High clonal diversity and spatial genetic admixture in early prostate cancer and surrounding normal tissue.

Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: 'pseudo-diploid' cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.

Prostate cancer disease recurrence after radical prostatectomy is associated with HLA type and local cytomegalovirus immunity.

Prostate cancer is a heterogeneous disease with a need for new prognostic biomarkers. Human leukocyte antigen (HLA) genes are highly polymorphic genes central to antigen presentation to T-cells. Two alleles, HLA-A*02:01 and HLA-A*24:02, have been associated with prognosis in patients diagnosed with de novo metastatic prostate cancer. We leveraged the next-generation sequenced cohorts CPC-GENE and TCGA-PRAD to examine HLA alleles, antiviral T-cell receptors and prostate cancer disease recurrence after prostatectomy. Carrying HLA-A*02:01 (111/229; 48% of patients) was independently associated with disease recurrence in patients with low-intermediate risk prostate cancer. HLA-A*11 (carried by 42/441; 10% of patients) was independently associated with rapid disease recurrence in patients with high-risk prostate cancer. Moreover, HLA-A*02:01 carriers in which anti-cytomegalovirus T-cell receptors (CMV-TCR) were identified in tumors (13/144; 10% of all patients in the cohort) had a higher risk of disease recurrence than CMV-TCR-negative patients. These findings suggest that HLA-type and CMV immunity may be valuable biomarkers for prostate cancer progression.

Diploid hepatocytes drive physiological liver renewal in adult humans.

Physiological liver cell replacement is central to maintaining the organ's high metabolic activity, although its characteristics are difficult to study in humans. Using retrospective radiocarbon (14C) birth dating of cells, we report that human hepatocytes show continuous and lifelong turnover, allowing the liver to remain a young organ (average age <3 years). Hepatocyte renewal is highly dependent on the ploidy level. Diploid hepatocytes show more than 7-fold higher annual birth rates than polyploid hepatocytes. These observations support the view that physiological liver cell renewal in humans is mainly dependent on diploid hepatocytes, whereas polyploid cells are compromised in their ability to divide. Moreover, cellular transitions between diploid and polyploid hepatocytes are limited under homeostatic conditions. With these findings, we present an integrated model of homeostatic liver cell generation in humans that provides fundamental insights into liver cell turnover dynamics.

Low temperature induces von-willebrand factor expression via increased early growth response 1 transcriptional activity in splenic sinusoidal endothelial cells.

von Willebrand factor (vWF) is a large plasma glycoprotein that plays an important role in hemostasis by forming molecular bridges with platelets following vascular injury. Previously, we reported that hypothermia enhanced vWF production in the spleen, which resulted in the activation of the platelet pool in a hypothermia-induced murine model. However, the mechanisms that regulate vWF expression under hypothermic conditions remain unclear. In this study, we focused on vWF expression under hypothermic conditions in splenic endothelial cell culture. Human splenic endothelial cells (HSEC) were incubated at 20 °C for 1 h. Total RNA was extracted from the cells, and cDNA microarray gene expression analysis was performed. Genes that may be associated with vWF expression in low temperature culture conditions were then selected for further analysis. Gene expression analysis showed that low temperature conditions increased the expression of FOS and EGR1. We then hypothesized that these factors upregulate vWF mRNA expression in HSEC. The transcriptional inhibitors of EGR1 significantly inhibited vWF mRNA expression in HSEC cultured at a low temperature. Our analysis revealed that low temperatures enhance the gene expression of EGR1, which transcriptionally increases vWF expression. This acute-phase reaction may play an important role in platelet activation in the spleen during hypothermia.

NF-κB-Associated Pain-Related Neuropeptide Expression in Patients with Degenerative Disc Disease.

