Purpose: We aimed to assess the effect of age on survival according to estrogen receptor (ER) and progesterone receptor (PR)-defined lobular breast cancer subtype in a wide age range.Methods: 43,230 invasive lobular breast cancer women without comorbidities diagnosed between 2004 and 2011 in the National Cancer Database (NCDB) were analyzed. The effects of age on overall survival (OS) among different age groups were evaluated by log-rank test and Cox proportional model.Results: Multivariate analysis showed that patients diagnosed at both young (<35 years) and old  ages had worse prognosis compared with those in the middle ages. We further analyzed the interaction between age and molecular subtype for predicting OS: in ER+PR+ subtype, the HR of OS declined with age from 1.55  in the group younger than 35 years to 1.38  in the 35–39 group, but increased with age to 10.1  in the group older than 79. While in ER+PR- and ER-PR- subtypes, the HRs showed no statistical differences among women diagnosed before 60 (P > 0.1); and in ER-PR+ subgroup, the HRs were similar in patients younger than 70 (P > 0.1); thus, the plots of HRs in these three subtypes remained steady until the age of 60 or 70.Conclusions: Our findings identified that the effect of age on OS in lobular breast cancer varied with ER/PR-defined subtypes. Personalized treatment strategies should be developed to improve outcomes of breast cancer patients with different ages and ER/PR statuses. Breast cancer is known to be a heterogeneous disease, which exhibits distinct clinical presentations, aggressiveness, treatment response, as well as outcomes among different subtypes of breast cancer. Accurate prognostic factors play a critical role in treatment decision-making during the management of this life-threating disease. Besides the classic biologic predictors, age at diagnosis has been proved as an independent prognostic factor in several studies –9. AlthoInvasive lobular carcinoma accounts for approximately 10% of all breast cancer cases, and it differs from invasive ductal carcinoma . SeveralIndeed, little is known about the prognostic value of age according to ER/PR-defined subtypes for invasive lobular breast cancer . A singlHere, we analyzed a large national cohort of invasive lobular breast cancer patients using the National Cancer Database (NCDB) to assess the prognostic value of age on OS of different ER/PR-defined breast cancer subtypes in a wide age range. The large sample size and available systemic therapy information in this database enabled us to better quantify the impact of age on survival and provide high-quality evidence for individualized treatment decision-making of lobular breast cancer.P < 0.001 for all comparisons), which might be partially explained by the large sample size of the study. ER-PR+ and ER-PR- subtypes had more patients who were diagnosed at a very young age (<35 years) compared with ER+PR+ or ER+PR- subgroup  patients had ER+PR+ tumors, 5,000 11.5%) patients had ER+PR-disease, 585 (1.4%) women had ER-PR+ cancers, and 9,683 (22.4%) women had ER-PR- subtype of tumors. Table 1.5% patin = 43,230), a total of 4,511 (10.4%) patients died. Kaplan–Meier curves were used to investigate OS in different age groups among the four ER/PR-defined subtypes . And surgery type (lumpectomy vs. mastectomy ± reconstruction) had a marginal effect on OS  in the group younger than 35 years old to 1.17 in the 35–39 group, and kept statistically flat through 1.09  in the 50–59 group, but then elevated to 6.50  in the group older than 79 years  in the group younger than 35 years old to 1.38  in the 35–39 group, but increased with age to 10.1  in the group older than 79. The curve of the HRs in this subtype declined slightly first and then elevated sharply , which was consistent with the previous log-rank test; and in ER-PR+ subgroup, the HRs of patients younger than 70 years old were not significantly different , which was also in accordance with the log-rank analysis. Thus, the plots of the HRs in these three subtypes remained steady until the patients reached an older age  between distinct combinations of age and ER/PR subtypes regarding OS Table . The trey Figure . While iIn this large, registry-based, national cohort study using data from NCDB, we quantified the prognostic impact of age on OS of different ER/PR-defined lobular breast cancer subtypes in a wide age range. After adjusting for known breast cancer prognostic factors and multiple treatment variables, we demonstrated that patients who were diagnosed with invasive lobular breast cancer at both very young (<35 years) and old (≥70 years) ages had significantly worse prognosis compared with those in the middle age groups. The more important finding was that different patterns of the effect that age had on OS, according to the ER/PR-defined breast cancer subtype were observed in our study.n = 9) young cases (<35 years) in their study cohort, and no in-depth analysis has been performed to assess the effect of age on OS according to the ER/PR status [To the best of our knowledge, the present study is the first and largest study that assessed the prognostic values of age on survival according to the ER/PR-defined molecular subtype in lobular breast cancer. Regarding the impact of age on outcomes in breast cancer, most of the studies found that the