Epigenetic Effects of Resveratrol on Oncogenic Signaling in Breast Cancer.

The crosstalk between oncogenic signaling pathways plays a crucial role in driving cancer development. We previously demonstrated that dietary polyphenols, specifically resveratrol (RSV) and other stilbenoids, epigenetically target oncogenes for silencing via DNA hypermethylation in breast cancer. In the present study, we identify signal transduction regulators among RSV-hypermethylated targets and investigate the functional role of RSV-mediated DNA hypermethylation in the regulation of Hedgehog and Wnt signaling. Non-invasive ER-positive MCF-7 and highly invasive triple-negative MCF10CA1a human breast cancer cell lines were used as experimental models. Upon 9-day exposure to 15 µM RSV, pyrosequencing and qRT-PCR were performed to assess DNA methylation and expression of GLI2 and WNT4, which are upstream regulators of the Hedgehog and Wnt pathways, respectively. Our results showed that RSV led to a DNA methylation increase within GLI2 and WNT4 enhancers, which was accompanied by decreases in gene expression. Consistently, we observed the downregulation of genes downstream of the Hedgehog and Wnt signaling, including common targets shared by both pathways, CCND1 and CYR61. Further analysis using chromatin immunoprecipitation identified increased H3K27 trimethylation and decreased H3K9 and H3K27 acetylation, along with abolishing OCT1 transcription factor binding. Those changes indicate a transcriptionally silent chromatin state at GLI2 and WNT4 enhancers. The inhibition of the Wnt signal transduction was confirmed using a phospho-antibody array that demonstrated suppression of positive and stimulation of negative Wnt regulators. In conclusion, our results provide scientific evidence for dietary polyphenols as epigenetics-modulating agents that act to re-methylate and silence oncogenes, reducing the oncogenic signal transduction. Targeting such an action could be an effective strategy in breast cancer prevention and/or adjuvant therapy.

Policing of Drug Safety Information Dissemination Under the False Claims Act.

On July 25, 2017, the second largest multi-million dollar settlement was pursued with the assistance of the Department of Justice and alleging inappropriate marketing strategies utilized by the pharmaceutical industry came to an end.

DNA hypermethylation of Kiss1r promoter and reduction of hepatic Kiss1r in female rats with type 2 diabetes.

Kisspeptin, produced from the brain and peripheral tissues, may constitute an important link in metabolic regulation in response to external cues, such as diet. The kisspeptin system is well described in the brain. However, its function and regulation in the peripheral tissues, especially in relation to metabolic disease and sex differences, remain to be elucidated. As Kiss1 and Kiss1r, encoding for kisspeptin and kisspeptin receptors, respectively, are altered by overnutrition/fasting and regulated by DNA methylation during puberty and cancer, epigenetic mechanisms in metabolic disorders are highly probable. In the present study, we experimentally induced type 2 diabetes mellitus (DM2) in female Wistar rats using high-fat diet/streptozocin. We analysed expression and DNA methylation of Kiss1 and Kiss1r in the peripheral tissues, using quantitative-reverse-transcription PCR (qRT-PCR) and pyrosequencing. We discovered differential expression of Kiss1 and Kiss1r in peripheral organs in DM2 females, as compared with healthy controls, and the profile differed from patterns reported earlier in males. DM2 in females was linked to the increased Kiss1 mRNA in the liver and increased Kiss1r mRNA in the liver and adipose tissue. However, Kiss1r promoter was hypermethylated in the liver, suggesting gene silencing. Indeed, the increase in DNA methylation of Kiss1r promoter was accompanied by a reduction in Kiss1r protein, implying epigenetic or translational gene repression. Our results deliver novel evidence for tissue-specific differences in Kiss1 and Kiss1r expression in peripheral organs in DM2 females and suggest DNA methylation as a player in regulation of the hepatic kisspeptin system in DM2.

Real-world overall survival of patients receiving cetuximab in later lines of treatment for metastatic colorectal cancer.

Aim: To examine characteristics of and treatment duration and real-world overall survival (rwOS) in patients receiving cetuximab as second-line (2L) or third-line (3L) treatment for metastatic colorectal cancer. Materials & methods: This was a retrospective study of 1096 and 684 patients in 2L and 3L cohorts, respectively. Results: The most common cetuximab-based regimens were cetuximab + folinic acid, fluorouracil and irinotecan (2L: 44%; 3L: 32%) and cetuximab + irinotecan (2L: 28%; 3L: 35%). Kaplan-Meier survival estimates and stepwise Cox regression model analysis demonstrated median treatment duration and rwOS of 3.7 and 14.4 months, respectively, in patients receiving treatment in the 2L cohort. In the 3L cohort, treatment duration was 3.3 months and rwOS was 12.0 months. Conclusion: This large real-world study provides evidence of rwOS in patients with metastatic colorectal cancer receiving cetuximab-based regimens as 2L or 3L treatment.

