Colocalization of ANCA-antigens and fibrinoid necrosis in ANCA-associated vasculitis

Colocalization of ANCA-antigens and fibrinoid necrosis in ANCA-associated vasculitis. A variety of antineutrophil cytoplasmic auto-antibodies (ANCAs) are known to be associated with small vessel vasculitides such as Wegener's granulomatosis and microscopic polyangiitis. To visualize colocalization patterns of the fibrinoid necrotic lesions and ANCA-antigens more accurately, we have developed a double staining technique in which an immunohistochemical staining is followed by a histological staining. Instead of using sequential biopsy slides of histologically and immunohistochemically stained sections, which may lead to an underestimation of the number and size of the lesions, our technique permits the visualization of the colocalized patterns of fibrinoid necrosis with an ANCA-antigen in a single slide. The double staining procedure is presented in this Technical Note

Btk Is Required for an Efficient Response to Erythropoietin and for SCF-controlled Protection against TRAIL in Erythroid Progenitors

Regulation of survival, expansion, and differentiation of erythroid progenitors requires the well-controlled activity of signaling pathways induced by erythropoietin (Epo) and stem cell factor (SCF). In addition to qualitative regulation of signaling pathways, quantitative control may be essential to control appropriate cell numbers in peripheral blood. We demonstrate that Bruton's tyrosine kinase (Btk) is able to associate with the Epo receptor (EpoR) and Jak2, and is a substrate of Jak2. Deficiency of Btk results in reduced and delayed phosphorylation of the EpoR, Jak2, and downstream signaling molecules such as Stat5 and PLCγ1 as well as in decreased responsiveness to Epo. As a result, expansion of erythroid progenitors lacking Btk is impaired at limiting concentrations of Epo and SCF. In addition, we show that SCF induces Btk to interact with TNF-related apoptosis-inducing ligand (TRAIL)–receptor 1 and that lack of Btk results in increased sensitivity to TRAIL-induced apoptosis. Together, our results indicate that Btk is a novel, quantitative regulator of Epo/SCF-dependent expansion and survival in erythropoiesis

RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells

Tumor escape mechanisms for immunotherapy include deficiencies in antigen presentation, diminishing adaptive CD8+ T cell antitumor activity. Although innate natural killer (NK) cells are triggered by loss of MHC class I, their response is often inadequate. To increase tumor susceptibility to both innate and adaptive immune elimination, we performed parallel genome-wide CRISPR-Cas9 knockout screens under NK and CD8+ T cell pressure. We identify all components, RNF31, RBCK1, and SHARPIN, of the linear ubiquitination chain assembly complex (LUBAC). Genetic and pharmacologic ablation of RNF31, an E3 ubiquitin ligase, strongly sensitizes cancer cells to NK and CD8+ T cell killing. This occurs in a tumor necrosis factor (TNF)-dependent manner, causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I. A small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF and greatly enhances bystander killing of MHC antigen-deficient tumor cells. These results merit exploration of RNF31 inhibition as a clinical pharmacological opportunity for immunotherapy-refractory cancers

Differentiation between chronic rejection and chronic cyclosporine toxicity by analysis of renal cortical mRNA

Differentiation between chronic rejection and chronic cyclosporine toxicity by analysis of renal cortical mRNA.BackgroundIn kidney transplantation, chronic allograft nephropathy (CAN) is the major cause of graft loss. Causes of CAN include chronic rejection and chronic cyclosporine A (CsA) nephrotoxicity. It is necessary to differentiate between these two entities in order to apply the appropriate therapeutic regimen for the individual patient, but this is hampered by the lack of discriminating functional and morphologic parameters. We investigated whether renal cortical mRNA levels for several matrix proteins can serve as discriminating parameters.MethodsPatients with chronic rejection (N = 19) and chronic CsA toxicity (N = 17) were selected by clinical and histologic criteria. Protocol biopsies without histologic abnormalities, taken at 6 months after transplantation from patients receiving CsA, were used as controls (N = 6). Total RNA was extracted from the renal biopsy tissue, and mRNA levels of transforming growth factor-β (TGF-β) and the extracellular matrix (ECM) molecules collagen Iα1, IIIα1, IVα3, decorin, fibronectin, and laminin β2 were measured by real-time polymerase chain reaction (PCR).ResultsIn both patient groups, the mean collagen IVα3 and fibronectin mRNA levels were significantly elevated compared to those in controls, whereas only in CsA toxicity were the laminin β2 and TGF-β mRNA levels significantly increased. The increase of laminin β2 and TGF-β mRNA levels was significantly higher in the CsA toxicity group than in the chronic rejection group (P < 0.001 and P = 0.004, respectively). Receiver-operating characteristic (ROC) curve analysis showed that with a 15.6-fold increase in laminin β2 mRNA expression as cut-off point, the presence of CsA toxicity could be predicted with an 87% sensitivity and an 88% specificity.ConclusionRenal laminin β2 and TGF-β mRNA levels can be used to differentiate between chronic rejection and chronic CsA toxicity in renal transplants. The method of mRNA quantification might be applicable as an additional diagnostic tool in clinical practice