survival difference based on age was observed only in patients with HR-positive cancers , 10, 11,R status . AlthougR status , 25.More interestingly, we identified that the effect of age at diagnosis on OS differed by ER/PR-defined subtype. The HRs for the ER+PR+ subtype declined slightly first and then elevated sharply when plotted against age, whereas the HRs for the ER-PR- patients remained steady until they reached an elder age (>60 years old). This is partially in accordance with the findings of above-mentioned SEER study, although their study population was invasive ductal breast cancer patients . Indeed,P = 0.051). Thus, lobular carcinoma patients who received breast-conserving surgery seemed to have better prognosis than those who were treated with mastectomy. Several recent population-based studies that included lobular breast cancer cases also identified an OS benefit of breast-conserving surgery vs. mastectomy, however, the percentages of lobular carcinomas in these studies were either very small (≤10%) or unknown, and no subset analysis was performed to assess the impact of surgery type on survival in lobular cancers [In addition, besides the well-defined independent prognostic factors of lobular breast cancer including age, race, histologic grade, pathologic tumor and nodal stage, receiving of radiotherapy, chemotherapy and hormone therapy, for the first time, we found that the surgery type had a marginal effect on OS , menopausal status and lymphovascular invasion information, as well as the information regarding pleomorphic lobular carcinomas, which are known to be associated with survival. However, in a retrospective analysis of a large randomized controlled trial of women with early-stage HER2-positive breast cancer, age was not significantly correlated with risk of early recurrence or prediction of benefit from trastuzumab therapy . Third, We used data from the NCDB, which is a national hospital-based cancer registry jointly sponsored by the American College of Surgeons and the American Cancer Society, and collects data on about 70% of newly diagnosed breast cancer cases from approximately 1,450 Commission on Cancer-approved hospitals across the United States. Data are coded and reported according to nationally established protocols coordinated under the auspices of the North American Association of Central Cancer Registries.A total of 146,290 pure lobular breast cancer cases  diagnosData within the NCDB were rendered anonymous, so the study was exempt from review by the Johns Hopkins Medicine Institutional Review Board, and no consent was needed in this study.The study outcome was OS, which was calculated from the date of diagnosis to the date of death from any cause, with surviving patients censored at date of last contact. To investigate the impact of age on OS, we treated age at diagnosis as a categorical variable classified into the following age groups: younger than 35 years, 35–39 years, 40–49 years, 50–59 years, 60–69 years, 70–79 years, and older than 79 years. The demographic statistics of patient included age at diagnosis, race, and year of diagnosis. The clinicopathological characteristics included histologic grade, laterality, pathologic tumor/node stage, number of examined regional lymph nodes, ER and PR status. The treatment information comprised surgery type, receiving of radiotherapy, and receiving of systemic chemotherapy or hormone therapy. The lobular breast cancer subtypes were defined as ER+PR+, ER+PR-, ER-PR+, and ER-PR- according to the ER and PR statuses of tumor. ER or PR positive groups included those with borderline results .To assess whether there was an interaction between age and ER/PR-defined subtypes in predicting OS, we created an interaction term. Pairwise comparisons were performed between different combinations of age and ER/PR-defined subtypes. Patient demographics, clinicopathological and treatment characteristics by breast cancer subtypes were compared using the χ2 test. The Kaplan–Meier method was used to analyze survival curves. Multivariate Cox proportional hazard model was applied to determine whether the independent prognostic effect of age on OS vary with distinct ER/PR-defined subtypes of lobular breast cancer.P < 0.05.Statistical analyses were conducted using STATA 13.0 software . All statistical tests were two-sided, and the statistical significance was defined as In summary, we demonstrated that the prognostic effect of age on OS in lobular breast cancer varied with different ER/PR-defined subtypes. The HRs for ER+PR+ subtype declined slightly first and then elevated sharply when plotted against age, whereas the HRs for ER+PR-, ER-PR+, and ER-PR- subgroups remained steady until they reached an older age (>60 or 70 years). Personalized management strategies should be developed to improve outcomes of breast cancer patients with different ages and ER/PR statuses. For example, young (<40yrs) ER+PR+ invasive lobular breast cancer women might be undertreated if they receive only chemotherapy or endocrine therapy. Adjuvant chemotherapy followed by endocrine therapy may be recommended to these patients. Moreover, our findings can help clinicians with the patient consultation about the long term prognosis according to their age and ER/PR status. Further prospective studies are needed to confirm our findings.


