Prolongation of allograft survival by artemisinin treatment is associated with blockade of OX40-OX40L.

OBJECTIVES: It has been demonstrated that artemisinin (ART) possesses multiple immune modulatory effects. However, its role as immunosuppressant in allogeneic transplantation is undetermined. Here, we investigated the effect of ART on co-stimulatory signaling in OX40+ T cells and evaluated ART as a potential immunosuppressant in transplantation. MATERIALS AND METHODS: Allogeneic skin transplantation was performed in C57BL/6 to BALB/c mice. Recipient mice were administrated with vehicle, ART or cyclosporine A daily from day 0 to day 19 post transplantation. Proportions of splenic CD4+OX40+ and CD4+CD44hiCD62Lhi cells, and serum IgG was measured by using flow cytometry. An in vitro lymphocyte stimulation with Con A or LPS under various concentrations of ART was performed, expression of CD4+OX40+ and CD4+CD44hiCD62Lhi cells was evaluated, and interleukin(IL)-6 production was measured by ELISA. RESULTS: In in vivo allogeneic skin transplant model, ART significantly prolongs allogeneic skin survival. Furthermore, our in vitro studies demonstrate that the immune suppression of ART on T cells is associated with a reduction in OX40+ T cells and inhibition of IL-6 secretion. CONCLUSION: Our data indicate that the OX40-OX40L pathway and IL-6 are possibly involved in ART-induced immunosuppression, and ART is a potential novel immunosuppressant.

Artemisinin attenuates IgM xenoantibody production via inhibition of T cell-independent marginal zone B cell proliferation.

Artemisinin (ART) has been shown to suppress B cell activation and plasma cell formation. However, its effect on splenic marginal zone (MZ) B cells is unknown. Splenic MZ B cells play a critical role in rapidly induced Ab production against blood-borne foreign Ags. Dysfunction of MZ B cells, due to inhibition of its proliferation or displacement of its homing, results in an attenuated adaptive humoral response. Here, we investigate the effect of ART on splenic MZ B (CD19+ CD21high CD23low ) and B10 (CD19+ CD1dhigh CD5+ ) B cells to explore the mechanisms of ART-induced immunosuppression in T cell-deficient nude mice challenged with hamster xenoantigens. In this study, we demonstrate that ART decreases T cell-independent xenogeneic IgM Ab production and, this is associated with a strong suppression of MZ B cell proliferation and a relative increase of CD21low CD23+ follicular and B10 B cells. In addition, this suppression impairs IL-10 production. Taken together, our data indicate that ART suppresses B cell immune responses through a distinctive effect on splenic MZ B and other B cells. This represents a new mechanism of ART-induced immunosuppression.

Discovery of a Potent, Orally Bioavailable PI4KIIIß Inhibitor (UCB9608) Able To Significantly Prolong Allogeneic Organ Engraftment in Vivo.

The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.

Police work stressors and cardiac vagal control.

OBJECTIVES: This study examines relationships between the frequency and intensity of police work stressors and cardiac vagal control, estimated using the high frequency component of heart rate variability (HRV). METHODS: This is a cross-sectional study of 360 officers from the Buffalo New York Police Department. Police stress was measured using the Spielberger police stress survey, which includes exposure indices created as the product of the self-evaluation of how stressful certain events were and the self-reported frequency with which they occurred. Vagal control was estimated using the high frequency component of resting HRV calculated in units of milliseconds squared and reported in natural log scale. Associations between police work stressors and vagal control were examined using linear regression for significance testing and analysis of covariance for descriptive purposes, stratified by gender, and adjusted for age and race/ethnicity. RESULTS: There were no significant associations between police work stressor exposure indices and vagal control among men. Among women, the inverse associations between the lack of support stressor exposure and vagal control were statistically significant in adjusted models for indices of exposure over the past year (lowest stressor quartile: M = 5.57, 95% CI 5.07 to 6.08, and highest stressor quartile: M = 5.02, 95% CI 4.54 to 5.51, test of association from continuous linear regression of vagal control on lack of support stressor ß = -0.273, P = .04). CONCLUSIONS: This study supports an inverse association between lack of organizational support and vagal control among female but not male police officers.

Molecular mechanisms of pulmonary response progression in crystalline silica exposed rats.