The role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) has been highlighted in mechanisms underlying inflammatory and neuropathic pain processes. The present study was designed to investigate whether NF-κB signaling is associated with pain-related neuropeptide expression in patients with chronic back pain related to degenerative disc disease (DDD). Intervertebral disc (IVD) tissues were collected from forty DDD patients undergoing disc replacement or fusion surgery, and from eighteen postmortem (PM) control subjects. RELA, NFKB1, CGRP, TAC1, TRPV1, and MMP-3 gene expression were analyzed by RT-qPCR, while NF-κB subunit RelA and NF-κB1⁻DNA binding in nuclear extracts and calcitonin gene related peptide (CGRP), substance P (SP), and transient receptor potential, subfamily V, member 1 (TRPV1) protein levels in cytosolic extracts of tissues were assessed by enzyme-linked immunosorbent assay (ELISA). An upregulated NF-κB1⁻DNA binding, and higher CGRP and TRPV1 protein levels were observed in DDD patients compared to PM controls. In DDD patients, NF-κB1⁻DNA binding was positively correlated with nuclear RelA levels. Moreover, NF-κB1⁻DNA binding was positively associated with TRPV1 and MMP-3 gene and SP and TRPV1 protein expression in DDD patients. Our results indicate that the expression of SP and TRPV1 in IVD tissues was associated with NF-κB activation. Moreover, NF-κB may be involved in the generation or maintenance of peripheral pain mechanisms by the regulation of pain-related neuropeptide expression in DDD patients.

Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease.

Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.

Activation of NF-κB in Synovium versus Cartilage from Patients with Advanced Knee Osteoarthritis: A Potential Contributor to Inflammatory Aspects of Disease Progression.

The aim was to assess the activation and association of the NF-κB system across synovial membrane (SM) and articular cartilage (AC) in patients with knee osteoarthritis (OA) and ascertain its potential effects on catabolic mediator expression in advanced OA. SM and AC were obtained from 40 OA patients undergoing total knee arthroplasty and from 19 postmortem control subjects. NF-κB subunit RelA in nuclear and cytosolic fractions and NF-κB1-DNA binding in nuclear extracts was assessed by ELISA, whereas NFKB1, RELA, IL-8, IL-6, and MMP3 gene expression were analyzed by reverse transcriptase-quantitative PCR in tissues. We observed higher SM nuclear RelA protein levels and upregulated NF-κB1-DNA binding in OA patients compared with postmortem controls. However, in AC, lower nuclear RelA levels were observed compared with cytosolic extracts in patients. Nuclear RelA levels correlated positively with NF-κB1-DNA binding in SM and AC in patients. SM RELA and MMP3 mRNA levels were upregulated, whereas IL-8 and IL-6 as well as AC RELA were downregulated in patients compared with controls. In SM, nuclear RelA levels correlated positively with MMP3 gene expression in patients. A negative correlation was observed between SM nuclear RelA levels and AC NF-κB1-DNA binding, and SM nuclear NF-κB1-DNA binding correlated negatively with AC MMP3 and NFKB1 mRNA levels in patients. These findings highlight NF-κB-triggered cross-talk and feedback mechanisms between SM and AC in OA. Further, our findings strongly support a role for an activated NF-κB system in the transcriptional mechanism of inflammatory processes, especially in SM of patients with advanced OA.

Comorbidities in relation to fatality of first myocardial infarction.

INTRODUCTION: Present knowledge concerning potential associations between comorbidities and the fatality of a first myocardial infarction (MI) is limited. AIM: To identify comorbidities in 45-70-year-old individuals who suffered a first MI and died within 7 days in Stockholm County from 1992-1994. In addition, to assess how each of the comorbidities identified, as well as the number of hospitalizations during the 10-year period prior to the MI, was associated with MI fatality. METHODS: The data collected on our inception cohort of 1984 first MI, of which 524 were fatal within 7 days, were primarily self-reported, proxy-reported by questionnaire and/or extracted from comprehensive national registers. Comorbidities among fatal cases with a prevalence >2% were identified. Risk ratios (with 95% confidence intervals) for the association of MI fatality with number of prior hospitalizations and specific comorbidities were calculated using binomial regression with log link. A structured review of autopsy reports on fatal cases was performed in order to identify additional indicators of comorbidities. RESULTS: After adjusting for sex, age and disposable income, the number of previous hospitalizations was associated with 7-day MI fatality. Of the comorbidities identified as prevalent in fatal cases, the following were associated with 7-day fatality in crude analysis: epilepsy, heart failure, stroke, alcoholism, cancer, renal diseases, asthma, psychiatric diseases, diabetes, and rheumatoid arthritis. Indicators of comorbidities identified from autopsy data included a silent MI, severe atherosclerosis of the abdominal aorta, and hepatic steatosis. Adjustments for sex and age (although not possible for epilepsy and alcoholism), did not substantially alter results. CONCLUSIONS: Our current findings indicate that in connection with a first MI, particular attention should be paid to those with repeated prior hospitalizations and/or epilepsy, heart failure, stroke, alcoholism, cancer, renal diseases, asthma, psychiatric diseases, diabetes and rheumatoid arthritis.