School-based health centers (SBHCs) offering a wide scope of health services are an expanding model of healthcare in the US. SBHCs are potential venues for brief intervention (BI), but little is known about the best way to deliver BI in these settings. The objective is to describe pilot data collected in preparation for a randomized trial of nurse- vs. computer-delivered BI for high school SBHC patients with risky alcohol or cannabis use.In preparation for the randomized trial, we (1) reviewed electronic medical record (EMR) data on patient volume and risk behaviors, and (2) conducted a longitudinal survey with an assessment-only cohort of 50 adolescents representing the target population for the forthcoming trial. Patients were eligible if they endorsed alcohol or cannabis use and scored positive on the CRAFFT, a brief substance use screening tool. Audio computer-assisted self-interviewing was used to assess lifetime and past 30 day frequency of substance use and sex risk behaviors, with follow-up assessments at 3- and 6-months.In the year prior to initiating study activities, the two participating SBHCs logged 5,967 visits with 1,083 unique patients, of whom 11% had alcohol and/or drug use recorded in the EMR. Participants with risky substance use in the assessment-only cohort reported baseline rates of past 30-day cannabis and alcohol use of 88% and 50%, respectively. Base rates of past 30 day unprotected sex and sex while high/drunk were 44% and 34%, respectively. Follow-ups will examine the short-term natural trajectories of these behaviors.Our pilot data supports SBHCs as promising sites for delivering BIs to adolescents. Our forthcoming trial will examine the comparative effectiveness of two BI approaches at these SBHCs.


The prognosis of intensive care patients with acute kidney injury (AKI), which is associated with increased mortality rates (up to 30 %), is still poor. In the long run, patients with acute kidney injury on ICU have a high risk of maintaining advanced chronic kidney disease (CKD) or even end-stage renal disease (ESRD).The aim of this study was to investigate the prevalence of AKI in adult critically ill patients in the University Hospital, Regensburg, Germany. Furthermore, we searched for the most common ICU conditions associated with AKI, evaluated the length of stay in the intensive care unit (ICU) and in the hospital and calculated the additional in-hospital costs associated with AKI in Diagnosis Related Groups (DRG) based reimbursement system in Germany.In this retrospective study all finished admissions in 5 adults ICUs during 2011-2013 were evaluated. AKI and the 3 ICU-diagnosis commonly associated with AKI were identified using ICD-10 classification. Using regression analyses, the length of stay in hospital and ICU for patients with and without AKI was examined. AKI-associated in-hospital costs were calculated for 2013 according to DRG-related calculation for single diagnosis/ procedure and adjusted to nationwide base case value for reimbursement.In total 891 ICU-patients AKI was classified according to ICD-10 code. Acute respiratory distress syndrome (ARDS), myocardial infarction (MI) and sepsis were the three most common ICU-conditions associated with AKI. 1103 patients were admitted with one of these three diagnoses during 2011-2013 and 249 (22.6%) of these developed an AKI. Patients with AKI had a significantly longer ICU and hospital stay compared to patients without AKI . In-hospital costs for septic patients with AKI were higher than for septic patients without AKI (25 216 € vs. 13 486 €). Patients with ARDS or MI without AKI had an in-hospital costs of 12 387 € compared to 41 949 € for patients with ARDS or MI and AKI. Presence of AKI in critically ill patients with ARDS, MI or sepsis was associated with additional costs of 2 019 120 € in the University Hospital, Regensburg in 2013.Occurrence of AKI in critically ill patients is associated with significant longer ICU- and hospital stay. Moreover, it causes considerable higher in-hospital costs in DRG-based reimbursement. Early recognition of patients at risk, coordinated research for novel interventions and establishment of national AKI-network for implantation of evidence-based therapies may decrease the incidence of AKI, improve outcome and save costs for national health care system.


