Transient CDK4/6 inhibition protects hematopoietic stem cells from chemotherapy-induced exhaustion

Conventional cytotoxic chemotherapy is highly effective in certain cancers, but causes dose-limiting damage to normal proliferating cells, especially hematopoietic stem and progenitor cells (HSPCs). Serial exposure to cytotoxics causes a long-term hematopoietic compromise (‘exhaustion’), which limits the use of chemotherapy and success of cancer therapy. Here, we show that the co-administration of G1T28 (trilaciclib), a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), contemporaneously with cytotoxic chemotherapy protects murine hematopoietic stem cells (HSCs) from chemotherapy-induced exhaustion in a serial 5-fluorouracil (5FU) treatment model. Consistent with a cell intrinsic effect, we show directly preserved HSC function resulting in a more rapid recovery of peripheral blood counts, enhanced serial transplantation capacity and reduced myeloid skewing. When administered to healthy human volunteers, G1T28 demonstrated excellent in vivo pharmacology and transiently inhibited bone marrow (BM) HSPC proliferation. These findings suggest that the combination of CDK4/6 inhibitors (CDK4/6i) with cytotoxic chemotherapy should provide a means to attenuate therapy-induced BM exhaustion in patients with cancer

Reactivity against Complementary Proteinase-3 Is Not Increased in Patients with PR3-ANCA-Associated Vasculitis

The etiology of anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides (AAV) is unknown, but the association between infections and autoimmunity has been studied extensively. In 2004, a novel theory was proposed that could link infection and autoimmunity. This ‘theory of autoantigen complementarity’ was based on the serendipitous finding of antibodies against complementary-PR3 (cPR3) in patients with PR3-ANCA-associated vasculitis. cPR3 demonstrated homology to several bacterial proteins, and it was hypothesized that PR3-ANCA develop in response to anti-cPR3 antibodies, as a consequence of the anti-idiotypic network. These data have not been confirmed in other patient cohorts. We investigated the presence of anti-cPR3 antibodies in a Dutch cohort of PR3-ANCA-associated vasculitis patients. Anti-cPR3 reactivity was determined in serum using ELISA. Two separate batches of cPR3 were used to determine reactivity in two separate cohorts of PR3-ANCA-associated vasculitis patients. We found that anti-cPR3-reactivity was not increased in our PR3-ANCA-associated vasculitis patients, in comparison to control groups. Further research will be necessary to prove the concept of autoantigen complementarity in autoimmune diseases

Increased Expression of Toll-Like Receptors by Monocytes and Natural Killer Cells in ANCA-Associated Vasculitis

INTRODUCTION: Toll-like receptors (TLRs) are a family of receptors that sense pathogen associated patterns such as bacterial cell wall proteins. Bacterial infections are associated with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Here, we assessed the expression of TLRs 2, 4, and 9 by peripheral blood leukocytes from patients with AAV, and investigated TLR mediated responses ex vivo. METHODS: Expression of TLRs was determined in 38 AAV patients (32 remission, 6 active disease), and 20 healthy controls (HC). Membrane expression of TLRs 2, 4, and 9, and intracellular expression of TLR9 by B lymphocytes, T lymphocytes, NK cells, monocytes and granulocytes was assessed using 9-color flowcytometry. Whole blood from 13 patients and 7 HC was stimulated ex vivo with TLR 2, 4 and 9 ligands and production of cytokines was analyzed. RESULTS: In patients, we observed increased proportions of TLR expressing NK cells. Furthermore, patient monocytes expressed higher levels of TLR2 compared to HC, and in a subset of patients an increased proportion of TLR4(+) monocytes was observed. Monocytes from nasal carriers of Staphylococcus aureus expressed increased levels of intracellular TLR9. Membrane expression of TLRs by B lymphocytes, T lymphocytes, and granulocytes was comparable between AAV patients and HC. Patients with active disease did not show differential TLR expression compared to patients in remission. Ex vivo responses to TLR ligands did not differ significantly between patients and HC. CONCLUSIONS: In AAV, monocytes and NK cells display increased TLR expression. Increased TLR expression by these leukocytes, probably resulting from increased activation, could play a role in disease (re)activation