In this retrospective study, the authors examined baseline characteristics of and treatment duration and real-world overall survival (rwOS) in 1096 and 684 patients with metastatic colorectal cancer receiving cetuximab as second-line (2L) and third-line (3L) treatment, respectively. The most common cetuximab-based regimens were cetuximab + folinic acid, fluorouracil and irinotecan (2L: 44%; 3L: 32%) and cetuximab + irinotecan (2L: 28%; 3L: 35%). Median treatment duration and rwOS were 3.7 and 14.4 months, respectively, in patients receiving treatment in the 2L cohort. In the 3L cohort, median treatment duration was 3.3 months and rwOS was 12.0 months. This large real-world study provides evidence of rwOS in patients with metastatic colorectal cancer receiving cetuximab-based regimens as 2L or 3L treatment.

Epigenetic aberrations of gene expression in a rat model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is mostly triggered by environmental and life-style factors and may involve epigenetic aberrations. However, a comprehensive documentation of the link between the dysregulated epigenome, transcriptome, and liver carcinogenesis is lacking. In the present study, Fischer-344 rats were fed a choline-deficient (CDAA, cancer group) or choline-sufficient (CSAA, healthy group) L-amino acid-defined diet. At the end of 52 weeks, transcriptomic alterations in livers of rats with HCC tumours and healthy livers were investigated by RNA sequencing. DNA methylation and gene expression were assessed by pyrosequencing and quantitative reverse-transcription PCR (qRT-PCR), respectively. We discovered 1,848 genes that were significantly differentially expressed in livers of rats with HCC tumours (CDAA) as compared with healthy livers (CSAA). Upregulated genes in the CDAA group were associated with cancer-related functions, whereas macronutrient metabolic processes were enriched by downregulated genes. Changes of highest magnitude were detected in numerous upregulated genes that govern key oncogenic signalling pathways, including Notch, Wnt, Hedgehog, and extracellular matrix degradation. We further detected perturbations in DNA methylating and demethylating enzymes, which was reflected in decreased global DNA methylation and increased global DNA hydroxymethylation. Four selected upregulated candidates, Mmp12, Jag1, Wnt4, and Smo, demonstrated promoter hypomethylation with the most profound decrease in Mmp12. MMP12 was also strongly overexpressed and hypomethylated in human HCC HepG2 cells as compared with primary hepatocytes, which coincided with binding of Ten-eleven translocation 1 (TET1). Our findings provide comprehensive evidence for gene expression changes and dysregulated epigenome in HCC pathogenesis, potentially revealing novel targets for HCC prevention/treatment.

Characteristics and Clinical Outcomes of Patients With Relapsed/Refractory Diffuse Large B-cell Lymphoma Who Received At Least 3 Lines of Therapies.

INTRODUCTION: The treatment landscape for diffuse large B-cell lymphoma (DLBCL) has recently changed. We examined characteristics and clinical outcomes of DLBCL patients who initiated a third (3L) and fourth (4L) line of therapy during a contemporary time frame. MATERIALS AND METHODS: Adult patients diagnosed with DLBCL who received ≥ 3L after January 1, 2014 were selected from the COTA database. Patients were grouped into cohorts by 3L or 4L initiation and further stratified by type of treatment received: chemotherapy or chemoimmunotherapy (CT/CIT), targeted therapy (TT), chimeric antigen receptor T cells (CAR-T), or salvage therapy consolidated with hematopoietic cell transplant (HCT). Patient characteristics, response rates, and overall survival (OS) were examined. RESULTS: Among adult patients with relapsed/refractory (r/r) DLBCL, 212 (mean age; 61.8 years; 59.0% male) received their 3L and 127 (mean age: 61.0 years; 61.4% male) their 4L. Among those treated with their 3L and 4L, 55.2% and 50.4%, respectively, received CT/CIT; 26.9% and 34.6% received TT. The complete response rate of 3L patients was 9.4% for CT/CIT, 10.5% for TT, and 60% for CAR-T. Similar findings were seen with 4L patients (CT/CIT: 6.3%; TT: 15.9%; CAR-T: 53.8%). For those who received pharmacological treatment in 3L and 4L, median OS times were 7.7 and 4.4 months, respectively. Median OS times of patients who received cell-based therapies (CAR-T/HCT) were not reached. CONCLUSION: In this study, a majority of r/r DLBCL patients were treated with CT/CIT or TT in 3L and 4L settings and had poor clinical outcomes, underscoring the need for more effective treatments.

