HistoNet: A Deep Learning-Based Model of Normal Histology.

We introduce HistoNet, a deep neural network trained on normal tissue. On 1690 slides with rat tissue samples from 6 preclinical toxicology studies, tissue regions were outlined and annotated by pathologists into 46 different tissue classes. From these annotated regions, we sampled small 224 × 224 pixels images (patches) at 6 different levels of magnification. Using 4 studies as training set and 2 studies as test set, we trained VGG-16, ResNet-50, and Inception-v3 networks separately at each magnification level. Among these model architectures, Inception-v3 and ResNet-50 outperformed VGG-16. Inception-v3 identified the tissue from query images, with an accuracy up to 83.4%. Most misclassifications occurred between histologically similar tissues. Investigation of the features learned by the model (embedding layer) using Uniform Manifold Approximation and Projection revealed not only coherent clusters associated with the individual tissues but also subclusters corresponding to histologically meaningful structures that had not been annotated or trained for. This suggests that the histological representation learned by HistoNet could be useful as the basis of other machine learning algorithms and data mining. Finally, we found that models trained on rat tissues can be used on non-human primate and minipig tissues with minimal retraining.

Classic and evolving approaches to evaluating cross reactivity of mAb and mAb-like molecules - A survey of industry 2008-2019.

Monoclonal antibodies (mAbs) and mAb derivatives have become mainstay pharmaceutical modalites. A critical assessment is to ascertain the specificity of these molecules prior to human clinical trials. The primary technique for determining specificity has been the immunohistochemistry (IHC)-based "Tissue Cross-Reactivity" (TCR) assay, where the candidate molecule is applied to > 30 tissues to look for unexpected staining. In the last few years, however, non-IHC array-based platforms have emerged that allow for screening 75-80% of the human membrane proteome, indicating a viable alternative and/or addition to the IHC methods. The preclinical sciences subcommittee of the Biotechnology Innovation Organization (BIO), "BioSafe", conducted a survey of 26 BIO member companies to understand current sponsor experience with the IHC and array techniques. In the last ten years, respondents noted they have conducted more than 650 IHC TCR assays, largely on full length mAbs, with varying impacts on programs. Protein/cell arrays have been utilized by almost half of the companies and sponsors are gaining familiarity and comfort with the platform. Initial experience with recent versions of these arrays has been largely positive. While most sponsors are not prepared to eliminate the IHC TCR assay, growing experience with these alternatives allows them to confidently choose other approaches with or without TCR assays.

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf-/- mouse model.

Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf-/- mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.

Colony-Stimulating Factor-1 Antibody Lacnotuzumab in a Phase 1 Healthy Volunteer Study and Mechanistic Investigation of Safety Outcomes.

The colony-stimulating factor-1 (CSF-1) receptor pathway has been implicated in a variety of diseases, and CSF-1-dependent mechanisms are also involved in bloodborne protein clearance. Lacnotuzumab is a novel, high-affinity, humanized, anti-CSF-1 monoclonal antibody that prevents CSF-1-mediated receptor activation. This phase 1, two-part, double-blind study in healthy volunteers assessed the safety and tolerability of lacnotuzumab and its pharmacokinetics (PK) and pharmacodynamic properties. Part A (n = 36) was a single, ascending-dose assessment of eight lacnotuzumab doses (0.01-20 mg/kg); in part B (n = 16), lacnotuzumab was administered at either 5 or 10 mg/kg. In each study cohort, individuals were randomized 3:1 to lacnotuzumab or placebo. Lacnotuzumab was generally well tolerated. At higher doses (10 and 20 mg/kg), creatine kinase (CK) elevations (>5× the upper limit of normal, but asymptomatic and reversible) and mild transient periorbital swelling were reported. Most adverse events (AEs) were low-grade, no unexpected or novel AEs were observed, and there were no discontinuations for AEs. Free, unbound lacnotuzumab serum concentration-time profiles showed nonlinear PK across doses from 0.01 to 20 mg/kg, with faster apparent elimination at lower doses or concentrations; this finding was consistent with apparent target-mediated drug disposition. Lacnotuzumab also showed dose-dependent, on-target effects on multiple downstream biomarkers. Preclinical investigations of the CK elevation and periorbital swelling observed after lacnotuzumab administration suggest that these are reversible, nonpathological events linked to inhibition of the CSF-1 pathway. These data support further evaluation of lacnotuzumab in clinical studies.

