Ghostwriting of medical journal articles raises serious ethical and legal concerns, bearing on the integrity of medical research and scientific evidence used in legal disputes.Medical journals, academic institutions, and professional disciplinary bodies have thus far failed to enforce effective sanctions.The practice of ghostwriting could be deterred more effectively through the imposition of legal liability on the “guest authors” who lend their names to ghostwritten articles.We argue that a guest author's claim for credit of an article written by someone else constitutes legal fraud, and may give rise to claims that could be pursued in a class action based on the Racketeer Influenced and Corrupt Organizations Act (RICO).The same fraud could support claims of “fraud on the court” against a pharmaceutical company that has used ghostwritten articles in litigation. This claim also appropriately reflects the negative impact of ghostwriting on the legal system.There are persistent concerns about the influence of the pharmaceutical and device industries on the medical literature, and particularly on the reporting of clinical trials, which can include the distortion of the true evidence base of medical interventions and overestimation of the clinical benefit of a drug used to treat patients Commentators have condemned the practice as unethical and unacceptable and have discussed the harms resulting from this form of medical ghostwriting, recommending that journal submissions be policed more aggressively and that the “guest authors” be suitably sanctioned by journals, academic institutions, and regulatory agencies Guest authorship is a disturbing violation of academic integrity standards, which form the basis of scientific reliability Daubert v. Merrell Dow PharmaceuticalsIn the legal setting, peer-reviewed articles are credible sources of evidence that may be used in lawsuits to support claims about safety and effectiveness, and hence to determine liability Daubert standard, and in the process they credentialize themselves as expert witnesses who can speak authoritatively about a product's efficacy and safety.Publications on which academics appear as guest authors also give credibility to these authors in the legal setting. These articles are sometimes used to establish an expert witness's authority, even when the validity of the research in the article is the very issue under dispute. As a result, the treatment of the guest author as a legal expert may prevent scrutiny of the practice that is being challenged for contributing to serious harm. Numerous studies have shown that industry-sponsored clinical trials are often biased in favor of the sponsor, sometimes in ways that can be detected only with access to the original data and study protocol The International Committee of Medical Journal Editors (ICMJE), in establishing leading standards for biomedical publications, has sought to curb inappropriate and unethical authorship practices by requiring that journals ask detailed questions about what exactly each author has contributed to an article PLoS Medicine, have called for bans on future submissions by authors who act as guests, formal retraction if unacknowledged ghostwriting is discovered after publication, and reporting of authors' misconduct to institutions Some journals, such as Commentators have also called for academic sanction Moreover, institutions may decide not to act because the practices involved in ghost and guest authorship may not be far removed from other common publication practices in academic medicine where laboratory directors, departmental chairs, and supervisors often claim authorship on publications because of those institutional roles rather than by standard authorship criteria Professional organizations, such as State Medical Boards in the US, Colleges of Physicians and Surgeons in Canada, and the General Medical Council in the United Kingdom, could also intervene when evidence of guest authorship by licensed heath care professionals is uncovered, particularly if it involves outright misrepresentation of data However, these professional organizations have so far failed to issue serious sanctions in the rare cases when an organization has looked into allegations of authorship violations In light of the lack of institutional responses to curb the practices of ghostwriting and guest authorship and in light of the significance of these practices for the legal system, we suggest that a firm legal response is appropriate.An important starting point for a legal response involves the ICMJE uniform guidelines Substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data;Drafting the article or revising it critically for important intellectual content; andFinal approval of the version to be published Medical journals typically require all authors to confirm in writing that they have satisfied these criteria. “Guest authors” often fail both of the first two requirements, as suggested by evidence that has been revealed in recent class actions involving drugs such as Vioxx (rofecoxib), Prempro (combined estrogen/progestin), and Paxil (paroxetine) The authorship requirements are known not only to named authors but also to readers. The warranty of authorship is an important factor in ascertaining an article's integrity and quality. To see this, we need only ask how readers would react to an article prefaced with a statement that a lead author has refused to sign (or has repudiated) the authorship warranty, and now wishes to clarify the contributions of an industry-based medical writer. Such a statement would significantly undermine the article's credibility The above thought experiment, involving a guest author who admits to playing that role, shows that a false affirmation of authorship is an example of fraud. Fraud occurs when a person makes a knowingly false representation in order to acquire something of value, and harm occurs as a result Strom ex rel. U.S. v. Scios, Inc., the US government alleged that the defendants' activities led to the presentation of false Medicare claims Strom, it appears that the unwarranted claims made in the ghostwritten articles, rather than their fraudulent authorship, helped to support the allegations of fraud. This approach has great potential, but it will not always be easy to prove the falsity of ghostwritten research.The characterization of guest authorship as fraud has received limited but important recognition in suits involving the False Claims Act (FCA), which imposes liability on those who cause fraudulent claims to be presented to the US government Strom shows, the fraud underlying these articles cannot be attributed solely to the guest author, who after all has responded to an invitation. Pharmaceutical companies and medical communications agencies are well aware of the journals' publication requirements. Soliciting and facilitating fraud may amount to conspiracy, and may incur liability on the same grounds as the fraud itself As Because a journal's readers are all harmed by the fraud, they may sue the guest in a civil RICO class action In addition, some readers access articles on a pay-per-view basis. These readers, too, will assume that the article meets the journal's requirements, and they would also be unlikely to pay if they first saw a disclaimer of authorship responsibility. These purchasers might constitute a distinct subclass in a RICO class action, with damages based on the cost of the download.To prevail, the plaintiffs would not have to prove individually that they relied on the guest's fraudulent claim. In 2008, the US Supreme Court held that when plaintiffs allege fraud under RICO, they are not required to show that they relied on the defendant's assertions, so long as they were harmed because someone else relied on the fraud  Why should this approach be directed against guest authors, rather than the others who are complicit in the same fraud? RICO fraud could be added to the claims raised against pharmaceutical companies in negligence suits, but the damages would be low, as against those already available in such cases. But the combination of monetary sanctions and reputational harm might deter academics, and might also deter the medical communications agencies that design these studies and seek impressive names for the byline. Here is a case where the threat of liability—and the uncertainties and distractions that it brings—may be sufficient to discourage those who are not normally sued for harmful drugs, but who help to legitimate the studies that publicize these products.As to the pharmaceutical companies, we propose another approach, also grounded in fraud. Just as the integrity of medical research is a key factor in recognizing false authorship warranties as fraud, the courts' concern about the integrity of their proceedings is key to the doctrine of “fraud on the court.” This doctrine takes a similar form in England, Australia, Canada, India, and many other countries Hazel-Atlas Glass v. Hartford-Empire Co. (1944), which seems to be the only ghostwriting case decided by the US Supreme Court For a more concrete sense of the doctrine, consider Daubert requirements, as noted above In explaining why Hartford's actions merited sanction, the Supreme Court offered several observations that apply with equal force to current examples of medical ghostwriting. The Court stated that using spurious claims of authorship to legitimate claims before the Patent Office and the courts “is a wrong against the institutions set up to protect and safeguard the public” Daubert purposes, or to credentialize an expert witness. Each of those uses, if attempted by a party that had helped to create the article, could risk sanction. The articles could still be used to promote drugs, but if litigation should arise, the defendant's arsenal of responses would be limited.Ghostwritten articles are not created and developed primarily for legal purposes; rather, they are used to publicize and market drugs. However, a restriction on the legal use of articles to which guest authors have added their name could significantly diminish their overall value. They are often used in litigation to support the manufacturer's arguments about a drug's efficacy and safety, or to establish a record of scientific acceptance for Hazel-Atlas, Hartford insisted that the article's claims were true, attribution issues notwithstanding. The Supreme Court found this argument unavailing: “Truth needs no disguise. The article, even if true, should have stood or fallen under the only title it could honestly have been given—that of a brief in behalf of Hartford, prepared by Hartford's agents, attorneys, and collaborators” The false respectability afforded to claims of safety and effectiveness through the use of academic investigators risks undermining the integrity of biomedical research and patient care. This integrity also underpins the use of scientific evidence in the courtroom. Whether publications with academic guest authors are factually accurate is irrelevant. In