Pterostilbene Changes Epigenetic Marks at Enhancer Regions of Oncogenes in Breast Cancer Cells.

Epigenetic aberrations are linked to sporadic breast cancer. Interestingly, certain dietary polyphenols with anti-cancer effects, such as pterostilbene (PTS), have been shown to regulate gene expression by altering epigenetic patterns. Our group has proposed the involvement of DNA methylation and DNA methyltransferase 3B (DNMT3B) as vital players in PTS-mediated suppression of candidate oncogenes and suggested a role of enhancers as target regions. In the present study, we assess a genome-wide impact of PTS on epigenetic marks at enhancers in highly invasive MCF10CA1a breast cancer cells. Following chromatin immunoprecipitation (ChIP)-sequencing in MCF10CA1a cells treated with 7 µM PTS for 9 days, we discovered that PTS leads to increased binding of DNMT3B at enhancers of 77 genes, and 17 of those genes display an overlapping decrease in the occupancy of trimethylation at lysine 36 of histone 3 (H3K36me3), a mark of active enhancers. We selected two genes, PITPNC1 and LINC00910, and found that their enhancers are hypermethylated in response to PTS. These changes coincided with the downregulation of gene expression. Of importance, we showed that 6 out of 17 target enhancers, including PITPNC1 and LINC00910, are bound by an oncogenic transcription factor OCT1 in MCF10CA1a cells. Indeed, the six enhancers corresponded to genes with established or putative cancer-driving functions. PTS led to a decrease in OCT1 binding at those enhancers, and OCT1 depletion resulted in PITPNC1 and LINC00910 downregulation, further demonstrating a role for OCT1 in transcriptional regulation. Our findings provide novel evidence for the epigenetic regulation of enhancer regions by dietary polyphenols in breast cancer cells.

Pterostilbene leads to DNMT3B-mediated DNA methylation and silencing of OCT1-targeted oncogenes in breast cancer cells.

Transcription factor (TF)-mediated regulation of genes is often disrupted during carcinogenesis. The DNA methylation state of TF-binding sites may dictate transcriptional activity of corresponding genes. Stilbenoid polyphenols, such as pterostilbene (PTS), have been shown to exert anticancer action by remodeling DNA methylation and gene expression. However, the mechanisms behind these effects still remain unclear. Here, the dynamics between oncogenic TF OCT1 binding and de novo DNA methyltransferase DNMT3B binding in PTS-treated MCF10CA1a invasive breast cancer cells has been explored. Using chromatin immunoprecipitation (ChIP) followed by next generation sequencing, we determined 47 gene regulatory regions with decreased OCT1 binding and enriched DNMT3B binding in response to PTS. Most of those genes were found to have oncogenic functions. We selected three candidates, PRKCA, TNNT2, and DANT2, for further mechanistic investigation taking into account PRKCA functional and regulatory connection with numerous cancer-driving processes and pathways, and some of the highest increase in DNMT3B occupancy within TNNT2 and DANT2 enhancers. PTS led to DNMT3B recruitment within PRKCA, TNNT2, and DANT2 at loci that also displayed reduced OCT1 binding. Substantial decrease in OCT1 with increased DNMT3B binding was accompanied by PRKCA promoter and TNNT2 and DANT2 enhancer hypermethylation, and gene silencing. Interestingly, DNA hypermethylation of the genes was not detected in response to PTS in DNMT3B-CRISPR knockout MCF10CA1a breast cancer cells. It indicates DNMT3B-dependent methylation of PRKCA, TNNT2, and DANT2 upon PTS. Our findings provide a better understanding of mechanistic players and their gene targets that possibly contribute to the anticancer action of stilbenoid polyphenols.