An understanding of the mechanisms underlying diseases is critical for their prevention. Excessive exposure to crystalline silica is a risk factor for silicosis, a potentially fatal pulmonary disease. Male Fischer 344 rats were exposed by inhalation to crystalline silica (15 mg/m3, six hours/day, five days) and pulmonary response was determined at 44 weeks following termination of silica exposure. Additionally, global gene expression profiling in lungs and BAL cells and bioinformatic analysis of the gene expression data were done to understand the molecular mechanisms underlying the progression of pulmonary response to silica. A significant increase in lactate dehydrogenase activity and albumin content in BAL fluid (BALF) suggested silica-induced pulmonary toxicity in the rats. A significant increase in the number of alveolar macrophages and infiltrating neutrophils in the lungs and elevation in monocyte chemoattractant protein-1 (MCP-1) in BALF suggested the induction of pulmonary inflammation in the silica exposed rats. Histological changes in the lungs included granuloma formation, type II pneumocyte hyperplasia, thickening of alveolar septa and positive response to Masson's trichrome stain. Microarray analysis of global gene expression detected 94 and 225 significantly differentially expressed genes in the lungs and BAL cells, respectively. Bioinformatic analysis of the gene expression data identified significant enrichment of several disease and biological function categories and canonical pathways related to pulmonary toxicity, especially inflammation. Taken together, these data suggested the involvement of chronic inflammation as a mechanism underlying the progression of pulmonary response to exposure of rats to crystalline silica at 44 weeks following termination of exposure.

Transcriptional Pathways Altered in Response to Vibration in a Model of Hand-Arm Vibration Syndrome.

OBJECTIVE: The aim of this study was to use an established model of vibration-induced injury to assess frequency-dependent changes in transcript expression in skin, artery, and nerve tissues. METHODS: Transcript expression in tissues from control and vibration-exposed rats (4 h/day for 10 days at 62.5, 125, or 250 Hz; 49 m/s, rms) was measured. Transcripts affected by vibration were used in bioinformatics analyses to identify molecular- and disease-related pathways associated with exposure to vibration. RESULTS: Analyses revealed that cancer-related pathways showed frequency-dependent changes in activation or inhibition. Most notably, the breast-related cancer-1 pathway was affected. Other pathways associated with breast cancer type 1 susceptibility protein related signaling, or associated with cancer and cell cycle/cell survivability were also affected. CONCLUSION: Occupational exposure to vibration may result in DNA damage and alterations in cell signaling pathways that have significant effects on cellular division.

Leptin, adiponectin, and heart rate variability among police officers.

OBJECTIVES: Police officers have a high prevalence of cardiovascular disease (CVD). Reduced heart rate variability (HRV) is known to increase CVD risk. Leptin and adiponectin may be related to CVD health. Therefore, our objective was to investigate the relationship between these variables and HRV. METHODS: Leptin and adiponectin levels were measured in 388 officers from the Buffalo Cardio-Metabolic Occupational Police Stress study. HRV was assessed according to methods published by the Task Force of the European Society of Cardiology and the North American Society of Pacing Electrophysiology for measurement and analysis of HRV. Mean values of high-frequency (HF) and low-frequency (LF) HRV were compared across tertiles of leptin and adiponectin using analysis of variance and analysis of covariance; trends were assessed using linear regression models. RESULTS: Leptin, but not adiponectin, was significantly and inversely associated with HRV. Body mass index (BMI) and percent body fat significantly modified the association between leptin and LF (but not HF) HRV. Among officers with BMI < 25 kg/m(2) , leptin was not significantly associated with HRV. However, among officers with BMI ≥ 25 kg/m(2) , leptin was inversely associated with HRV, after adjustment for age, gender, and race/ethnicity; HF HRV, P = 0.019 and LF HRV, P < 0.0001. Similarly, among officers with percent body fat ≥ 25.5%, leptin and LF HRV showed significant, inverse associations (adjusted P = 0.001). CONCLUSIONS: Leptin levels were inversely associated with LF HRV, especially among officers with increased adiposity. Increased leptin levels may be associated with CVD-related health problems.

Molecular insights into the progression of crystalline silica-induced pulmonary toxicity in rats.