Reduced Activity of Sphingosine-1-Phosphate Lyase Induces Podocyte-related Glomerular Proteinuria, Skin Irritation, and Platelet Activation.

Sphingosine-1-phosphate (S1P) lyase is considered as a drug target in autoimmune diseases based on the protective effect of reducing activity of the enzyme in animal models of inflammation. Since S1P lyase deficiency in mice causes a severe, lethal phenotype, it was of interest to investigate any pathological alterations associated with only partially reduced activity of S1P lyase as may be encountered upon pharmacological inhibition. Both genetic reduction of S1P lyase activity in mice and inhibition of S1P lyase with a low-molecular-weight compound in rats consistently resulted in podocyte-based kidney toxicity, which is the most severe finding. In addition, skin irritation and platelet activation were observed in both instances. The similarity of the findings in both the genetic model and the pharmacological study supports the value of analyzing inducible partially target-deficient mice for safety assessment. If the findings described in rodents translate to humans, target-related toxicity, particularly podocyte dysfunction, may limit chronic systemic treatment of autoimmune diseases with S1P lyase inhibitors. Furthermore, partial deficiency or inhibition of S1P lyase appears to provide an in vivo rodent model to enable studies on the mechanism of podocyte dysfunction.

Shear stress modulates the expression of the atheroprotective protein Cx37 in endothelial cells.

High laminar shear stress (HLSS) is vasculoprotective partly through induction of Kruppel-like factor 2 (KLF2). Connexin37 (Cx37) is highly expressed in endothelial cells (ECs) of healthy arteries, but not in ECs overlying atherosclerotic lesions. Moreover, Cx37 deletion in apolipoprotein E-deficient (ApoE(-/-)) mice increases susceptibility to atherosclerosis. We hypothesized that shear stress, through KLF2 modulation, may affect Cx37 expression in ECs. Cx37 expression and gap-junctional intercellular (GJIC) dye transfer are prominent in the straight portion of carotid arteries of ApoE(-/-) mice, but are reduced at the carotid bifurcation, a region subjected to oscillatory flow. Shear stress-modifying vascular casts were placed around the common carotid artery of ApoE(-/-) mice. Whereas Cx37 expression was conserved in HLSS regions, it was downregulated to ~50% in low laminar or oscillatory flow regions. To study the mechanisms involved, HUVECs or bEnd.3 cells were exposed to flow in vitro. Cx37 and KLF2 expression were increased after 24h of HLSS. Interestingly, shear-dependent Cx37 expression was significantly reduced after silencing of KLF2. Moreover after exposure to simvastatin, a well-known KLF2 inducer, KLF2 binds to the Cx37 promoter region as shown by ChIP. Finally, GJIC dye transfer was highly reduced after KLF2 silencing and was increased after exposure to simvastatin. HLSS upregulates the expression of Cx37 in ECs by inducing its transcription factor KLF2, which increases intercellular communication. Therefore, this effect of shear stress on Cx37 expression may contribute to the synchronization of ECs and participate in the protective effect of HLSS.

Connexin 37 limits thrombus propensity by downregulating platelet reactivity.

BACKGROUND: Formation of platelet plug initiates hemostasis after vascular injury and triggers thrombosis in ischemic disease. However, the mechanisms leading to the formation of a stable thrombus are poorly understood. Connexins comprise a family of proteins that form gap junctions enabling intercellular coordination of tissue activity, a process termed gap junctional intercellular communication. METHODS AND RESULTS: In the present study, we show that megakaryocytes and platelets express connexin 37 (Cx37). Deletion of the Cx37 gene in mice shortens bleeding time and increases thrombus propensity. Aggregation is increased in murine Cx37(-/-) platelets or in murine Cx37(+/+) and human platelets treated with gap junction blockers. Intracellular microinjection of neurobiotin, a Cx37-permeant tracer, revealed gap junctional intercellular communication in platelet aggregates, which was impaired in Cx37(-/-) platelets and in human platelets exposed to gap junction blockers. Finally, healthy subjects homozygous for Cx37-1019C, a prognostic marker for atherosclerosis, display increased platelet responses compared with subjects carrying the Cx37-1019T allele. Expression of these polymorphic channels in communication-deficient cells revealed a decreased permeability of Cx37-1019C channels for neurobiotin. CONCLUSIONS: We propose that the establishment of gap junctional communication between Cx37-expressing platelets provides a mechanism to limit thrombus propensity. To our knowledge, these data provide the first evidence incriminating gap junctions in the pathogenesis of thrombosis.

