CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis

Background Wegener's Granulomatosis and Microscopic Polyangiitis are life-threatening systemic necrotizing vasculitides of unknown aetiology. The appearance of circulating antibodies to neutrophil cytoplasmic antigens (ANCA) is strongly associated with the development of the disease. A link between infection and disease has long been suspected, and the appearance of ANCA antibodies has been reported following bacterial and viral infections. The depletion of circulating B cells with monoclonal antibody therapy can induce remission, and this observation suggests a pathogenic role for B cells in this disease. As bacterial DNA is known to induce B cell proliferation and antibody production via TLR-9 stimulation, we have explored the possibility that unmethylated CpG oligodeoxynucleotide, as found in bacterial and viral DNA, may play a role in stimulating circulating autoreactive B cells to produce ANCA in patients with vasculitis. Results We have confirmed that unmethylated CpG oligonucleotide is a potent stimulator of antibody production by PBMC in vitro. The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls. In spite of this, PR3 ANCA+ patients synthesised significantly higher amount of IgG ANCA than normal controls. In MPO ANCA+ patients, there was a tendency for patients to produce higher amount of ANCA than controls, however, the difference did not reach significance. Furthermore, we were able to detect circulating MPO-reactive B cells by ELISpot assay from the peripheral blood of 2 MPO+ ANCA vasculitis patients. Together, this indicates that circulating anti-neutrophil autoreactive B cells are present in ANCA+ vasculitis patients, and they are capable of producing antibodies in response to CpG stimulation. Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases. Conclusion Circulating ANCA autoreactive B cells are present in patients with ANCA+ vasculitis. The production of ANCA from these cells in response to unmethylated CpG stimulation lead us to propose that stimulation of these cells by immunostimulatory DNA sequences such as CpG oligodeoxynucleotide during infection may provide a link between infection and ANCA associated vasculitis. This phenomenon may also apply to other antibody mediated autoimmune diseases.Plinio R Hurtado, Lisa Jeffs, Jodie Nitschke, Mittal Patel, Ghafar Sarvestani, John Cassidy, Pravin Hissaria, David Gillis and Chen Au Pe

GDNF gene delivery via the the P75NTR receptor rescues injured motor neurons

The retrograde axonal transport mechanism of motor neurons has been exploited to deliver the gene encoding Glial cell line-derived neurotrophic factor (GDNF) into the central nervous system to provide trophic support following injury. A nonviral gene delivery system, consisting of a monoclonal antibody (MC192) that binds the neurotrophic receptor, p75NTR, coupled to poly-L-lysine, was constructed and used to deliver the gene via a receptor-mediated mechanism. The MC192-poly-l-lysine/pGDNF complex was injected into the hind limb of newborn rats to allow gene expression within motor neurons prior to sciatic nerve transection. In adult rats, the gene delivery complex was administrated in gel foam placed on a transected hypoglossal nerve. We show that the delivered construct is internalized following binding to p75NTR and is transported into the brain and spinal cord, bypassing the blood-brain barrier. The presence of the GDNF transgene and its transcript could be detected for up to 8 weeks in spinal cord and brain stem. Expression of the GDNF protein rescued 38% of the targeted motor neurons 1 week postinjury in newborn rats while the survival rate in control group was below 12%. In adult rats, neuronal death induced by axotomy was almost completely reversed by the introduction of the transgene (95 ᠳ%). Thus, the significant functional outcomes of this novel gene delivery system are demonstrated both in postnatal and adult motor neurons.No Full Tex

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC–derived EVs are a heterogeneous population, ranging in size from that of micro–vesicles (150 nm–1 μm) down to that of exosomes (60–150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60–150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL–6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)–derived EVs previously described, suppressing T cell response to activation. The finding that USC–derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer

CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis-5

D mitogen (PWM) + IL2, or inactivated + IL2. A representative set of results is shown from a PR3ANCA patient (and a MPOANCA patient ().<p><b>Copyright information:</b></p><p>Taken from "CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis"</p><p>http://www.biomedcentral.com/1471-2172/9/34</p><p>BMC Immunology 2008;9():34-34.</p><p>Published online 14 Jul 2008</p><p>PMCID:PMC2483256.</p><p></p

CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis-4

G-B and IL-2. Each patient assay was paired with a healthy control. After 12 days of culture, supernatants were harvested. IgG concentration and supernatant reactivity to either PR3 or MPO was measured by ELISA. The amount of IgG detected in the supernatants was of 5.5 ± 2.2 μg mLin the patients compared to 4.1 ± 1.2 μg mLin the control group (. Figure shows the reactivity of the supernatants from PR3ANCA patients towards PR3 antigen. The difference against control individuals was highly significant (P = 0.0082). Figure shows the reactivity of the supernatants from MPOANCA patients towards MPO antigen. The difference was not significant (P = 0.072) although their supernatants showed a clear tendency towards higher reactivity compared to controls. There was no correlation between patients' serum ANCA titre at the time of the assay and their production of ANCA in response to CpG-B as shown in (r= 0.172).<p><b>Copyright information:</b></p><p>Taken from "CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis"</p><p>http://www.biomedcentral.com/1471-2172/9/34</p><p>BMC Immunology 2008;9():34-34.</p><p>Published online 14 Jul 2008</p><p>PMCID:PMC2483256.</p><p></p

CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis-1

D mitogen (PWM) + IL2, or inactivated + IL2. A representative set of results is shown from a PR3ANCA patient (and a MPOANCA patient ().<p><b>Copyright information:</b></p><p>Taken from "CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis"</p><p>http://www.biomedcentral.com/1471-2172/9/34</p><p>BMC Immunology 2008;9():34-34.</p><p>Published online 14 Jul 2008</p><p>PMCID:PMC2483256.</p><p></p

CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis-0

G-B and IL-2. Each patient assay was paired with a healthy control. After 12 days of culture, supernatants were harvested. IgG concentration and supernatant reactivity to either PR3 or MPO was measured by ELISA. The amount of IgG detected in the supernatants was of 5.5 ± 2.2 μg mLin the patients compared to 4.1 ± 1.2 μg mLin the control group (. Figure shows the reactivity of the supernatants from PR3ANCA patients towards PR3 antigen. The difference against control individuals was highly significant (P = 0.0082). Figure shows the reactivity of the supernatants from MPOANCA patients towards MPO antigen. The difference was not significant (P = 0.072) although their supernatants showed a clear tendency towards higher reactivity compared to controls. There was no correlation between patients' serum ANCA titre at the time of the assay and their production of ANCA in response to CpG-B as shown in (r= 0.172).<p><b>Copyright information:</b></p><p>Taken from "CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis"</p><p>http://www.biomedcentral.com/1471-2172/9/34</p><p>BMC Immunology 2008;9():34-34.</p><p>Published online 14 Jul 2008</p><p>PMCID:PMC2483256.</p><p></p