For almost two decades, cell-based therapies have been tested in modern regenerative medicine to either replace or regenerate human cells, tissues, or organs and restore normal function. Secreted paracrine factors are increasingly accepted to exert beneficial biological effects that promote tissue regeneration. These factors are called the cell secretome and include a variety of proteins, lipids, microRNAs, and extracellular vesicles, such as exosomes and microparticles. The stem cell secretome has most commonly been investigated in pre-clinical settings. However, a growing body of evidence indicates that other cell types, such as peripheral blood mononuclear cells (PBMCs), are capable of releasing significant amounts of biologically active paracrine factors that exert beneficial regenerative effects. The apoptotic PBMC secretome has been successfully used pre-clinically for the treatment of acute myocardial infarction, chronic heart failure, spinal cord injury, stroke, and wound healing. In this review we describe the benefits of choosing PBMCs instead of stem cells in regenerative medicine and characterize the factors released from apoptotic PBMCs. We also discuss pre-clinical studies with apoptotic cell-based therapies and regulatory issues that have to be considered when conducting clinical trials using cell secretome-based products. This should allow the reader to envision PBMC secretome-based therapies as alternatives to all other forms of cell-based therapies. The concept of using paracrine factors in a therapeutic context in regenerative medicine can be seen as a consequence of the stem cell theory in the beginning of the twenty-first century. Here we will discuss the beginnings of stem cell research, with its promising initial results and subsequent disillusion, followed by the development of the secretome concept in regenerative medicine. We will discuss the term “secretome”, characterize the components of the cell secretome, describe its effects in pre-clinical and clinical studies, and then outline the challenges and opportunities of secretome-based therapies in regenerative medicine.The origin of modern cell-free therapies can be traced back to the late nineteenth and early twentieth centuries. The Swiss physician Paul Niehans 1882–1971) coined the term “cell therapy”. His cell therapy patients included celebrities such as Winston Churchill, Pope Pius XII, Agha Kahn, and Charles de Gaulle. Niehans extensively evaluated the effects of endogene rejuvenation via injection of xenogeneic cell suspensions derived from the endocrine glands, heart, kidney, liver, bone marrow, intestinal mucosa, and reticulo-endothelial system into the corresponding organ, studying whether normal function was restored. He also studied blood enriched with leukocytes and hypothesized that this form of treatment could be used for a myriad of degenerative diseases and cancer. Niehans used either fresh xenogeneic cells in suspension or lyophilized xenogeneic cells (vacuum freeze-dried) derived from young animals, such as sheep or animal fetuses, or human fetal cells 882–1971 .Prior to Niehans, physicians experimented with stem cell-like cell types, including Alexander Maksimov in 1909 during his work on blood formation . In addiThe current concept of using autologous or allogeneic stem cells for replacing or engineering injured tissues to re-establish normal biological function was re-envisioned in the beginning of the twenty-first century in the clinical setting of acute myocardial infarction (AMI) and chronic heart failure (HF). Initially, it was thought that the human heart is a post-mitotic and terminally developed organ with no cell renewal capacity, but this dogma was refuted in 2001 when enhanced myocyte cell proliferation was demonstrated following AMI  and thenBased on these encouraging pre-clinical experiments, several groups rapidly transferred the “stem cell potential” to the realm of clinical application. Initial trials, such as the randomized controlled trial TOPCARE-AMI, were able to show minor improvements in left ventricular function following intracoronary injection of autologous progenitor cells . HoweverHow can we interpret the data generated during the last two decades in stem cell research and, more importantly, what lessons have been learned from these investigations? Given that the injection of diverse types of stem cells can promote cardiomyocyte cytoprotection/regeneration pre-clinically, it was initially thought that these stem cells incorporate into the injured organ and transdifferentiate into cardiomyocytes . Later, In contrast, only a small percentage of injected stem cells engraft in the heart . PenickaIf stem cells do not engraft in a biologically relevant manner or demonstrate long-term survival in injured heart , how canCell necrosis-induced inflammatory reactions are an integral part of the pathophysiology of myocardial ischemia. These inflammatory processes augment cardiomyocyte dysfunction and ventricular remodeling. In 2005, Thum et al. proposed that immunomodulatory signals released by injected apoptotic stem cells may be responsible for the beneficial effects observed in the initial clinical trials utilizing ex vivo manipulated stem cells . They drThum’s hypothesis was proven by Ankersmit et al., who sought to answer two major questions using a wild-type rodent model of acute AMI: are PBMCs capable of attenuating myocardial damage when injected intravenously after coronary ligation and does the induction of apoptosis in PBMCs prior to injection alter their biological activity?Using in vitro assays, Ankersmit et al. were able to show that gamma-irradiated apoptotic PBMCs attenuated lipopolysaccharide (LPS)-induced spillage of IL-6 and IL-1β in co-cultured monocytes and PBMCs . In addiIn in vivo experiments using a rodent model of acute AMI induced by ligation of the left anterior descending artery (LAD), the infusion of irradiated PBMC suspensions (apoptotic PBMC-S) reduced infarct size compared to animals receiving non-irradiated PBMCs (living PBMC-S) or cell culture medium alone. Functional parameters  were improved in animals treated with apoptotic PBMC-S . In lineapo = off, away + ptosis = a falling) was coined in 1972 [11 human neutrophils undergo apoptosis [At first glance, the statement “Dying cells can augment cell survival” seems contradictory, but after detailed consideration has an evolutionary justification and rationale. Apoptosis  . WhetherInitially, Ankersmit’s group was able to show that the injection of paracrine factors released from apoptotic PBMCs attenuates myocardial damage and preserves myocardial function following AMI in a wild type rodent model and clinically more relevant porcine large animal model . Pigs reHoetzenecker et al. showed that, in addition to an effect on intracellular myocardial signaling cascades, apoptotic PBMC-S preserved porcine epi/myocardial microvascular perfusion in a closed chest reperfusion infarction model . MicrovaThis work was extended by Pavo et al., who were able to demonstrate that apoptotic PBMC-S exerts regenerative effects in a latent porcine chronic post-myocardial infarction model . In thesThe pathophysiology and molecular processes following ischemic stroke are somewhat similar to those of AMI. Following vascular obstruction due to a ruptured atherosclerotic plaque or arterial thrombus, the downstream cells become hypoxic and eventually apoptotic/necrotic. As in myocardial infarction, immediate restoration of blood flow is the first line treatment in patients with acute ischemic stroke. Altmann et al. showed that, in a rodent model of cerebral stroke, the administration of apoptotic cell secretome reduced the ischemic area by 36 % and improved the functional neurological outcome . In vitrSimilar to stroke, the neurological damage following acute spinal cord injury initiates a short initial trauma followed by a second injury caused by the endogenous immune response to the traumatic injury. This second undesirable phase of spinal cord injury consists of a multilayered and complex combination of inflammatory pathways that are thought to drive ongoing tissue damage via enzymatic degradation, oxidative stress, and blood–brain barrier dysfunction . Haider The PBMC secretome has been shown to enhance wound healing in vivo and in vitro , 105. MiRecently, Hoetzenecker et al. investigated the capacity of apoptotic PBMC-S to modulate myocardial inflammation in a CD4-positive T-cell-dependent murine model of autoimmune myocarditis. Mice receiving apoptotic PBMC–S at the clinical onset of disease had reduced lymphocytic infiltrate, offering a novel treatment option in an experimental myocarditis model. The authors showed that PBMC-S induced caspse-8-dependent apoptosis of autoreactive CD-4-positive T-cells, leading to remission of the decorative autoimmune processes  in a welThe immunomodulatory effects of PBMC-S were further evaluated by Kasiri et al. . In thisBased on these data, we concluded that apoptotic PBMC-S initiates multiple modes of action (MOAs) that eventually lead to cytoprotection and prevention of hypoxic tissue in vivo.A plethora of unresolved questions are still unanswered regarding which component(s), concentration(s), and mode(s) of delivery are responsible for the effects of the PBMC secretome. A single molecule may regulate a broad spectrum of different processes, including platelet inhibition, vasodilation, neo-angiogenesis, and cytoprotection. Another option is a pleiotropy of factors alone or in combination that orchestrate different molecular cascades, leading to the beneficial effects. In order to determine the MOA of the PBMC secretome, we first have to define the secretome itself.Using a systemic approach, Beer et al. tried to classify the secretome components of gamma–irradiated apoptotic PBMCs  based onA growing body of evidence indicates that lipids released from apoptotic cells exert paracrine biological activity , 110. UsIn addition to proteins and lipids, the cell supernatant contains extracellular vesicles that can be isolated using an ultracentrifugation protocol, ultrafiltration, or immunoprecipitation technologies with antibody-tagged magnetic beads . ExtraceUsing both ultracentrifugation and a magnetic bead-based protocol, Beer et al. characterized exosomes and microparticles of irradiated and non-irradiated PBMCs. Gamma-irradiation induced the release of exosomes and microparticles and altered the exosomal protein content. Functional analysis revealed that exosomes and proteins are the two major biological components that induce keratinocyte and fibroblast migration and the secretion of pro-angiogenic proteins.Although the principle components released from apoptotic PBMCs have been