TAFI (thrombin-activatable fibrinolysis inhibitor) is a carboxypeptidase zymogen originally identified in plasma. The TAFI pathway helps to regulate the balance between the coagulation and fibrinolytic cascades. Activated TAFI (TAFIa) can also inactivate certain pro-inflammatory mediators, suggesting that the TAFI pathway may also regulate communication between coagulation and inflammation. Expression in the liver is considered to be the source of plasma TAFI. TAFI has also been identified in platelets and CPB2 (the gene encoding TAFI) mRNA has been detected in megakaryocytic cell lines as well as in endothelial cells. We have undertaken a quantitative analysis of CPB2 mRNA and TAFI protein in extrahepatic cell types relevant to vascular disease. Using RT-PCR and quantitative RT-PCR, we detected CPB2 mRNA in the human megakaryoblastic cell lines MEG-01 and Dami, the human monocytoid cell line THP-1 as well as THP-1 cells differentiated into a macrophage-like phenotype, and in primary human umbilical vein and coronary artery endothelial cells. CPB2 mRNA abundance in MEG-01, Dami, and THP-1 cells was modulated by the state of differentiation of these cells. Using a recently developed TAFIa assay, we detected TAFI protein in the lysates of the human hepatocellular carcinoma cell line HepG2 as well as in MEG-01 and Dami cells and in the conditioned medium of HepG2 cells, differentiated Dami cells, and THP-1 macrophages. We have obtained clear evidence for extrahepatic expression of TAFI, which has clear implications for the physiological and pathophysiological functions of the TAFI pathway.

The list of pharmacological agents that can modify the gut microbiome or be modified by it continues to grow at a high rate. The greatest amount of attention on drug-gut microbiome interactions has been directed primarily at pharmaceuticals used to treat infection, diabetes, cardiovascular conditions and cancer. By comparison, drugs of abuse and addiction, which can powerfully and chronically worsen human health, have received relatively little attention in this regard. Therefore, the main objective of this study was to characterize how selected synthetic psychoactive cathinones (aka "Bath Salts") and amphetamine stimulants modify the gut microbiome. Mice were treated with mephedrone (40 mg/kg), methcathinone (80 mg/kg), methamphetamine (5 mg/kg) or 4-methyl-methamphetamine (40 mg/kg), following a binge regimen consisting of 4 injections at 2h intervals. These drugs were selected for study because they are structural analogs that contain a β-keto substituent (methcathinone), a 4-methyl group (4-methyl-methamphetamine), both substituents (mephedrone) or neither (methamphetamine). Mice were sacrificed 1, 2 or 7 days after treatment and DNA from caecum contents was subjected to 16S rRNA sequencing. We found that all drugs caused significant time-and structure-dependent alterations in the diversity and taxonomic structure of the gut microbiome. The two phyla most changed by drug treatments were Firmicutes (methcathinone, 4-methyl-methamphetamine) and Bacteriodetes (methcathinone, 4-methyl-methamphetamine, methamphetamine, mephedrone). Across time, broad microbiome changes from the phylum to genus levels were characteristic of all drugs. The present results signify that these selected psychoactive drugs, which are thought to exert their primary effects within the CNS, can have profound effects on the gut microbiome. They also suggest new avenues of investigation into the possibility that gut-derived signals could modulate drug abuse and addiction via altered communication along the gut-brain axis.

Gulf War Illness (GWI) is a chronic health condition that appeared in Veterans after returning home from the Gulf War. The primary symptoms linked to deployment are posttraumatic stress disorder, mood disorders, GI problems and chronic fatigue. At first glance, these symptoms are difficult to ascribe to a single pathological mechanism. However, it is now clear that each symptom can be linked individually to alterations in the gut microbiome. The primary objective of the present study was to determine if gut microbiome dysbiosis was evident in a mouse model of GWl. Because the majority of Gulf War Veterans are overweight, a second objective was to determine if a high fat diet (HF) would alter GWI outcomes. We found that the taxonomic structure of the gut microbiome was significantly altered in the GWI model and after HF exposure. Their combined effects were significantly different from either treatment alone. Most treatment-induced changes occurred at the level of phylum in Firmicutes and Bacteroidetes. If mice fed HF were returned to a normal diet, the gut microbiome recovered toward normal levels in both controls and GWI agent-treated mice. These results add support to the hypotheses that dysbiosis in the gut microbiome plays a role in GWI and that lifestyle risk factors such as an unhealthy diet can accentuate the effects of GWI by impacting the gut microbiome. The reversibility of the effect of HF on the gut microbiome suggests new avenues for treating GWI through dietary intervention. Soon after the end of hostilities in the Gulf War (August 1990-April 1991), a series of health issues began emerging in Gulf War Veterans and have persisted to the present day. The health issues reported are a perplexing and complex constellation of symptoms now known as Gulf War Illness (GWI). Over the past two decades, the Institute of Medicine has completed a series of studies on GWI and Health and the most recent review concluded that "Evidence is sufficient to conclude that a causal relationship exists between being deployed to the Gulf War and a health outcome" (p. 3 1). When considering all symptoms that have been reported to be part of GWI, posttraumatic stress disorder was the only condition judged to have sufficient evidence of a causal relationship. The other symptoms for which evidence was sufficient to establish an association with deployment were mood disorders (anxiety, depression), GI symptoms (irritable bowel syndrome [IBS], dyspepsia) and chronic fatigue syndrome 1. These disparate outcomes make it difficult to attribute GWI to a single mechanism until consideration is given to the gut microbiome. The GI system of humans and most other mammals is inhabited by a very large number of bacteria, viruses, fungi and archaea. Collectively, these microorganisms make up the gut microbiome. It has been estimated that the gut contains 100 trillion cells and these cells express >150-fold more unique genes than the human genome 2. The commensal members of the gut microbiome support human health but disruption in it has been...