Identification of molecular target(s) and mechanism(s) of silica-induced pulmonary toxicity is important for the intervention and/or prevention of diseases associated with exposure to silica. Rats were exposed to crystalline silica by inhalation (15 mg m(-3), 6 h per day, 5 days) and global gene expression profile was determined in the lungs by microarray analysis at 1, 2, 4, 8 and 16 weeks following termination of silica exposure. The number of significantly differentially expressed genes (>1.5-fold change and <0.01 false discovery rate P-value) detected in the lungs during the post-exposure time intervals analyzed exhibited a steady increase in parallel with the progression of silica-induced pulmonary toxicity noticed in the rats. Quantitative real-time PCR analysis of a representative set of 10 genes confirmed the microarray findings. The number of biological functions, canonical pathways and molecular networks significantly affected by silica exposure, as identified by the bioinformatics analysis of the significantly differentially expressed genes detected during the post-exposure time intervals, also exhibited a steady increase similar to the silica-induced pulmonary toxicity. Genes involved in oxidative stress, inflammation, respiratory diseases, cancer, and tissue remodeling and fibrosis were significantly differentially expressed in the rat lungs; however, unresolved inflammation was the single most significant biological response to pulmonary exposure to silica. Excessive mucus production, as implicated by significant overexpression of the pendrin coding gene, SLC26A4, was identified as a potential novel mechanism for silica-induced pulmonary toxicity. Collectively, the findings of our study provided insights into the molecular mechanisms underlying the progression of crystalline silica-induced pulmonary toxicity in the rat. Published 2012. This article is a US Government work and is in the public domain in the USA.

Transcriptomics analysis of lungs and peripheral blood of crystalline silica-exposed rats.

Minimally invasive approaches to detect/predict target organ toxicity have significant practical applications in occupational toxicology. The potential application of peripheral blood transcriptomics as a practical approach to study the mechanisms of silica-induced pulmonary toxicity was investigated. Rats were exposed by inhalation to crystalline silica (15 mg/m(3), 6 h/day, 5 days) and pulmonary toxicity and global gene expression profiles of lungs and peripheral blood were determined at 32 weeks following termination of exposure. A significant elevation in bronchoalveolar lavage fluid lactate dehydrogenase activity and moderate histological changes in the lungs, including type II pneumocyte hyperplasia and fibrosis, indicated pulmonary toxicity in the rats. Similarly, significant infiltration of neutrophils and elevated monocyte chemotactic protein-1 levels in the lungs showed pulmonary inflammation in the rats. Microarray analysis of global gene expression profiles identified significant differential expression [>1.5-fold change and false discovery rate (FDR) p < 0.01] of 520 and 537 genes, respectively, in the lungs and blood of the exposed rats. Bioinformatics analysis of the differentially expressed genes demonstrated significant similarity in the biological processes, molecular networks, and canonical pathways enriched by silica exposure in the lungs and blood of the rats. Several genes involved in functions relevant to silica-induced pulmonary toxicity such as inflammation, respiratory diseases, cancer, cellular movement, fibrosis, etc, were found significantly differentially expressed in the lungs and blood of the silica-exposed rats. The results of this study suggested the potential application of peripheral blood gene expression profiling as a toxicologically relevant and minimally invasive surrogate approach to study the mechanisms underlying silica-induced pulmonary toxicity.

Type I interferon and pattern recognition receptor signaling following particulate matter inhalation.

BACKGROUND: Welding, a process that generates an aerosol containing gases and metal-rich particulates, induces adverse physiological effects including inflammation, immunosuppression and cardiovascular dysfunction. This study utilized microarray technology and subsequent pathway analysis as an exploratory search for markers/mechanisms of in vivo systemic effects following inhalation. Mice were exposed by inhalation to gas metal arc - stainless steel (GMA-SS) welding fume at 40 mg/m3 for 3 hr/d for 10 d and sacrificed 4 hr, 14 d and 28 d post-exposure. Whole blood cells, aorta and lung were harvested for global gene expression analysis with subsequent Ingenuity Pathway Analysis and confirmatory qRT-PCR. Serum was collected for protein profiling. RESULTS: The novel finding was a dominant type I interferon signaling network with the transcription factor Irf7 as a central component maintained through 28 d. Remarkably, these effects showed consistency across all tissues indicating a systemic type I interferon response that was complemented by changes in serum proteins (decreased MMP-9, CRP and increased VCAM1, oncostatin M, IP-10). In addition, pulmonary expression of interferon α and ß and Irf7 specific pattern recognition receptors (PRR) and signaling molecules (Ddx58, Ifih1, Dhx58, ISGF3) were induced, an effect that showed specificity when compared to other inflammatory exposures. Also, a canonical pathway indicated a coordinated response of multiple PRR and associated signaling molecules (Tlr7, Tlr2, Clec7a, Nlrp3, Myd88) to inhalation of GMA-SS. CONCLUSION: This methodological approach has the potential to identify consistent, prominent and/or novel pathways and provides insight into mechanisms that contribute to pulmonary and systemic effects following toxicant exposure.