characterized, there are questions yet to answer. What is the biologically active component? Which factors interact with each other, whose separation would abolish the biological activity. Based on the complexity of pathological changes during an ischemic injury, it seems rather plausible to target variable pathological pathways using multifaceted drug compounds, such as apoptotic PBMC-S, instead of focusing on one single aspect. The identification of a specific cause and effect relationship on a cellular level is a matter of systems biology rather than to define a drug compound that is potentially able to treat multiple indications of unmet need.Basic science and pre-clinical research should ultimately aim to transfer knowledge into clinical practice. Though stem cell-based clinical trials at the beginning of the twentieth century have paved the way for regenerative medicine, the first clinical trials utilizing the PBMC secretome will successfully provide proof-of-concept in humans for several indications with unmet need. Diabetic foot ulcer, AMI, stroke, and spinal cord injury are the first such indications. Ankersmit’s group recently finished a phase I study utilizing autologous GMP PBMC secretome .In addition to these clinically oriented issues, fundamental basic research has to be done in order to identify the biologically active components in the cell secretome and the mode of action (MOA) of these forms of therapy. For example, exosomes are currently an intensively investigated component of the cell secretome. Future research should focus on a detailed description of the composition of exosomes, including exosomal lipids, mRNAs, miRNAs, proteins, and surface receptors. We should try to understand the biological distribution of systemically injected paracrine factors to further characterize their organ-specific function.In addition, whether apoptosis-induced lipid release has any beneficial effect in regenerative medicine is currently unknown. Oxidized phospholipids exert diverse biological functions in vitro and in vivo, suggesting that this class of lipids is responsible, at least in part, for the beneficial effect of cell secretome-based therapies.Moving towards clinical application, physicians, non-clinical researchers, and investors have collaborations with reciprocal advantages for each discipline. Clinically oriented researchers and non-clinical scientists are currently in fast-moving environments that provide great opportunities for both the development and implementation of novel therapeutic modalities . HoweverIn particular, knowledge of the MOA of a substance helps with regulatory approval. However, based on our experience in cell-based and paracrine research, a simplification of the monocausal effect of pleiotropic paracrine factors is considered absurd. Cell-free and cell-based therapies have multiple different molecular targets, and it is thought that only the interactions of these different effects lead to the beneficial clinical impacts observed in multiple pre-clinical studies.Thus, a closer interaction between blood banks that know all of these regulatory issues with major pre-clinical driving forces could accelerate scientific progress.Blood bank and transfusion units should not just follow urgent changes in clinical settings, but be actively incorporated into the process because they are familiar with GMP policy; they are familiar with handling inspections and experienced in testing biological samples . DetaileThe use of human PBMCs as bioreactors for secretome-based therapies can be seen as an extension of the original concept of stem cell-based therapies to enable clinical applications. A large number of studies of transplanted stem cells have failed to show any significant integration and differentiation of injected cells into mature (cardiac) cells. In addition, there is a considerable loss of injected cells immediately after translation. Using apoptotic PBMC-S instead of stem cells, we could promote tissue repair and regeneration in multiple pre-clinical indications with the identification of a variety of MOAs. However, as stem cells are rare, their isolation and cultivation in large numbers is neither simple nor practical. Therefore, a more easily obtainable source of cells as the “bioreactor” for the secretome may facilitate the introduction of this new secretome-based medicine.PBMCs release pleiotropic paracrine factors that augment tissue regeneration, and their usage has the following advantages Fig. : (I) PBMIn conclusion, the PBMC secretome may potentially revolutionize regenerative medicine when appropriate proof of concept studies in humans that meet the requirements of regulating agencies are finished.


We report the case of a patient with insidious onset and slowly progressive cognitive impairment, behavioral symptoms, temporal lobe seizures and delusional thoughts typical of delusional misidentification syndromes. Clinical presentation along with extensive diagnostic work-up revealed limbic encephalitis secondary to diffuse large B-cell lymphoma. The patient underwent immunotherapy with high-dose corticosteroid but no significant improvement was observed. No specific treatment for lymphoma was performed because the patient died of septic shock following a nosocomial respiratory infection. Delusional misidentification syndromes are an unusual and unique form of cognitive impairment in which a patient consistently misidentifies persons, places, objects, or events. Capgras syndrome is the most common subtype of this disorder, being defined by the recurrent and transient belief that someone close has been substituted by an imposter. These entities are generally associated with neurodegenerative diseases and psychiatric disturbances. Rare reports of associations between misidentification syndromes and autoimmune diseases such as multiple sclerosis have been published, but no papers address a correlation with limbic encephalitis or lymphoma. Delusional misidentification syndromes (DMS) are an uncommon group of disorders characterized by delusional beliefs that the people, objects, or places around the patient change or are made to change with one another.Limbic encephalitis (LE) is an uncommon inflammatory and autoimmune neuropsychiatric condition affecting the medial temporal lobe of the brain characterized by subacute cognitive symptoms, short-term memory loss, seizures, and affective changes.We describe the case of a patient who presented with the clinical picture of a DMS which, following a thorough investigation, was found to have LE associated with Diffuse Large B Cell Lymphoma.A 64-year-old right handed male with a university degree in arts presented to the neurology service with the chief medical complaint of behavioral changes slowly progressing over the course of the past seven years. Initially the patient experienced an insidious onset of decline in work productivity and gradually stopped working within the space of a year. Meanwhile, his family also noticed a short-term memory deficit primarily evidenced by difficulty remembering messages he was supposed to give to his wife, as well as not being able to name some specific objects. In the ensuing years the symptoms kept worsening while new behavioral and motor changes such as apathy, irritability, aggressiveness, delusional thoughts, psychomotor and gait slowing were contributing to progressive cognitive impairment. There were also reports of paroxysmal events characterized by self-limited episodes of experiencing unpleasant smells that lasted for seconds to a few minutes. The patient had consulted several physicians during the course of the disease. He was diagnosed with bipolar disorder by a psychiatrist with the report of accelerated thinking at the time, and was treated with olanzapine and valproate without any significant improvement. A neurosurgeon diagnosed normal pressure hydrocephalus and performed a ventriculoperitoneal shunt with no apparent symptomatic relief.At a later stage, he presented to the Neurology Department of the University of São Paulo with the main complaint of worsening delusional thoughts. The patient stated that his wife had been substituted by a perfect copy of herself, that the house he was living in only resembled his original property, and the city's most famous avenue had been duplicated in two identical copies. Past medical history was relevant for Pulmonary Abscess, Hypertension, Depression, Diabetes and former tobacco and alcohol abuse. He had an unremarkable family history. Prescribed medications were Carbamazepine, Olanzapine, Sertraline and Clonazepam.Neurological examination was remarkable for a cognitive impairment characterized by dysexecutive syndrome , psychomotor slowing and inappropriate behavior. The Mini-Mental State Examination test score was 26 out of 30 . Glabellar and palmomental primitive reflexes were markedly present as well as inhibitory paratonia, demonstrating frontal release signs. Based on initial impressions, the clinical presentation was attributed to LE  of protracted course associated with a delusional misidentification syndrome (Capgras Syndrome concomitant to Reduplicative Paramnesia).3), elevated protein levels (116mg/dl), normal glucose levels (56 mg/dl), negative flow cytometry for malignant cells, and negative infectious markers . Positron Emission Tomography (PET) with fluorodeoxyglucose (FDG) disclosed hypermetabolism in the left mesial temporal region and peri-insular cortex, as well as severe and diffuse hypometabolism in frontal, parietal and lateral temporal cortices. Electroencephalography showed diffuse slow waves (theta and delta) without epileptiform activity. The patient was then admitted to the neurology ward for extensive investigation. General laboratory tests were unremarkable, serology for common infectious diseases were negative , Antinuclear Antibodies (ANA) were positive in high titers (1:2560) but the remaining rheumatologic panel was unrevealing . Brain Magnetic Resonance Imaging (MRI) revealed high signal intensity on T2/ FLAIR in the left temporal pole and left mesial temporal region. Cerebral spinal fluid (CSF) was relevant for moderate lymphocytic pleocytosis . Computed Tomography (CT) of the neck, thorax, abdomen and pelvis disclosed no significant findings. Ultrasound of the testicles was also normal. However, whole-body FDG-PET revealed an anomalous increase in glycolytic metabolism in cervical external inguinal and iliac portacaval lymph nodes. A cervical lymph node biopsy was performed but anatomopathological study showed nonspecific neutrophilic inflammatory reaction and absence of signs suggestive of malignancy. The patient was then discharged for clinical follow-up scheduled over the next few months.Two months following previous hospital admission, the patient exhibited progressive worsening of cognitive and behavioral symptoms and therefore another work-up for occult malignancy was undertaken. The repeat whole-body CT scan disclosed enlarged mediastinal, hilar, cardiophrenic and axillary lymph nodes. The hypothesis of lymphoproliferative disease was proposed and the patient was admitted to the hematology ward for fine-needle aspiration of the axillary lymph node. Papanicolaou and Giemsa-stained cytology slides, and the cell block preparation from the sample material, showed morphologic features of a large cell lymphoma: intermediate-to-large sized cells, nuclei of different sizes and shapes, clumping artifact of naked nuclei, frequent mitosis and apoptotic bodies. Immunophenotyping showed a B-cell phenotype with a high Ki-67 labelling index . The tumDelusional Misidentification Syndromes (DMS) are among the most fascinating and puzzling forms of cognitive problems that can result from psychiatric or neurological disease.Epidemiological studies of the prevalence of misidentification syndromes are scarce but show a prevalence of 0.12% in the general population. Capgras' syndrome has a high incidence 15%) in patients diagnosed with schizophrenia and also has an incidence of 2%-30% in those with Alzheimer's disease.