of techniques are available for antimicrobial susceptibility testing, disk diffusion methods remain the most widely used. We compared the accuracy of disk diffusion zone diameters as obtained by manual zone measurements in a low resource country (Indonesia) to that by automated zone measurements (Oxoid aura image system) in a high resource setting (the Netherlands) to determine susceptibility categories (sensitive, intermediate susceptible or resistant). A total of 683 isolates were studied, including 294 Staphylococcus aureus, 195 Escherichia coli and 194 other Enterobacteriaceae. Antimicrobial agents included tetracycline, oxacillin, gentamicin, erythromycin, trimethoprim/sulfamethoxazole and chloramphenicol for S. aureus and ampicillin, gentamicin, cefotaxime, ciprofloxacin, trimethoprim/sulfamethoxazole, and chloramphenicol for E. coli and other Enterobacteriaceae. Of the 4098 drug-organism combinations, overall category agreement (CA), major discrepancy (MD) and minor discrepancy (mD) between the two methods were 82.4% (3379/4098), 6.0% (244/4098) and 11.6% (475/4098), respectively. One hundred and sixty three of 244 MDs were resolved using reference broth microdilution method. Overall very major error (VME), major error (ME) and minor error (mE) of manual zone measurement were 28.8%, 45.4% and 4.9%, respectively and for the aura image system 4.9%, 16.0% and 4.9%, respectively. The results of this study indicate that the disk diffusion method with manual zone measurement in Indonesia is reliable for susceptibility testing. The use of an automated zone reader, such as the aura image system, will reduce the number of errors, and thus improve the accuracy of susceptibility test results for medically relevant bacteria.     

Infectious Cellular Load in Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and Susceptibility of Peripheral Blood Mononuclear Cells from Their Exposed Partners to Non-Syncytium-Inducing HIV-1 as Major Determinants for HIV-1 Transmission in Homosexual Couples

To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4⁺ T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 Δ32). Three of these individuals (all nonrecipients) had a CCR5 Δ32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 Δ32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.     

Ultrastructure of Draparnaldia glomerata (Chaetophorales, Chlorophyceae) II. Mitosis and cytokinesis

The mitosis and cytokinesis of Draparnaldia glomerata as examined here by transmission electron microscopy are in many aspects similar to those described earlier for other chaetophoralean algae. The standard chaetophoralean model of the mechanism of mitosis/cytokinesis is described in detail. Characteristic in this pattern is the movement of sets of centrioles towards the nuclear poles followed by a proliferation of extranuclear microtubules at prophase, the (partial) fusion of centrioles with the spindle poles at metaphase and anaphase, the simultaneous separation of chromosomes apparently caused by both spindle elongation and shortening of the chromosomal microtubules at anaphase, the expulsion of the centrioles by daughter nuclei and finally the non–persistent spindle at telophase. Cytokinesis takes place by formation of a cell plate associated with phycoplast microtubules. The possible function of the phycoplast in cytokinesis in Draparnaldia is discussed.     

The chimpanzee major histocompatibility complex class II DR subregion contains an unexpectedly high number of beta-chain genes

The major histocompatibility complex (MHC) class II DR subregion of the chimpanzee was studied by restriction fragment length polymorphism (RFLP) analysis. Genomic DNA obtained from a panel of 94 chimpanzees was digested with the restriction enzyme Taq I and hybridized with an HLA-DR probe specific for the 3' untranslated (UT) region. Such a screening revealed the existence of 14 distinct DRB/Taq I gene-associated fragments allowing the definition of 11 haplotypes. Segregation studies proved that the number of chimpanzee class II DRB/Taq I fragments is not constant and varies from three to six depending on the haplotype. Comparison of these data with a human reference panel manifested that some MHC DRB/Taq I fragments are shared by man and chimpanzee. Moreover, the number of HLA-DRB/Taq I gene-associated fragments detected in a panel of homozygous typing cells varies from one to three and corresponds with the number of HLA-DRB genes present for most haplotypes. However, a discrepancy is observed for the HLA-DR4,-DR7, and -DR9 haplotypes since a fourth HLA-DRB pseudogene present within these haplotypes lacks its 3' UT region and thus is not detected with the probe used. These results suggest that chimpanzees have a higher maximum number of DRB genes per haplotype than man. As a consequence, some chimpanzee haplotypes must show a dissimilar organization of the MHC DR subregion compared to their human equivalents. The implications of these findings are discussed in the context of the trans-species theory of MHC polymorphism. Address correspondence and offprint requests to: R. E. Bontrop.     

Modeling human arthritic diseases in nonhuman primates

Models of rheumatoid arthritis (RA) in laboratory animals are important tools for research into pathogenic mechanisms and the development of effective, safe therapies. Rodent models (rats and mice) have provided important information about the pathogenic mechanisms. However, the evolutionary distance between rodents and humans hampers the translation of scientific principles into effective therapies. The impact of the genetic distance between the species is especially seen with treatments based on biological molecules, which are usually species-specific. The outbred nature and the closer anatomical, genetic, microbiological, physiological, and immunological similarity of nonhuman primates to humans may help to bridge the wide gap between inbred rodent strain models and the heterogeneous RA patient population. Here we review clinical, immunological and pathological aspects of the rhesus monkey model of collagen-induced arthritis, which has emerged as a reproducible model of human RA in nonhuman primates.