Effect of ofatumumab on pregnancy, parturition, and lactation in cynomolgus monkeys.

Knowledge of the impacts of the anti-CD20 monoclonal antibody ofatumumab on the developing immune system is limited. This study examined the effects of intravenous ofatumumab on pregnancy, parturition, and lactation, and on pre- and postnatal survival and development in cynomolgus monkeys, an established model for developmental toxicity assessment. Pregnant cynomolgus monkeys (n = 42) were randomized to receive vehicle only (control group; n = 14), low-dose ofatumumab (n = 14), or high-dose ofatumumab (n = 14). Survival, clinical outcomes, and clinical pathology investigations were evaluated regularly until lactation day (maternal animals) and postnatal day 180±1 (infants). Anatomic pathology was investigated in euthanized infants and unscheduled terminations of maternal animals and infants. Ofatumumab treatment was not associated with maternal toxicity or embryotoxicity and had no effect on the growth and development of offspring. As expected, B-cell depletion occurred in maternal animals and their offspring, with a reduced humoral immune response in infants of mothers on high-dose ofatumumab. Both effects were reversible. In the high-dose group, perinatal deaths of 3 infants were attributed to infections, potentially secondary to pharmacologically induced immunosuppression. The no-observed adverse-effect level for initial/maintenance ofatumumab doses was 100/20 mg, and 10/3 mg/kg for pharmacological effects in infant animals, which are associated with exposures significantly higher than those following therapeutic doses in humans. In this study with cynomolgus monkeys, ofatumumab treatment was not associated with maternal toxicity or embryotoxicity and had no effect on the growth and development of offspring.

Unexpected role for the human Cx37 C1019T polymorphism in tumour cell proliferation.

Connexins are a large family of proteins that form gap junction channels allowing exchange of ions and small metabolites between neighboring cells. They have been implicated in pathological processes such as tumourigenesis in which they may act as tumour suppressors. A polymorphism in the human connexin37 (Cx37) gene (C1019T), resulting in a non-conservative amino acid change in the regulatory C-terminus (CT) of the Cx37 protein (P319S) has been suggested to be implicated in predisposition to angiosarcomas. In this study, we have used communication-deficient HeLa and SK-HEP-1 cells transfected with Cx37-319S, Cx37-319P or empty vector. We showed that the expression of Cx37-319P limited proliferation of HeLa and SK-HEP-1 cells, whereas Cx37-319S expression was without effect. Using an in vitro kinase assay, we demonstrated phosphorylation of Cx37 CT by glycogen synthase kinase-3 (GSK-3), a kinase known to be implicated in cell proliferation and cancer. GSK-3-induced phosphorylation was associated with reduced gap junctional intercellular communication (GJIC) as measured by microinjection of the tracer neurobiotin. Inhibition of GSK-3 by LiCl or SB415286 reduced phosphorylation of Cx37-319P and increased GJIC. This latter effect on GJIC involved the beta and not the alpha isoform of GSK-3. In contrast, GSK-3 inhibitors were without effect on HeLa cells expressing Cx37-319S. In conclusion, our data indicate functional effects of the Cx37 C1019T polymorphism on GJIC that might contribute to tumour cell growth.

Rapid cytopathic effects of Clostridium perfringens beta-toxin on porcine endothelial cells.

Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C beta-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

Exploring the nuclear envelope of herpes simplex virus 1-infected cells by high-resolution microscopy.