% in patiThere is no consensus on specific lesion topography responsible for the clinical manifestations of DMS, but it is believed to arise from structural lesions of the very complex face processing areas,There are no formal guidelines currently outlining the standards for assessment or treatment of these syndromes. Once identified, organic, structural, and metabolic conditions must be ruled out and adequately treated. Comorbid substance use must also be identified. Routine blood work should address metabolic parameters of hepatic and renal function (to identify causes or indicators of delirium), thyroid function and when pertinent, levels of drugs such as lithium. Neuropsychological testing can be conducted, as deemed necessary.,,LE was initially identified as a paraneoplastic neurologic syndrome characterized by subacute onset of short-term memory loss, seizures, psychiatric changes, and neuroradiological or pathologic evidence of involvement of the amygdala and medial aspect of temporal lobes.Paraneoplastic LE is preferentially associated with small cell lung cancer 40%), germ cell tumors of the testis (20%), breast cancer 8%), Hodgkin's lymphoma, thymoma, and immature teratoma.%, Hodgki%, germ cIn conclusion, we described the extremely rare association of DMS and Capgras Syndrome with LE secondary to diffuse large B-cell lymphoma. Capgras syndrome is a complex disorder defined as misidentification of persons by a patient generally in the course of neurodegenerative or psychiatric disorders. It is rarely linked to other conditions such as autoimmune neurologic disease. LE is an autoimmune entity characterized by inflammatory disturbance of medial temporal lobes usually associated with cancer. Non-Hodgkin lymphomas associated with LE have only been described in a few case reports. To our knowledge, there are no previous publications describing DMS in the context of LE and lymphoma diagnosis.


To make blood components and blood products safe, many safety measures are applied to avoid transfusion-transmitted infections. Defining a balanced safety policy is not easy, we face several dilemmas: How safe should blood be? Should we opt for maximal or optimal safety? Are perceived threats real and relevant? Should blood be clean while food, air, or mosquitoes are not? Is vCJD still a threat? It seems wise to discuss these issues more in the open. Treponema pallidum); specific processing ; quarantine of plasma (preventing window infections); bacterial culturing (detecting contaminated platelets); and post-donation and post-transfusion notification . This impressive preventive machinery does not mean that the problem of transfusion-transmitted infections has been solved. New infectious threats arise or are being recognized. Threats may arise locally, such as hepatitis E virus genotype 3 in parts of Europe and Babesia in parts of the USA; or abroad, causing temporary deferral of traveling donors, returning from an ever changing set of affected areas. This dynamic situation necessitates ongoing monitoring, and discussion of safety measures that could be introduced.In developed countries, a variety of safety measures ensures a low risk of transfusion-transmitted infections. These safety measures include donor selection (limiting imported and window infections); skin disinfection and diversion bags ; the screening of donations  or cost-ineffective . At the same time an entirely new, additional layer of safety is becoming available: pathogen reduction based on photo-chemical treatment of plasma, platelets, or whole blood. Should this technique be implemented on top of existing safety measures, or should pathogen reduction replace (some) existing safety measures? In either scenario: what would be the cost per prevented transmission? Considering the complex mixture of old and new infectious threats, and of old and new safety measures, it is hard to determine at which stage the blood supply is safe enough. Discussing the safety of the blood supply, several dilemmas can be recognized.Sometimes clear-cut economic guidelines are applied regarding preventive measures in public health. For example, recently universal vaccination of Dutch children against rotavirus was considered cost-ineffective as long as costs per quality adjusted life year (QALY) gained are more than € 20,000 . At the Among the dilemmas surrounding the prevention of transfusion-transmitted infections, the dilemma whether one can accept a certain residual risk , or whether one should cover each threat (maximum safety), is an easy one. Maximum safety does not exist. Even if all available safety tests and procedures for all agents would have been implemented, one could perform each screening test not once, but twice on each donation, thus increasing the sensitivity of detection. Input volumes for PCR extractions and reactions could be increased, etcetera. Hence, implicitly we always implement a reasonable set of safety measures , not a maximum set of safety measures.via transfusion. This assumption may be wrong. The apparent lack of significant disease caused by transfusion-transmitted dengue-, zika-, and chikungunyavirus, even in immunosuppressed patients, suggests that these viruses need transmission via a mosquito bite to cause disease. Of course, one must be careful because sound epidemiological data on this topic is scarce. On the other hand, we must realize that tick-bore encephalitis historically seems assumed to be irrelevant for blood safety. Before automatically assuming that zika-, dengue-, chikungunya-, and usutuvirus necessitate blood safety measures like WNV does, we must study the actual threat they pose to blood safety. We must consider to give these viruses the “benefit of the doubt”: as long as significant post-transfusion pathology (in the number of cases and in the nature of pathology) seems absent, blood safety measures may be avoided. In the “post-post-HIV era,” it is too easy to automatically apply the precautionary principle again, which demands that preventive measures should be taken even if cause and effect relationships are not established scientifically (Transfusion-transmitted West Nile virus (WNV) infection can cause serious disease in affected recipients . WNV andifically .Coxiella burnetii DNA by PCR  via air? History repeats itself: since July 2017 Dutch blood donations are being screened for the presence of hepatitis E virus, another zoonotic agent. During the first 11 weeks of screening, 48 of 85,023  donations were found confirmed positive for HEV. We calculated that in Holland only 1 of 700 HEV infections is caused by blood products. In multitransfused at-risk patients, one of 3.5 cases of chronic hepatitis E is caused by blood products  for codon 129 of the human prion gene. This is alarming, because so far all tested vCJD patients were methionine homozygous. Possibly, this first heterozygous patients reflects the start of a second wave of vCJD cases, with longer incubation times than the former homozygous cases. Apparently, people may harbor the infection during many years, and, regarding blood banking, could be seen as asymptomatic, but possibly infectious carriers. On the other hand, no more cases of blood-transmitted vCJD have surfaced. Studies of archived appendices suggest that roughly 1 in 2,500 British appendices tests positive for the vCJD agent. The first and second appendix study involved 12,674, respectively, 32,441 appendices, from persons born between 1961 and 1985, respectively, 1941–1985 (Since the outbreak of BSE in Great Britain with subsequent cases of vCJD in humans, many blood banks maintain safety measures to prevent transmission of vCJD 941–1985 . The finIn summary, several dilemmas exist concerning the desired level of safety of blood transfusion. Perhaps it is good to discuss these issues more in the open, and keep the struggle with difficult and expensive decisions not confined to blood transfusion services and ministries of health.