The Swedish cause of death register.

Sweden has a long tradition of recording cause of death data. The Swedish cause of death register is a high quality virtually complete register of all deaths in Sweden since 1952. Although originally created for official statistics, it is a highly important data source for medical research since it can be linked to many other national registers, which contain data on social and health factors in the Swedish population. For the appropriate use of this register, it is fundamental to understand its origins and composition. In this paper we describe the origins and composition of the Swedish cause of death register, set out the key strengths and weaknesses of the register, and present the main causes of death across age groups and over time in Sweden. This paper provides a guide and reference to individuals and organisations interested in data from the Swedish cause of death register.

Dynamic Changes in Brain Mesenchymal Perivascular Cells Associate with Multiple Sclerosis Disease Duration, Active Inflammation, and Demyelination.

Vascular changes, including blood brain barrier destabilization, are common pathological features in multiple sclerosis (MS) lesions. Blood vessels within adult organs are reported to harbor mesenchymal stromal cells (MSCs) with phenotypical and functional characteristics similar to pericytes. We performed an immunohistochemical study of MSCs/pericytes in brain tissue from MS and healthy persons. Post-mortem brain tissue from patients with early progressive MS (EPMS), late stage progressive MS (LPMS), and healthy persons were analyzed for the MSC and pericyte markers CD146, platelet-derived growth factor receptor beta (PDGFRß), CD73, CD271, alpha-smooth muscle actin, and Ki67. The MS samples included active, chronic active, chronic inactive lesions, and normal-appearing white matter. MSC and pericyte marker localization were detected in association with blood vessels, including subendothelial CD146+ PDGFRß+ Ki67+ cells and CD73+ CD271+ PDGFRß+ Ki67- cells within the adventitia and perivascular areas. Both immunostained cell subpopulations were termed mesenchymal perivascular cells (MPCs). Quantitative analyses of immunostainings showed active lesions containing increased regions of CD146+ PDGFRß+ Ki67+ and CD73+ CD271+ PDGFRß+ Ki67- MPC subpopulations compared to inactive lesions. Chronic lesions presented with decreased levels of CD146+ PDGFRß+ Ki67+ MPC cells compared to control tissue. Furthermore, LPMS lesions displayed increased numbers of blood vessels harboring greatly enlarged CD73+ CD271+ adventitial and perivascular areas compared to control and EPMS tissue. In conclusion, we demonstrate the presence of MPC subgroups in control human brain vasculature, and their phenotypic changes in MS brain, which correlated with inflammation, demyelination and MS disease duration. Our findings demonstrate that brain-derived MPCs respond to pathologic mechanisms involved in MS disease progression and suggest that vessel-targeted therapeutics may benefit patients with progressive MS. Stem Cells Translational Medicine 2017;6:1840-1851.

The Lifespan and Turnover of Microglia in the Human Brain.

The hematopoietic system seeds the CNS with microglial progenitor cells during the fetal period, but the subsequent cell generation dynamics and maintenance of this population have been poorly understood. We report that microglia, unlike most other hematopoietic lineages, renew slowly at a median rate of 28% per year, and some microglia last for more than two decades. Furthermore, we find no evidence for the existence of a substantial population of quiescent long-lived cells, meaning that the microglia population in the human brain is sustained by continuous slow turnover throughout adult life.

Opioid precursor protein isoform is targeted to the cell nuclei in the human brain.

BACKGROUND: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain. METHODS: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus. RESULTS: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging. CONCLUSIONS AND GENERAL SIGNIFICANCE: High levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.

Dynamics of Cell Generation and Turnover in the Human Heart.

The contribution of cell generation to physiological heart growth and maintenance in humans has been difficult to establish and has remained controversial. We report that the full complement of cardiomyocytes is established perinataly and remains stable over the human lifespan, whereas the numbers of both endothelial and mesenchymal cells increase substantially from birth to early adulthood. Analysis of the integration of nuclear bomb test-derived (14)C revealed a high turnover rate of endothelial cells throughout life (>15% per year) and more limited renewal of mesenchymal cells (<4% per year in adulthood). Cardiomyocyte exchange is highest in early childhood and decreases gradually throughout life to <1% per year in adulthood, with similar turnover rates in the major subdivisions of the myocardium. We provide an integrated model of cell generation and turnover in the human heart.