Bacterial infection and ANCA-associated vasculitis - connecting mechanisms

ABSTRACT: ANCA-associated vasculitides are a group of chronic, autoimmune mediated inflammatory disorders characterised by the presence of anti-neutrophil cyoplasmic autoantibodies (ANCA). When untreated, the disease progresses fast and will cause severe damage to the airways and kidneys. Although the disease mechanisms are increasingly being understood, it is still unclear why certain people develop autoimmunity. Likely a certain genetic background is involved. Also there are indications that bacterial infection could play a role. The focus of this thesis was on mechanisms involved. First, it was investigated whether or not antibodies against complementary Proteinase-3 are present in patients with PR3-ANCA associated vasculitis. Furthermore, the possible role of Toll-like receptors was investigated. Toll-like receptors are important detectors of bacterial proteins and DNA, and we studied the expression of these receptors in white blood cells from patients with ANCA-associated vasculitis. In addition, the capacity of bacterial proteins to stimulate the production of ANCA was studied. KORTE SAMENVATTING: ANCA-geassocieerde vasculitis (AAV) is een zeldzame auto-immuunziekte, waarbij de kleinste bloedvaten ontstoken raken. De ontsteking is het gevolg van een ontsporing van het immuunsysteem, dat een klein deel van het eigen lichaam als ‘vreemd’ beschouwt en hiertegen in actie komt. Als het immuunsysteem niet met medicijnen wordt onderdrukt, kan ernstige schade aan nieren en longen optreden. Promovendus Henko Tadema ontdekte dat de productie van de antistoffen die zich tegen het eigen lichaam richten, in het laboratorium kan worden gestimuleerd met CpG-DNA. In een levend organisme lijkt hetzelfde mogelijk met bacterieel DNA. Ook ontdekte Tadema dat bij AAV-patiënten op de celwand van monocyten en NK-cellen (beide witte bloedcellen) meer Toll-like receptoren actief worden. Deze receptoren zijn gespecialiseerd in het herkennen van bacteriële structuren. Ook vond hij dat niet bij alle patiënten met PR3-AAV antistoffen tegen complementair PR3 voorkomen. Dit weerspreekt eerder onderzoek, dat suggereerde dat AAV wordt veroorzaakt door een infectie. Deze en andere conclusies dragen bij aan het begrip van de mechanismen die een rol spelen bij het ontstaan van AAV als gevolg van bacteriële infectie. Er werden echter geen bevindingen gedaan die op korte termijn zullen leiden tot bijvoorbeeld de ontwikkeling van een medicijn.

Vasculitis and infections: Contribution to the issue of autoimmunity reviews devoted to "autoimmunity and infection"

Infections are associated with secondary forms of vasculitis. However, there is increasing evidence that microbial agents play a role also in primary systemic vasculitides. For a long time it has been noted that Hepatitis B virus (HBV) is involved in polyarteritis nodosa (PAN) although the incidence of HBV-associated PAN seems to decline. Cryoglobulinemic vasculitis has been shown to be strongly associated with Hepatitis C Virus (HCV) infection, but this is most striking in Southern Europe and less in Northern Europe. Different microbial agents have been suggested to influence disease expression in other primary vasculitides but no specific association has been established. In Wegener's Granulomatosis (WG) chronic carriage of Staphylococcus aureus (S. aureus) is associated with a strongly increased risk for relapsing disease. Various pathogenic pathways for this association have been suggested by clinical and experimental observations. Recent studies even suggest that S. aureus derived peptides, amongst others, may induce proteinase 3-ANCA via idiotypic-anti-idiotypic interactions. Treatment with co-trimoxazole in WG localized to the upper airways may result in (temporary) remission of the disease. (C) 2008 Elsevier B.V. All rights reserved