Random KNN feature selection - a fast and stable alternative to Random Forests.

BACKGROUND: Successfully modeling high-dimensional data involving thousands of variables is challenging. This is especially true for gene expression profiling experiments, given the large number of genes involved and the small number of samples available. Random Forests (RF) is a popular and widely used approach to feature selection for such "small n, large p problems." However, Random Forests suffers from instability, especially in the presence of noisy and/or unbalanced inputs. RESULTS: We present RKNN-FS, an innovative feature selection procedure for "small n, large p problems." RKNN-FS is based on Random KNN (RKNN), a novel generalization of traditional nearest-neighbor modeling. RKNN consists of an ensemble of base k-nearest neighbor models, each constructed from a random subset of the input variables. To rank the importance of the variables, we define a criterion on the RKNN framework, using the notion of support. A two-stage backward model selection method is then developed based on this criterion. Empirical results on microarray data sets with thousands of variables and relatively few samples show that RKNN-FS is an effective feature selection approach for high-dimensional data. RKNN is similar to Random Forests in terms of classification accuracy without feature selection. However, RKNN provides much better classification accuracy than RF when each method incorporates a feature-selection step. Our results show that RKNN is significantly more stable and more robust than Random Forests for feature selection when the input data are noisy and/or unbalanced. Further, RKNN-FS is much faster than the Random Forests feature selection method (RF-FS), especially for large scale problems, involving thousands of variables and multiple classes. CONCLUSIONS: Given the superiority of Random KNN in classification performance when compared with Random Forests, RKNN-FS's simplicity and ease of implementation, and its superiority in speed and stability, we propose RKNN-FS as a faster and more stable alternative to Random Forests in classification problems involving feature selection for high-dimensional datasets.

Mechanisms of crystalline silica-induced pulmonary toxicity revealed by global gene expression profiling.

A proper understanding of the mechanisms underlying crystalline silica-induced pulmonary toxicity has implications in the management and potential prevention of the adverse health effects associated with silica exposure including silicosis, cancer and several auto-immune diseases. Human lung type II epithelial cells and rat lungs exposed to crystalline silica were employed as experimental models to determine global gene expression changes in order to understand the molecular mechanisms underlying silica-induced pulmonary toxicity. The differential gene expression profile induced by silica correlated with its toxicity in the A549 cells. The biological processes perturbed by silica exposure in the A549 cells and rat lungs, as identified by the bioinformatics analysis of the differentially expressed genes, demonstrated significant similarity. Functional categorization of the differentially expressed genes identified cancer, cellular movement, cellular growth and proliferation, cell death, inflammatory response, cell cycle, cellular development, and genetic disorder as top ranking biological functions perturbed by silica exposure in A549 cells and rat lungs. Results of our study, in addition to confirming several previously identified molecular targets and mechanisms involved in silica toxicity, identified novel molecular targets and mechanisms potentially involved in silica-induced pulmonary toxicity. Further investigations, including those focused on the novel molecular targets and mechanisms identified in the current study may result in better management and, possibly, reduction and/or prevention of the potential adverse health effects associated with crystalline silica exposure.

IL-7 is required for the development of the intrinsic function of marginal zone B cells and the marginal zone microenvironment.

