Louisville VA Medical Center

BACKGROUND: Idiopathic membranous nephropathy is an autoimmune disease. In approximately 70% of patients, it is associated with autoantibodies against the phospholipase A2 receptor 1 (PLA2R1). Antigenic targets in the remaining patients are unknown. METHODS: Using Western blotting, we screened serum samples from patients with idiopathic membranous nephropathy, patients with other glomerular diseases, and healthy controls for antibodies against human native glomerular proteins. We partially purified a putative new antigen, identified this protein by means of mass spectrometry of digested peptides, and validated the results by analysis of recombinant protein expression, immunoprecipitation, and immunohistochemical analysis. RESULTS: Serum samples from 6 of 44 patients in a European cohort and 9 of 110 patients in a Boston cohort with anti-PLA2R1-negative idiopathic membranous nephropathy recognized a glomerular protein that was 250 kD in size. None of the serum samples from the 74 patients with idiopathic membranous nephropathy who were seropositive for anti-PLA2R1 antibodies, from the 76 patients with other glomerular diseases, and from the 44 healthy controls reacted against this antigen. Although this newly identified antigen is clearly different from PLA2R1, it shares some biochemical features, such as N-glycosylation, membranous location, and reactivity with serum only under nonreducing conditions. Mass spectrometry identified this antigen as thrombospondin type-1 domain-containing 7A (THSD7A). All reactive serum samples recognized recombinant THSD7A and immunoprecipitated THSD7A from glomerular lysates. Moreover, immunohistochemical analyses of biopsy samples from patients revealed localization of THSD7A to podocytes, and IgG eluted from one of these samples was specific for THSD7A. CONCLUSIONS: In our cohort, 15 of 154 patients with idiopathic membranous nephropathy had circulating autoantibodies to THSD7A but not to PLA2R1, a finding that suggests a distinct subgroup of patients with this condition. (Funded by the French National Center for Scientific Research and others.).

Daily exposure of humans to nanoparticles from edible plants is inevitable, but significant advances are required to determine whether edible plant nanoparticles are beneficial to our health. Additionally, strategies are needed to elucidate the molecular mechanisms underlying any beneficial effects. Here, as a proof of concept, we used a mouse model to show that orally given nanoparticles isolated from ginger extracts using a sucrose gradient centrifugation procedure resulted in protecting mice against alcohol-induced liver damage. The ginger-derived nanoparticle (GDN)-mediated activation of nuclear factor erythroid 2-related factor 2 (Nrf2) led to the expression of a group of liver detoxifying/antioxidant genes and inhibited the production of reactive oxygen species, which partially contributes to the liver protection. Using lipid knock-out and knock-in strategies, we further identified that shogaol in the GDN plays a role in the induction of Nrf2 in a TLR4/TRIF-dependent manner. Given the critical role of Nrf2 in modulating numerous cellular processes, including hepatocyte homeostasis, drug metabolism, antioxidant defenses, and cell-cycle progression of liver, this finding not only opens up a new avenue for investigating GDN as a means to protect against the development of liver-related diseases such as alcohol-induced liver damage but sheds light on studying the cellular and molecular mechanisms underlying interspecies communication in the liver via edible plant-derived nanoparticles.