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Among the dramatic increased internal migration in China in past three decades, a considerable proportion of young females migrated to urban areas and found employment in “entertainment venues”, who may be vulnerable to psychological distress. This study examines the prevalence of depression and explores its associations with health-risk behaviors and social capital among this subgroup.358 female migrants were recruited from entertainment venues in a rapidly growing urban city in China. A survey which included measures of depressive symptoms, health-risk behaviors, social capital, and socio-demographic characteristics was administered. Multivariable logistic regression was conducted to identify the independent correlates of depression.Of participants, 31.0% had clinically significant depressive symptoms (CES-D score ≥ 16). In multivariable models, greater likelihood of depressive symptoms was associated with working in massage centers/hotels , having probable alcohol dependence , self-reported lifetime use of illicit drugs , growing up in a non-nuclear family , and poor social capital .Intervention strategies to address the high prevalence of depression among female migrants are needed, and should also aim to reduce problematic alcohol and drug use, improve social capital, and target women working in massage centers or hotels. Stimulated by rapid modernization and industrialization, China has witnessed a dramatic increase in internal migration in the past three decades. The estimated number of internal migrants in China increased from 6.75 million in 1982 to 253 million in 2014 [A study of internal migrants in five major cities in China revealed that females accounted for 56% of among young migrants between the ages of 15–29 years old . PreviouWith the growing recognition of the social determinants of health, the concept of social capital has become an increasingly important concept to explain relationships between the social environment, social relationships, and mental health –21. AlthPrevious studies of Chinese female migrants have examined risk outcomes such as abortion, sexually transmitted infections, and HIV in China , 35, no From March to July 2012 we conducted a cross-sectional study among young migrant women employed in entertainment venues in Hefei, the capital city of Anhui Province, China. The study site represents a rapidly growing urban setting in central China (estimated population of 7.8 million) and is developing into a national hub for science and technology industries, and is a site for migration for many people in rural central China who cannot or choose not to migrate to the larger mega-cities on China’s eastern coast. In general, migrants to Hefei are young (53% younger than 29 years old), seeking job opportunities (62%), and have low levels of education (78% have not completed high school) . EligibiIndividuals who expressed initial interest were scheduled to meet outside of work hours in a private location to conduct the survey. After confirming their eligibility and providing informed consent, participants completed a structured survey which was administered verbally by a trained staff member; research staff (comprising a team of young women) received training in non-coercing recruitment, ethics, consent procedures, and on strategies to minimize social desirability and maximize fidelity to study procedures. During screening, we enquired whether any women felt forced or coerced to participate, and we planned to exclude these women from participation; no women reported force or coercion. Of 374 women who responded to outreach efforts and expressed an interest in the study, 16 did not meet eligibility criteria. A total of 358 women were enrolled and completed a structured questionnaire interview. The questionnaire took about 30–45 minutes to complete. Each participant was compensated with a small gift worth 50 Chinese Yuan (approximately 7.30 U.S. dollars) after completion of the survey, and all participants were informed about health services provided by the local CDC including sexual and reproductive health and mental health services.We assessed socio-demographic characteristics, including: age; types of workplace ; education level ; marital status, family status ; monthly income (less or greater than 4000 RMB), and length of time working in Hefei (in years). We assessed sexual health and substance use behaviors using measured validated or administered previously in studies of migrants and vulnerable populations in China. Sexual behaviors assessed in this study were limited to vaginal sex; anal and oral sex behaviors were not assessed. We assessed whether participants engaged during the past 6 months in commercial sexual behavior, casual sexual behavior , sex with multiple partners. Condom use with each sexual partner type  in the previous 6 months was assessed. Alcohol use was measured using the Alcohol Use Disorder Identification Test (AUDIT) ; a scorep < 0.10) based on procedures described in Hosmer and Lemeshow [We conducted descriptive analyses for socio-demographic characteristics and levels of depressive symptoms, health-risks, and social capital. Pearson Chi-square tests were employed to explore the associations of depressive symptoms (above or below the 16-score cutoff) with socio-demographic characteristics, health risks, and social capital. We conducted multivariable logistic regression to identify the independent correlates of depressive symptoms. We entered variables into the model if they had a moderate bivariate association with depressive symptoms  of participants had clinically significant depressive symptoms . Correlations between participant characteristics and depressive symptoms are reported in As shown in Participants’ endorsements of the social capital statements are shown in Given trends in rural-to-urban migration, modernization, and social-economic development in China, emerging health challenges related to the mobile status and living/working situations of migrant populations warrant examination. The current study adds to a growing body of research on rural-to-urban female migrants in China ,10,12,13Due to the social conditions and employment expectations of entertainment venues, female migrants working in these venues have high exposure to and risk for engagement in hazardous alcohol use, sexual risk behaviors, and illicit drug use –17. The Although economic factors are widely acknowledged as primary drivers of rural-to-urban migration in China, there is a need for further research to explore more fully the range of social, familial, personal, as well as economic drivers of migration and potential determinants of depression for young women who work in entertainment venues. Two demographic characteristics were correlated with clinically significant depressive symptoms in this study. First, compared with women working in karaoke parlors or nightclubs, those working in massage centers or hotels had three-fold greater odds of having clinically significant depressive symptoms. Reasons for this association are unclear, but may be due in part to the lower status of these venues and higher vulnerability for engagement in commercial sex work compared with working in karaoke and nightclub settings, which may contribute to increased depressive symptoms. Female migrants who work in massage centers and hotels might also have different migration trajectories into these employment venues compared with those who work in karaoke bars or nightclubs, and the working conditions  between these employment venues might also differ in ways that contribute to employees’ depression. Although this finding demands further research, it suggests that massage centers and hotels warrant particular attention for venue-based interventions that aim to reduce depressive symptoms of female migrant workers. Second, female migrants who grew up in a non-nuclear family were more likely to have clinically significant depressive symptoms. This finding corresponds with other research showing that emotional distress or health-risk behaviors are more prevalent among children and adolescents from single-parent families or step-families compared with those growing up in nuclear families , 47. ThiPoor levels of social capital were revealed in this sample, with approximately half of the participants reporting having no satisfying social participation, social trust, or social support in the city, and less than 20% reporting all four social capital indicators. These findings might reflect the working conditions and social status of these women. Due to institutional barriers associated with the Hukou system, migrants have limited access to services and socioeconomic opportunities in their new city of residence. This issue highlights a structural tension and a potential health risk in the motive for migration—that despite the desire for new economic opportunities in larger urban cities, migrants face exclusion from services and legitimate opportunities for advancement due to their non-permanent Hukou status. Employment in entertainment venues can bring further social problems due to the perception that these work venues are sites for commercial sex work . ConsequSeveral limitations to this research should be considered. First, due to the stigmatized and marginalized status of female migrants working in entertainment venues in China, our data were subject to volunteer bias and socially desirable reporting bias. Second, our data were also subject to recall bias, as participants were asked to recall their previous sexual behaviors during the past six months. Third, due to the non-representative sampling methods and geographic recruitment settings, findings from this study might not be generalizable to female migrants elsewhere in China. Fourth, information on additional factors that can contribute to depression among female migrants, such as discrimination and housing conditions, were not assessed. Due to sample size restrictions and restricted range, we were unable to test moderators of the association of social capital on depression  which might reveal specific population subtypes with greater risk. Fifth, the measure of social capital was exploratory and findings from this measure must be interpreted with caution. Additional measurement development is needed to refine strategies for assessing social capital in China. Finally, we cannot make casual or temporal interpretations due to the nature of cross-sectional data. Other research designs, such as longitudinal cohort studies, and more complex measurement and analytic strategies are needed to understand syndemic processes by which depressive symptoms are associated and determined by co-occurring health risks behaviors, social capital, and “upstream” social factors that contribute to health vulnerability in these women.In conclusion, the current study contributes additional knowledge toward understanding the prevalence of depression and its co-occurrence with health-risk factors and social capital among female migrants working in entertainment venues in China. Our study underscores the high prevalence of depressive symptoms and call for more attentions on depressive symptoms and mental health needs of this marginal and vulnerable population. Female migrants working in massage centers or hotels have higher prevalence of depressive symptoms, which implies that mental health interventions targeting these workplaces are needed. Potential intervention strategies may also focus on addressing alcohol and illicit drug use and increasing levels of social capital among female migrants working in entertainment venues. On a structural level, efforts to reform the Hukou system, in order to reduce inequalities between urban and rural populations, and to encourage entertainment venue owners/managers to support the social integration and well-being of female migrant workers may benefit this population. Future research and mobilization efforts are needed to understand factors that can motivate entertainment venue owners/managers to participate in public health efforts for supporting the well-being of their employees.S1 Dataset(SAV)Click here for additional data file.