Herpesviruses are composed of capsid, tegument, and envelope. Capsids assemble in the nucleus and exit the nucleus by budding at the inner nuclear membrane, acquiring tegument and the envelope. This study focuses on the changes of the nuclear envelope during herpes simplex virus 1 (HSV-1) infection in HeLa and Vero cells by employing preparation techniques at ambient and low temperatures for high-resolution scanning and transmission electron microscopy and confocal laser scanning microscopy. Cryo-field emission scanning electron microscopy of freeze-fractured cells showed for the first time budding of capsids at the nuclear envelope at the third dimension with high activity at 10 h and low activity at 15 h of incubation. The mean number of pores was significantly lower, and the mean interpore distance and the mean interpore area were significantly larger than those for mock-infected cells 15 h after inoculation. Forty-five percent of nuclear pores in HSV-1-infected cells were dilated to more than 140 nm. Nuclear material containing capsids protrude through them into the cytoplasm. Examination of in situ preparations after dry fracturing revealed significant enlargements of the nuclear pore diameter and of the nuclear pore central channel in HSV-1-infected cells compared to mock-infected cells. The demonstration of nucleoporins by confocal microscopy also revealed fewer pores but focal enhancement of fluorescence signals in HSV-1-infected cells, whereas Western blots showed no loss of nucleoporins from cells. The data suggest that infection with HSV-1 alters the number, size, and architecture of nuclear pores without a loss of nucleoporins from altered nuclear pore complexes.

Targeting connexin 43 prevents platelet-derived growth factor-BB-induced phenotypic change in porcine coronary artery smooth muscle cells.

We previously reported that reducing the expression of the gap junction protein connexin (Cx)43 in mice restricts intimal thickening formation after acute vascular injury by limiting the inflammatory response and the proliferation and migration of smooth muscle cells (SMCs) toward the damaged site. SMC populations isolated from porcine coronary artery exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). S-SMCs are predominant in the normal media, whereas R-SMCs are recovered in higher proportion from stent-induced intimal thickening, suggesting that they participate in the restenotic process. Here, we further investigate the relationship between connexin expression and SMC phenotypes using porcine coronary artery SMCs. Cx40 was highly expressed in normal media of porcine coronary artery in vivo, whereas Cx43 was barely detectable. In contrast, Cx40 was downregulated and Cx43 was markedly upregulated in stent-induced intimal thickening. In vitro, S-SMCs expressed Cx40 and Cx43. In R-SMCs, Cx43 expression was increased and Cx40 was absent. We confirmed that S-SMCs treated with platelet-derived growth factor-BB acquire an R phenotype. This was accompanied by an upregulation of Cx43 and a loss of Cx40. Importantly, platelet-derived growth factor-BB-induced S-to-R phenotypic change was prevented by a reduction of Cx43 expression with antisense, ie, S-SMCs retained their typical elongated appearance and the expression of alpha-smooth muscle actin, a well-known SMC differentiation marker, whereas the expression of S100A4, a typical marker of R-SMCs, was prevented. In conclusion, limiting Cx43 expression in S-SMCs prevents platelet-derived growth factor-BB-induced S-to-R modulation. This suggests that Cx43 may be an additional target for local delivery strategies aimed at reducing restenosis.

Pharmacological Inhibition of MALT1 Protease Leads to a Progressive IPEX-Like Pathology.

Genetic disruption or short-term pharmacological inhibition of MALT1 protease is effective in several preclinical models of autoimmunity and B cell malignancies. Despite these protective effects, the severe reduction in regulatory T cells (Tregs) and the associated IPEX-like pathology occurring upon congenital disruption of the MALT1 protease in mice has raised concerns about the long-term safety of MALT1 inhibition. Here we describe the results of a series of toxicology studies in rat and dog species using MLT-943, a novel potent and selective MALT1 protease inhibitor. While MLT-943 effectively prevented T cell-dependent B cell immune responses and reduced joint inflammation in the collagen-induced arthritis rat pharmacology model, in both preclinical species, pharmacological inhibition of MALT1 was associated with a rapid and dose-dependent reduction in Tregs and resulted in the progressive appearance of immune abnormalities and clinical signs of an IPEX-like pathology. At the 13-week time point, rats displayed severe intestinal inflammation associated with mast cell activation, high serum IgE levels, systemic T cell activation and mononuclear cell infiltration in multiple tissues. Importantly, using thymectomized rats we demonstrated that MALT1 protease inhibition affects peripheral Treg frequency independently of effects on thymic Treg output and development. Our data confirm the therapeutic potential of MALT1 protease inhibitors but highlight the safety risks and challenges to consider before potential application of such inhibitors into the clinic.

Fibromatosis in a young Bernese Mountain Dog: clinical, imaging, and histopathological findings.