Enteric dysbiosis plays an essential role in the pathogenesis of alcoholic liver disease (ALD). Detailed characterization of the alterations in the gut microbiome is needed for understanding their pathogenic role in ALD and developing effective therapeutic approaches using probiotic supplementation. Mice were fed liquid Lieber-DeCarli diet without or with alcohol (5% v/v) for 6 weeks. A subset of mice were administered the probiotic Lactobacillus rhamnosus GG (LGG) from 6 to 8 weeks. Indicators of intestinal permeability, hepatic steatosis, inflammation and injury were evaluated. Metagenomic analysis of the gut microbiome was performed by analyzing the fecal DNA by amplification of the V3-V5 regions of the 16S rRNA gene and large-scale parallel pyrosequencing on the 454 FLX Titanium platform. Chronic ethanol feeding caused a decline in the abundance of both Bacteriodetes and Firmicutes phyla, with a proportional increase in the gram negative Proteobacteria and gram positive Actinobacteria phyla; the bacterial genera that showed the biggest expansion were the gram negative alkaline tolerant Alcaligenes and gram positive Corynebacterium. Commensurate with the qualitative and quantitative alterations in the microbiome, ethanol caused an increase in plasma endotoxin, fecal pH, hepatic inflammation and injury. Notably, the ethanol-induced pathogenic changes in the microbiome and the liver were prevented by LGG supplementation. Overall, significant alterations in the gut microbiome over time occur in response to chronic alcohol exposure and correspond to increases in intestinal barrier dysfunction and development of ALD. Moreover, the altered bacterial communities of the gut may serve as significant therapeutic target for the prevention/treatment of chronic alcohol intake induced intestinal barrier dysfunction and liver disease.

Acrolein, a highly reactive unsaturated aldehyde, is a ubiquitous environmental pollutant and its potential as a serious environmental health threat is beginning to be recognized. Humans are exposed to acrolein per oral (food and water), respiratory (cigarette smoke, automobile exhaust, and biocide use) and dermal routes, in addition to endogenous generation (metabolism and lipid peroxidation). Acrolein has been suggested to play a role in several disease states including spinal cord injury, multiple sclerosis, Alzheimer's disease, cardiovascular disease, diabetes mellitus, and neuro-, hepato-, and nephro-toxicity. On the cellular level, acrolein exposure has diverse toxic effects, including DNA and protein adduction, oxidative stress, mitochondrial disruption, membrane damage, endoplasmic reticulum stress, and immune dysfunction. This review addresses our current understanding of each pathogenic mechanism of acrolein toxicity, with emphasis on the known and anticipated contribution to clinical disease, and potential therapies.

There are few data evaluating plasma and/or peripheral blood monocyte cytokine concentrations/production or attempts to manipulate proinflammatory cytokines in nonalcoholic steatohepatitis (NASH). A pilot project in a general clinical research center evaluated the effects of a step 1 American Heart Association diet plus aerobic exercise with or without 800 IU of vitamin E daily on cytokine profiles and liver enzyme levels in 16 patients with biopsy-proven NASH. Biochemical assessment of liver function, lipid profiles, and body mass index significantly improved during the first 6 weeks of therapy and remained stable during the following 6 weeks. Plasma hyaluronic acid (HA) concentrations decreased in parallel with weight loss. Plasma tumor necrosis factor (TNF) concentrations were significantly elevated in patients with NASH and similar to patients with stable alcoholic cirrhosis but not as elevated as in patients with acute alcoholic steatohepatitis (AH). Although plasma TNF, interleukin 8 (IL-8), and IL-6 concentrations were all significantly elevated compared with control values, only plasma IL-6 levels significantly decreased with therapy. Peripheral blood monocyte TNF, IL-8, and IL-6 production was significantly elevated in patients with NASH but did not significantly decrease. Independent effects of vitamin E were not observed in this small sample. In conclusion, patients with NASH have dysregulated cytokine metabolism similar to, but less pronounced than abnormalities documented in AH. Cytokine values generally did not decrease significantly with weight loss with or without vitamin E over the duration of the study. Lifestyle modifications (low-fat diet and exercise) were associated with improvement in liver enzymes, cholesterol, and plasma HA levels in patients with NASH, whereas the level of vitamin E supplementation used in this short-term pilot study provided no apparent added benefit.

Exosomes are emerging mediators of intercellular communication; whether the release of exosomes has an effect on the exosome donor cells in addition to the recipient cells has not been investigated to any extent. Here, we examine different exosomal miRNA expression profiles in primary mouse colon tumour, liver metastasis of colon cancer and naive colon tissues. In more advanced disease, higher levels of tumour suppressor miRNAs are encapsulated in the exosomes. miR-193a interacts with major vault protein (MVP). Knockout of MVP leads to miR-193a accumulation in the exosomal donor cells instead of exosomes, inhibiting tumour progression. Furthermore, miR-193a causes cell cycle G1 arrest and cell proliferation repression through targeting of Caprin1, which upregulates Ccnd2 and c-Myc. Human colon cancer patients with more advanced disease show higher levels of circulating exosomal miR-193a. In summary, our data demonstrate that MVP-mediated selective sorting of tumour suppressor miRNA into exosomes promotes tumour progression.