Introduction: Adolescence and young adulthood are periods of continued biological and psychosocial maturation. Thus, there may be deleterious effects of consuming large quantities of alcohol on neural development and associated cognition during this time. The purpose of this mini review is to highlight neuroimaging research that has specifically examined the effects of binge and heavy drinking on adolescent and young adult brain structure and function.Methods: We review cross-sectional and longitudinal studies of young binge and heavy drinkers that have examined brain structure  and investigated brain response using functional magnetic resonance imaging (fMRI).Results: Binge and heavy-drinking adolescents and young adults have systematically thinner and lower volume in prefrontal cortex and cerebellar regions, and attenuated white matter development. They also show elevated brain activity in fronto-parietal regions during working memory, verbal learning, and inhibitory control tasks. In response to alcohol cues, relative to controls or light-drinking individuals, binge and heavy drinkers show increased neural response mainly in mesocorticolimbic regions, including the striatum, anterior cingulate cortex (ACC), hippocampus, and amygdala. Mixed findings are present in risky decision-making tasks, which could be due to large variation in task design and analysis.Conclusions: These findings suggest altered neural structure and activity in binge and heavy-drinking youth may be related to the neurotoxic effects of consuming alcohol in large quantities during a highly plastic neurodevelopmental period, which could result in neural reorganization, and increased risk for developing an alcohol use disorder (AUD). Magnetic resonance imaging (MRI) studies have highlighted ongoing brain maturation through young adulthood . Thus, we excluded samples based on diagnostic criteria , treatment studies, and those that characterized drinking based on non-binge or heavy-drinking criteria .Structural MRI assesses the metrics  of specific brain tissues at the macrostructure level. Additional techniques utilize the diffusion of water molecules  to characterize the microstructure of GM and WM. The majority of studies present cross-sectional data using retrospective reports of drinking experience, while a few recent studies have reported longitudinal changes in brain structure associated with binge drinking Table .n = 134) exhibited smaller volumes and thinner cortical tissue in total, frontal, and temporal GM as well as thinner cingulate cortex compared to controls (n = 674). In addition, within the BD group the number of binges in the previous year was negatively related to frontal and parietal cortical thickness  associated with the onset of binge drinking , and there is some evidence that male and female BD may exhibit an inverse relationship in some frontal and subcortical regions.Among alcohol dependent adults WM integrity tends to be weakened , ACC, and the anterior insula than LD during NoGo trials, suggesting greater reliance on executive functioning, error monitoring, and emotional interoception regions during inhibitory control , the ability to maintain and manipulate information during a short time span Diamond, . WrkM haSome studies have reported that sex differences may also be present in WrkM-related activation between male and female BD. Female BD had less spatial WrkM activation in several frontal, temporal, and cerebellar regions compared to female controls and this was linked to poor behavioral performance in the BD, a pattern opposite to what was seen in male BD relative to male controls (Squeglia et al., While longitudinal research is sparse among fMRI studies of BD/HD youth, one study reported reduced baseline fronto-parietal activity in adolescents who later transitioned into heavy drinking. However, HD showed significantly increased activity in these areas at a 3-year follow-up relative to baseline brain response (Squeglia et al., Deficits in learning and memory have been previously reported in individuals with AUD (Pitel et al., Pictorial as opposed to verbal stimuli were used in a study of college HD who demonstrated similar patterns of brain activity to previous studies of adolescents, namely greater fronto-parietal activity during encoding of novel stimuli, as well as greater hippocampal response relative to LD (Dager et al., A number of studies have investigated the neural correlates of risky decision making and reward processing across monetary decision making tasks in young BD. A study using the Iowa Gambling Task found that compared with their peers, adolescent BD had greater insular and amygdala activity, suggesting greater emotion-driven decision making in the BD (Xiao et al., Alcohol cue reactivity studies have found greater neural response in reward and emotional processing brain regions among individuals with AUD (Heinz et al., Research on the effects of binge and heavy drinking on the developing brain are limited in other domains, such as socio-cognitive and socio-emotional processing. While recent meta-analyses highlight deficits in social cognition in individuals with AUD (Onuoha et al., Binge drinking among youth is associated with smaller/thinner cortical and subcortical structures and decreased WM integrity. Consistent across many fMRI studies of cognitive control, WrkM, and verbal learning, young BD and HD show greater reliance on fronto-parietal systems while performing these tasks (Schweinsburg et al., AC conducted literature searches, wrote, edited, and revised the section on fMRI findings, wrote the conclusions, and created the table of fMRI findings. TB conducted literature searches, wrote, edited, and revised the section on structural MRI findings, wrote the introduction, and created the table of MRI structural findings. AC edited the final version of the manuscript and wrote the abstract.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sex differences in life history, physiology, and behavior are nearly ubiquitous across taxa, owing to sex-specific selection that arises from different reproductive strategies of the sexes. The pace-of-life syndrome (POLS) hypothesis predicts that most variation in such traits among individuals, populations, and species falls along a slow-fast pace-of-life continuum. As a result of their different reproductive roles and environment, the sexes also commonly differ in pace-of-life, with important consequences for the evolution of POLS. Here, we outline mechanisms for how males and females can evolve differences in POLS traits and in how such traits can covary differently despite constraints resulting from a shared genome. We review the current knowledge of the genetic basis of POLS traits and suggest candidate genes and pathways for future studies. Pleiotropic effects may govern many of the genetic correlations, but little is still known about the mechanisms involved in trade-offs between current and future reproduction and their integration with behavioral variation. We highlight the importance of metabolic and hormonal pathways in mediating sex differences in POLS traits; however, there is still a shortage of studies that test for sex specificity in molecular effects and their evolutionary causes. Considering whether and how sexual dimorphism evolves in POLS traits provides a more holistic framework to understand how behavioral variation is integrated with life histories and physiology, and we call for studies that focus on examining the sex-specific genetic architecture of this integration.The online version of this article (10.1007/s00265-018-2462-1) contains supplementary material, which is available to authorized users. Sex differences in life history, behavior, and physiology are nearly ubiquitous in nature , each consisting of genetic variances and covariances of traits within each sex, and the cross-sex genetic trait covariances   will constrain changes in the trait means within sexes. Recent studies exemplify these tenets in terms of the evolution POLS in a seed beetle Callosobruchus maculatus, where sexually antagonistic selection operates on an integrated phenotype  favoring a “fast” life history strategy in males and a “slow” in females. Artificial male-limited selection on lifespan revealed an intra-locus sexual conflict due to genetic correlations between the traits within and between the sexes. In addition to these constraints, evolutionary response in activity levels in the direction of male-specific selection  was limited by lack of sufficient additive genetic variance with consequences on sex differences in POLS trait covariance , for example, wing length is under sexually antagonistic selection and despite higher fitness in females with shorter wings, female wing length is expected to increase due to a correlated response to selection in males  Lande . Both thx) Lande . Strong,Given these constraints, how can sexual dimorphism be so commonplace and how do we expect it to evolve in POLS? There are a number of mechanisms that may facilitate the process. Tightly integrated traits, as predicted by the POLS framework, can more readily respond to selection as a unit, so that when selection acts on one trait, a correlated response is obtained in coadapted traits Futuyma . Howevermale and Gfemale) is considered to be more stable over time than the cross-sex covariance matrix (B), which suggests that there is more constraint to change POLS trait covariances within the sexes than it is between them  for example influences the expression of many autosomal and X-linked male-biased genes with roles in male reproduction and metabolism, despite having only few genes  is also expected to influence the genomic distribution of loci under sex-specific selection  showed how testosterone regulates changes in sex-biased expression in genes involved in growth and metabolism associated with the development of sexual size dimorphism , in which sexual dimorphism in the gene expression correlates with dimorphism in behavior