traumatic brain injury (tBi) is often accompanied by gastrointestinal and metabolic disruptions. these systemic manifestations suggest possible involvement of the gut microbiota in head injury outcomes. Although gut dysbiosis after single, severe tBi has been documented, the majority of head injuries are mild, such as those that occur in athletes and military personnel exposed to repetitive head impacts. therefore, it is important to determine if repetitive, mild tBi (rmtBi) will also disrupt the gut microbiota. Male mice were exposed to mild head impacts daily for 20 days and assessed for cognitive behavior, neuropathology and disruptions in the gut microbiota at 0, 45 or 90 days after injury. Deficits in recognition memory were evident at the late post-injury points. Brains show an early increase in microglial activation at the 0-day time point that persisted until 90 days post-injury. This was compounded by substantial increases in astrocyte reactivity and phosphorylated tau at the 90day time point. in contrast, changes in the microbial community were minor and transient, and very few differences were observed in mice exposed to rmTBI compared to controls. While the progressive emergence of white matter damage and cognitive alterations after rmtBi resembles the alterations observed in athletes and military personnel exposed to rmtBi, these changes could not be linked to systematic modifications in the gut microbiota. Traumatic brain injury (TBI) results from a blow to the head and its severity can range along a continuum from mild (e.g., brief change in mental status or consciousness) to severe (e.g., extended unconsciousness, coma, prolonged amnesia) to fatal. TBI is one of the most common neurological diagnoses in the US 1 , with a CDC estimate of approximately 2.5 million people on an annual basis. About one-third of all injury deaths arise from TBI and roughly 90,000 persons experience long-term disability yearly as a result of brain injury 2. The cost of lifetime care for survivors of TBI has been estimated at $0.6-$1 million per individual with a total annual cost to the United States of ~$60 billion 3 , reflecting the severity of the dysfunction that results from this injury. Perhaps the form of TBI that has garnered the greatest scrutiny recently, in the public eye as well as within military, scientific and medical communities, is repetitive, mild TBI (rmTBI). Military operations in Iraq and Afghanistan are revealing that TBI accounts for about 28% of all combat casualties 4 approximately 85% of which are mild 4-6. More than 2.4 million US military personnel have served on multiple combat tours, and the US Defense and Veterans Brain Injury Center has estimated that ~413,000 military service members have been diagnosed with mTBI from 2001-2011 (https://dvbic.dcoe.mil/dod-worldwide-numbers-tbi). Besides the military,

TAFI (thrombin-activatable fibrinolysis inhibitor) is a pro-carboxypeptidase, encoded by the CPB2 gene in humans that links the coagulation cascade to fibrinolysis and inflammation. The liver is the main source for plasma TAFI, and TAFI expression has been documented in platelets and monocyte-derived macrophages. A recent study reported an alternatively spliced CPB2 mRNA variant lacking exon 7 (∆7) in HepG2 cells and liver. Another study identified a CPB2 mRNA variant lacking exon 7 and a 52 bp deletion in exon 11 (∆7+11) in human hippocampus. We have examined alternative splicing of CPB2 mRNA in various cell types by RT-PCR and have assessed the functional properties of TAFI variants encoded by these transcripts by recombinant expression in mammalian cells. We identified the Δ7 exon skipping event in liver, Dami megakaryoblasts, THP-1-derived macrophages, peripheral blood mononuclear cells, platelets, testis, cerebellum, and SH-SY5Y neuroblastoma cells. The Δ11 alternative splicing event was notably absent in liver cells. We also detected a novel exon Δ7+8 skipping event in liver and megakaryocytes. Of note, we detected non-alternatively spliced CPB2 transcripts in brain tissues, suggesting the expression of full-length TAFI in brain. Experiments using cultured mammalian cells transfected with wild-type CPB2-, ∆7-, ∆7+11-, and ∆11-cDNA revealed that alternatively spliced TAFI is stored inside the cells, cannot be activated by thrombin-thrombomodulin, and does not have TAFIa activity. The alternative splicing events clearly do not give rise to a secreted protein with basic carboxypeptidase activity, but the intracellular forms may possess novel functions related to intracellular proteolysis.

No abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-carboxypeptidase B-like pro-enzyme that upon activation attenuates fibrinolysis and inflammatory processes. There is a large inter-individual variability in plasma TAFI levels within the population which is primarily due to epigenetic factors. Novel associations between plasma TAFI levels and sex steroids have triggered interest in determining the role of TAFI as a mediator of the cardioprotective effects of estrogens and progestins, or as a mediator of the increased thrombotic risk that accompanies use of oral contraceptives or hormone replacement therapy (HRT). In this study, we measured the effect of sex steroids on hepatic expression of CPB2, the human gene encoding TAFI. Using human hepatocellular carcinoma cells cultured in the presence of progesterone and 17β-estradiol, we demonstrated that the level of TAFI protein is decreased by those sex steroids. These changes in protein expression were paralleled by decreases in CPB2 mRNA abundance and promoter activity. We did not find evidence of estrogen or progesterone receptor binding sites in the CPB2 promoter region, suggesting that the genomic effects of progesterone and 17β-estradiol are mediated indirectly, without receptor binding to the CPB2 promoter. Indeed, we found that the effect of estrogen was independent of the estrogen receptor and was mediated through a novel signaling pathway dependent on phosphatidylinositol 3-kinase and Akt that did not involve GPR30. Our findings provide the molecular explanation for the ability of female sex steroids to decrease plasma TAFI concentrations.

No abstract