, DCs pre-pulsed with SFN prevented DSS-induced colitis in B6 mice, further supporting the DC AMPK role in SFN-mediated prevention of DSS-induced colitis. This finding could open new preventive or therapeutic avenues to address intestinal-related inflammatory diseases via activating AMPK.

Background and Aims Cholestatic liver disease is characterized by gut dysbiosis and excessive toxic hepatic bile acids (BAs). Modification of gut microbiota and repression of BA synthesis are potential strategies for the treatment of cholestatic liver disease. The purpose of this study was to examine the effects and to understand the mechanisms of the probiotic Lactobacillus rhamnosus GG (LGG) on hepatic BA synthesis, liver injury, and fibrosis in bile duct ligation (BDL) and multidrug resistance protein 2 knockout ( Mdr2−/− ) mice. Approach and Results Global and intestine‐specific farnesoid X receptor (FXR) inhibitors were used to dissect the role of FXR. LGG treatment significantly attenuated liver inflammation, injury, and fibrosis with a significant reduction of hepatic BAs in BDL mice. Hepatic concentration of taurine‐β‐muricholic acid (T‐βMCA), an FXR antagonist, was markedly increased in BDL mice and reduced in LGG‐treated mice, while chenodeoxycholic acid, an FXR agonist, was decreased in BDL mice and normalized in LGG‐treated mice. LGG treatment significantly increased the expression of serum and ileum fibroblast growth factor 15 (FGF‐15) and subsequently reduced hepatic cholesterol 7α‐hydroxylase and BA synthesis in BDL and Mdr2−/− mice. At the molecular level, these changes were reversed by global and intestine‐specific FXR inhibitors in BDL mice. In addition, LGG treatment altered gut microbiota, which was associated with increased BA deconjugation and increased fecal and urine BA excretion in both BDL and Mdr2−/− mice. In vitro studies showed that LGG suppressed the inhibitory effect of T‐βMCA on FXR and FGF‐19 expression in Caco‐2 cells. Conclusion LGG supplementation decreases hepatic BA by increasing intestinal FXR–FGF‐15 signaling pathway–mediated suppression of BA de novo synthesis and enhances BA excretion, which prevents excessive BA‐induced liver injury and fibrosis in mice.

Inflammation is a hallmark of cancer. Activated immune cells are intrinsically capable of homing to inflammatory sites. Using three inflammatory-driven disease mouse models, we show that grapefruit-derived nanovectors (GNV) coated with inflammatory-related receptor enriched membranes of activated leukocytes (IGNVs) are enhanced for homing to inflammatory tumor tissues. Blocking LFA-1 or CXCR1 and CXCR2 on the IGNVs significantly inhibits IGNV homing to the inflammatory tissue. The therapeutic potential of IGNVs was further demonstrated by enhancing the chemotherapeutic effect as shown by inhibition of tumor growth in two tumor models and inhibiting the inflammatory effects of dextran sulfate sodium-induced mouse colitis. The fact that IGNVs are capable of homing to inflammatory tissue and that chemokines are overexpressed in diseased human tissue provides the rationale for using IGNVs to more directly deliver therapeutic agents to inflammatory tumor sites and the rationale for the use of IGNVs as treatment for certain cancers in personalized medicine.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