Opioid-related mortality and filled prescriptions for buprenorphine and methadone.

INTRODUCTION AND AIMS: To assess opioid-related mortality and correlation with filled prescriptions for buprenorphine and methadone. DESIGN AND METHODS: A register study, including data from the Swedish Forensic Pathology and Forensic Toxicology databases 2003-2010, the Prescribed Drug Register and the National Patient Register. RESULTS: A total of 1301 deaths, assessed as related to buprenorphine, methadone or heroin, or a combination of them, were studied. The largest number of fatalities was related to intake of heroin (n = 776), followed by methadone (n = 342) and buprenorphine (n = 168). The total annual number of fatal cases related to the studied drugs more than doubled (116 to 255) during the study period. There were increases in mortality related to both buprenorphine and methadone: from 1 to 49 cases for buprenorphine, and from 19 to 81 cases for methadone. Only one-fifth of the fatal cases had a filled prescription for the maintenance drug assessed as the cause of death. DISCUSSION AND CONCLUSION: This study showed that most fatalities were not related to filled prescriptions of maintenance drugs, and a substantial illicit use of buprenorphine and methadone resulting in deaths was revealed. To prevent opioid toxicity deaths it is important to make efforts not only to reduce drug diversion from maintenance programs, but also to improve the control of drug trafficking and other illegal sources.

Pathophysiological effects and changes in potassium, ionised calcium, glucose and haemoglobin early after severe blunt chest trauma.

BACKGROUND: Severe lung contusion is often observed after blunt chest trauma due to traffic accidents or fall from heights, but may also occur after a non-penetrating ballistic impact against body armour. Such trauma has been designated behind armour blunt trauma (BABT). Our aim in the present study has been to evaluate pathophysiological changes and compensatory mechanisms that occur early after such severe lung contusion. METHODS: Twelve pigs wearing body armour were shot with a 7.62mm assault rifle to produce a standardised pulmonary contusion. Exposed animals were compared with five control animals shot with blank ammunition. Physiological parameters and levels of potassium, glucose, haemoglobin, calcium, lactate and pH were monitored for two hours after the shot. RESULTS: The impact induced severe pulmonary contusion with apnoea, desaturation and hypotension in all exposed animals. Increased haemoglobin, glucose and severe hyperkalaemia were seen shortly after impact. Seven of twelve animals died due to the trauma. Dense cardiac tissue was observed during post mortem examination in six of the animals that died during the experimental course. CONCLUSION: In conclusion, this study has shown that life-threatening hyperkalaemia occurs early after severe lung contusion. Moreover, dense cardiac tissue and early increase of haemoglobin and glucose are intriguing findings that should be investigated in future studies.

The desmoplastic stroma plays an essential role in the accumulation and modulation of infiltrated immune cells in pancreatic adenocarcinoma.

Tumor microenvironment is composed of tumor cells, fibroblasts, and infiltrating immune cells, which all work together and create an inflammatory environment favoring tumor progression. The present study aimed to investigate the role of the desmoplastic stroma in pancreatic ductal adenocarcinoma (PDAC) regarding expression of inflammatory factors and infiltration of immune cells and their impact on the clinical outcome. The PDAC tissues examined expressed significantly increased levels of immunomodulatory and chemotactic factors (IL-6, TGFß, IDO, COX-2, CCL2, and CCL20) and immune cell-specific markers corresponding to macrophages, myeloid, and plasmacytoid dendritic cells (DCs) as compared to controls. Furthermore, short-time survivors had the lowest levels of DC markers. Immunostainings indicated that the different immune cells and inflammatory factors are mainly localized to the desmoplastic stroma. Therapies modulating the inflammatory tumor microenvironment to promote the attraction of DCs and differentiation of monocytes into functional DCs might improve the survival of PDAC patients.