and plumage  suggests that the different features of male sexual behavior, male aggression, maternal behavior, and female sexual behavior are controlled by many sex-specifically regulated genes  with highly specific effects on components of behaviors, rather than few genes with broad pleiotropic effects on whole behaviors  behavioral syndromes and (II) life history trade-offs that have shared effects on multiple POLS traits, and discuss evidence for sex specificity in these effects Fig.  and 2. WIn the POLS framework systematic differences in risk-taking behavior, including traits like aggressiveness, boldness, and exploration are expected to coevolve with the trade-off between current and future reproduction , there is a sex-specific genetic correlation between melanic coloration, immunity, and body mass , while its effects on corticosterone levels are sexually monomorphic , physiology  and also morphology  Fig. , Table 1ts Table . One of Rattus norvegicus), DRD4 affects sexual desire, arousal, and function  shows how a number of single nucleotide polymorphisms (SNPs) in both DRD4 and SERT are sex-specifically associated with mating behavior (whether birds breed in monogamous pairs or in promiscuous groups), as well as risk taking  is another candidate gene in the dopaminergic and serotonin systems and associated with variation in aggression human and mouse males (Shih and Chen nitric oxidase synthase 1 (NOS1) which has pleiotropic effects also on sexual function  and deer mice (P. maniculatus), that differ markedly in their mating system and level of offspring care   are an important candidate mediator of POLS because they orchestrate energy allocation and storage and allow individuals to respond to the environment adaptively to maximize fitness through their influence on investment into reproduction, immunity, and resource acquisition  shows how divergence in the pace-of-life is associated with metabolic rate in a sex-specific way . The mechanisms behind these effects in female Drosophila are not yet known, but hormonal control via juvenile hormone may play a role , plumage coloration and sexually dimorphic polymorphism in reproductive strategies is determined by alternative alleles at an inversion-based “supergene” including ERα  is a good example of such a polymorphic reproductive strategy: Males exhibit three alternative mating tactics where they differ in aggression, hormone levels, mating behaviors, and morphology    Table , Fig. 1.The degree of integration  genetic studies on covariation between life history trade-offs between current and future reproduction and behavioral syndromes in each sex, which are currently rare. This entails studying correlations between traits in more than two POLS categories at a time , which is currently the dominating approach. Also, given that the POLS composition is expected to vary between the sexes and species depending, e.g., on the mating system and environment Fig. S1(DOCX 476 kb)


Excessive alcohol consumption is a global healthcare problem. The liver sustains the greatest degree of tissue injury by heavy drinking because it is the primary site of ethanol metabolism. Chronic and excessive alcohol consumption produces a wide spectrum of hepatic lesions, the most characteristic of which are steatosis, hepatitis, and fibrosis/cirrhosis. Steatosis is the earliest response to heavy drinking and is characterized by the deposition of fat in hepatocytes. Steatosis can progress to steatohepatitis, which is a more severe, inflammatory type of liver injury. This stage of liver disease can lead to the development of fibrosis, during which there is excessive deposition of extracellular matrix proteins. The fibrotic response begins with active pericellular fibrosis, which may progress to cirrhosis, characterized by excessive liver scarring, vascular alterations, and eventual liver failure. Among problem drinkers, about 35 percent develop advanced liver disease because a number of disease modifiers exacerbate, slow, or prevent alcoholic liver disease progression. There are still no FDA-approved pharmacological or nutritional therapies for treating patients with alcoholic liver disease. Cessation of drinking  is an integral part of therapy. Liver transplantation remains the life-saving strategy for patients with end-stage alcoholic liver disease. Excessive alcohol consumption is a global healthcare problem with enormous social, economic, and clinical consequences, accounting for 3.3 million deaths in 2012 . Excessi2O2) to water and oxygen. However, when ethanol is present, catalase has an accessory role in ethanol metabolism by using H2O2 to oxidize ethanol to acetaldehyde. Ethanol oxidation by catalase is a relatively minor pathway in the liver, but has a larger ethanol-oxidizing function in the brain  is chiefly metabolized in the main parenchymal cells of the liver  that make up about 70 percent of the liver mass . These che brain .+) as a cofactor, generating reduced NAD+ (NADH) and acetaldehyde. The latter compound is highly reactive and toxic. It can covalently bind to proteins  and hydroxyl radicals (·OH). Continuous generation of these reactive molecules in problem drinkers eventually creates the condition known as oxidant stress or oxidative stress. Under these conditions, the rate of ROS generation exceeds the liver’s capacity to neutralize them with natural antioxidants, such as glutathione and vitamins E, A, and C, or to remove them using antioxidant enzymes, including those listed in CYP2E1 is the other major hepatic enzyme that catalyzes ethanol oxidation to acetaldehyde. Although the catalytic efficiency of CYP2E1 is considerably slower than that of ADH, CYP2E1 has a 10-fold higher capacity for binding ethanol, becoming half-saturated at 46 to 92 milligrams per deciliter. Also important is that CYP2E1 is an inducible enzyme; its hepatocellular content rises during chronic ethanol consumption . EthanolAlthough hepatocytes comprise most of the liver mass, nonparenchymal cells, including Kupffer cells (KCs), sinusoidal endothelial cells, hepatic stellate cells (HSCs), and liver-associated lymphocytes make up the remaining 15 to 30 percent of the liver mass. These nonparenchymal cells interact with hepatocytes and with each other via soluble mediators and by direct cell-to-cell contact. Each liver cell type plays a specific role not only in normal hepatic physiology but also in initiating and perpetuating liver injury.Heavy ethanol consumption produces a wide spectrum of hepatic lesions, the most characteristic being fatty liver , hepatitis, and fibrosis/cirrhosis see . SteatosAlcoholic hepatitis is a more severe, inflammatory type of liver injury characterized by swollen, dying hepatocytes , neutrophilic infiltration, and the development of tangled aggregates of insoluble proteins called Mallory-Denk bodies within hepatocytes. Central to hepatitis development is the activation of KCs, the resident liver macrophages.Global Status Report on Alcohol and Health estimates that 50 percent of all deaths caused by cirrhosis were attributable to alcohol abuse.Fibrosis and its terminal or late stage, cirrhosis, refer to the deposition of abnormal amounts of extracellular matrix proteins, principally by activated HSCs. Patients initially exhibit active pericellular fibrosis, which may progress to cirrhosis, the late stage of hepatic scarring. However, some degree of hepatitis likely is always present in cirrhotic patients, whereas hepatic fat usually is not prominent in these individuals. The The following sections provide a detailed description of the mechanisms involved in the development of these major lesions.As the preceding section on ethanol metabolism stated, ethanol and acetaldehyde oxidations generate higher levels of NADH, which alters the cellular redox potential and enhances lipid synthesis . However, ethanol-induced redox change alone does not fully explain why the liver rapidly accumulates fat. More recent studies now strongly support the notion that ethanol-induced steatosis is multifactorial as discussed below see .Enhanced lipid synthesis results from a higher expression of lipogenic enzymes and cytokines see  that areIn addition to enhanced hepatic lipogenesis, fat  tissue contributes to the development of steatosis. Adipose tissue normally is an important energy depot, storing excess calories derived from food consumption as fat. When necessary, high-energy fat then can be used to fulfill energy requirements during times of low nutrition  or high calorie utilization . Research with rodents subjected to chronic alcohol feeding has shown that ethanol consumption reduces adipose tissue mass by enhancing fat breakdown  in adipose tissue . The freBecause most lipids in hepatocytes are stored in lipid droplets, these organelles must first be degraded to extract the lipids for their subsequent oxidation. Breakdown of lipid droplets is accomplished by lipophagy, a specialized form of the intracellular process that degrades cytoplasmic components . During lipophagy, lipid droplets are engulfed within double- membrane–bound vacuoles called autophagosomes. These vacuoles transport the lipid-droplet cargo to lysosomes, where they are degraded by lipid-digesting enzymes , releasing free fatty acids that then undergo β-oxidation inside mitochondria. The rates of autophagy reportedly are retarded by chronic ethanol consumption, at least in part because ethanol is thought to cause faulty lysosome biogenesis. This results in fewer, more defective lysosomes , 1996, tIt also is quite clear that once fatty acids are released from lipid droplets, heavy alcohol consumption reduces their rates of β-oxidation. There are several reasons for the slowdown: First, the enhanced generation of NADH by ethanol oxidation inhibits mitochondrial β-oxidation. Second, metabolically generated acetaldehyde inactivates the peroxisome proliferator activated receptor alpha (PPAR-α), a transcription factor that acts in concert with the retinoid X receptor (RXR) and