Obesity and its metabolic complications are major health problems in the United States and worldwide, and increasing evidence implicates the microbiota in these important health issues. Indeed, it appears that the microbiota function much like a metabolic "organ," influencing nutrient acquisition, energy homeostasis, and, ultimately, the control of body weight. Moreover, alterations in gut microbiota, increased intestinal permeability, and metabolic endotoxemia likely play a role in the development of a chronic low-grade inflammatory state in the host that contributes to the development of obesity and associated chronic metabolic diseases such as nonalcoholic fatty liver disease. Supporting these concepts are the observations that increased gut permeability, low-grade endotoxemia, and fatty liver are observed in animal models of obesity caused by either high-fat or high-fructose feeding. Consistent with these observations, germ-free mice are protected from obesity and many forms of liver injury. Last, many agents that affect gut flora/permeability, such as probiotics/prebiotics, also appear to affect obesity and certain forms of liver injury in animal model systems. Here the authors review the role of the gut microbiota and metabolic endotoxemia-induced inflammation in the development of obesity and liver injury, with special reference to the intensive care unit setting.

BACKGROUND: High-level occupational exposures to some industrial chemicals have been associated with liver diseases, including nonalcoholic fatty liver disease (NAFLD). However, the potential role of low-level environmental pollution on liver disease in the general population has not been evaluated. OBJECTIVE: We determined whether environmental pollutants are associated with an elevation in serum alanine aminotransferase (ALT) activity and suspected NAFLD in U.S. adults. METHODS: This cross-sectional cohort study evaluated adult participants without viral hepatitis, hemochromatosis, or alcoholic liver disease from the National Health and Nutrition Examination Survey (NHANES) for 2003-2004. ALT elevation was defined in men as ≥ 37 IU/L (age 18-20 years) and ≥ 48 IU/L (age ≥ 21 years) and in women as ≥ 30 IU/L (age 18-20 years) and ≥ 31 IU/L (age ≥ 21 years). Adjusted odds ratios (ORs) for ALT elevation were determined across exposure quartiles for 17 pollutant subclasses comprising 111 individual pollutants present with at least a 60% detection rate. Adjustments were made for age, race/ethnicity, sex, body mass index, poverty income ratio, and insulin resistance. Individual pollutants from subclasses associated with ALT elevation were subsequently analyzed. RESULTS: The overall prevalence of ALT elevation was 10.6%. Heavy metals and polychlorinated biphenyls (PCBs) were associated with dose-dependent increased adjusted ORs for ALT elevation. Within these subclasses, increasing whole-blood levels of lead and mercury and increasing lipid-adjusted serum levels of 20 PCBs were individually associated with ALT elevation. CONCLUSIONS: PCB, lead, and mercury exposures were associated with unexplained ALT elevation, a proxy marker of NAFLD, in NHANES 2003-2004 adult participants.

PTH and Vitamin D are two major regulators of mineral metabolism. They play critical roles in the maintenance of calcium and phosphate homeostasis as well as the development and maintenance of bone health. PTH and Vitamin D form a tightly controlled feedback cycle, PTH being a major stimulator of vitamin D synthesis in the kidney while vitamin D exerts negative feedback on PTH secretion. The major function of PTH and major physiologic regulator is circulating ionized calcium. The effects of PTH on gut, kidney, and bone serve to maintain serum calcium within a tight range. PTH has a reciprocal effect on phosphate metabolism. In contrast, vitamin D has a stimulatory effect on both calcium and phosphate homeostasis, playing a key role in providing adequate mineral for normal bone formation. Both hormones act in concert with the more recently discovered FGF23 and klotho, hormones involved predominantly in phosphate metabolism, which also participate in this closely knit feedback circuit. Of great interest are recent studies demonstrating effects of both PTH and vitamin D on the cardiovascular system. Hyperparathyroidism and vitamin D deficiency have been implicated in a variety of cardiovascular disorders including hypertension, atherosclerosis, vascular calcification, and kidney failure. Both hormones have direct effects on the endothelium, heart, and other vascular structures. How these effects of PTH and vitamin D interface with the regulation of bone formation are the subject of intense investigation.

