Differences in inflammatory pain in nNOS-, iNOS- and eNOS-deficient mice.

To assess the relative importance of the isoforms of nitric oxide synthase (NOS) in inflammatory pain, we directly compared pain behaviour and paw thickness after intraplantar injection of complete Freund's adjuvant (CFA) in wild-type (WT) mice and in mice lacking either inducible (iNOS), endothelial (eNOS) or neuronal NOS (nNOS). In mice deficient for nNOS, thermal hyperalgesia was reduced by approximately 50% compared to wild type mice at 4 and 8h after CFA injection, and mechanical hypersensitivity was absent. The only change in pain behaviour in iNOS and eNOS deficient mice compared to WT mice was a more rapid recovery from thermal hyperalgesia. A compensatory up-regulation of nNOS in dorsal root ganglia (DRG) and spinal cords of iNOS and eNOS knockout mice was excluded using RT-PCR. However, an increase of iNOS gene expression was found in spinal cords of eNOS and nNOS deficient mice. To study the downstream effects of nNOS deficiency on DRG neurones, we assessed their immunoreactivity for calcitonin gene-related peptide (CGRP) and cytokines. We found a significant reduction in the CFA induced increase in CGRP immunoreactive neurones as well as in CGRP gene expression in nNOS deficient mice, whereas the percentage of cells immunopositive for tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was unchanged. These results support the proposed role of nNOS in sensitization of DRG neurones, and might indicate that CGRP is involved in this process.     

In vitro induction of cytochrome P4503A1-mRNA and testosterone hydroxylation in precision-cut liver slices from male and female rats

Cytochrome P450 (CYP) 3A is constitutively highly expressed in the liver. Thus detection of induction might be more difficult than shown for scarcely expressed CYP families. In this paper the suitability of rat liver slices to prove CYP3A inducibility was demonstrated. CYP3A dependent basal testosterone hydroxylation (TH) at positions 15β, 6β and 2β was lower in liver slices from female than male rats, but was more markedly induced by 10⁻⁶ M dexamethasone (DEX) within 24 h (mean induction factors 12.5, 18.3 and 140, respectively, for female slices and 3.7, 2.3 and 3.5, respectively, for male slices). Basal expression of CYP3A1-mRNA was stable in vitro until 24 h and did not differ between male and female rats. In liver slices from male rats this mRNA was induced about 14fold by both DEX and pregnenolone 16-carbonitrile (PCN) within 24 h. In one sample of a female rat a similar range of CYP3A1-mRNA induction was reached by DEX.

Altogether, CYP3A induction can be detected more sensitively in liver slices from female than male rats, if TH rates are used as indicators. With liver slices from male rats CYP3A1-mRNA reacts more sensitively to inducers than TH.     

Cleavage of Osteopontin by Matrix Metalloproteinase-12 Modulates Experimental Autoimmune Encephalomyelitis Disease in C57BL/6 Mice

A role for osteopontin (OPN) in promoting disease activity of multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) has recently been suggested. As the biological activity of OPN is heavily influenced by posttranslational processing, we investigated the capacity of matrix metalloproteinase (MMP)-12 to cleave OPN and determined whether this influenced disease activity. We found that OPN mRNA and protein expression in the spinal cord increased with EAE disease in C57BL/6 mice concurrently with MMP-12 expression. A Western blot of EAE and control spinal cords revealed different OPN-immunoreactive bands, with a pattern that was similar to MMP-12 cleavage of recombinant OPN in vitro. In addition, OPN fragments in the spinal cord of EAE-afflicted mice were reduced in MMP-12^−/− mice compared with wild-type controls. However, examination of OPN^−/− mice in short- and long-term experiments revealed no difference in EAE outcomes from wild-type animals. OPN/MMP-12 double null mice were generated, and it was revealed that MMP-12^−/− mice had a worsening of disease compared with wild-type mice, which returned to wild-type levels in the OPN/MMP-12 double null mice. These results suggest that EAE disease activity may be modulated by the cleavage of OPN by MMP-12.Multiple sclerosis (MS) is a disease in which peripheral T cells infiltrate the central nervous system (CNS), where they become re-activated by presentation of CNS antigens by local antigen-presenting cells including microglia and dendritic cells to result in demyelination.^1,2,3,4 In attempts to find putative antigens and mediators of disease, studies using gene array analyses revealed that osteopontin (OPN) was highly up-regulated in lesions from patients with MS compared with control subjects.⁵ This result has prompted the investigations of OPN expression and function in MS and experimental autoimmune encephalomyelitis (EAE).OPN, also known as early T cell activation gene 1 (Eta-1), is a calcium binding phosphorylated acidic glycoprotein.^6,7 OPN has pluripotent activity and is involved in various biological roles such as extracellular matrix remodeling, tumor invasion, angiogenesis, cell-mediated immunity, and the regulation of urokinase and matrix metalloproteinase (MMP) production.⁸ Chabas et al⁵ found that OPN was expressed during EAE in various cell types and that OPN^−/− mice had reduced EAE clinical disease, which was associated with a shift toward a Th2 cytokine profile. Jansson et al⁹ also found that OPN^−/− mice had reduced mean maximal score, fewer days of paralyzing disease, and no spontaneous relapses on proteolipid protein-induced EAE. In that study, OPN^−/− mice showed reduced production of pro-inflammatory cytokines, interferon-γ (IFN-γ), and tumor necrosis factor-α. In contrast to these studies, Blom et al¹⁰ found no difference between OPN^−/− and wild-type mice for EAE outcomes.The EAE results sparked an interest in the expression of OPN in MS. Increased levels of OPN protein is reported in the serum and plasma in patients with relapsing-remitting MS compared with controls,^11,12,13,14 particularly during relapses, and in their cerebrospinal fluid.^15,16 However, there is little evidence for a genetic link between OPN and MS disease susceptibility and disease course.^{11,13,17,18,19,20} Elevated OPN levels have also been documented by immunohistochemistry around MS lesions^21,22 although this was not observed by others.²³ The role that OPN plays in EAE and MS remains uncertain, but a prevailing view is that OPN plays a destructive role during EAE since it reduces apoptosis of effector T cells and because the exogenous administration of recombinant OPN exacerbates EAE clinical disease.²⁴The biological functions of OPN are heavily influenced by posttranslational modifications such as phosphorylation, glycosylation, sulfation, and proteolytic cleavage.^25,26 Currently, there is very little knowledge about the role that proteolytic cleavage of OPN has on MS and EAE. In other disease states, the cleavage of OPN by MMPs and thrombin alters the biological functions of OPN.^27,28 For example, OPN that is cleaved by MMP-3 and MMP-7 significantly increases adhesion of tumor cells compared with full length OPN.²⁹ Recombinant MMP-12 has been demonstrated to cleave OPN, a protein that strongly influences osteoclasts activities, including attachment, spreading, and resorption.³⁰The family of MMPs is implicated in MS and EAE as several MMP members are elevated in biological samples from these conditions. These include MMP-2, -3, -8, -9, -10, -11, -12, -13, -14, and -25.^{31,32,33,34,35,36,37,38} Although the majority of these MMP members appear to have detrimental roles in MS and EAE,^39,40,41 MMP-12 is unique since its absence causes an exacerbation in EAE disease scores that is associated with up-regulation of pro-inflammatory cytokines.^31,42 Since the MMP-12^−/− mice phenotype is opposite to the reduced levels of pro-inflammatory cytokines in OPN^−/− mice with EAE,⁵ we considered the possibility that MMP-12 and OPN are inversely associated. In this article, we have tested the hypothesis that MMP-12 processes OPN to reduce its pro-inflammatory potential in EAE. We describe OPN mRNA and protein expression during EAE and have used a combination of MMP-12 and OPN single and OPN/MMP-12 double null mice to profile their EAE phenotypes.     

Extended runs of homozygosity at 17q11.2: an association with type‐2 NF1 deletions?

Large deletions in the NF1 gene region at 17q11.2 are caused by nonallelic homologous recombination (NAHR). The recurrent type‐2 NF1 deletions span 1.2 Mb, with breakpoints in the SUZ12 gene and SUZ12P. Type‐2 NF1 deletions occur preferentially during mitosis and are associated with somatic mosaicism. A panel of 16 type‐2 NF1 deletions was used as a model system in which to investigate whether extended homozygosity across 17q11.2 might be associated with somatic deletion. Using SNP arrays, a 3.2 Mb interval encompassing the NF1 deletion region was found to harbor runs of homozygosity (ROHs) in different human populations. However, ROHs ≥500 kb directly flanking the NF1 deletion region on both sides were not found to occur disproportionately in NF1 patients harboring type‐2 deletions compared to controls. Although low allelic diversity in 17q11.2 is unlikely to be a key factor in promoting NAHR‐mediated somatic type‐2 deletions, a specific ROH of 588 kb (roh1), located some 525 kb proximal to the deletion interval, was found to occur more frequently (P=0.012) in the type‐2 deletion patients compared with controls. We postulate that roh1 may act remotely, via an as yet unknown mechanism, to increase the frequency of somatic recombination between the distally duplicated SUZ12 sequences. Hum Mutat 30:1–10, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of a 28 kDa lychee allergen as a triose-phosphate isomerase

The aim of this study was to investigate the allergenic potency of the exotic fruit lychee (Litchi chinensis SONN.). For this purpose the lychee protein extract was separated by ion exchange chromatography and a purified allergen with a molecular weight of 28 kDa was identified by N-terminal sequencing and peptide mass fingerprinting. Both methods determined the lychee allergen as a triose-phosphate isomerase. To this protein 67% of patients showed allergic reactions respectively IgE binding of their sera. Similar enzymes from other plants were also recognized as allergens.     

Identification of a peptide mimicking the binding pattern of an antiphospholipid antibody

Our objective was to characterize monoclonal antiphospholipid antibodies (APL) and identify disease-associated antigens in patients with the antiphospholipid syndrome (APS). We used the monoclonal antibody HL-5B, derived from a patient with APS suffering from multiple ischemic events, to screen a 12-mer peptide phage display library (New England Biolabs, London, England). The identified phage clones were sequenced and the derived consensus peptide was synthesized. The peptide was used to perform competitive inhibition experiments for their ability to inhibit the binding of the monoclonal antibody and of serum antibodies to cardiolipin and phosphatidylserine. Additionally patients and control sera were screened for their binding reactivities to this peptide. Using this 12-mer phage display library the peptide APHKHKASLSIY as consensus peptide for the monoclonal antiphospholipid antibody HL-5B could be identified. In competitive inhibition studies we showed that this peptide is able to inhibit the binding of HL-5B to cardiolipin and phosphatidylserine and furthermore another antiphospholipid antibody used as control was also inhibited in its binding to phospholipids. Using 21 sera from APS patients 67% showed a binding to the peptide in a specific ELISA above the cutoff level, generated with sera from 20 healthy controls. Out of the reactive patients' sera we used two exemplarily to perform inhibition studies. Both sera could be inhibited more than 40% in their binding to cardiolipin in a commercially available antiphospholipid antibody assay (Aescu.diagnostics, Wendelsheim, Germany). The identified peptide APHKHKASLSIY simulates the antigenic structure recognized from a subpopulation of serum antiphospholipid antibodies. This might indicate that the diversity of the antiphospholipid antibodies is limited and only few epitopes or few common structures are responsible for the development of those antibodies. Tests using these epitopes will strongly improve laboratory diagnosis of the APS.     

Spontaneous B cell hyperactivity in autoimmune-prone MRL mice

The MRL-lpr/lpr mouse strain is a commonly used model of the human autoimmune disease systemic lupus erythematosus (SLE). Although much is known about the contribution of the lpr Fas mutation to B cell tolerance breakdown, the role of the genetic background of the MRL strain itself is less well explored. In this study, we use the MD4 anti-hen egg lysozyme Ig (IgHEL) transgenic system to explore B cell function in MRL+/+ and non-autoimmune mice. We demonstrate that MRL IgHEL B cells show spontaneous hyperactivity in the absence of self-antigen, which is associated with low total B cell numbers but an expansion of the marginal zone B cell population. However, B cell anergy is normal in the presence of soluble lysozyme [soluble hen egg lysozyme (sHEL)], and MRL IgHEL B cells undergo normal elimination in the presence of sHEL when competing with a polyclonal C57BL/6 B cell repertoire. We conclude that B cell hyperactivity may contribute to the autoimmune phenotype of MRL+/+ and MRL-lpr/lpr strains when it initiates antibody responses to rare or sequestered antigens that are below the threshold for tolerance induction, but that there is no B cell intrinsic defect in anergy in MRL mice.     

Mitotic CDKs control the metaphase-anaphase transition and trigger spindle elongation

Mitotic cyclin-dependent kinases (CDKs) control entry into mitosis, but their role during mitotic progression is less well understood. Here we characterize the functions of CDK activity associated with the mitotic cyclins Clb1, Clb2, and Clb3. We show that Clb-CDKs are important for the activation of the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C)-Cdc20 that triggers the metaphase-anaphase transition. Furthermore, we define an essential role for Clb-CDK activity in anaphase spindle elongation. Thus, mitotic CDKs serve not only to initiate M phase, but are also needed continuously throughout mitosis to trigger key mitotic events such as APC/C activation and anaphase spindle elongation.     

Stereotactic imaging for radiotherapy: accuracy of CT,MRI, PET and SPECT

CT, MRI, PET and SPECT provide complementary information for treatment planning in stereotactic radiotherapy. Stereotactic correlation of these images requires commissioning tests to confirm the localization accuracy of each modality. A phantom was developed to measure the accuracy of stereotactic localization for CT, MRI, PET and SPECT in the head and neck region. To this end. the stereotactically measured coordinates of structures within the phantom were compared with their mechanically defined coordinates. For MRI, PET and SPECT, measurements were performed using two different devices. For MRI, T1- and T2-weighted imaging sequences were applied. For each measurement, the mean radial deviation in space between the stereotactically measured and mechanically defined position of target points was determined. For CT, the mean radial deviation was 0.4 +/- 0.2 mm. For MRI, the mean deviations ranged between 0.7 +/- 0.2 mm and 1.4 +/- 0.5 mm, depending on the MRI device and the imaging sequence. For PET, mean deviations of 1.1 +/- 0.5 mm and 2.4 +/- 0.3 mm were obtained. The mean deviations for SPECT were 1.6 +/- 0.5 mm and 2.0 +/- 0.6 mm. The phantom is well suited to determine the accuracy of stereotactic localization with CT, MRI, PET and SPECT in the head and neck region. The obtained accuracy is well below the physical resolution for CT, PET and SPECT, and of comparable magnitude for MRI. Since the localization accuracy may be device dependent, results obtained at one device cannot be generalized to others.