CD45 Isoform Expression in Microglia and Inflammatory Cells in HIV-1 Encephalitis

CD45 is a membrane tyrosine phosphatase that modulates the function of the hematopoietic cells. In vitro, agonist antibodies to CD45RO or CD45RB isoforms have been shown to suppress microglial activation, but whether microglia in vivo express these isoforms in HIV encephalitis (HIVE) is unknown. Brain sections from control and HIVE were immunostained for CD45 isoforms using exon-specific antibodies (RA, RB, RC and RO). RA and RC were limited to rare lymphocytes, while RB expression was robust in microglia and inflammatory cells. RO was low in control microglia, but increased in HIVE. RO was also localized to macrophages and CD8+ T cells. Targeting CD45 in vivo with isoform-specific antibodies remains a therapeutic option for neuroinflammatory diseases

CC chemokine receptor 2 is relevant for CD8-induced graft-versus- host disease but not for graft-versus-tumor activity

I Summary Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapy for a variety of malignant and non-malignant disorders of the hematopoietic system and for certain solid tumors. One of the major complications limiting the success and wider application of allogeneic HSCT is the occurrence of acute graft-versus-host disease (GVHD), which is a rapidly progressive illness with epithelial damage of gut, liver, skin, and lung, immunosuppression and cachexia. GVHD is mediated by alloreactive donor T cells contained in the graft and can be prevented by depletion of these T cells prior to transfer. However, alloreactive donor T cells also mediate the so-called graft-versus-tumor (GVT) effect, which is increasingly being recognized as an important component of the overall anti-tumor effect of an allogeneic HSCT. Therefore, a major focus of current research is to ameliorate GVHD without reducing GVT activity. Recent murine bone marrow transplantation studies suggest that interfering with T cell migration represents an attractive therapeutic approach towards this goal. In the present study, the role of the inflammatory chemokine receptor CCR2 for donor CD8 + T cell migration during GVHD was analyzed in well-established murine bone marrow transplantation models. It was found that recipients of CCR2-deficient (CCR2 -/-) CD8 + T cells develop significantly less GVHD morbidity and mortality than recipients of wild type CD8 + T cells and that this correlates with reduced target organ damage to the gut and liver. A competitive in vivo migration assay revealed that CCR2 -/-CD8 + T cells have an intrinsic migratory defect to the gut and liver, which was previously unknown. Other causes for the reduction in GVHD could be excluded, as alloreactive proliferation, activation, IFN-γ production and in vitro cytotoxicity of CCR2 -/-CD8 + T cells were intact. Importantly, the GVT effect of CCR2 -/-CD8 + T cells against murine P815 mastocytoma and A20 B cell lymphoma was preserved, which demonstrates that interference with T cell migration by blockade of CCR2 signaling can separate GVHD from GVT activity. These data provide first evidence for a critical role of CCR2 for the control of CD8 + T cell migration in a pre-clinical disease model and establish the rationale for the use of CCR2 antagonists possibly in combination with other chemokine receptor antagonists as novel therapeutic tools in GVHD

Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age

Cadherin-9 Is a Novel Cell Surface Marker for the Heterogeneous Pool of Renal Fibroblasts

BACKGROUND: Interstitial fibroblasts are a minor, but nevertheless very important, component of the kidney. They secrete and remodel extracellular matrix and they produce active compounds such as erythropoietin. However, studying human renal fibroblasts has been hampered by the lack of appropriate surface markers. METHODS AND FINDINGS: The expression of cadherin-9 in various human renal cell lines and tissues was studied on the mRNA level by RT-PCR and on the protein level with the help of newly generated cadherin-9 antibodies. The classical type II cadherin-9, so far only described in the neural system, was identified as a reliable surface marker for renal fibroblasts. Compared to FSP1, a widely-used cytosolic renal fibroblast marker, cadherin-9 showed a more restricted expression pattern in human kidney. Under pathological conditions, cadherin-9 was expressed in the stroma of renal cell carcinoma, but not in the tumor cells themselves, and in renal fibrosis the percentage of cadherin-9-positive cells was clearly elevated 3 to 5 times compared to healthy kidney tissue. Induction of epithelial mesenchymal transition in renal epithelial cells with cyclosporin-A, which causes renal fibrosis as a side effect, induced cadherin-9 expression. Functional studies following siRNA-mediated knockdown of cadherin-9 revealed that it acts in the kidney like a typical classical cadherin. It was found to be associated with catenins and to mediate homophilic but not heterophilic cell interactions. CONCLUSIONS: Cadherin-9 represents a novel and reliable cell surface marker for fibroblasts in healthy and diseased kidneys. Together with the established marker molecules FSP1, CD45 and alpha smooth muscle actin, cadherin-9 can now be used to differentiate the heterogenic pool of renal fibroblasts into resident and activated fibroblasts, immigrated bone marrow derived fibroblast precursors and cells in different stages of epithelial mesenchymal transition

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs

We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action

Catalytic cleavage of HEAT and subsequent covalent binding of the tetralone moiety by the SARS-CoV-2 main protease

Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

The human cytomegalovirus ul11 protein interacts with the receptor tyrosine phosphatase cd45, resulting in functional paralysis of t cells

Human cytomegalovirus (CMV) exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR) is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV

X ray screening identifies active site and allosteric inhibitors of SARS CoV 2 main protease

The coronavirus disease COVID 19 caused by SARS CoV 2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID 19, we have performed a high throughput x ray crystallographic screen of two repurposing drug libraries against the SARS CoV 2 main protease Mpro , which is essential for viral replication. In contrast to commonly applied x ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS CoV