governs expression of genes that regulate fatty-acid transport and oxidation. Acetaldehyde likely inactivates PPAR-α by covalently binding to the transcription factor , therebyIt is well known that the liver exports triglycerides and cholesterol only as constituents of very low density lipoprotein (VLDL) particles; any impairment in either the synthesis or export of VLDL particles therefore contributes to fat accumulation within hepatocytes. VLDL assembly is regulated by the availability of triglycerides (which make up more than 50 percent of the VLDL lipids) stored in cytoplasmic lipid droplets. Up to 70 percent of the triglycerides in VLDLs are derived from the pool of triglycerides stored in lipid droplets that first undergo lipolysis and then are re-esterified to constitute VLDL triglycerides. Although earlier reports implicated altered VLDL secretion in the development of alcoholic steatosis , exactlyAlcoholic hepatitis occurs in about 30 to 40 percent of individuals reporting chronic alcohol abuse. It represents the most serious form of ALD and is associated with high short-term mortality. Ballooning degeneration of hepatocytes containing Mallory-Denk bodies, infiltrating neutrophils, and fibrosis are characteristic pathologic findings indicative of hepatitis . CentralThe ability of macrophages to regulate inflammation depends on their polarization—that is, their ability to develop into one of two different functional states, namely M1  or M2  macrophages. The polarization to either phenotype depends on the microenvironment, including circulating growth factors, cytokines, and pathogen-associated molecular pattern (PAMP) as well as damage-associated molecular pattern (DAMP) molecules. Because the liver is exposed to countless antigens, pathogens, and toxic substances that come from the intestine via the portal circulation, it must be protected from developing an immune response to such exposure. As a result, KCs usually have tolerogenic properties, meaning that they do not respond to all antigens with an immune response. However, excessive alcohol exposure can switch KCs to a proinflammatory M1 phenotype. Usually, ALD progression from liver steatosis to inflammation requires a second insult in addition to the alcohol exposure, such as another toxic insult, nutritional factor, or viral infection . More imWhat factors trigger KC activity in patients with alcohol use disorder? One major factor is endotoxin, also called lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria that translocates from the gut lumen into the portal circulation to reach the liver . AccumulOther factors can exacerbate liver inflammation. Prominent among these are MAA adducts that are produced in alcohol-exposed hepatocytes. These adducts are taken up by scavenger receptors on KCs , furtherRecently, it was reported that HSCs also play a dual  role in the regulation of liver inflammation . An impoHSCs are the key players in the development of fibrosis. These cells normally reside in the space of Disse as quiescent, lipid (retinyl-ester)-storing cells . FollowiHepatic fibrosis is a transient and reversible wound-healing response, which may be restored to normal in some patients if alcohol intake ceases. However, if drinking continues, chronic inflammation and sustained fibrogenesis progress, resulting in the substitution of liver parenchyma by scar tissue that severely compromises the liver’s vascular architecture. The main pathological feature of cirrhosis is the formation of regenerative nodules of hepatic parenchyma surrounded by fibrous septa. Cirrhosis development progresses from a compensated phase, in which part of the liver remains undamaged and functionally compensates for the damaged regions, to a decompensated phase, in which scar tissue fully envelops the organ. The latter is characterized by development of portal hypertension and/or liver failure.Among problem drinkers, only about 35 percent develop advanced liver disease. This is because modifiers, as listed below, exist that exacerbate, slow, or prevent ALD disease progression.Pattern of Consumption and Beverage Type. The most important factors determining the progression of liver disease are the beverage type consumed and the amount and pattern of drinking . Intake of 40 to 80 grams ethanol/day by males and of 20 to 40 grams/day by females for 10 to 12 years is a general predictor of more severe cases of ALD, including alcoholic steatohepatitis, fibrosis, and cirrhosis  in genes encoding alcohol-metabolizing enzymes, cytokines, and antioxidant enzymes that are related to the progression of ALD  interact to enhance hepatotoxicity. For example, as described earlier, acetaminophen hepatotoxicity can be exacerbated by alcohol abuse.Obesity. Population-based studies have indicated a significant correlation between the risk of liver damage and alcohol consumption in people with a high body mass index  and hepatitis B (HBV) viral infections is worsened in alcohol-abusing patients, causing rapid progression to fibrosis, cirrhosis, and even hepatocellular carcinoma  . CoupledEthanol metabolites appear to stimulate HCV replication. CYP2E1-positive hepatoma cells exposed to ethanol show an increase in HCV RNA . HoweverInnate immunity is the first line of antiviral protection in the liver. HCV commandeers this line of defense, and ethanol metabolism potentiates its takeover. For example, activation of antiviral IFNβ production in liver cells occurs via the interferon regulatory factor 3 pathway, which requires participation of a protein called mitochondrial antiviral signaling protein (MAVS). HCV evades this innate-immunity protection by cleaving MAVS , and ethThere are no FDA-approved therapies for treating patients with ALD. The following therapies currently are used for optimal ALD management.Drinking cessation is considered the most effective therapy in patients with ALD. Abstinence from alcohol not only resolves alcoholic steatosis but also improves survival in cirrhotic patients . The effCorticosteroid treatment, including the use of prednisolone, has been the most extensively used form of therapy, especially for moderate to severe alcoholic hepatitis, based on their ability to suppress the immune response and proinflammatory cytokine response . HoweverNearly all patients with severe alcoholic hepatitis and cirrhosis are malnourished and their degree of malnutrition correlates with disease severity and complications, such as variceal bleeding, ascites, infections, encephalopathy, and hepatorenal syndrome . DeficieThis procedure remains the standard of care for patients with end-stage liver disease. Some patients with ALD are not listed for the replacement of their own liver by a donor organ  for reasons such as continued alcohol consumption, improvement in liver function after abstinence, and a higher incidence of cancers of the upper airways and upper digestive tract. As a result, transplantation candidates with ALD often are screened for common malignancies and must undergo a formal medical and psychiatric evaluation. They also must abstain from alcohol for 6 months before being considered for liver transplantation. Data show that fewer than 20 percent of patients with histories of alcohol use as the primary cause of end-stage liver disease receive liver transplants . HoweverPatients often turn to natural and herbal therapies based on their potential for hepatoprotection. A U.S. survey revealed that 41 percent of patients with liver disease used some form of complementary and alternative medicine. An extract of milk-thistle seeds (silymarin) and garlic were reported as the most commonly used herbs for liver disease, followed by ginseng, green tea, gingko, echinacea, and St. John’s wort . As indi


A recent international workshop, organized by the authors, analyzed the obstacles facing the ambitious goal of eliminating viral hepatitis globally. We identified several policy areas critical to reaching elimination targets. These include providing hepatitis B birth-dose vaccination to all infants within 24 hours of birth, preventing the transmission of blood-borne viruses through the expansion of national hemovigilance schemes, implementing the lessons learned from the HIV epidemic regarding safe medical practices to eliminate iatrogenic infection, adopting point-of-care testing to improve coverage of diagnosis, and providing free or affordable hepatitis C treatment to all. We introduce Egypt as a case study for rapid testing and treatment scale-up: this country offers valuable insights to policy makers internationally, not only regarding how hepatitis C interventions can be expeditiously scaled-up, but also as a guide for how to tackle the problems encountered with such ambitious testing and treatment programs. Viral hepatitis was responsible for 1.3 million deaths globally in 2015 and is now the seventh leading cause of mortality, rising from the 10th cause in 1990 , , 4, 3, 4Scaling-up of infant vaccination has already had demonstrable impacts on global HBV prevalence . Infant A key component of a comprehensive PMTCT strategy is birth-dose vaccination. Modeling studies have suggested that an 80% global scale-up of birth-dose vaccination plus infant vaccination, compared with scaling-up of infant vaccination alone, could avert 18.7 million new chronic infections over the next 15 years, highlighting its importance as a PMTCT tool  but significantly greater in high-risk populations. The most studied population is PWID, in which anti-HCV prevalence can exceed 90% [S64], but men who have sex with men (MSM) and other recreational drug users are increasingly being recognized as significant at-risk populations . Such populations are often difficult to reach for a variety of reasons, including stigma and the possibility of prosecution . Programs specifically designed to address epidemics within these populations are critical to the success of disease burden reduction efforts.Qualitative  and quantitative [S71–S73] research has shown that PWID