The characteristic microarchitecture of the marginal zone (MZ), formed by locally interacting MZ-specific B cells, macrophages, and endothelial cells, is critical for productive marginal zone B cell (MZB cell) Ab responses. Reportedly, IL-7-deficient mice, although severely lymphopenic, retain small numbers of CD21(high)CD23(low) B cells consistent with MZB cell phenotype, suggesting that IL-7 signaling is not exclusively required for MZB cell lymphopoiesis. In this study, we investigated the function of IL-7(-/-) MZB cells and the IL-7(-/-) microenvironment using a model of hamster heart xenograft rejection, which depends exclusively on MZB cell-mediated production of T cell-independent IgM xenoantibodies (IgMXAb). C57BL/6-IL-7(-/-) mice accepted xenografts indefinitely and failed to produce IgMXAb, even after transfer of additional IL-7(-/-) or wild-type C57BL/6 MZB cells. Transfer of wild-type but not IL-7(-/-) B cells enabled SCID mice to produce IgMXAb. When transferred to SCID mice, wild-type but not IL-7(-/-) B cells formed B cell follicles with clearly defined IgM(+), MOMA-1(+), and MAdCAM-1(+) MZ structures. Conversely, adoptively transferred GFP(+) C57BL/6 B cells homed to the MZ area in a SCID but not an IL-7(-/-) environment. Naive IL-7(-/-) mice showed absent or aberrant splenic B cell structures. We provide evidence that IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against T cell-independent type II Ags, for their homing potential, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.

Blood gene expression profiling detects silica exposure and toxicity.

Blood gene expression profiling was investigated as a minimally invasive surrogate approach to detect silica exposure and resulting pulmonary toxicity. Rats were exposed by inhalation to crystalline silica (15 mg/m³, 6 h/day, 5 days), and pulmonary damage and blood gene expression profiles were determined after latency periods (0-16 weeks). Silica exposure resulted in pulmonary toxicity as evidenced by histological and biochemical changes in the lungs. The number of significantly differentially expressed genes in the blood, identified by microarray analysis, correlated with the severity of silica-induced pulmonary toxicity. Functional analysis of the differentially expressed genes identified activation of inflammatory response as the major biological signal. Induction of pulmonary inflammation, as suggested by the blood gene expression data, was supported by significant increases in the number of macrophages and infiltrating neutrophils as well as the activity of pro-inflammatory chemokines observed in the lungs of the silica-exposed rats. A gene expression signature developed using the blood gene expression data predicted the exposure of rats to lower, minimally toxic and nontoxic concentrations of silica. Taken together, our findings suggest the potential application of peripheral blood gene expression profiling as a minimally invasive surrogate approach to detect pulmonary toxicity induced by silica in the rat. However, further research is required to determine the potential application of our findings specifically to monitor human exposure to silica and the resulting pulmonary effects.

Response of the mouse lung transcriptome to welding fume: effects of stainless and mild steel fumes on lung gene expression in A/J and C57BL/6J mice.

BACKGROUND: Debate exists as to whether welding fume is carcinogenic, but epidemiological evidence suggests that welders are an at risk population for the development of lung cancer. Recently, we found that exposure to welding fume caused an acutely greater and prolonged lung inflammatory response in lung tumor susceptible A/J versus resistant C57BL/6J (B6) mice and a trend for increased tumor incidence after stainless steel (SS) fume exposure. Here, our objective was to examine potential strain-dependent differences in the regulation and resolution of the lung inflammatory response induced by carcinogenic (Cr and Ni abundant) or non-carcinogenic (iron abundant) metal-containing welding fumes at the transcriptome level. METHODS: Mice were exposed four times by pharyngeal aspiration to 5 mg/kg iron abundant gas metal arc-mild steel (GMA-MS), Cr and Ni abundant GMA-SS fume or vehicle and were euthanized 4 and 16 weeks after the last exposure. Whole lung microarray using Illumina Mouse Ref-8 expression beadchips was done. RESULTS: Overall, we found that tumor susceptibility was associated with a more marked transcriptional response to both GMA-MS and -SS welding fumes. Also, Ingenuity Pathway Analysis revealed that gene regulation and expression in the top molecular networks differed between the strains at both time points post-exposure. Interestingly, a common finding between the strains was that GMA-MS fume exposure altered behavioral gene networks. In contrast, GMA-SS fume exposure chronically upregulated chemotactic and immunomodulatory genes such as CCL3, CCL4, CXCL2, and MMP12 in the A/J strain. In the GMA-SS-exposed B6 mouse, genes that initially downregulated cellular movement, hematological system development/function and immune response were involved at both time points post-exposure. However, at 16 weeks, a transcriptional switch to an upregulation for neutrophil chemotactic genes was found and included genes such as S100A8, S100A9 and MMP9. CONCLUSIONS: Collectively, our results demonstrate that lung tumor susceptibility may predispose the A/J strain to a prolonged dysregulation of immunomodulatory genes, thereby delaying the recovery from welding fume-induced lung inflammation. Additionally, our results provide unique insight into strain- and welding fume-dependent genetic factors involved in the lung response to welding fume.