Lung inflammation is a hallmark of coronavirus disease 2019 (COVID-19). In this study, we show that mice develop inflamed lung tissue after being administered exosomes released from the lung epithelial cells exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp12 and Nsp13 (exosomes Nsp12Nsp13 ). Mechanistically, we show that exosomes Nsp12Nsp13 are taken up by lung macrophages, leading to activation of nuclear factor κB (NF-κB) and the subsequent induction of an array of inflammatory cytokines. Induction of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β from exosomes Nsp12Nsp13 -activated lung macrophages contributes to inducing apoptosis in lung epithelial cells. Induction of exosomes Nsp12Nsp13 -mediated lung inflammation was abolished with ginger exosome-like nanoparticle (GELN) microRNA (miRNA aly-miR396a-5p. The role of GELNs in inhibition of the SARS-CoV-2-induced cytopathic effect (CPE) was further demonstrated via GELN aly-miR396a-5p- and rlcv-miR-rL1-28-3p-mediated inhibition of expression of Nsp12 and spike genes, respectively. Taken together, our results reveal exosomes Nsp12Nsp13 as potentially important contributors to the development of lung inflammation, and GELNs are a potential therapeutic agent to treat COVID-19.

The lack of access to the brain is a major obstacle for central nervous system drug development. In this study, we demonstrate the capability of a grapefruit-derived nanovector (GNV) to carry miR17 for therapeutic treatment of mouse brain tumor. We show that GNVs coated with folic acid (FA-GNVs) are enhanced for targeting the GNVs to a folate receptor-positive GL-26 brain tumor. Additionally, FA-GNV-coated polyethylenimine (FA-pGNVs) not only enhance the capacity to carry RNA, but the toxicity of the polyethylenimine is eliminated by the GNVs. Intranasal administration of miR17 carried by FA-pGNVs led to rapid delivery of miR17 to the brain that was selectively taken up by GL-26 tumor cells. Mice treated intranasally with FA-pGNV/miR17 had delayed brain tumor growth. Our results demonstrate that this strategy may provide a noninvasive therapeutic approach for treating brain-related disease through intranasal delivery.

High-fat diet (HFD) decreases insulin sensitivity. How high-fat diet causes insulin resistance is largely unknown. Here, we show that lean mice become insulin resistant after being administered exosomes isolated from the feces of obese mice fed a HFD or from patients with type II diabetes. HFD altered the lipid composition of exosomes from predominantly phosphatidylethanolamine (PE) in exosomes from lean animals (L-Exo) to phosphatidylcholine (PC) in exosomes from obese animals (H-Exo). Mechanistically, we show that intestinal H-Exo is taken up by macrophages and hepatocytes, leading to inhibition of the insulin signaling pathway. Moreover, exosome-derived PC binds to and activates AhR, leading to inhibition of the expression of genes essential for activation of the insulin signaling pathway, including IRS-2, and its downstream genes PI3K and Akt. Together, our results reveal HFD-induced exosomes as potential contributors to the development of insulin resistance. Intestinal exosomes thus have potential as broad therapeutic targets.

BACKGROUND: A low level of HDL cholesterol has been identified as a risk factor for stroke in observational studies. METHODS AND RESULTS: Our objective was to determine whether treatment aimed at raising HDL cholesterol and lowering triglycerides reduces stroke in men with coronary heart disease and low levels of both HDL and LDL cholesterol. The study was a placebo-controlled, randomized trial conducted in 20 Veterans Affairs medical centers. A total of 2531 men with coronary heart disease, with mean HDL cholesterol 0.82 mmol/L (31.5 mg/dL) and mean LDL cholesterol 2.9 mmol/L (111 mg/dL), were randomized to gemfibrozil 1200 mg/d or placebo and were followed up for 5 years. Strokes were confirmed by a blinded adjudication committee. Relative risks were derived from Cox proportional hazards models. There were 134 confirmed strokes, 90% of which were ischemic. Seventy-six occurred in the placebo group (9 fatal) and 58 in the gemfibrozil group (3 fatal), for a relative risk reduction, adjusted for baseline variables, of 31% (95% CI, 2% to 52%, P=0.036). The reduction in risk was evident after 6 to 12 months. Patients with baseline HDL cholesterol below the median may have been more likely to benefit from treatment than those with higher HDL cholesterol. CONCLUSIONS: In men with coronary heart disease, low HDL cholesterol, and low LDL cholesterol, gemfibrozil reduces stroke incidence.