Interleukin 1α sustains the expression of inflammatory factors in human pancreatic cancer microenvironment by targeting cancer-associated fibroblasts.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) is dynamic, with an extensive interaction between the stroma and tumor cells. The aim of this study was to delineate the cross talk between PDAC and cancer-associated fibroblasts (CAFs), with a focus on the mechanism creating the chronic inflammatory tumor milieu. We assessed the effects of the cross talk between PDAC and CAF cell lines on the creation and sustenance of the inflammatory tumor microenvironment in pancreatic cancer. The coculture of PDAC and CAF cell lines enhanced the levels of inflammatory factors including IL-1α, IL-6, CXCL8, VEGF-A, CCL20, and COX-2. CAFs were superior to tumor cells regarding the production of most inflammatory factors, and tumor cell-associated IL-1α was established as the initiator of the enhanced production of inflammatory factors through the binding of IL-1α to IL-1 receptor 1 (IL-1R1) expressed predominantly by CAFs. Furthermore, we found a correlation between IL-1α and CXCL8 expression levels in PDAC tissues and correlation between IL-1α expression and the clinical outcome of the patients. This confirmed an important role for the IL-1 signaling cascade in the creation and sustenance of a tumor favorable microenvironment. Neutralization of the IL-1α signaling efficiently diminished the cross talk-induced production of inflammatory factors. These data suggest that the cross talk between PDAC cells and the main stroma cell type, i.e. CAFs, is one essential factor in the formation of the inflammatory tumor environment, and we propose that neutralization of the IL-1α signaling might be a potential therapy for this cancer.

Differential forms of p53 in medulloblastoma primary tumors, cell lines and xenografts.

Medulloblastoma is the most common malignant brain tumor in childhood. The most prevalent chromosomal abnormalities are isochromosome 17q and loss of 17p, the location of the tumor suppressor gene p53. Mutations in the p53 gene in medulloblastoma are relatively infrequent but have recently been correlated to poor prognosis. Furthermore, the p53 gene encodes nine different isoforms, which may have a profound impact on p53 tumor suppressor activity. Nine medulloblastoma primary biopsy samples, six cell lines from medulloblastoma, and one from a supratentorial PNET, and a medulloblastoma xenograft, along with human brain and visceral tissues, were analyzed by Western blotting, using monoclonal p53 antibodies against two regions in the N-terminus or the central domain. Medulloblastoma primary tissue and xenografts present low molecular weight proteins recognized by both N-terminal p53 antibodies that are absent in all cell lines including the one used for xenografts. Normal visceral organs display short forms of p53, and low levels of canonical p53. Normal brain structures, including cerebellum, contained only canonical size p53 at high levels. In conclusion, our results indicate that the presence of p53 isoforms may play a functional role in medulloblastoma. The observed differences in their presence in cell lines and derived xenografts, suggest that p53 should be investigated in in vivo models rather than in cell lines.

Effects of fluid resuscitation with hypertonic saline dextrane or Ringer's acetate after nonhemorrhagic shock caused by pulmonary contusion.

BACKGROUND: Injured lungs are sensitive to fluid resuscitation after trauma. Such treatment can increase lung water content and lead to desaturation. Hypertonic saline with dextran (HSD) has hyperosmotic properties that promote plasma volume expansion, thus potentially reducing these side effects. The aim of this study was to (1) evaluate whether fluid treatment counteracts hypotension and improves survival after nonhemorrhagic shock caused by lung contusion and (2) analyze whether resuscitation with HSD is more efficient than treatment with Ringer's acetate (RA) in terms of blood oxygenation, the amount of lung water, circulatory effects, and inflammatory response. METHODS: Twenty-nine pigs, all wearing body armor, were shot with a 7.62-mm assault rifle to produce a standardized pulmonary contusion. These animals were allocated into three groups: HSD, RA, and an untreated shot control group. Exposed animals were compared with animals not treated with fluid and shot with blank ammunition. For 2 hours after the shot, the inflammatory response and physiologic parameters were monitored. RESULTS: The impact induced pulmonary contusion, desaturation, hypotension, increased heart rate, and led to an inflammatory response. No change in blood pressure was observed after fluid treatment. HSD treatment resulted in significantly less lung water (p < 0.05) and tended to give better Pao2 (p = 0.09) than RA treatment. Tumor necrosis factor-α release and heart rate were significantly lower in animals given fluids. CONCLUSION: Fluid treatment does not affect blood pressure or mortality in this model of nonhemorrhagic shock caused by lung contusion. However, our data indicate that HSD, when compared with RA, has advantages for the injured lung.