Characterization of frequency-dependent responses of the vascular system to repetitive vibration.

OBJECTIVE: The current frequency weighting proposed in the International Standards Organization-5349 standard may underestimate the risk of injury associated with exposure to vibration >100 Hz. The goal of this study was to assess the frequency-dependent responses of the peripheral vascular system to repeated bouts of vibration. METHODS: The effects of exposure to vibration at 62.5, 125, or 250 Hz (constant acceleration of 49 m/s2) on vascular morphology, oxidative stress, inflammation, and gene expression were examined in the ventral tail artery of rats. RESULTS: Vascular responses indicative of dysfunction (eg, remodeling and oxidative activity) became more pronounced as the frequency of the exposure increased. CONCLUSION: Exposure to vibration frequencies that induce the greatest stress and strain on the tail (ie, >100 Hz) result in vascular changes indicative of dysfunction.

Blood gene expression markers to detect and distinguish target organ toxicity.

The purpose of this study was to investigate whether the expression of specific genes in peripheral blood can be used as surrogate marker(s) to detect and distinguish target organ toxicity induced by chemicals in rats. Rats were intraperitoneally administered a single, acute dose of a well-established hepatotoxic (acetaminophen) or a neurotoxic (methyl parathion) chemical. Administration of acetaminophen (AP) in the rats resulted in hepatotoxicity as evidenced from elevated blood transaminase activities. Similarly, administration of methyl parathion (MP) resulted in neurotoxicity in the rats as evidenced from the inhibition of acetyl cholinesterase activity in their blood. Administration of either chemical also resulted in mild hematotoxicity in the rats. Microarray analysis of the global gene expression profile of rat blood identified distinct gene expression markers capable of detecting and distinguishing hepatotoxicity and neurotoxicity induced by AP and MP, respectively. Differential expressions of the marker genes for hepatotoxicity and neurotoxicity were detectable in the blood earlier than the appearance of the commonly used clinical markers (serum transaminases and acetyl cholinesterase). The ability of the marker genes to detect hepatotoxicity and neurotoxicity was further confirmed using the blood samples of rats administered additional hepatotoxic (thioacetamide, dimethylnitrobenzene, and carbon tetrachloride) or neurotoxic (ethyl parathion and malathion) chemicals. In summary, our results demonstrated that blood gene expression markers can detect and distinguish target organ toxicity non-invasively.

Xenotransplantation: role of natural immunity.

Hyperacute rejection, mediated by natural anti-Galalpha1,3Galbeta1,4GlcNAc (alphaGal) antibodies and the classically activated complement pathway, was identified as the first major barrier to the survival of porcine organs in humans. Subsequently, discordant pig-to-nonhuman primate and concordant rodent models revealed key roles for T and B lymphocytes in the second form of rejection, acute vascular rejection (AVR) or delayed xenograft rejection (DXR). As significant progress was made in strategies to circumvent or suppress xenoreactivity of the adaptive immune system, it became clear that, apart from natural antibodies, other innate immune system elements actively participate in AVR/DXR and represent a barrier to xenograft acceptance that may be particularly difficult to overcome. Observations in pig-to-primate and semi-discordant and concordant rodent models indicate that Natural Killer (NK) cells play a more prominent role in xenograft than in allograft rejection. Several mechanisms through which human NK cells recognize porcine endothelial cells have been elucidated and these appear to be more diverse than those involved in NK cell alloreactivity. Further, it has been demonstrated that human macrophages and neutrophils can directly recognize pig derived cells and can mediate direct xenograft damage. Here, we review the recent progress in the understanding of the xenoreactivity of the natural immune system, focussing on preclinical pig-to-(non)human primate systems, and discuss the proposed strategies to overcome these barriers.