The s908 mutation leads to a leucine to arginine substitution in the ectodomain of the hepatocyte growth factor (HGF) tyrosine kinase receptor, Met. This missense mutation impedes the proteolytic maturation of the receptor, its trafficking to the plasma membrane, and diminishes the phospho-activation of its kinase domain. Interestingly, during pancreatogenesis, met and its hgf ligands are expressed in pancreatic epithelia and mesenchyme, respectively. Although Met signaling elicits mitogenic and migratory responses in varied contexts, normal proliferation rates in donut mutant pancreata together with dysmorphic, mislocalized ductal cells suggest that met primarily functions motogenically in pancreatic tail formation. Treatment with PI3K and STAT3 inhibitors, but not with MAPK inhibitors, phenocopies the donut pancreatic defect, further indicating that Met signals through migratory pathways during pancreas development. Chimera analyses showed that Met-deficient cells were excluded from the duct, but not acinar, compartment in the pancreatic tail. Conversely, wild-type intrapancreatic duct and “tip cells” at the leading edge of the growing pancreas rescued the donut phenotype. Altogether, these results reveal a novel and essential role for HGF signaling in the intrapancreatic ducts during exocrine morphogenesis.In a forward genetic screen for regulators of pancreas development in zebrafish, we identified  donut because the body of the pancreas fails to elongate and thus remains rounded. The mutation leading to this phenotype affects the extracellular domain of Met, the hepatocyte growth factor (HGF) receptor, impairing its maturation, plasma membrane localization and phospho-activation. Although HGF/Met signaling may elicit many context-dependant cellular responses, our data indicate that HGF/Met signaling triggers the migration, but not the proliferation, of the pancreatic ductal cells to drive the extension of the pancreatic tail.The pancreas functions as an endocrine and exocrine gland that secretes hormones regulating blood glucose homeostasis, and pancreatic juice that aids the digestion and absorption of nutrients, respectively. Contrary to endocrine tissue development, that of the exocrine pancreas has received less attention. We conducted a forward genetic screen in zebrafish and identified HGF/Met signaling as a key regulator of exocrine development. We called the mutant  The vertebrate pancreas is an endodermal organ that is part endocrine, releasing hormones that regulate glucose metabolism, and part exocrine, releasing pancreatic juices that aid in digestion. Pancreatic endocrine and exocrine developmental dysmorphogenesis and dysregulation, including diabetes mellitus and pancreatic adenocarcinoma, can result in human diseases with high morbidity and mortality. Thus, a more sophisticated understanding of molecular mechanisms mediating pancreatic development and homeostasis will certainly refine the treatment of these diseases.In zebrafish as in mammals, all pancreatic endocrine and exocrine tissues derive from the fusion of a dorsal and ventral bud arising at the level of somites 2–9 Hepatocyte Growth Factor (HGF) is a stromally-produced ligand which binds Met, a receptor tyrosine kinase that is predominantly expressed in epithelia. Upon receptor dimerization and autophosphorylation, Met activates a bevy of cellular processes including motogenesis, tubulogenesis, mitosis, chemotaxis, and cell survival 2-color liver, insulin, exocrine pancreas) zebrafish with EGFP-expressing pancreatic acinar cells and DsRed-expressing pancreatic beta-cells and hepatocytes s908donut mutants, which show shortened (type 1) or absent (type 2) exocrine pancreatic (xp) tails. jh1Tg(ptf1a:GFP)s908donut background to reveal the short exocrine pancreas phenotype at 3 days post fertilization  domain in the Met ectodomain, which together with IPT4 comprises a high affinity HGF binding site . We found that even low doses of metMO (2 ng) could reproduce the shortened pancreatic tail phenotype observed in donut mutants with high penetrance (s908+/−donut embryos were injected with a near-threshold (1 ng) dose of MO, 53.4% displayed a short pancreas phenotype, as compared to 29.1% of injected wild-type embryos  also developed shortened pancreata , during (52 hpf), and after its caudal extension  between 52 and 80 hpf :GFP) line (hereafter duct:GFP) which highlights the intrapancreatic ducts (IPDs) met was expressed in both IPD and acinar cells at 60 hpf, a time of active pancreatic tail extension . Alternately, residual Met protein, which is expressed in endoderm prior to the onset of Pdx1 expression, or a parallel/redundant mechanism could drive exocrine outgrowth in mammals. Additional targeted studies are needed in mice to determine whether the role of Met in exocrine pancreas growth is conserved.To clarify how HGF signaling is implemented in pancreatogenesis, we examined the expression of  anlagen . Additio 80 hpf; . We found 80 hpf . Importat 75 hpf , indicatxtension . A role donut mutation affects Met function, we generated an I776R point mutation in murine Met, an analogous lesion to the s908donut mutation, and then transfected either WTMet or I776RMet into TOV112D cells. This cell line lacks endogenous Met activity, but expresses the intracellular components of the Met signaling pathway WT, most of the nascent polypeptide was cleaved to the mature form (150 kDa+30 kDa), while a small proportion of the receptor remained unprocessed (190 kDa). Since the I776R lesion lies far from the furin cleavage site (residues 302–307) I776R precursor polypeptide failed to be cleaved (R306A) can signal normally WT and MetI776R using specific anti-phosphorylated Met antibodies directed against several key tyrosine residues. Upon HGF binding, Met dimerizes and its cytoplasmic catalytic region is activated by trans-phosphorylation of tyrosines Y1234 and Y1235. Subsequently, residues Y1349 and Y1356 are phosphorylated in the Met multifunctional docking site, which connects Met to multiple downstream branches, including Ras/MAPK and PI3K, through several adapter proteins. In addition, phosphorylation of tyrosine Y1003 negatively regulates Met signaling by promoting receptor turnover. Compared to MetWT, phosphorylation of Y1234-5, Y1349, and Y1003 were significantly diminished in MetI776R . Antibody staining was evident at the membrane of unfixed, unpermeabilized HEK293T cells expressing MetWT or cleavage incompetent MetR306A, but not MetI776R . Together, these data support the hypothesis that deficient signaling through MetL775R causes the hypomorphic effect observed in s908donut mutants. Likely, reduced Met signaling is due to (1) localization of MetL775R away from the plasma membrane, (2) impaired binding of HGF to the high-affinity binding site in IPT3-4, and/or (3) impaired assembly of Met co-receptor moieties, that would then lead to defective phospho-activation of Met.To model how the MetI776R . FinallyMetI776R . HoweverMetI776R . Thus, ta stages . As obsedonut mutants, we first assayed cell proliferation using 30 min incorporation of the thymidine analog EdU at two time points during pancreatic tail growth: 56 and 75 hpf  and metMO-injected  (see Methods) embryos were directed toward an endodermal fate by cas mRNA coinjection ptf1a:GFP+ acinar or ptf1a:GFP− ductal cell compartments in the distal or proximal portion of the exocrine pancreas  hosts (data not shown). Next, we assessed the rescue of exocrine pancreatic tail outgrowth by transplanting varying numbers of WT Tg(hs:mCherry) cells into the endoderm of metMO-injected hosts s892Tg(ins:CFP-NTR)m1018Tg(ins:dsRed)ia3Tg(nkx2.2a(-3.5 kb):GFP) (a.k.a. “duct:GFP”) jh1Tg(ptf1a:eGFP)Tg line (a.k.a. Tg(hs:mCherry)), H2B-GFP was replaced in the plasmid hsp70l:loxP-mCherry-loxP-H2B-GFP-cryaa:CeruleanFish were raised and maintained under standard conditions donut mutant embryos. donut mutants were subsequently genotyped using a dCAPS strategy: a PCR product amplified by the primer set: 5′-TCCAG CCCAA ACATT CTTTC and 5′- CGTTT GTGTG GGTTG TATAG ACTCA CCACT TGGAA GAGTT TGCCC TCAGT GGCGG CAGcG was digested with HhaI.An ENU mutagenesis screen was executed as described Pymol software (Schrödinger) was used to manipulate the PDB model of human IPT1-4 in situ hybridization, and proliferation analysis with EdU were performed as described in situ hybridizations were performed as described met: CGGAG AGAGA GGGAG GAAG and TAATA CGACT CACTA TAGGG AGACA TTGAT GTCCG TGATG GAG; hgfa: TGTGT GCTTG AGAAA GAGAG AGA and TAATA CGACT CACTA TAGGG AGATC GACAA ATTGC CACGA TAA; hgfb: AGCCA CTGCA GGGAG ACTAC and TAATA CGACT CACTA TAGGG AGAGG GGTAC CTTTT AGGGT GGA; furina: GTGTC GGAGT GGCCT ACAAT and TAATA CGACT CACTA TAGGG AGGGT CTTCA TCCCA GGAGT; furinb: TGACC TGGAG AGACA TGCAG and TAATA CGACT CACTA TAGGG AATGC TGGGG GATTT TCTCT. For cytoimmunofluorescence, human embryonic kidney cells (HEK293T) were maintained in DMEM supplemented with 10% FBS. HEK293T cells were plated on polylysine-coated coverslips and transfected with lipofectamine with either murine pBabe-puro-MetWT (Addgene) or site directed mutagenesis-generated pBabe-puro-MetI776R. Cells were transfected 48 h before immunostaining: unfixed cells, as well as 4% paraformaldehyde-fixed and 0.1% Triton-permeabilized cells were incubated with indicated antibodies for 2 hours prior to incubation with appropriate fluorescent secondary antibodies.Whole mount antibody staining, WT and MetI776R were transfected into TOV112D cells with lipofectamine. Coimmunoprecipitations and protein blot analyses were performed as previously described TOV112D human ovarian carcinoma cells were obtained from ATCC  and cultured using a 1∶1 mixture of MCDB 105 medium and medium 199 supplemented with 15% FBS. Metia3; Tg(ptf1a:dsRed)jh1Tg(nkx2.2a(-3.5 kb):GFP) embryos, distributed in 12-well plates (20 embryos per well), were incubated with different chemicals from 60 to 76 hpf, during the growth of the pancreatic tail. Chemicals were specific inhibitors of MEK , p38MAPK , PI3K , Src kinase  and furin .in vitro, by kit (Ambion), and 150 pg of mRNA was injected. Live imaging of embryos was performed 5 hours post-injection. For transplantation, capped cas/sox32in vitro, and donor Tg embryos, which exhibit strong, ubiquitous RFP expression in the absence of Cre recombinase, were injected with 200 pg of cas mRNA alone or with 4 ng of met morpholino. Cell transplantations were performed as described cas-injected putative met mutant cells were transplanted, donors were genotyped after transplantation.The morpholinos (Gene Tools) used for gene knockdown were described in Figure S1donut mutants variably lack muscle tone in pectoral fins. (A–C) 9 dpf Tg(ptf1a:GFP) larvae imaged from the dorsal aspect to show fin morphology (arrows). In WT larvae, fins are folded closely against the body (A), while in type 1 (B) and type 2 (C) donut mutant larvae, the pectoral fins lack tone, and display an open wing configuration. Bar = 0.5 mm.(TIF)Click here for additional data file.Figure S2s908donut sensitizes embryos injected with met morpholino. Clutches resulting from donut heterozygote to WT crosses were injected with 1 ng of metMO and scored for pancreatic morphology. Embryos presenting type 1 and 2 phenotypes were pooled separately from WT clutchmates, and were retrospectively genotyped. Penetrance of short pancreas was increased from 29.1% in WT embryos to 53.4% in heterozygotes. Significance determined using 2-sided Chi squared test, p = 0.0002.Heterozygosity for (TIF)Click here for additional data file.Figure S3furina and furinb expression during pancreatogenesis by whole-mount in situ hybridization. Both furina and furinb are expressed in endoderm (brackets); furina is expressed in liver (Li) and pancreas (Pa) at 75 hpf. Dorsal views, anterior to the top.(TIF)Click here for additional data file.Figure S4hgfMO-injected larvae. (A–B) 84 hpf hgfMO-injected host larvae with a high contribution of transplanted WT endodermal cells (red). Extensive contribution of WT cells to both duct and acinar compartments did not rescue type 1 (A) or type 2 (B) exocrine pancreas tail outgrowth.WT endoderm cannot rescue exocrine tail formation in (TIF)Click here for additional data file.


This paper reports on the transformation that has occurred in the care of people living with HIV/AIDS in a Toronto Hospice. Casey House opened in the pre-HAART era to care exclusively for people with HIV/AIDS, an incurable disease. At the time, all patients were admitted for palliative care and all deaths were due to AIDS-defining conditions. AIDS-defining malignancies accounted for 22 percent of deaths, mainly, Kaposi sarcoma and lymphoma. In the post-HAART era, AIDS-defining malignancies dropped dramatically and non-AIDS-defining malignancies became a significant cause of death, including liver cancer, lung cancer and gastric cancers. In the post-HAART era, people living with HIV/AIDS served at Casey House have changed considerably, with increasing numbers of patients facing homelessness and mental health issues, including substance use. Casey House offers a picture of the evolving epidemic and provides insight into changes and improvements made in the care of these patients. Remarkable changes have taken place since AIDS was first described in 1981. The face and complexity of both living and dying with HIV have evolved significantly over the last three decades. Today, incidence is increasing in new subpopulations, new treatments are becoming available and individuals are living longer on treatment. There are also new and unanticipated complications of this virus which result in changing causes of death. In order to effectively research, plan, and provide the best care possible for people living with HIV/AIDS, it is essential that we understand both trends and the current state of the epidemic today, including differing causes of mortality.P. carinii pneumonia (now referred to as P. jirovecii pneumonia) and Kaposi sarcoma in 82% of the cases [P. carinii from 187 to 29 [In a classic paper examining deaths of individuals who were diagnosed with AIDS prior to 1986, the one-year mortality rate was 51.2% and the 5-year rate was 84.8%. The principal causes of death were he cases . EffectiThe changes in prognosis and associated diagnoses have resulted in a new burden of care on organizations that serve HIV-infected persons. One type of healthcare organization dramatically affected is the hospice that initially served those dying with HIV. In the early years of the epidemic, individuals arrived at these institutions with a devastated immune system that resulted in multiple HIV-related opportunistic infections and death within months. With the advent of combination antiretroviral therapy and an increased understanding of the clinical and medical management of HIV-related conditions, HIV/AIDS patients are now frequently discharged to return home to their community. However, this population requires a range of healthcare services to manage the extensive medication regimes and complex medical and psychiatric comorbidities, many of which are non-AIDS defining –9. With As a means of tracing this historical change, we performed a retrospective study to compare the causes of death of those who died in a Toronto hospice, known as Casey House, between 1988 and 2006–2008. These two time-periods were chosen to represent the first year of operation of the hospice, before the development of antiretroviral therapies, and a recent portrayal of patients in a time where highly active antiretroviral therapies (HAART) are widely available in developed countries. A longer time-frame in the post-HAART period was necessary to capture similar numbers of deaths. In another report we have described the demographics and medical, and psychiatric comorbidity of the entire population of patients admitted to the facility in 2008 . We inclCasey House was originally established in 1988 as Canada's first hospice for individuals with HIV infection. Today, Casey House is a 13-bed hospital which provides both in-patient and home care services to people living with HIV/AIDS. There are approximately 100 in-patient admissions a year. Individuals with HIV may be admitted for sub-acute rehabilitative care, medical and psychiatric symptom control (including pain management), post-hospitalization support, end-of-life palliative care, or respite care. Care is provided by an inter-professional team including primary and consultant specialist physicians, nurses, social workers, and rehabilitation therapists.A retrospective review of patient charts and death certificates was undertaken at Casey House, Toronto, Canada. The first set of the analyses focused on patients who had died at Casey House. We defined the first 10 months of Casey House operation as the “pre-HAART” period, thus including records of all patients who had died between the first day Casey House opened its doors, March 1, 1988, and December 31, 1988. In this era, Casey House operated solely as a hospice and patients were admitted only for end-of-life care. We compared characteristics of these pre-HAART patients who died to all Casey House patients who died during a 36-month time period from January 1, 2006 to December 31, 2008. The years 2006–2008 were chosen to represent the current epidemic in an environment where highly-active antiretroviral therapies are widely available. We describe differences in causes of death in the two time-periods. Uneven time-periods were necessary to obtain comparable numbers of deaths in each time.In addition to focusing on patients who died during their tenure at Casey House, we describe the characteristics of all patients admitted to Casey House during the entire year of 2008 (January 1–December 31) to demonstrate how the facility had evolved as the HIV epidemic, and consequently survival, changed .Clinical and sociodemographic characteristics were collected from patient charts and death certificates. Due to developments in medical knowledge and changes in the scope of practice, not all variables were available or collected for all deaths. Causes of death and types of cancer death were identified and classified using the International Classification of Diseases, the 10th edition. Causes of death were identified as AIDS-defining conditions according to the Centers for Disease Control and Prevention (CDC) . MalignaThis research project was approved by the Research Ethics Board at St. Michael's Hospital, Toronto, Canada.Fifty-nine patients died at Casey House in the 10 months between March 1 and December 31, 1988. All patients were male and all but one individual (98.3%) self-identified as men having sex with men (MSM). Individuals lived an average of 1.5 years (SD = 0.9) with an HIV/AIDS diagnosis before dying. The mean age at death was 39.0 years (SD = 9.3). Forty-eight patients died at Casey House in the 36 months between January 1, 2006 and December 31, 2008. Forty-three (89.6%) patients who died were male. Thirty-four (80.9%) persons identified MSM. The average age at death was 48.1 years (SD = 8.6) after living an average of 13.5 years (SD = 6.5) since HIV/AIDS diagnosis.Characteristics of patients who died in the pre- and post-HAART period are summarized in n = 13) were due to AIDS-defining malignancies. Forty-two percent (n = 25) of deaths were due to AIDS-defining opportunistic infections, most commonly disseminated Mycobacterium avium complex disease (MAC) (n = 5), toxoplasmosis (n = 5), and Pneumocystis pneumonia (PCP) (n = 3). Thirty-six percent (n = 21) of deaths were identified as other AIDS-defining conditions. In 19 of these cases the cause of death was listed only as “AIDS.”The primary cause of death was identified as an AIDS-defining condition in all 59 deaths in 1988. Twenty-two percent (n = 15) of individuals died of AIDS-defining opportunistic infections; 6% (n = 3) from AIDS-defining malignancies; 10% (n = 5) from other AIDS-defining conditions. The remaining 25 deaths (52%) were attributed to: non-AIDS-defining malignancies ; liver disease ; other causes  including multi-organ failure (n = 3) and respiratory failure (n = 2). Fourteen patients had hepatitis B or C listed as a contributing cause of death.Forty-eight percent of deaths in the post-HAART era were attributed to AIDS-defining conditions. Thirty-one percent (n = 13) of deaths in the pre-HAART era. All of these were AIDS-defining malignancies: 11 were attributed to Kaposi sarcoma and 2 to lymphoma. An additional 4 deaths had Kaposi sarcoma listed as a contributing factor. In the post-HAART era, malignancies accounted for the primary cause of death in 33% (n = 16) of deaths: 6% (n = 3) were due to AIDS-defining malignancies and 27% (n = 13) were due to non-AIDS-defining malignancies. Kaposi sarcoma was not listed as the primary cause of death for any patients in the post-HAART period but was a contributing factor in one death. Malignancies accounted for 22%  died during their stay at Casey House: seventy-percent of whom had a CD4 count below 200 cells/mm3. Of the individuals who died with decreased immune system function, 25% were homeless and 25% lived in supportive housing. All individuals who died with a CD4 count above 200 cells/mm3 reported renting their own housing (excluding supportive housing). P < .05 level) between patients who died and those who were discharged, although there were slightly more individuals with a CD4 count below the median in those who died (P = .07). Eighty percent of patients admitted were male. The average age at admission was 48.9 years (SD = 10.5) and the median CD4 count was 150 cells/mm3. The average number of medical comorbidities experienced by patients at the time of admission was 5.9 (SD = 2.3). The most common being: AIDS-defining opportunistic infections ; respiratory conditions, such as chronic obstructive pulmonary disease, , and non-AIDS-defining malignancies .Between January 1 and December 31, 2008, 87 individuals were admitted to Casey House. Of the 87 individuals who were admitted, 17 , aging, HAART, and traditional risk factors for cancer, such as smoking and sun exposure . There iCertainly there are complex factors at play in the patients seen at Casey House, who may have as many as 6 medical comorbidities, including coinfection with hepatitis, as well as risk factors such as unstable housing, injection drug-use, and smoking . IndiviWe acknowledge that this retrospective study has several important limitations. Data extracted from charts are susceptible to variations in reporting and diagnostic criteria and missing data are common. The changes in reporting practices are amplified in this study where data were collected from time-points 20 years apart in a disease that has been described for only 30 years. In 1988, many primary causes of death were written as “AIDS.” It is most likely that these individuals and deaths would have been evaluated and recorded differently today. This may underestimate the number of individuals who died of AIDS-defining malignancies and possibly other non-AIDS-related causes of death. In this study, death certificates and primary cause of death were identified for all individuals but data were incomplete for many clinical and demographic variables. Our ability to clinically describe these patients is also limited by the state-of-knowledge and treatments available in 1988. Many tests routinely issued to HIV-positive individuals today, such as CD4 counts and viral loads, either did not exist or were not widely used in 1988. Hepatitis C was not yet identified [In summary, our findings are consistent with other studies in identifying changing trends in both the demographics and causes of death in individuals dying from HIV/AIDS. This study focuses on the patients and care provided at an AIDS hospice that evolved with the epidemic into a subacute hospital, giving some perspective to the ensuing changes that have occurred in organizations and the care supporting individuals with HIV/AIDS. As our knowledge and treatments for HIV improve, individuals are living longer but often with substantial psychosocial risk factors and significant medical and psychiatric comorbidities. Fewer HIV-positive individuals are dying from AIDS-defining conditions and increasing numbers of individuals are dying from non-AIDS-defining malignancies and other non-AIDS-defining conditions such as liver disease. The palliative care model of symptom management remains important for these patients. In order to plan and provide the care that is needed, it is crucial that we address both the medical and psychiatric complications and the social and psychosocial needs and risks of living with a chronic disease that has such a devastating and stigmatizing history. And, as long as HIV has no cure, we must also continue to acknowledge and provide care for individuals with HIV that embraces the palliative care model.


Objective. To analyze the expenses of HIV/AIDS and acute myocardial infarction (AMI) treatment in Brazil. Methods. A search in the Brazilian epidemiological database (DATASUS) on AMI and AIDS hospitalizations and their costs was done from January 1998 to December 2011. The number of HIV/AIDS cases and antiretroviral treatment (ART) costs was obtained from public Brazilian databases. Results. In 5 years, HIV/AIDS cases increased 38.5%, mainly in patients aged 25–49. There were 180,640 patients in ART in 2007 at a cost of R$ 3,920 per patient/year. The hospitalizations due to AIDS were stable over the last 13 years; however, the hospitalizations due to AMI have increased 78%. In 2007, the expenses with hospitalizations for HIV/AIDS and AMI (25–49 years) were approximately 0.12 and 1.52% of the Ministry of Health budget allocated to reimburse inpatient costs. The expenses on ART totaled 1.5% of the total budget . Conclusion. The prevalence of HIV/AIDS is still increasing in Brazil. There are scientific evidences suggesting an increased incidence of AIM in this population. Considering the high costs for the treatment of both diseases, an economic analysis is important to alert health managers to strengthen the preventive measures to guarantee the financial sustainability of such treatment. The Brazilian HIV/AIDS Program was established in May 1985 .Following the development of drug resistance in HIV patients, the Brazilian Ministry of Health had to provide new drugs at higher costs, investing up to R$ 551 million in antiretroviral treatment in 2003. There was an increase in the investment in subsequent years due to the acquisition of patented drugs (up to R$ 960 million in 2006). The introduction of the first fusion inhibitor, enfuvirtide, in the therapeutic regimen of multidrug-resistant patients had a strong impact on total costs. It was provided to 1,030 patients at a daily cost of US$ 22.19, the accounting for 4.4% of antiretroviral expenditure in 2006 [It remains controversial whether the exposure to combination antiretroviral (ART) treatment increases the risk of acute myocardial infarction (AMI). Friis-Møller et al. conducted a prospective observational study with 23,468 patients with HIV from 11 previously established cohorts to evaluate the risk factors and the incidence of myocardial infarction. The authors concluded that ART therapy was independently associated with a 26-percent relative increase in the rate of myocardial infarction per year of exposure during the first four to six years of use . Islam eGiven the importance and the high costs of treating both conditions in Brazil, this analysis aimed to estimate and analyze the costs of HIV/AIDS and AMI treatment and their impact on the public budget of the Brazilian Unified Health System.A search in the Brazilian epidemiological database (DATASUS) on AMI and AIDS hospitalizations and their costs was done from January 1998 to December 2011. The survey was restricted to patients aged 25–49 . Data were registered as the number of hospitalizations and reimbursed expenditures in Reais (R$). The annual number of HIV/AIDS reported cases was obtained from the TABNET database of the Brazilian Ministry of Health .Data from a previously published study about the antiretroviral treatment costs were analyzed. The authors analyzed the Ministry of Health's total spending on drugs and its programs between 2002 and 2007 . The FedThe Brazilian Public Health System perspective, which considers only direct costs that are reimbursed to health care providers was adopted. In this scenario, indirect costs are not computed in the database.From 1980 to June 2007, 474,273 HIV/AIDS cases were reported. In June 2012, this number increased to 656,701 registered cases . Data onThe number of hospitalizations for AMI and AIDS and their costs  is shown in Figures In Brazil, the age-standardized mortality from cardiovascular diseases decreased by 26% in the last decade, and this decrease was partially due to the successful implementation of health policies that led to the reduction of smoking and the expansion of access to basic health care . HoweverCardiovascular diseases represented the third leading cause of hospitalizations in the Brazilian Unified Health System, having been responsible for the greater spending on hospitalization, accounting for a total of R$ 1.9 billion or 19% of the total cost of hospitalizations. Acute coronary syndrome  accounted for 7% of all deaths in Brazil, associated with substantial direct and indirect costs to healthcare systems and society .In the last 5 years, the number of HIV/AIDS registered cases increased 38.5% . The ageThe number of hospitalizations due to AIDS related conditions was stable over the last 13 years; however, the number of hospitalizations due to AMI in patients aged 25 to 49 has increased approximately 78% in the same period. This age group has a low risk of cardiovascular events, but the combination of HIV exposure and HIV/AIDS treatment could be a possible reason for this increase in the incidence of AIM.The total costs with hospitalization have increased in both conditions, but with a more substantial growth in the AMI hospitalizations costs . This cost increase can be due to changes in clinical practice, such as the use of thrombolytic agents and revascularization procedures during admissions.The total budget of the Brazilian Ministry of Health in 2007 was R$ 44.3 billion and of this total, R$ 23 billion were allocated to reimburse outpatient and inpatient treatment costs. In the same year, the expenses with hospitalizations for HIV/AIDS-related conditions and AIM (25–49 years) were approximately 0.12 and 1.52% of this budget, respectively. Its worth mentioning that, in the same year, spending on antiretroviral treatment totaled 1.5% of the total budget .There are 18 antiretroviral drugs and 37 formulations available in Brazil for the treatment of people living with HIV (for children and adults). Ten of these drugs are produced by multinational companies and eight are locally produced (one private and six state-run Brazilian laboratories). It has been noted that for most of these medications  there was a significant reduction in the prices between 2006 and 2007, reaching 50% reduction in some cases .This study has some limitations. Firstly it adds only the indirect evidence that the rising cost of hospitalization for AMI among the low coronary risk may be due to HIV infection and the use of antiretroviral agents. Secondly, it was not possible to compare the costs of AMI in both HIV positive and negative populations, mainly due to the lack of information available in the Brazilian databases. Further work is needed to clarify this important and relevant issue.The prevalence of HIV/AID is still increasing among young adults and all these individuals will have access to long-term antiretroviral treatment in Brazil. There are scientific evidences suggesting an increased incidence of myocardial infarction in this population. Considering the high costs for the treatment of both diseases, health managers and decision-makers should be aware of strengthening preventive measures in order to guarantee the financial sustainability of the treatment in public health units in Brazil.


Respiratory tract infections are an important burden in primary care and it’s known that they are usually self-limited and that antibiotics only alter its course slightly. This together with the alarming increase of bacterial resistance due to increased use of antimicrobials calls for a need to consider strategies to reduce their use. One of these strategies is the delayed prescription of antibiotics.Multicentric, parallel, randomised controlled trial comparing four antibiotic prescribing strategies in acute non-complicated respiratory tract infections. We will include acute pharyngitis, rhinosinusitis, acute bronchitis and acute exacerbation of chronic bronchitis or chronic obstructive pulmonary disease (mild to moderate). The therapeutic strategies compared are: immediate antibiotic treatment, no antibiotic treatment, and two delayed antibiotic prescribing (DAP) strategies with structured advice to use a course of antibiotics in case of worsening of symptoms or not improving .Delayed antibiotic prescription has been widely used in Anglo-Saxon countries, however, in Southern Europe there has been little research about this topic. The DAP trial wil evaluate two different delayed strategies in Spain for the main respiratory infections in primary care.http://NCT01363531.This trial is registered with ClinicalTrials.gov, number  Infectious diseases are among the most common complaints for family physicians. About 70% of these are respiratory tract infections, and the most common infections are acute bronchitis, pharyngitis and rhinitis . Most ofBesides, antibiotic treatment is no free of downsides including adverse events, increasing the costs for the same episode, and reinforcing beliefs about their usefulness. It has also been observed that antibiotic prescribing in respiratory tract infections correlates with a higher frequency of visits to the physician in the future, thus rising patients’ expectations about their use . More imOn the other hand, there is great variability in antibiotic prescribing among professionals, communities, and countries. Spain is one of the countries with the highest rate of antibiotic prescribing  and the Nevertheless, sometimes avoidance of antibiotic prescribing is still a challenge in patients with uncomplicated respiratory tract infections. Firstly, because it is not known which patients are at a higher risk of developing complications that, though uncommon, might be serious. Secondly, there are patients who wish and expect to be prescribed an antibiotic if symptoms do not subside , and phySeveral studies have shown that the interventions aimed at reducing antibiotic prescribing may result in lower prevalence of resistant strains . One of A systematic review , concludDespite the advantages seen in other parts of the world, no studies have evaluated the effectiveness and the acceptability of delayed prescription in Southern Europe. Only a single observational study  showed tThe aim of this study is to establish the effectiveness of delayed antibiotic prescribing strategies versus antibiotic therapy and no therapy in terms of duration and severity of symptoms. Therefore, the study will establish the effectiveness of delayed antibiotic prescribing strategies versus antibiotic therapy and no therapy in terms of: antibiotic use at 30 days, satisfaction and belief in antibiotic effectiveness.Multicenter, randomized controlled clinical trial (Phase IV) comparing four therapy strategies for uncomplicated acute respiratory tract infections .Stratified by condition of interest . Centralized using a Web-based platform.Open-label study given the type of strategies used and given that an effect on their beliefs and the antibiotic use is expected.Four treatment arms:a) Group 1: immediate antibiotic therapy.b) Group 2: no antibiotic therapy.c) Group 3: delayed prescription by handing the prescription to the patient.d) Group 4: delayed prescribing by collecting the prescription at the health care center from the third day after the initial visit.The inclusion of no antibiotic and immediate antibiotic strategies is due to the need to evaluate their relative effectiveness compared with the delayed strategies. Potential differences among the delayed strategies are assessed by comparing the two delayed prescription strategies.The choice of antibiotic therapy will depend on the specific respiratory tract condition and will be up to the physicians’ judgement, according to their usual clinical practice. Physicians will also handle symptomatic treatment and complications as needed.Patients over 18 years old with uncomplicated acute respiratory tract infections will be enrolled: acute pharyngitis, rhinosinusitis, acute bronchitis, and exacerbations of chronic bronchitis/mild to moderate chronic obstructive pulmonary disease. Patients with these infections will be included by the physicians as long as they are unsure of whether to use antibiotics or not. See details of the general and specific study criteria for each condition under [see Additional file The sample size calculated is 600 patients. This is an estimate based on an average duration of 12 days (s.d. 6) for the main symptoms . Consideantibiotic use, the study will have a 100% for antibiotic use as an outcome, since the percentage of antibiotic use in the antibiotic therapy arm is likely to exceed 90%. For the calculations we used Sample Power Release 2.0 software.The patients will be equally distributed among the four evaluation groups to maximize power in the comparison. In addition, the four groups will be stratified in terms of the included conditions. Besides, focusing on the three groups except for the immediate antibiotic therapy, the 450 patients distributed in the three arms would allow to detect a difference of at least 13% in antibiotic use with the same indicated power (assuming that the figures for antibiotic use in the two delayed strategies are very similar: delayed strategy group 27% versus 14% in the group with no antibiotic therapy). Finally, in the comparison of four strategies for the variable Criteria for withdrawals and expected analysis of withdrawals and dropouts:•Serious adverse event•Unsatisfactory therapeutic effect•Patient clinical conditions prevent compliance with the protocol•Protocol violation•Informed consent withdrawalPatients can choose to interrupt the medication any time during the course of the study. However, they will be followed up in the same way as the other patients. Analyses will follow the intention-to-treat principle, with patients being analysed in the groups to which they were randomized.A) Main effectiveness endpointDuration and severity of symptoms. Patients will complete a validated symptom questionnaire daily [see Additional file B) Secondary effectiveness endpointsThe following variables will be assessed:Use of antibiotics: patients will be asked about antibiotic use in their visit at 30 days and this will be crosschecked with the corresponding pharmacy units in the sanitary areas.Satisfaction with the strategy .Belief in the effectiveness of antibiotics .Perception about safety and effectiveness .C) Safety endpointsComplications related to the infections will be registered during the first 30 days after randomization. These complications will be prospectively recorded by the physicians by means of a standardized questionnaire and will be reported in a maximum of 48 hours to the study clinical coordinator. This researcher will report to the Safety and Data Monitoring Committee, whose aim is to assess the safety of the evaluated strategies. The Clinical Event Allocation Committee - blinded to treatment allocation - will assess the primary outcomes. The decisions of the Assessment Committee will be used in all the statistical analyses of the primary outcome. This Committee will meet quarterly.Enrollment will be carried out competitively at the participating physicians’ clinics and is expected to take one year. The study will be carried out in five Spanish Autonomous Communities .Baseline data will be collected in the clinic by the physician or with the help of the nursing staff. To standardize data collection, participants will be trained by the coordinating center. Patients will receive information about the study and, if they are interested in participating, an informed consent to read and sign will be handed to them. A maximum length of 10–15 minutes is expected for the interview and the introduction of the data.After the randomization, information on the strategy to which they have been allocated will be given to the participants, and they will be informed on the appropriate measures to take in case of worsening or no improvement. Besides, they will be given a diary with a validated questionnaire of symptoms for each condition  which thPatients will be interviewed by telephone after 48 hours of their inclusion in the study. Monitoring calls are also planned at day 7, 15 and 22 if symptoms are still reported by the patient on the calls. After 30 days, patients will visit their physician and then will submit the completed diary. We will also include a one year follow-up.http://www.grupopreada.com).All the study data will be entered in an electronic platform ; inferencing will be made by means of an exact Fisher test or Chi-square test. The Kruskall-Wallis test will be used for ordinal or quantitative variables with heteroscedasticity or abnormality issues. In case of significance, the decision on the groups will be based on the 2 × 2 Mann–Whitney test; no corrections will be applied for multiple comparisons, calculated the means and the higher and lower values for each group. For quantitative variables, the analysis of variance (ANOVA) test will be used, resorting to post-hoc in case of significance, thus showing the means and the standard deviations for each group.IDIAP Jordi Gol i Gurina and by the Ethical Committees of Clinical Research in the corresponding health care areas and Spanish Agency of Medicines and Health Products. Each patient should give their written consent to participate in the study after being informed in intelligible language for him on the nature, scope and possible consequences of the trial. After consent is submitted, the patients will be randomized.The protocol was approved by the Ethics Committee of Clinical Investigation Data confidentiality will be ensured at all times, as it is stated in the researcher’s commitment sheet, as well as the compliance with current legislation regarding the protection of personal data. The clinical trial will be insured by the health provider to which the different participating health centers belong.This is a clinical trial based on the outpatient setting, and no monetary compensation is given to either patients or researchers. Sponsorship is only reserved for the purposes of organization and implementation of the trial.The main issue addressed in our study is the effectiveness of the delayed antibiotic prescribing strategy in uncomplicated respiratory infections. To achieve this we will compare two delayed prescription strategies with immediate antibiotic prescribing and no prescription. Specifically, symptom duration and severity, patient satisfaction, belief in the effectiveness of antibiotics and antibiotic use in the different strategies will be assessed.There are two main types delayed strategies. One where patients are asked to return to the reception of the primary health center in order to collect the prescription, and another one in which the prescription is handed in the initial visit. The rationale for the former being that asking the patient to return could further reduce subsequent use of antibiotics ,23.Delayed antibiotic prescribing has been used not only for respiratory tract infections but also for other types of diseases ,19. So fIn our study the two delayed strategies will be compared with other alternatives and with each other. We also wish to find out whether they are effective reducing the number of new visits. Antibiotic prescribing in the clinic leads to a vicious circle in which the patient perceives that an antibiotic treatment is necessary, it increases the number of new visits for the same reason and increases the chance of requesting a new antibiotic in similar episodes in the future . ConsequDelayed strategies are also criticized for an increased possibility of complications. In order to avoid them, it is crucial to bear in mind that these are not used in the case of patients presenting with supposedly bacterial infections, and reserved in the event of reasonable uncertainty. They should not be used in critically ill patients or those meeting criteria for admission .Two systematic reviews have studied the use of delayed strategies and both have showed significant advantages. One of them  showed tFurther studies  concludeOur study has several strengths but also some limitations. For example, the open-label design of the trial could imply a placebo effect favoring antibiotics. This effect is minimized given the structured approach that physicians will apply to support and increase the confidence in each strategy. In previous studies this placebo effect has been minimal in favor of antibiotics or has not been recorded ,25. AnotOne of the important strengths of our study is the inclusion of all the main respiratory tract infections, considering for the first time the exacerbations of mild to moderate COPD. Besides, it will be the first study to compare two delayed strategies directly. The pragmatic character of the study increases the external validity of the results and this is of special interest to our health system priorities.The confirmation of our hypothesis will likely reassure physicians about the effectiveness and safety of these delayed strategies. This fact could facilitate their implementation in our country and, thus, help optimizing antibiotic prescribing. We believe that our results will likely help to change patients’ perception about the role and need of antibiotics.The impact of our trial is likely to be important since the available evidence in this field is still limited, and new larger studies, adequately designed and conducted, are warranted to clarify the effectiveness of these strategies. Similarly, including most of the uncomplicated respiratory infections will increase the external validity of the results. Comparing, for the first time, two delayed strategies will provide information on the possible differences within this modality. These characteristics make this study innovative and relevant to this field.The authors declare that they have no competing interests.PAC conceived the study. PAC, MPA, RRC, CL, SHC, MMB, AIG, PTM, ANP, GP, EBT, IG, PL and GMD designed the study. PAC, MPA, GMD are responsible for conducting the trial. All other authors critically commented on the study design. MPA, CL, SHC, MMB, AIG, PTM, ANP, EBT, IG and all other authors who are family practitioners or nurses, belonging to the participant health centers, will recruit patients. All authors read critically and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2296/14/63/prepubInclusion and exclusion criteria.Click here for fileDiary (Symptom Questionnaire).Click here for file


Recently, it has been demonstrated that high cholesterol diet induced hypercholesterolemia and vascular lipid oxidation and accumulation in zebrafish larvae, suggesting that zebrafish is a new promising atherosclerosis model in addition to mouse models. However, up to date, there was no report regarding inflammatory cytokine expression during the lipid accumulation in zebrafish larva and adult fish. In this study, we first demonstrated the expression levels of IL-1β and TNF-α in high cholesterol diet (HCD)-fed zebrafish larvae, and found that although HCD induced vascular lipid accumulation, the cytokine expressions in the larvae were not changed by HCD. Furthermore, there was no significant difference in leukocyte accumulation in vessels between control and HCD fed group. But prolonged HCD induced IL-1β expression in spleen and liver compared to those of control zebrafish, and produced very early stage of fatty streak lesion in dorsal aorta of 19 week HCD-fed zebrafish. These results indicate that HCD induced hypercholesterolemia and atherosclerotic changes in zebrafish are very early stage, and suggest the necessity of the generation of mutant zebrafish having a disruption in a lipid metabolism-related gene leading to severe hypercholesterolemia and advanced atherosclerosis. Atherosclerosis is a chronic multifactoral inflammatory disease involving elastic and muscular arteries The atherosclerotic processes in human continue for long time with high complexity. Furthermore, it is almost impossible to perform direct analysis of the atherosclerotic lesion from an individual patient. Therefore, several animal models including mouse, rat, rabbit, and pig have been used to understand the complicated processes of atherosclerosis fli1:EGFP zebrafish, which express enhanced green fluorescent protein (GFP) in the vascular endothelium, is very useful to visualize the whole vascular system in live animals, and widely used to analyze angiogenesis In addition to mouse model, Miler and colleagues recently developed a zebrafish model of hyperlipidemia and atherosclerosis To date, although high cholesterol diet induced lipid and myeloid cell accumulations in larva and adult zebrafish, there was no report on the expression profiles of inflammatory cytokines in these fishes. In this study, we analyzed the expressions of inflammatory cytokines in zebrafish larva and in adult zebrafish fed high cholesterol diet. Especially, we focused on the analysis of the expression of IL-1β and TNF-α, mainly produced during innate immune response induced by high cholesterol diet in larvae. And then, we also continued to see the cytokine expressions in adult zebrafish fed same diet for longer period.Fli:EGFP transgenic zebrafish (Danio rerio) was from Zebrafish Oraganogenesis Mutant Bank (ZOMB) in Korea. Cd45:DsRed transgenic zebrafish was a generous gift of Dr. David Traver from university of California, San Diego. Zebrafish embryos were obtained from natural spawning and grown in tank water at 28°C on a 14-hour light, 10-hour-dark cycle. A high-cholesterol diet (HCD) for zebrafish was made in a diethyl ether solution of cholesterol (Sigma) to achieve a content of 4% (wt/wt) cholesterol in the artificial artemia (Azoo) after ether evaporation. Zebrafish was fed the diet 3 times a day for 19 weeks. To visualize vascular lipid accumulation in larvae, both control and HCD food were supplemented with 10 µg/g of a fluorescent cholesteryl ester analog (cholesteryl BODIPY 576/589-C11) (Invitrogen).All animal studies were approved by the Animal Care and Use Committee of the Seoul National University (SNU-120102-7). More than two microliters of blood was extracted from the hearts of zebrafish of control and HCD groups, after HCD feeding for 19 weeks. PBS-EDTA was added and plasma extracted from 10 zebrafish from each group was obtained by centrifugation. Total Cholesterol was determined using commercial assay kits .Salmonella typhimurium (Sigma) at 50 µg/ml for 3 hours, then washed in sterilized water for positive control of pro-inflammatory cytokines expression. The sequences of PCR primers for messenger RNA detection were as followings; 5′-GAGGAGCACCCCGTCCTGCTCAC-3′ and 5′-GATGGCTGGACCAGGGCCTCTGG-3′ for beta actin (Genbank: AF057040), 5′-TGGCGA ACGTCATCCAAG-3′ and 5′-GGAGCACTGGGCGACGCATA-3′ for IL-1β (Genbank: AY340959), and 5′-GCTTATGAGCCATGCAGTGA-3′ and 5′-TGCCCAGTCTGTCTCCTTCT-3′ for TNF-α (GenBank: AY427649). PCR was performed with an initial denaturation of 5 minutes at 95°C, then 35 cycles were run for 30 seconds of denaturation at 95°C, 30 seconds of annealing at 55°C, and 30 seconds of extension at 72°C. The reacted products were electrophoresed on a 1.5% agarose gel. Quantitative real-time PCR (SYBRgreen) assays were performed using a Rotor-Gene Q (QIAGEN) with zebrafish IL-1β, TNF-α and IL-6. The primer sequences of IL-1β and TNF-α are same to the primers used in semiquantitative RT-PCR. The sequence of PCR primer for IL-6 mRNA detection is as following; 5′-TCAACTTCTCCAGCGTGATG-3′ 5′-TCTTTCCCTCTTTTCCTCCT G-3′.At 8, 10, 15 and 20 days after control and HCD feeding from 4 day post fertilization (dpf), larvae (n = 15∼20) were pooled with same concentration. Those larvae were homogenized in 1.0 ml Trizol reagent , and total RNA was extracted from the homogenization, according to the manufacturer’s protocol. RNA was used for cDNA synthesis with SuperScript3 Reverse Transcriptase kit (Invitrogen). Total RNA from spleen and liver from 19 week old zebrafish were extracted using Trizol reagent. 20 larvae at 48 hour post fertilization (hpf) were stimulated with LPS from For in vivo confocal microscopy, anesthetized fish larvae were housed in a sealed, temperature controlled room in a small drop of tricaine containing water. Zebrafish larvae were examined with an Olympus FV1000 confocal laser scanning microscope . Images of zebrafish larvae were captured every 200 nm, and all images were analyzed and processed using the Olympus Fluoview software (Olympus).2)/2, a = long length, b = short length Zebrafish was anesthetized by putting them in low-temperature, and then cut off a head of each samples to exsanguinate. Spleens of zebrafish fed a HCD or control diet after 19 week feeding were weighed by .fli1:EGFP zebrafish larvae were fed HCD or control diet for 10 days, both supplemented with a 10 µg/g red fluorescent lipid. We observed that caudal vasculature of live anesthetized zebrafish larvae  larvae were fed HCD or control diet for 10 days. We observed that caudal vasculature of live anesthetized zebrafish larvae from control and HCD groups  for an additional 8 to 19 weeks. Compared to a control diet-fed zebrafish, a HCD-fed zebrafish had an enlarged belly, but the weight gain was not statistically different . HoweverP = 0.0603, Feeding HCD to zebrafish, in which spleen enlargement was observed starting at 19 weeks . Spleen Recently, HCD-fed zebrafish has been shown to be a promising animal model of atherosclerosis According to the previous studies using human cells and mouse models, it is widely-accepted that retained lipoproteins in subintimal space can be oxidatively modified and the oxidized lipoproteins can induce the production of proinflammatory cytokines in endothelial cells, smooth muscle cells and monocytes leading to initiation of atherosclerosis The mice are quite resistant to atherosclerosis because of their high blood levels of anti-atherogenic HDL and low levels of proatherogenic LDL and VLDL These technologies are highly valuable to understand the function of certain gene in zebrafish. LDLR and ApoE, known as important molecules in mammalian lipid metabolism, are also conserved in zebrafish. Moreover, several lipid metabolism-related genes are conserved in zebrafish including ApoA1, ABCA1, and oxysterol-binding proteins so on In conclusion, although HCD effectively induced vascular lipid accumulation, the expressions of IL-1β and TNF-α were not increased in HCD-fed larvae. The prolonged HCD for 19 weeks induced IL-1β expression, but only produced very early fatty streak lesion in dorsal aorta. These results indicate that HCD induced hypercholesterolemia and atherosclerotic changes in zebrafish are very early stage, and suggest the necessity of the generation of mutant zebrafish having a disruption in a lipid metabolism-related gene leading to severe hypercholesterolemia and advanced atherosclerosis.


It also identifies breast cancer patients who are at high risk to develop an aggressive disease in six large published breast cancer patient cohorts . Moreover, we have identified novel putative Met pathways, which correlate with Met activity and patient prognosis. This signature may facilitate personalized therapy by identifying patients who will respond to anti-Met therapy. Moreover, this novel approach may be applied for other tyrosine kinases and other malignancies.To determine the signaling pathways leading from Met activation to metastasis and poor prognosis, we measured the kinetic gene alterations in breast cancer cell lines in response to HGF/SF. Using a network inference tool we analyzed the putative protein-protein interaction pathways leading from Met to these genes and studied their specificity to Met and prognostic potential. We identified a Met kinetic signature consisting of 131 genes. The signature correlates with Met activation and with response to anti-Met therapy (p<0.005) in  Met-HGF/SF signaling is crucial for normal development Targeting HGF/SF-Met pathway is becoming an attractive approach for developing anti-cancer agents In this work, we generated a distinct Met signature based on kinetic mRNA expression alteration following treatment with HGF/SF on a cellular model. We used Met activation and inhibition cellular and animal models to demonstrate the signatures specificity to Met. Moreover, we have shown the signature’s ability to predict survival in over 1,000 breast cancer patients. Using a protein-protein interaction network analysis tool, we demonstrated the association between Met and its signature genes and identified novel putative Met signaling pathways, which correlate with Met activity as well as with breast cancer patient prognosis.in vivo and in predicting response to anti-Met therapy in cellular models, potentially serving to personalize anti-Met therapy.Our main contributions are: (i) using data derived from a cellular model of TKR activation we identify novel signaling pathways that are specific to the TKR (Met) and correlate with patient survival (ii) we demonstrate the utility of the kinetic signature in determining tyrosine kinase activity To characterize the effects of Met induction on breast cancer, we studied a cellular model consisting of five human breast cancer cell lines and one normal breast epithelium cell line (MCF10). Three of the cell lines , designated as high-Met, had significantly higher Met levels than the other cell lines  designated as low-Met, as shown by their HGF/SF binding capacity  p<1e-4,.http://www.ebi.ac.uk/arrayexpress/).To model the kinetic effect of Met activation in low  and high-Met  cell lines, we measured the relative mRNA levels using cDNA array at four different time points . These time points represent immediate and late responses to HGF/SF  were partitioned into six groups according to the time and direction of their initial response . The onlEnriched gene ontology (GO) terms and KEGG pathways of Met kinetic signature genes are listed in the Supplementary Results .We further assessed whether the immediate (10 min.) changes in gene expression in response to HGF/SF treatment can be explained by the mRNA half-life of Met kinetic signature genes  [27]. Wemt), described by Ponzo et al. The ability of the Met kinetic signature to differentiate Met activation/inhibition was assessed in several cellular and animal models. We first used mRNA levels from a transgenic mouse model, expressing an oncogenic variant of Met  from Met kinetic signature and demonstrated that the truncated signature differentiates between: 1) normal and tumor samples in the MetTaken together, these results may indicate that Met signature may be able to identify Met activation and inhibition in cellular and animal models , S4.To validate the cDNA array data expression levels, reverse transcription polymerase chain reaction (qRT-PCR) was performed on selected genes from the Met kinetic signature: Survivin, Pbk, Cyclin E1 and Ki67  Seven of the 32 genes included in the Met canonical pathway are included in the ANAT-derived Met pathways. Moreover, the most common GO annotation is HGF/SF receptor signaling pathway (23%). Other significantly enriched GO annotations are detailed in To validate the ANAT network, we examined if genes that were found to be a part of a specific Met pathway by ANAT are significantly co-regulated Appendix . We founTo validate the specificity of ANAT’s pathways to Met, we measured the capability of each pathway to differentiate between high and low-Met cell-line samples. We compared the results to a large set of network models derived with randomized data . The expTo assess the prognostic ability of ANAT’s pathways, we measured the capability of each pathway to predict patient survival in three breast cancer patient cohorts . We compared the results to a large set of network models derived with randomized data .The expression score of 18 (17.3%) pathways correlated with patient survival in all three data sets (p<1e-4). The expression score of 11 of these pathways was significantly higher in high-Met as compared to low-Met samples in the cellular model as well as correlated with poor prognosis in three large breast cancer cohorts . Interestingly, the expression of pathways whose genes have similar kinetic response to HGF/SF (pathway with high coherency ranking) in the cellular model, correlate with poor patient outcome (spearman rank correlation was -0.316 with p = 0.001). These results indicate that the coherent pathways play a critical role in Met signaling, affecting tumorigenesis and metastasis.To further validate the specificity of ANAT network to Met, we used ANAT to compute the pathways leading from four alternative different tyrosine kinases serving as anchors  to the 131 genes composing the Met kinetic signature. Met and ERbB2 ANAT derived network showed similar number of prognostic pathways . However, significantly more Met pathways (17.3%) correlated with patient prognosis, as compared to the EGFR, ERbB2, INSR and PDGFRA derived ANAT pathways respectively .These results demonstrate the prognostic value of the Met anchored ANAT derived pathways, the similarity between Met and ERbB2 signaling pathways and it’s specificity as compared to the other pathways.mRNA levels and clinical data from three large human breast cancer patient data sets , estrogen receptor (ER) status  and histological grade . Moreover, the Met kinetic signature correlated with patient survival even after removal of cell-cycle genes  and is comparable to the 70 gene signature Thus Met kinetic signature is associated with triple negative basal like tumors, and identifies patients with reduced metastasis-free, disease-related and overall survival.These results suggest that Met kinetic signature may serve to identify breast cancer patients with activated Met pathways and high risk of an aggressive disease, who would benefit from personalized anti-Met therapy.In the past decade, several microarray-based prognostic signatures were developed, that combined with other traditional clinical factors, facilitate management of cancer. Currently, however, cDNA based signatures do not predict response to targeted therapies. In this work we lay the grounds for the identification of breast cancer patients most likely to benefit from anti-Met therapy.We have used, a cellular model which simulates a specific TKR activation to generate a molecular signature which is then applied on other cellular and animal models combined with several large scale patient cohorts, strongly supporting its validity. Moreover, the signature relation to the TKR was validated by activation and inhibition models as well as by protein-protein interaction network analysis. We postulate that this methodology may be used to reveal new signaling pathways and predict response for other oncogenes.Careful design of the cellular model is critical for its ability to best differentiate between tumors with low and high Met activity. To this aim we: 1) used breast cancer cell lines with low and high Met expression and activation; 2) used several cell lines in each group to highlight Met activation features which are not cell line specific; 3) measured short and intermediate temporal changes in gene expression following Met activation by HGF/SF and 4) used multiple replications to ensure data integrity. Taken together, these measures ensured our cellular model identifies differences in Met activation between low and high Met samples with high specificity.Although the cells were selected only based on their Met expression levels and their response to HGF/SF treatment, the high-Met cell lines were assigned to the Claudin-low subtype by the Perou group while the low-Met cell lines are either of the luminal cancer subtype or normal cells (MCF10) We started by generating Met kinetic signature, based on the kinetic response to HGF/SF of cell lines with high or low Met expression. The fact that Met kinetic signature classified the cell lines to Met-level groups indicates that these genes play a major role in regulating the biological activities induced by Met.We do not fully understand why genes which expression is down regulated shortly after HGF/SF treatment, on one hand differentiate between High and Low Met samples in the cellular model, and on the other hand high expression of these genes correlate with poor prognosis. One possible explanation relates to the role of many of these gene products in cell cycle regulation and DNA replication and repair. Such genes’ expression is tightly regulated with regard to the cell cycle phase. In the cellular mode, the kinetics measured hint towards short-term HGF/SF induced cell cycle synchronization as opposed to the altered expression in the unsynchronized tumor cells. These immediate changes in the expression of Met kinetic signature genes may also relate to the relatively short half life of Met kinetic signature genes. Moreover, the immediate response might be more specific to Met while the long term response may represent the similarity in all TKR signaling pathways.The classification between High and Low Met kinetic signature correlates with histopathological and clinical features. Thus, most of the tumors in the “High Met kinetic signature” group were basal-like tumors and the patients had reduced disease free intervals and poor prognosis independent of its Claudin classification . These rTo assess the relation between Met and its kinetic signature genes, we used ANAT, a new interactive software tool for elucidating functional networks of proteins We have then showed that the ANAT derived Met network is enriched with pathways whose gene values correlate with patient prognosis. Moreover, networks recalculated the after replacing Met with EGFR, ERbB2, INSR and PDGFRA, yielded far less prognostic pathways, indicating the signatures specificity to Met and patient prognosis.The ANAT derived network linking Met and its kinetic signature genes revealed eleven novel putative pathways which correlate with Met activity and breast cancer patient prognosis. These pathways contain genes that are involved in cell migration, invasion, proliferation, cell cycle and some correlate with anti-estrogen resistance and p53 deactivation. Genes from all eleven pathways were found to be associated with Met and with cancer progression . DetaileImportantly, we found a correlation between the coherencies of the ANAT derived Met pathways in the cellular model and their prognostic value in breast cancer patients, indicating biological co-regulation of prognosis-associated Met pathway genes.Multiple analyses were used to demonstrate the signature’s specificity to Met and its robustness as prognostic marker for breast cancer. Specifically, by demonstrating its prognostic capabilities on more than 1000 patients in six different breast cancer patient cohorts.mt animal model; 2) differentiates Met inhibited cells from control in a Met-addicted cellular model and 3) predicts long term prognosis on a large cohort of patients. Met pathway signature was found to be comparable to other classification methodologies  identifies high Met activity in a cellular model as well as Met-specific tumors in a MetMet kinetic signature may also open a new spectrum of therapy targets, through understanding of the molecular mechanism of Met activity in the tumor. We hypothesize that this signature will enable personalized therapy by identifying patients in which anti-Met therapy will suppress Met downstream signaling and delay tumor progression. Clinical trials are needed to study Met pathway signature’s role as a clinical tool to identify breast cancer patients most likely to benefit from personalized anti-Met therapy.Details regarding the preparation and characterization of the cellular model, generation of the cDNA arrays its validation by qRT-PCR, immunofluorescence and Western blot, as well as description of Met kinetic signature generation are listed in Human breast cancer cell lines expressing low Met ,or high constitutively activated Met  and a human cell line derived from normal breast epithelium MCF10  . Cells were incubated with purified HGF/SF mut mammary tumor model, described by Graveel et al We used six published large data sets of genome-wide expression measurements taken from breast cancer patients: (i) The Miller data Met canonical expression score), including a list of Met canonical pathway genes according to published literature  network (described below) in the same manner.We evaluated the predictive power of the signature with respect to several phenotypes  by clustering the patients into two groups using hierarchical clustering as previously described.Met canonical expression score was then calculated for patients in each group. The group with the higher score average was designated “High Met kinetic signature” and the other group was designated “Low Met kinetic signature”  sampled from the cDNA array. We evaluated the prognostic value of each signature by taking a lower bound on its performance across all three  data sets . To further validate Met kinetic signature, we evaluated its association with classical  tumor characteristics and patient prognosis, on additional three human breast cancer data sets ANAT  is a new interactive software tool for elucidating functional networks of proteins We used ANAT to construct the most likely network that connects the proteins encoded by the newly found signature to Met (acting as an anchor). After an initial construction of the network, we performed minor adjustments by forcing directionality on several of the edges , and by removing FGFR1 and FGF3 . If a specific pathway was found to be included in another, the longer pathway was excluded from the network.The specificity of the ANAT predicted Met pathways’ was assessed by comparing each pathway’s expression score in high versus low-Met cell lines. To evaluate its prognostic value in breast cancer patients, we compared the expression score of each pathway between patients with good prognosis  and patients with bad prognosis (those who died from the disease or developed metastasis at any time during follow-up). On both tests, significance is estimated using a two-tailed t-test.To evaluate the specificity of the results, we performed two analyses using (i) another tyrosine kinase growth factor receptor as an anchor to the Met signature genes and (ii) using ANAT to construct 100 Met anchored networks using 100 random signatures of the same size as the Met kinetic signature. We compared the significance levels obtained for the pathways of the original network to those of the random pathways, determining a pathway as significant if its level scored higher than 95% of the random pathways. To evaluate the significance of the Gene Ontology (GO) t-test.To further validate the ANAT network, we examined if genes that were found to be a part of a specific Met pathway by ANAT are significantly co-regulated. Indeed, it was recently shown that two proteins that share the same pathway and are co-regulated by specific factors/elements are also co-expressed and their expression levels are correlated 10 of the worst p-value between all data sets was designated as the pathway’s prognosis ranking. The relation between the coherency and the prognosis ranking of ANAT derived pathways was determined using Spearman’s rank correlation.The coherency ranking of a pathway is defined as the average correlation of consecutive pairs of genes along the pathway. For each pathway we calculated: 1) the coherency ranking of the ANAT derived pathways based on the cellular model , 2) pathway expression score for each patient in the breast cancer patient data sets. We calculated each pathway’s predictive value by comparing the pathway expression score between good and bad prognosis patients using student’s t-test on each data set. The logFigure S1Hierarchical clustering of the cell line model according to Met canonical pathway genes. Hierarchical clustering of the breast cancer cell line model according to Met canonical pathway genes, perfectly segmented the cell array samples into low and high-Met samples (p<1e-4) (A). Met canonical pathway score correlates with patient survival in only one of three breast cancer patient data sets (B). Met mRNA levels did not correlate with patient survival in all three breast cancer patient data sets (C).(PDF)Click here for additional data file.Figure S2Met kinetic signature after removal of cell cycle genes, identifies Met activity and predicts survival. Cell cycle genes (according to their GO annotation) were removed from the Met kinetic signature, resulting in a 96 gene signature. The reduced signature significantly correlated with Met activation animal model (A) and Met inhibition cellular model (B) and predicted survival in five of six large breast cancer patient cohorts: van ‘t Veer (C), Miller (D), Chang (E), GSE3165 (F), GSE1456 (G) and GSE11121 (H).(PDF)Click here for additional data file.Figure S3Hierarchical clustering of mutationally activated Met mouse model according to Met kinetic signature genes. We used mRNA levels from a mutationally activated Met mouse model and found that using Met kinetic signature, hierarchical clustering significantly segmented the samples into a “normal” and “tumor” groups (p<1e-4).(PDF)Click here for additional data file.Figure S4Hierarchical clustering of Met inhibition cellular model according to Met kinetic signature genes. Using the Met inhibition cellular model described by Bertotti et al., we found that Met kinetic signature perfectly separated Met inhibited samples in the high-Met cell line and also the EGFR inhibited samples in the EGFR-addicted samples (p<0.005).(PDF)Click here for additional data file.Figure S5Quantification of Western blot analysis. Basal levels of Met are 1.56 times higher in MDA231 as compared to MCF7 cells and treatment with HGF/SF did not significantly change Met basal levels in either both cell lines (A). Levels of pMet are 6.4 times higher in MDA231 as compared to MCF7 cells. Sixty minutes following treatment with HGF/SF, pMet levels are 1.86 higher in MCF7 as compared to base line, but are still significantly lower than in MDA231, whose pMet levels did not significantly change following treatment with HGF/SF (B). Levels of ERK in MCF7 and MDA231 are similar (C). Levels of pERK are 15 times higher in MDA231 as compared to MCF7 cells. Levels of pERK did not significantly change following treatment with HGF/SF (D). As expected, E-cadherin levels are almost undetected in MCF7 cells and in MDA231, its levels are elevated following treatment with HGF/SF (E). Survivin levels are 1.9 higher in MDA231 as compared to MCF7 cells and do not significantly change following treatment with HGF/SF (F).(PDF)Click here for additional data file.Figure S6Survivin immuno-fluorescence in MCF7 and MDA231 cells. MCF7 and MDA231 cells were incubated with the primary antibody anti-Survivin . Slides were analyzed using a 510 Meta Zeiss confocal laser scanning microscope (CLSM). When comparing fluorescence intensities, identical CLSM parameters  were used. To compare the relative levels of protein expression, we used the average area intensity (AAI) image analysis procedure for cells immunostaining. The image analysis calculations were performed on five to ten microscopic fields. Cell outline was drawn based on DIC images; nuclei were defined based on the DAPI staining. Average pixel intensity was calculated separately for the nucleus and cytoplasm areas. . Variance was analyzed by student’s T-test.(PDF)Click here for additional data file.Figure S7Gene-pair correlation distribution comparison of ANAT derived Met network. The gene-pair correlation distribution comparison of ANAT derived Met network is significantly higher then that of all the interacting genes in the ANAT database (p = 0.0006).(PDF)Click here for additional data file.Figure S8Expression score of ANAT-derived pathways. The expression score of 38 out of the 104 calculated pathways (36.5%) significantly differentiated High vs. Low Met cell lines as compared to a random expectation of 5% .(PDF)Click here for additional data file.Figure S9Subgroup analysis of Met kinetic signature by ER status. Subgroup analysis of Met kinetic signature by ER status in van ‘t Veer, Miller, Chang and GSE3165 data sets showed that high-Met kinetic signature correlated with poor prognosis in ER+ patients, but not in ER- patients.(PDF)Click here for additional data file.Table S1Met Kinetic Signature genes.(PDF)Click here for additional data file.Table S2Primers for qRT-PCR. The following primers are used for the quantification of gene expression.(PDF)Click here for additional data file.Table S3ANAT derived pathways originating from Met to the kinetic signature genes.(PDF)Click here for additional data file.Table S4Differences in Met canonical pathway expression score between patient groups segmented by Met kinetic signature.(PDF)Click here for additional data file.Table S5Cox proportional hazards regression survival analysis using Met kinetic signature and basal-like classification on three breast cancer patient cohorts.(PDF)Click here for additional data file.Text S1Supplemental Methods.(DOC)Click here for additional data file.Text S2Supplemental Results.(DOC)Click here for additional data file.