are interested in engaging with health care services for HCV testing and treatment; there is clear evidence of successful treatment outcomes in this group [S74], yet treatment in PWID remains suboptimal . Health care provider concerns can act as barriers to HCV treatment. Such concerns may include the presence of comorbidities, the belief that there will be adherence issues, and difficulties managing side effects [S77]. From a patient perspective, there are several factors that may reduce the likelihood of accessing HCV treatment, including negative experiences within health care systems , low literacy rates , inadequate communication with health care providers regarding the nature of the disease and treatment options [S79], and social factors such as discrimination, the threat of stigmatization, and criminalization [S81].Reviews of treatment models in high-income countries suggest that effective approaches to improving rates of HCV diagnosis and treatment integrate HCV services into addiction care units [S82]. Focusing on treatment of addiction [S83] and encouraging positive feedback between health care staff and PWID [S84] improve outcomes. Such multidisciplinary approaches reduce noncompliance even among homeless or active illicit drug users [S85]. Similar approaches can be utilized in general practitioner services; a study in the United Kingdom demonstrated how specialist nurses seconded to primary care facilities can offer screening to at-risk individuals, considerably improving HCV detection [S86]. An alternative approach may be treatment as prevention: Modeling suggests that it may be effective at curbing transmission in both PWID [S87] and MSM [S88].In LMIC, many patients, in addition to those discussed above, are hard to reach through lack of health care services. In such scenarios, HCV screening could be integrated into existing care delivery models for HIV and tuberculosis (TB) . Success of HIV treatment roll-out offers valuable lessons : CommuniAchieving ambitious WHO testing and treatment targets for HBV requires careful consideration of the challenges of scale-up; discussion should focus on simplified models of care and methods of wide-scale testing and treatment, particularly in LMICs.Treatment for CHB targets individuals with, or at risk for, advanced liver disease, and aims to suppress virus replication, halt disease progression, prevent complications, and avert HBV-related deaths S92–S94]. Several antiviral agents approved for the treatment of CHB are available in developed countries, with regional guidelines outlining a continuum of care and recommendations on who to treat, how to treat, and when to stop treatment [S95–S96]. These documents, while based on evidence of drug efficacy and benefits of treatment, are not adapted for use in countries where CHB is endemic and HBV-related mortality is highest . All cirrhotics should be treated regardless of ALT levels, HBeAg status, or HBV DNA levels. The simplicity of administration of these antivirals, their tolerability and safety profiles, and their high barrier to resistance make them ideal for long-term use in regions where close monitoring and management of adverse effects may not be feasible.While there is consensus that cirrhotics require lifelong treatment, the safety of stopping therapy in noncirrhotic patients remains less clear. Most international guidelines recommend discontinuation of therapy for noncirrhotic HBeAg-positive individuals who show evidence of HBeAg loss and seroconversion to antibody to HBeAg (anti-HBe), undetectable HBV DNA, and who complete at least 12 months of consolidation therapy [S94]. Discontinuation is only recommended when ALT and HBV DNA levels can be monitored, as a high proportion relapse after stopping treatment , placing an additional financial strain on health care systems. Antiviral therapy can be stopped in noncirrhotic HBeAg-negative individuals at least 12 months following loss of HBsAg and achieving anti-HBs status [S98]. This needs to be accompanied by sustained virological suppression and normalization of ALT, with off-treatment monitoring for relapse. With only a small proportion of patients achieving HBsAg loss, and with the need for regular monitoring for reactivation and flares after cessation of antiviral therapy in both HBeAg-positive and -negative individuals, we believe it is advisable at present to treat all patients indefinitely [S98]. The additional financial burden that such lifelong treatment imposes, however, as well as challenges to maintaining people in the cascade of care, must be acknowledged when adopting this approach.An additional complication is access to treatment in LMICs: despite generic tenofovir costing less than US$50 per annum and being accessible to HIV-HBV co-infected individuals as part of antiretroviral therapy, many HBV-monoinfected individuals cannot access antiviral therapy or have to pay out of pocket. National health care systems need to ensure funded, sustainable access to antiviral therapy for HBV-mono-infected individuals, implemented in concert with diagnosis scale-up strategies.Treatment programs need to be accompanied by wide-scale HBV testing, particularly in LMICs, where screening rates are low. Community-based testing and treatment for chronic HBV infection has been shown to be feasible: In The Gambia, the Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) study showed that POC HBV tests perform well in African community settings [S99], and subsequently demonstrated that community-based screening could achieve high coverage and good linkage to care [S100] while remaining cost-effective [S101].A 12-week DAA course costs in excess of US$70 000 in the United States today [S102]. With an estimated 71 million active HCV infections globally . India has used this approach to facilitate production of generics, but a recent decision to grant a patent to Gilead for Sofosbuvir may damage India’s role as an HCV generics producer [S105]. Pharmaceutical companies have, in places, awarded voluntary license agreements to permit local companies to produce generics. As of August 2015, Gilead had 11 such agreements with Indian companies [S106]. Additionally, the originator company of Daclatasvir has signed an agreement with the Medicines Patent Pool to enable sublicensing to multiple generic manufacturers in 112 LMICs [S103]. Many middle-income countries are viewed as having market potential and are excluded from these agreements, including China, Brazil, and Thailand [S106]. More generally, complexities of voluntary licensing fragment the market, ensuring that pricing power remains in the hands of pharmaceutical companies [S107]. This problem can be circumvented by development of new therapies: The Drugs for Neglected Diseases Initiative (DNDi) has obtained the license for a new HCV DAA, ravidasvir. This has allowed it to begin production of the drug through an Egyptian firm, Pharco Pharmaceuticals, without the need to maximize profit [S108].High-income countries can bulk purchase to reduce costs; no country has attempted this more ambitiously than Australia. The government agreed to a AU$1 billion (US$0.73 billion) program to treat 62 000 individuals, corresponding to per-treatment costs of around US$12 000 [S109]. The true novelty of the Australian approach is that if expenditure exceeds up-front cost, the price of drugs decreases, potentially to 0 [S109]. Australia has, in effect, created a subscription system, paying a fixed amount and treating as many Australians as it can.What ultimately unites these approaches is their customized nature; some countries have had success in reducing prices, but others risk being left behind. A unified approach must be taken to ensure that high-quality, low-cost drugs are available regardless of location. Pooled procurement is one method for achieving this, an approach pioneered by the Global Fund to tackle the HIV, malaria, and TB epidemics [S110], and attempts are underway to apply this approach to viral hepatitis [S111]. Such schemes could be transformative; however, they will rely on capital and political will to achieve the scale required for the program to be a success.Egypt serves as a model for HCV diagnosis and treatment scale-up . This country, with the world’s highest HCV prevalence, has increased HCV treatment numbers to the hundreds of thousands [S103] and intends to treat 5 million patients by 2030 [S104]. It has achieved this by slashing HCV treatment costs and empowering dozens of diagnostic facilities. Furthermore, by simplifying the diagnostic process through implementing the first universal test and treat strategy, it has reduced the time from initial diagnosis to treatment to as little as one week in some centers [104]. Egypt offers an invaluable example to policy makers, not only as an exemplar of how viral hepatitis interventions can be scaled-up quickly and intensively, but also as a guide for how to tackle the problems that may be faced with such ambitious interventions.Egypt demonstrates what can be accomplished with regards to HCV diagnosis and treatment, given sufficient political will, state coordination, and investment. Across the world, efforts to address viral hepatitis, both HBV and HCV, should focus on providing universal HBV birth-dose vaccination within 24 hours of birth, establishing hemovigilance schemes to prevent transmission of blood-borne viruses, providing nonreusable syringes and educating health staff in injection safety, adopting new diagnostic technologies to expand access to screening, specializing intervention efforts for vulnerable high-risk groups to reduce transmission where it is highest, treating all HBV-infected patients indefinitely to minimize potential harm caused by discontinuation, and collaborating across the continent to drive down HCV drug costs to ensure that, once treatment has been accessed, it is affordable.Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ofx252_suppl_supplementary_appendixClick here for additional data file.