Author Correction: DNA methylation landscape of triple-negative ductal carcinoma in situ (DCIS) progressing to the invasive stage in canine breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

DNA methylation landscape of triple-negative ductal carcinoma in situ (DCIS) progressing to the invasive stage in canine breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer unresponsive to traditional receptor-targeted treatments, leading to a disproportionate number of deaths. Invasive breast cancer is believed to evolve from non-invasive ductal carcinoma in situ (DCIS). Detection of triple-negative DCIS (TN-DCIS) is challenging, therefore strategies to study molecular events governing progression of pre-invasive TN-DCIS to invasive TNBC are needed. Here, we study a canine TN-DCIS progression and investigate the DNA methylation landscape of normal breast tissue, atypical ductal hyperplasia (ADH), DCIS and invasive breast cancer. We report hypo- and hypermethylation of genes within functional categories related to cancer such as transcriptional regulation, apoptosis, signal transduction, and cell migration. DNA methylation changes associated with cancer-related genes become more pronounced at invasive breast cancer stage. Importantly, we identify invasive-only and DCIS-specific DNA methylation alterations that could potentially determine which lesions progress to invasive cancer and which could remain as pre-invasive DCIS. Changes in DNA methylation during TN-DCIS progression in this canine model correspond with gene expression patterns in human breast tissues. This study provides evidence for utilizing methylation status of gene candidates to define late-stage (DCIS and invasive), invasive stage only or DCIS stage only of TN-DCIS progression.

Failure to administer recommended chemotherapy: acceptable variation or cancer care quality blind spot?

BACKGROUND: Chemotherapy quality measures consider hospitals compliant when chemotherapy is recommended, even if it is not received. This may mask shortcomings in cancer care delivery. Objectives of this study were to (1) identify patient factors associated with failure to receive recommended chemotherapy without a documented contraindication and (2) assess hospital variation in failure to administer recommended chemotherapy. METHODS: Patients from 2005 to 2015 with breast, colon and lung cancers who failed to receive recommended chemotherapy were identified using the National Cancer Database. Hospital-level rates of failure to administer recommended chemotherapy were calculated, and patient and hospital factors associated with failure to receive recommended chemotherapy were identified by multivariable logistic regression. RESULTS: A total of 183 148 patients at 1281 hospitals were analysed. Overall, 3.5% of patients with breast, 6.6% with colon and 10.7% with lung cancers failed to receive recommended chemotherapy. Patients were less likely to receive recommended chemotherapy in all cancers if uninsured or on Medicaid (p<0.05), as were non-Hispanic black patients with both breast and colon cancer (p<0.001). Significant hospital variation was observed, with hospital-level rates of failure to administer recommended chemotherapy as high as 21.8% in breast, 40.2% in colon and 40.0% in lung cancers. CONCLUSIONS AND RELEVANCE: Though overall rates are low, failure to receive recommended chemotherapy is associated with sociodemographic factors. Hospital variation in failure to administer recommended chemotherapy is masked by current quality measure definitions and may define a significant and unmeasured difference in hospital quality.

Cost burden and resource utilization in patients with chronic rhinosinusitis and nasal polyps.

OBJECTIVES/HYPOTHESIS: Establish treatment patterns and economic burden in US patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) versus without chronic rhinosinusitis (CRS). Determine comparative costs of subgroups with high clinical burden. STUDY DESIGN: Observational, retrospective, case-control study. METHODS: This study matched patients with CRSwNP to patients without CRS (1:1) using the Truven Health MarketScan US claims database. Categorical and continuous variables were compared using McNemar test and paired t test (normal distribution) or Wilcoxon signed rank tests (non-normal distribution). Within subgroups, χ2 and Wilcoxon or t tests were used (normal distribution). RESULTS: There were 10,841 patients with CRSwNP and 10,841 patients without CRS included. Mean age in the CRSwNP cohort was 45.8 years; 56.2% were male. During follow-up, patients with CRSwNP had an increased diagnosis of asthma versus patients without CRS (20.8% vs. 8.1%, respectively; P < .001). Annual incremental costs were $11,507 higher for patients with CRSwNP versus those without CRS. Costs were higher in subgroups of patients with CRSwNP undergoing functional endoscopy sinus surgery (FESS), with a comorbid diagnosis of asthma, receiving oral corticosteroids, or macrolides versus the overall CRSwNP group. Patients with CRSwNP undergoing FESS had the highest costs of the four subgroups ($26,724, $22,456, $20,695, and $20,990, respectively). CONCLUSIONS: Annual incremental costs were higher among patients with CRSwNP versus without CRS. Patients with CRSwNP with high clinical burden had higher overall costs than CRSwNP patients without. LEVEL OF EVIDENCE: NA Laryngoscope, 129:1969-1975, 2019.