Endotoxemia is a contributing cofactor to alcoholic liver disease (ALD), and alcohol-induced increased intestinal permeability is one of the mechanisms of endotoxin absorption. Probiotic bacteria have been shown to promote intestinal epithelial integrity and protect barrier function in inflammatory bowel disease (IBD) and in ALD. Although it is highly possible that some common molecules secreted by probiotics contribute to this action in IBD, the effect of probiotic culture supernatant has not yet been studied in ALD. We examined the effects of Lactobacillus rhamnosus GG culture supernatant (LGG-s) on the acute alcohol-induced intestinal integrity and liver injury in a mouse model. Mice on standard chow diet were supplemented with supernatant from LGG culture (10(9) colony-forming unit/mouse) for 5 days, and one dose of alcohol at 6 g/kg body wt was administered via gavage. Intestinal permeability was measured by FITC-FD-4 ex vivo. Alcohol-induced liver injury was examined by measuring the activity of alanine aminotransferase (ALT) in plasma, and liver steatosis was evaluated by triglyceride content and Oil Red O staining of the liver sections. LGG-s pretreatment restored alcohol-induced reduction in ileum mRNA levels of claudin-1, intestine trefoil factor (ITF), P-glycoprotein (P-gp), and cathelin-related antimicrobial peptide (CRAMP), which play important roles on intestinal barrier integrity. As a result, LGG-s pretreatment significantly inhibited the alcohol-induced intestinal permeability, endotoxemia and subsequently liver injury. Interestingly, LGG-s pretreatment increased ileum mRNA expression of hypoxia-inducible factor (HIF)-2α, an important transcription factor of ITF, P-gp, and CRAMP. These results suggest that LGG-s ameliorates the acute alcohol-induced liver injury by promoting HIF signaling, leading to the suppression of alcohol-induced increased intestinal permeability and endotoxemia. The use of bacteria-free LGG culture supernatant provides a novel strategy for prevention of acute alcohol-induced liver injury.

Recent associations between oral health and systemic disease have led to renewed interest in the mouth and its contribution to health outcomes. Many pathways for this relationship have been postulated, among them the potential mediating role of nutrition. The link between various nutrients and systemic disease has been established, but relatively little work has been done in relating oral conditions with nutrition. We searched MEDLINE, from 1966 to July, 2001, to identify articles relating specific oral measures to nutrition outcomes. We included original articles written in English with a sample size greater than 30 that used objective oral health measures. We reviewed a total of 56 articles. Only a small proportion of these studies were methodologically sound. Although many studies were small and cross-sectional, the literature suggests that tooth loss affects dietary quality and nutrient intake in a manner that may increase the risk for several systemic diseases. The impact of tooth loss on diet may be only partially compensated for by prostheses. To date, there is little information relating periodontal disease and oral pain and nutrition. A few studies suggest poorer nutrition among individuals with xerostomia and altered taste. Further, impaired dentition may contribute to weight change, depending on age and other population characteristics. There is a paucity of well-designed studies addressing oral health and nutrition. Before we can acquire a better understanding of how nutrition and oral health interrelate, however, more studies will be required to confirm these associations-preferably longitudinal studies with larger sample sizes and better control of important confounders.

Cell-derived vesicles represent a recently discovered mechanism for intercellular communication. We investigated their potential role in interaction of microbes with host organisms. We provide evidence that different stimuli induced isolated neutrophilic granulocytes to release microvesicles with different biologic properties. Only opsonized particles initiated the formation of microvesicles that were able to impair bacterial growth. The antibacterial effect of neutrophil-derived microvesicles was independent of production of toxic oxygen metabolites and opsonization or engulfment of the microbes, but depended on β(2) integrin function, continuous actin remodeling, and on the glucose supply. Neutrophil-derived microvesicles were detected in the serum of healthy donors, and their number was significantly increased in the serum of bacteremic patients. We propose a new extracellular mechanism to restrict bacterial growth and dissemination.