A young, intact, male Bernese Mountain Dog was presented to the animal hospital for lameness and diffuse thickening of the soft tissue in the right hind limb. Magnetic resonance imaging revealed multiple, multilobular, space-occupying lesions within and between the muscles of the right femur. Biopsies taken from the lesions revealed an infiltrative mass composed mainly of collagen fibers and a low density of benign-appearing fibroblasts. These findings were compatible with a diagnosis of a fibromatosis. Taking the age of onset into account, infantile fibromatosis was most likely. A deep fibromatosis, similar to that seen in adults, could not be excluded based on histology.

Sex-related differences in wall remodeling and intraluminal thrombus resolution in a rat saccular aneurysm model.

OBJECTIVE: Intracranial aneurysms (IAs) are more often diagnosed in women. Hormones and vessel geometry, which influences wall shear stress, may affect pathophysiological processes of the arterial wall. Here, the authors investigated sex-related differences in the remodeling of the aneurysm wall and in intraluminal thrombus resolution. METHODS: A well-characterized surgical side-wall aneurysm model was used in female, male, and ovariectomized rats. Decellularized grafts were used to model highly degenerated and decellularized IA walls and native grafts to model healthy IA walls. Aneurysm growth and thrombus composition were analyzed at 1, 7, 14, and 28 days. Sex-related differences in vessel wall remodeling were compared with human IA dome samples of men and pre- and postmenopausal women. RESULTS: At 28 days, more aneurysm growth was observed in ovariectomized rats than in males or non-ovariectomized female rats. The parent artery size was larger in male rats than in female or ovariectomized rats, as expected. Wall inflammation increased over time in all groups and was most severe in the decellularized female and ovariectomized groups at 28 days compared with the male group. Likewise, in these groups the most elastin fragmentation was seen at 28 days. In female rats, on days 1, 7, and 14, the intraluminal thrombus was mainly composed of red blood cells and fibrin. On days 14 and 28, macrophage and smooth muscle cell invasion inside the thrombus was shown, leading to the removal of red blood cells and deposition of collagen and elastin. On days 14 and 28, similar profiles of thrombus reorganization were observed in male and ovariectomized female rats. However, collagen content in thrombi and vessel wall macrophage content were higher in aneurysms of male rats at 28 days than in those of female rats. On day 28, thrombus coverage by endothelial cells was lower in ovariectomized than in female or male rats. Finally, analysis of human IA domes showed that endothelial cell coverage was lower in men and postmenopausal women than in younger women. CONCLUSIONS: Aneurysm growth and intraluminal thrombus resolution show sex-dependent differences. While certain processes (endothelial cell coverage and collagen deposition) point to a strong hormonal dependence, others (wall inflammation and aneurysm growth) seem to be influenced by both hormones and parent artery size.

Correlating Clinical Risk Factors and Histological Features in Ruptured and Unruptured Human Intracranial Aneurysms: The Swiss AneuX Study.

Pathogenesis of intracranial aneurysm is complex and the precise biomechanical processes leading to their rupture are uncertain. The goal of our study was to characterize the aneurysmal wall histologically and to correlate histological characteristics with clinical and radiological factors used to estimate the risk of rupture. A new biobank of aneurysm domes resected at the Geneva University Hospitals (Switzerland) was used. Histological analysis revealed that unruptured aneurysms have a higher smooth muscle cell (SMC) content and a lower macrophage content than ruptured domes. These differences were associated with more collagen in unruptured samples, whereas the elastin content was not affected. Collagen content and type distribution were different between thick and thin walls of unruptured aneurysms. Classification of aneurysm domes based on histological characteristics showed that unruptured samples present organized wall rich in endothelial and SMCs compared with ruptured samples. Finally, aneurysm wall composition was altered in unruptured domes of patients presenting specific clinical factors used to predict rupture such as large dome diameter, dome irregularities, and smoking. Our study shows that the wall of aneurysm suspected to be at risk for rupture undergoes structural alterations relatively well associated with clinical and radiological factors currently used to predict this risk.

Reduced connexin43 expression limits neointima formation after balloon distension injury in hypercholesterolemic mice.