Esophageal Intramural Pseudodiverticulosis and Concomitant Eosinophilic Esophagitis: A Case Series.

Background. Esophageal intramural pseudodiverticulosis (EIPD) is an idiopathic benign chronic disease characterized by flask-like outpouchings of the esophageal wall. It is unknown whether there is a genuine association between EIPD and eosinophilic esophagitis (EoE). Aims. To investigate a possible relationship between EIPD and EoE. Methods. Patients with radiographic or endoscopic evidence of pseudodiverticulosis were identified from the database at a single academic center. Cases were analyzed in three areas: clinical information, endoscopic findings, and course. Results. Sixteen cases of esophageal pseudodiverticulosis were identified. Five patients had histologic evidence of eosinophilic esophagitis. Patients with EoE had pseudodiverticula in the mid-to-distal esophagus while those with EIPD had pseudodiverticula predominantly in the proximal esophagus (p < 0.001). EoE with pseudodiverticulosis occurred in younger patients (p < 0.019). Food bolus obstructions were more common in patients with EoE and pseudodiverticulosis than in EIPD (p < 0.034). Conclusions. This is the first case series supporting a potential association between EoE and pseudodiverticulosis. We also identify characteristic features of pseudodiverticulosis that may raise clinical suspicion of underlying eosinophilic esophagitis.

Family history of alcohol use disorders and neuromaturation: a functional connectivity study with adolescents.

BACKGROUND: A positive family history (FHP) of alcohol use disorders (AUD) is linked to increased risk for personal AUD, but the mechanisms behind this risk are unclear. Previous research suggests that a subtle neurodevelopmental lag in FHP adolescents may contribute to risk for future AUD. METHODS: Functional magnetic resonance imaging (fMRI) response to a spatial working memory (SWM) task was examined for markers of neuromaturational delay in 85 youth with and without FHP. It was hypothesized that FHP adolescents (n = 24, ages 12-14 years), as compared to matched FHN youth (n = 26, ages 12-14 years), would show less similarity to brain connectivity observed in older adolescents (n = 35, ages 16-20 years) and that statistical comparison of SWM functional connectivity models would differentiate FHN and FHP youth. Structural equation modeling tested the fit of brain response connectivity between FH groups and against the older-adolescent model. RESULTS: Patterns of connectivity were more similar between older adolescent and FHN than FHP adolescents; FHP youth demonstrated higher association between right posterior and left frontal brain regions than FHN and older adolescent youth. Comparison of FH groups indicated a significant difference on the pathway from the right superior parietal lobule to the left middle frontal gyrus. CONCLUSIONS: These findings provide additional support for the notion of a neuromaturational lag in FHP youth. Protracted neuromaturation may be a mechanism by which FH increases risk for alcohol dependence, and this less mature neural connectivity pattern may provide a novel endophenotype for identifying youth at risk for drinking problems.

Prefrontal cortex morphometry in abstinent adolescent marijuana users: subtle gender effects.

Adult human studies suggest frontal dysfunction associated with chronic marijuana (MJ) use, but due to continued neuromaturation, adult studies may not generalize to adolescents. This study characterized prefrontal cortex (PFC) morphometry in chronic MJ-using adolescents following 1 month of monitored abstinence. Data were collected from MJ users (n = 16) and controls (n = 16) aged 16-18. Extensive exclusionary criteria included co-morbid psychiatric and neurologic disorders. Substance use and anatomical measures were collected after 28 days of monitored abstinence. PFC volumes were ascertained from manual tracing by reliable raters on high-resolution magnetic resonance images. After controlling for lifetime alcohol use, gender and intracranial volume, MJ users did not differ from controls in PFC volume. However, marginal group-by-gender interactions were observed (P < 0.09): female MJ users demonstrated comparatively larger PFC volumes while male MJ users had smaller volumes compared with same-gender controls. Further, group status and total PFC volume interacted in predicting executive functioning (P < 0.05). Among MJ users, smaller PFC total volume was associated with better executive functioning while the opposite pattern was seen among the controls. These preliminary results indicate that gender may moderate the relationship between MJ use and PFC morphometry. Given the relationship between larger PFC total volumes and poorer executive functioning among MJ users, female MJ users may be at increased risk for neurocognitive consequences. Future research will measure PFC gray and white matter separately and follow boys and girls over adolescence to examine the influence of MJ use on neurodevelopment.

Critical negative regulation of type 1 T cell immunity and immunopathology by signaling adaptor DAP12 during intracellular infection.