The pathology of fresh and cryopreserved homograft heart valves: An analysis of forty explanted homograft valves

Objective: Tissue degeneration reduces the durability of aortic and pulmonary homograft heart valves. Homograft valves can evoke cellular and humoral immune responses that might be detrimental to the valve tissue. Analyzing explanted homograft valves helps in understanding the different factors that eventually lead to tissue degeneration. Methods: A total of 40 homografts was acquired from patients whose grafts had been explanted because of stenosis (n = 22). insufficiency (n = 8), paravalvular leakage (n = 4), other technical problems (n = 4), noncardiac death (n = 1), and stenosis with endocarditis (n = 1). The period of implantation varied from 14 days to 16 years (median, 4 years). Cryopreserved valves (n = 31) were, in the majority, derived from beating-heart donors, whereas the fresh valves were sterilized with antibiotics and stored at 4°C for an average of 32 days. Four unimplanted cryopreserved valves, 1 native aortic valve, and 1 native pulmonary valve were used as references. Analysis included macroscopy, light microscopy with routine hematoxylin and eosin staining (cellularity and tissue structure), and immunohistochemical studies to allow identification of macrophages (CD68) and T lymphocytes (CD3), endothelial cells, leukocyte adhesion molecules (CD54, CD106, and CD62E), and immunoglobulin (IgG) and complement factor (C3) depositions. In situ hybridization for the Y chromosome was performed in 10 cases, with host-donor sex mismatch, to distinguish between host and donor cells. The outcomes of histology and immunohistochemistry were related to clinical factors, such as implantation time and reason for explantation. Results: In the first year after implantation, a strong reduction in cellularity of the valve tissue was observed, with almost acellular tissues after 1 year. Trilaminar tissue architecture disappeared with the same speed, whereas endothelial cells were almost absent in all explants. Macrophages and T lymphocytes were encountered in 85% and 78% of the leaflets, respectively. Expression of leukocyte adhesion molecules was low in almost all grafts, and IgG and C3 depositions were not increased. Valve tissue cellularity consisted mainly of ingrown host cells when the implantation time exceeded 1 year. Conclusions: During the first year of implantation, homograft valves rapidly lose their cellular components and normal tissue architecture. A low-grade inflammatory response was observed, but no convincing evidence of immune-mediated injury was found

The pathology of fresh and cryopreserved homograft heart valves: An analysis of forty explanted homograft valves

Objective: Tissue degeneration reduces the durability of aortic and pulmonary homograft heart valves. Homograft valves can evoke cellular and humoral immune responses that might be detrimental to the valve tissue. Analyzing explanted homograft valves helps in understanding the different factors that eventually lead to tissue degeneration. Methods: A total of 40 homografts was acquired from patients whose grafts had been explanted because of stenosis (n = 22). insufficiency (n = 8), paravalvular leakage (n = 4), other technical problems (n = 4), noncardiac death (n = 1), and stenosis with endocarditis (n = 1). The period of implantation varied from 14 days to 16 years (median, 4 years). Cryopreserved valves (n = 31) were, in the majority, derived from beating-heart donors, whereas the fresh valves were sterilized with antibiotics and stored at 4°C for an average of 32 days. Four unimplanted cryopreserved valves, 1 native aortic valve, and 1 native pulmonary valve were used as references. Analysis included macroscopy, light microscopy with routine hematoxylin and eosin staining (cellularity and tissue structure), and immunohistochemical studies to allow identification of macrophages (CD68) and T lymphocytes (CD3), endothelial cells, leukocyte adhesion molecules (CD54, CD106, and CD62E), and immunoglobulin (IgG) and complement factor (C3) depositions. In situ hybridization for the Y chromosome was performed in 10 cases, with host-donor sex mismatch, to distinguish between host and donor cells. The outcomes of histology and immunohistochemistry were related to clinical factors, such as implantation time and reason for explantation. Results: In the first year after implantation, a strong reduction in cellularity of the valve tissue was observed, with almost acellular tissues after 1 year. Trilaminar tissue architecture disappeared with the same speed, whereas endothelial cells were almost absent in all explants. Macrophages and T lymphocytes were encountered in 85% and 78% of the leaflets, respectively. Expression of leukocyte adhesion molecules was low in almost all grafts, and IgG and C3 depositions were not increased. Valve tissue cellularity consisted mainly of ingrown host cells when the implantation time exceeded 1 year. Conclusions: During the first year of implantation, homograft valves rapidly lose their cellular components and normal tissue architecture. A low-grade inflammatory response was observed, but no convincing evidence of immune-mediated injury was found