Vasculitis and infections:Contribution to the issue of autoimmunity reviews devoted to "autoimmunity and infection"

Infections are associated with secondary forms of vasculitis. However, there is increasing evidence that microbial agents play a role also in primary systemic vasculitides. For a long time it has been noted that Hepatitis B virus (HBV) is involved in polyarteritis nodosa (PAN) although the incidence of HBV-associated PAN seems to decline. Cryoglobulinemic vasculitis has been shown to be strongly associated with Hepatitis C Virus (HCV) infection, but this is most striking in Southern Europe and less in Northern Europe. Different microbial agents have been suggested to influence disease expression in other primary vasculitides but no specific association has been established. In Wegener's Granulomatosis (WG) chronic carriage of Staphylococcus aureus (S. aureus) is associated with a strongly increased risk for relapsing disease. Various pathogenic pathways for this association have been suggested by clinical and experimental observations. Recent studies even suggest that S. aureus derived peptides, amongst others, may induce proteinase 3-ANCA via idiotypic-anti-idiotypic interactions. Treatment with co-trimoxazole in WG localized to the upper airways may result in (temporary) remission of the disease. (C) 2008 Elsevier B.V. All rights reserved

Bacterial infections in Wegener's granulomatosis:mechanisms potentially involved in autoimmune pathogenesis

Purpose of review Wegener's granulomatosis is associated with bacterial infection, in particular nasal carriage of Staphylococcus aureus. Infection may play a role in the induction of autoimmunity as well as in the effector phase of the disease. Here, the current hypotheses aiming to explain the link between infections and Wegener's granulomatosis immunopathogenesis are reviewed and discussed. Recent findings In recent years, studies suggested that molecular mimicry could play a role in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV), either via direct mimicry between human lysosome-associated membrane protein-2 and bacterial FimH or indirectly via the development of antibodies against a peptide complementary to proteinase 3 (cPr3). More recent work has focused on Toll-like receptors (TLRs), a family of receptors specialized in the recognition of pathogen-associated molecular patterns. In animal models, it has been shown that TLR ligands can aggravate anti-MPO antibody-mediated disease. Furthermore, it was shown that a TLR9 ligand can trigger the production of ANCA in vitro by peripheral blood-derived B lymphocytes from AAV patients. The newly described process of ANCA-mediated neutrophil extracellular trap formation may provide an endogenous TLR9 ligand. Finally, TLR2 signaling is involved in the development of a Th17-driven immune response, consistent with skewing towards a Th17 T cell phenotype that has been observed in Wegener's granulomatosis. Summary Although Wegener's granulomatosis pathophysiology is becoming better understood, the specific events leading to autoimmunity are not clear. Recent studies show that several mechanisms may be involved in linking infection to autoimmunity. Molecular mimicry may be involved, and a role for TLR signaling is suggested

Anti-cPR3m reactivity in serum of AAV patients and healthy controls.

<p>A) Anti-cPR3 reactivity in ANCA-associated vasculitis patients and controls, determined by ELISA using in-house produced cPR3m. Anti-cPR3m-reactivity in PR3-ANCA positive patient sera was significantly lower than reactivity in healthy controls (HC) (<i>P</i> = 0.04) and MPO-ANCA patients (<i>P</i> = 0.01). Anti-cPR3m reactivity in MPO-ANCA positive patients and HC was comparable. In neither PR3-ANCA nor MPO-ANCA positive patients, significant differences were found between samples at the time of disease diagnosis (closed circles) or during follow-up (open circles). B) Anti-cPR3 reactivity in PR3-ANCA positive Wegener's granulomatosis patients and HC, determined by ELISA using cPR3 provided by Dr. Preston. Anti-cPR3m reactivity was decreased in patients, compared to HC (<i>P</i> = 0.004). Reactivity in patients at the time of diagnosis (closed circles) did not differ from reactivity in patients in remission.</p