Transmembrane signaling adaptor DAP12 has increasingly been recognized for its important role in innate responses. However, its role in the regulation of antimicrobial T cell responses has remained unknown. In our current study, we have examined host defense, T cell responses, and tissue immunopathology in models of intracellular infection established in wild-type and DAP12-deficient mice. During mycobacterial infection, lack of DAP12 leads to pronounced proinflammatory and Th1 cytokine responses, overactivation of Ag-specific CD4 and CD8 T cells of type 1 phenotype, and heightened immunopathology both in the lung and lymphoid organs. DAP12-deficient airway APC display enhanced NF-kappaB activation and cytokine responses upon TLR stimulation or mycobacterial infection in vitro. Of importance, adoptive transfer of Ag-loaded DAP12-deficient APC alone could lead to overactivation of transferred transgenic or endogenous wild-type T cells in vivo. We have further found that the immune regulatory role by DAP12 is not restricted only to intracellular bacterial infection, since lack of this molecule also leads to uncontrolled type 1 T cell activation and severe immunopathology and tissue injury during intracellular viral infection. Our study thus identifies DAP12 as an important novel immune regulatory molecule that acts, via APC, to control the level of antimicrobial type 1 T cell activation and immunopathology.

Solitary fibrous tumor of the retropharynx causing obstructive sleep apnea.

Solitary fibrous tumors (SFTs) are rare, usually benign, spindle cell neoplasms that most often originate near mesothelium-lined surfaces of the pleural or peritoneal cavity. SFTs reported in the head and neck occur most commonly in the oral cavity, sinonasal tract, and orbit. We report a case of SFT of the retropharynx causing severe obstructive sleep apnea. The diagnostic and management strategies of SFTs are discussed.

TNF-alpha is a critical negative regulator of type 1 immune activation during intracellular bacterial infection.

TNF-alpha has long been regarded as a proimmune cytokine involved in antimicrobial type 1 immunity. However, the precise role of TNF-alpha in antimicrobial type 1 immunity remains poorly understood. We found that TNF-alpha-deficient (TNF(-/-)) mice quickly succumbed to respiratory failure following lung infection with replication-competent mycobacteria, because of apoptosis and necrosis of granuloma and lung structure. Tissue destruction was a result of an uncontrolled type 1 immune syndrome characterized by expansion of activated CD4 and CD8 T cells, increased frequency of antigen-specific T cells, and overproduction of IFN-gamma and IL-12. Depletion of CD4 and CD8 T cells decreased IFN-gamma levels, prevented granuloma and tissue necrosis, and prolonged the survival of TNF(-/-) hosts. Early reconstitution of TNF-alpha by gene transfer reduced the frequency of antigen-specific T cells and improved survival. TNF-alpha controlled type 1 immune activation at least in part by suppressing T cell proliferation, and this suppression involved both TNF receptor p55 and TNF receptor p75. Heightened type 1 immune activation also occurred in TNF(-/-) mice treated with dead mycobacteria, live replication-deficient mycobacteria, or mycobacterial cell wall components. Our study thus identifies TNF-alpha as a type 1 immunoregulatory cytokine whose primary role, different from those of other type 1 cytokines, is to keep an otherwise detrimental type 1 immune response in check.

Biosynthetic origin and functional significance of murine platelet factor V.

Factor V (FV), a central regulatory protein in hemostasis, is distributed into distinct plasma and platelet compartments. Although platelet FV is highly concentrated within the platelet alpha-granule, previous analysis of human bone marrow and liver transplant recipients has demonstrated that platelet FV in these individuals originates entirely from the uptake of plasma FV. In order to examine further the biosynthetic origins of the platelet and plasma FV pools, we performed bone marrow transplantations of Fv-null (Fv-/-) fetal liver cells (FLCs) into wild-type mice. Fractionation of whole blood from control mice demonstrated that approximately 14% of total blood FV activity is platelet-associated. Mice that received transplants of Fv-null FLCs displayed a high degree of engraftment and appeared grossly normal, with no evidence for spontaneous hemorrhage. Although total FV levels in Fv-null FLC recipients were only mildly decreased, the FV activity within the platelet compartment was reduced to less than 1% of that in normal mice. We conclude that the murine platelet FV compartment is derived exclusively from primary biosynthesis within cells of marrow origin, presumably megakaryocytes, and that an intact platelet FV pool is not required for protection from spontaneous hemorrhage or bleeding following minor trauma.