BACKGROUND: Reducing the expression of the gap junction protein connexin43 (Cx43) inhibits the progression of atherosclerosis, a chronic inflammatory disease. Furthermore, acute vascular injury induced by percutaneous coronary interventions is associated with increased Cx43 expression in neointimal smooth muscle cells (SMCs). However, the relevance of Cx43 after acute vascular injury remains unclear. METHODS AND RESULTS: To investigate whether reducing Cx43 expression would affect neointima formation in vivo, we subjected hypercholesterolemic Cx43+/- LDL receptor-deficient (LDLR-/-) mice and Cx43+/+LDLR-/- control littermates to carotid balloon distension injury, which induced marked endothelial denudation and activation of medial SMCs. We observed decreased macrophage infiltration in Cx43+/-LDLR-/- mice 7 days after injury. Similarly, peritoneal macrophages isolated from Cx43+/-LDLR-/- mice showed reduced migration in vitro compared with Cx43+/+LDLR-/- macrophages. Interestingly, Cx43+/-LDLR-/- macrophages also displayed decreased chemotactic activity for SMCs. In addition, we observed less SMC infiltration and proliferation in Cx43+/-LDLR-/- mice 7 and 14 days after balloon angioplasty. Likewise, Cx43+/-LDLR-/- SMCs showed decreased proliferation and migration in vitro compared with Cx43+/+LDLR-/- cells. All these events resulted in a reduction of neointimal thickening after vascular injury in Cx43+/-LDLR-/- mice. CONCLUSIONS: The present study shows for the first time that reducing Cx43 limits neointima formation after acute vascular injury by decreasing the inflammatory response and reducing SMC migration and proliferation. Thus, decreasing Cx43 expression may offer a novel therapeutic strategy for reducing restenosis after percutaneous coronary intervention.

Endothelial Cx40 limits myocardial ischaemia/reperfusion injury in mice.

AIMS: Gap junctions are indispensable for the function of heart and blood vessels by providing electrical coupling and direct cell-to-cell transfer of small signalling molecules. Gap junction channels between neighbouring cells are composed of 12 connexins (Cx). Changes in Cx43 expression, localization, and channel properties in cardiomyocytes contribute to infarction and reperfusion injury of the heart. It is increasingly recognized that deleterious consequences of ischaemia/reperfusion (IR) are modulated by the inflammatory response and endothelial function. The role of the endothelial connexins, i.e. Cx40 and Cx37, in cardiac IR injury is, however, not known. METHODS AND RESULTS: Following 30 min ischaemia and 24 h reperfusion, we found a significant increase in myocardial infarct size in mice with endothelial-specific deletion of Cx40 (Cx40del), but not in Cx37-deficient mice. The cardioprotective effect of endothelial Cx40 was associated with a decrease in neutrophil infiltration. Moreover, beneficial effects of endothelial Cx40 were not observed in isolated Langendorff-perfused hearts, suggesting direct involvement of endothelial-leucocyte interactions in the cardiac injury. Single-dose administration of methotrexate, a CD73 activator, reduced infarct size and neutrophil infiltration into the infarcted myocardium in Cx40del but not in control mice. Similar to Cx40del mice, CD73-deficient mice showed increased sensitivity to cardiac IR injury, which could not be conversed by methotrexate. CONCLUSION: Endothelial Cx40, but not Cx37, is implicated in resistance of the heart to IR injury by activation of the CD73 pathway. Thus, the Cx40-CD73 axis may represent an interesting target for controlling reperfusion damage associated with revascularization in coronary disease.

Herpes simplex virus 1 induces de novo phospholipid synthesis.

Herpes simplex virus type 1 capsids bud at nuclear membranes and Golgi membranes acquiring an envelope composed of phospholipids. Hence, we measured incorporation of phospholipid precursors into these membranes, and quantified changes in size of cellular compartments by morphometric analysis. Incorporation of [³H]-choline into both nuclear and cytoplasmic membranes was significantly enhanced upon infection. [³H]-choline was also part of isolated virions even grown in the presence of brefeldin A. Nuclei expanded early in infection. The Golgi complex and vacuoles increased substantially whereas the endoplasmic reticulum enlarged only temporarily. The data suggest that HSV-1 stimulates phospholipid synthesis, and that de novo synthesized phospholipids are inserted into nuclear and cytoplasmic membranes to i) maintain membrane integrity in the course of nuclear and cellular expansion, ii) to supply membrane constituents for envelopment of capsids by budding at nuclear membranes and Golgi membranes, and iii) to provide membranes for formation of transport vacuoles.

Vav1 GEF activity is required for T cell mediated allograft rejection.

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression.