Blocking of interleukin-17 during reactivation of experimental arthritis prevents joint inflammation and bone erosion by decreasing RANKL and interleukin-1

Rheumatoid arthritis is characterized by an intermittent course of disease with alternate periods of remission and relapse. T cells, and in particular the T-cell cytokine interleukin-17 (IL-17), are expected to be involved in arthritic flares. Here, we report that neutralizing endogenous IL-17 during reactivation of antigen-induced arthritis prevents joint inflammation and bone erosion. Synovial IL-17 mRNA expression was clearly up-regulated during primary arthritis and was further enhanced after antigen rechallenge. Neutralization of IL-17 significantly prevented joint swelling at day 1 of flare and significantly suppressed joint inflammation and cartilage proteoglycan depletion at day 4, as assessed by histology. Blocking IL-17 also clearly reduced bone erosions. Cathepsin K, a marker of osteoclast-like activity, and synovial RANKL mRNA expression were both suppressed. The degree of bone erosions strongly correlated with the severity of joint inflammation, suggesting that anti-IL-17 treatment reduced bone erosion by suppressing joint inflammation. Interestingly, blocking IL-17 suppressed synovial expression of both IL-1beta and tumor necrosis factor-alpha, whereas blocking IL-1 did not affect tumor necrosis factor-alpha levels. These data indicate that IL-17 is an important upstream mediator in joint pathology during flare-up of experimental arthritis.     

Acute phase proteins but not activated microglial cells are present in parenchymal deposits in the brains of patients with hereditary cerebral hemorrhage with amyloidosis-Dutch type

With immunohistochemical staining methods on cryostat sections we investigated the brains of three patients with hereditary cerebral hemorrhage with amyloidosis-Dutch type, one of the cerebral amyloid diseases. Immunostaining for protein revealed numerous non-fibrillar depositions (amorphous or diffuse plaques) in the brain parenchyma in addition to extensive vascular amyloid deposition. All amorphous plaques contain complement proteins and α₁-antichymotrypsin but activated microglial cells expressing major histocompatibility (MHC) class II antigens HLA-DR and leucocyte adhesion molecules belonging to the lymphocyte-function-associated antigen (LFA)-1 family are virtually absent in cortical gray matter. Our findings are discussed from the view that a cascade of events including acute phase proteins and activated microglial cells are involved in classical amyloid plaque formation.     

T-cell responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 in Brazilian tuberculosis patients

The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-gamma), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-gamma secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50% of the leprosy patients and 60% of the non-TB controls. Nevertheless, the levels of IFN-gamma in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-gamma in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-gamma production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity.     

Activated microglial cells and complement factors are unrelated to cortical Lewy bodies

Inflammatory mechanisms have been demonstrated in Alzheimer’s disease (AD) but their presence in other neurodegenerative disorders is not well documented. Complement factors and activated microglia have been reported in the substantia nigra of Parkinson’s disease (PD). In the present study we investigated the cingulate gyrus of 25 autopsied patients with clinically and neuropathologically well-documented PD, with or without dementia, for the presence of (activated) microglial cells and their relation with Lewy body (LB)-bearing neurons. In addition, we studied the presence of complement factors in LBs. Of the 25 patient, 15 were clinically demented, fulfilling criteria for dementia with LBs (DLB); 7 also fulfilled CERAD morphological criteria for probable or definite Alzheimer type of dementia. Microglia clustering was seen around congophilic plaques with or without tau pathology. Microglial cells were not associated with LB-bearing neurons or noncongophilic plaques. The cortex of DLB patients without AD plaques did not show more microglial cells than the cortex of non-demented controls. The number of microglia was the lowest in young control patients who died immediately after trauma. Complement factor C3d was occasionally seen in diffusely ubiquinated neurons but late complement factors were not detected in these neurons. Double staining for complement and α-synuclein was negative, suggesting the absence of complement in LBs. In contrast, AD plaques in the same sections showed complement factors C3c, C3d, C1q and C5–9. In conclusion, we have found no evidence that inflammatory mechanism are involved in LB formation in cerebral cortex. Received: 17 June 1999 / Revised: 20 December 1999 / Accepted: 15 February 2000     

In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein.

Multidrug resistance (MDR) remains a major obstacle to successful chemotherapeutic treatment of cancer and can be caused by overexpression of P-glycoprotein, the MDR1 gene product. To further validate a knockdown approach for circumventing MDR, we developed a P-glycoprotein inhibition strategy using short hairpin RNA interference (shRNAi) and now show efficacy and target specificity in vivo. Two of eight tested shRNAi constructs targeted against human MDR1 mRNA inhibited expression of P-glycoprotein by >90%, whereas control shRNAi had no effect. Ablation of P-glycoprotein in cells stably transduced with retroviral-mediated shRNAi was documented by Western blot and functionally confirmed by increased sensitivity of MDR1-transfected cells toward the cytotoxic drugs vincristine, paclitaxel, and doxorubicin as well as by transport of (99m)Tc-Sestamibi. shRNAi-mediated down-regulation of P-glycoprotein transport activity both in cultured cells and in tumor implants in living animals could be followed by direct noninvasive bioluminescence imaging using the Renilla luciferase fluorophore, coelenterazine, a known P-glycoprotein transport substrate. Furthermore, after somatic gene transfer by hydrodynamic infusion of a MDR1-Firefly luciferase (MDR1-FLuc) fusion construct into mouse liver, the effect of shRNAi delivered in vivo on P-glycoprotein-FLuc protein levels was documented with bioluminescence imaging using d-luciferin. ShRNAi against MDR1 reduced bioluminescence output of the P-glycoprotein-FLuc reporter 4-fold in vivo compared with mice treated with control or scrambled shRNAi. Targeted down-regulation of a somatically transferred P-glycoprotein-eGFP fusion reporter also was observed using fluorescence microscopy. Our results show that shRNAi effectively inhibited MDR1 expression and function in cultured cells, tumor implants and mammalian liver, documenting the feasibility of a knockdown approach to reversing MDR in vivo.     

Distribution pattern and functional state of α1-antichymotrypsin in plaques and vascular amyloid in Alzheimer's disease

Summary Monoclonal antibodies (mAbs) were raised against inactivated 1-antichymotrypsin (ACT) to study the presence and functional state of the serine protease inhibitor 1-antichymotrypsin in cerebral amyloid deposits in Alzheimer's disease. A panel of seven different mAbs was obtained; six of them were directed against neoepitopes that are expressed on ACT after interaction with proteases (inactivated ACT) and one mAb was directed against an epitope that is exposed both on native and inactivated ACT. The mAbs against neoepitopes could discriminate native ACT from complexed and inactivated ACT in vitro as shown in binding experiments in the presence of either native or inactivated ACT. With the mAbs against ACT we found that: (a) besides classical congophilic plaques, amorphous noncongophilic /A4-positive plaques were stained; (b) amorphous and classical plaques reacted with both types of mAbs against ACT indicating that this ACT was either complexed to a protease or proteolytically inactivated; (c) vascular amyloid was not stained for ACT. The presence of ACT in amorphous and classical plaques and its absence in vascular amyloid may indicate differences in the proteolytic degradation of preamyloid into amyloid fibrils. Our study strongly suggests that ACT is biologically active in amyloid plaques from an early stage.Supported by a grant of the Prevention Fund (grant no. 28-1945) and was partly supported by the Faculty of Medicine of the Free University, Amsterdam     

Mild neonatal hypoxia–ischemia induces long-term motor- and cognitive impairments in mice

To understand and potentially treat the lifelong cognitive and motor deficits in humans resulting from perinatal mild cerebral hypoxic–ischemic (HI) events, valid animal models are of high importance. Nowadays the murine model of neonatal cerebral HI-injury (unilateral carotid artery occlusion followed by hypoxia) is applied more frequently. In the present study we investigated motor, behavioral and cognitive functioning in mice with mild cerebral HI-injury (45 min of hypoxia; HI-45) in comparison to mice exposed to severe HI (HI-75) and sham-control mice. Lateralizing motor disturbances as measured using the cylinder rearing test developed in both HI-45 and HI-75 mice and was significantly more severe in HI-75 animals. To assess behavior and cognitive functions, we used the modified hole board (mHB) test in two stages. First, the ability of the animals to find the three food rewards in cued holes over time was determined. The results revealed an overall learning impairment in HI-75 mice, while HI-45 mice were not different from sham controls. In the second stage, a reversal test was performed with rewarded cylinders being non-cued and non-rewarded cylinders being cued. This reversal-task revealed impairments in cognitive flexibility in HI-45 mice as compared to sham-control animals. Our data indicate that both the cylinder rearing task and the two stages of the mHB are suitable behavioral approaches to differentiate consequences of neonatal mild and severe brain damage on executive functioning.     

Impact of iron sucrose therapy on leucocyte surface molecules and reactive oxygen species in haemodialysis patients.

BACKGROUND: It has been suggested that iron increases oxidative stress and that an excess of iron contributes to cardiovascular disease and infections in haemodialysis patients. In the present study, the effects of parenterally administered iron on leucocyte surface molecule expression and the production of reactive oxygen species (ROS) were evaluated. METHODS: Ten chronic haemodialysis (HD) patients without iron overload were studied. To each patient, four different regimens were applied: placebo; iron sucrose, either 30 or 100 mg, administered via the outflow dialyser line; and 100 mg of iron sucrose infused via the inflow dialyser line. Blood was sampled at different time points: before, during and after infusion and immediately before the next dialysis session. Levels of CD11b and CD45 expression on granulocytes and of CD11b, CD14 and CD36 on monocytes were determined using flow cytometric analysis. The generation of ROS was quantified using chemiluminescence with and without ex vivo stimulation by phorbol myristate acetate (PMA). RESULTS: No significant differences among the four different treatment regimes were found, neither in chemilumescence activity nor in the expression of CD11b and CD45 on granulocytes, and of CD11b, CD14 and CD36 on monocytes. CONCLUSIONS: Our results suggest that parenteral infusion of iron sucrose during haemodialysis in patients who have no signs of iron overload has no significant effect on the expression of leucocyte surface molecules and does not increase production of ROS.