The latest update of the miRTarBase expanded it to identify systematically Argonaute-miRNA-RNA interactions from 138 crosslinking and immunoprecipitation sequencing (CLIP-seq) data sets that were generated by 21 independent studies. The database contains 4966 articles, 7439 strongly validated MTIs (using reporter assays or western blots) and 348 007 MTIs from CLIP-seq. The number of MTIs in the miRTarBase has increased around 7-fold since the 2014 miRTarBase update. The miRNA and gene expression profiles from The Cancer Genome Atlas (TCGA) are integrated to provide an effective overview of this exponential growth in the miRNA experimental data. These improvements make the miRTarBase one of the more comprehensively annotated, experimentally validated miRNA-target interactions databases and motivate additional miRNA research efforts.MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides, which negatively regulate the gene expression at the post-transcriptional level. This study describes an update of the miRTarBase ( MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs with ∼22 nucleotides (nt) that play important roles at the post-transcriptional level in animals and plants . The mecmicroRNA.org  includes a total of 366 181 curated MTIs between 3786 miRNAs and 22 563 target genes, which were collected from 4966 articles. The number of MTIs in miRTarBase has increased ∼7-fold since the 2014 miRTarBase update (miRTarBase v4.5) . MTIs wiet al.  RNA-binding proteins have been collected. The CLIP-seq NGS raw data were downloaded from the Gene Expression Omnibus (GEO), Short Read Archive (SRA), European Nucleotide Archive (ENA) and websites hosted by individual researchers. A total of 109 HITS-CLIP and 29 PAR-CLIP data were collected from 21 studies. The samples were annotated with the following categories: Data set source, Reference, RNA binding protein (RBP), Species, Accession number, NGS methods, Tissue/cell line and Treatment (Supplementary Table S1).Number of MTIs validated by ‘NGS’’ in Table Six CLIP-seq studies do not provide NGS raw data but do provide MTIs in their supplementary data. Their results were directly included into our database. Most of the samples (138 samples) provide raw NGS data that were analyzed based on the miRTarCLIP pipeline  combinedDysregulated miRNAs are reportedly associated with many diseases. Recently, studies have reported that some circulating miRNAs are biomarkers for disease diagnosis and provide clues concerning potential disease therapies. Aside from experimentally validated miRNA disease information that was curated by our group, miRTarBase integrates the data of HMDD version 2.0, miR2Disease, ExcellmiRDB  and miRCP-value = 3.9e−7 and R = −0.639). Interestingly, Tsai et al. reported that miR-122 is a tumor suppressor and targets ALDOA in hepatocellular carcinoma  that are obtained using the array-based or NGS-based technique. The correlation of the expression of miRNA with that of mRNA importantly indicates the direct targets of miRNA. Large TCGA clinical data sets are used in clinical research to develop microRNA biomarkers or to help biologists study tumor-specific miRNA regulatory pathways. starBase v2.0  displaysarcinoma . starBasUnlike databases and prediction tools, miRNA-related information is largely available as unstructured text. As shown in Supplementary Figure S2 (in the Supplementary Document), the number of PubMed query results for ‘miRNA’ in the title or abstract has grown substantially since 2000. The number of miRNA related publications is estimated to grow to over 10 000 by 2016, and manual retrieval of associations between miRNA and target gene can be labor-intensive. Therefore, a two-step MTI retrieval system is developed herein to reduce the effort for curator. First, natural language processing (NLP) techniques are used to rapidly screen a large number of PubMed-indexed studies to select the correct miRNA and target gene pairs. Additionally, all of the screened documents are manually validated to extract other key elements such as terms concerning experimental methods, cell lines and descriptions of microRNA and target gene association.Named entity recognition (NER) and relation extraction (RE) are two important components of the proposed MTI retrieval system. NER is utilized to locate and classify elements in unstructured biomedical texts, such as the names of miRNA and target genes. Relation extraction is the other crucial component of extracting miRNA-target interactions. Accordingly, rule-based and machine learning approaches have been used to extract MTIs. Numerous public databases, such as miRCancer  and miRSAccordingly, this work developed an automatic text-mining module adopting a principle-based approach (PBA)  for the The proposed retrieval system has the following advantages over existing automatic methods for extracting miRNA targets; (i) based on an annotated training corpus , it consFigure Table MicroRNAs play essential roles in different diseases as either tumor suppressors or oncogenes. Their expression pattern is tissue-specific and holds potential for therapeutic targets and novel biomarkers. The word cloud of miRNA-disease information is improved in the updated version of miRTarBase. A user who searches for a microRNA of interest in miRTarBase can now learn about its association with different diseases. The cloud of disease-related words provides at a glance useful information about the role of microRNA in diseases. For example, hsa-miR-122–5p is the microRNA that is most frequently found in liver and associated with liver biology and liver diseases such as hepatitis and hepatocellular carcinoma Figure .CLIP is a method that combines ultraviolet (UV) cross-linking with immunoprecipitation to identify specific protein–RNA interactions. Large-scale MTIs have been recently identified using the CLIP-seq approach including HITS-CLIP, PAR-CLIP and CLASH. The first step in CLIP-seq data processing is to map all of the reads to the genome and transcriptome; it is followed by cluster detection and, finally, binding site detection . miRTarBP-value = 3.9e−7 and R = −0.639) (Figure TCGA collects large clinical data sets to develop microRNA biomarkers and to help biologists identify tumor-specific miRNA regulatory pathways. The correlation between the expression values of miRNA and mRNA provides an important indication of direct targets of miRNA. For instance, the change in miR-122–5p and ALDOA reveals the negative correlation significantly only in human hepatocellular carcinoma  Figure .in silico identification of confirmed targets of miRNA of gingival tissue in periodontitis in cases of obesity (Recently, many researchers have utilized miRTarBase. Four main applications of this database are summarized here. The first is in the elucidation of miRNA functions under different conditions and in different species. Notably, miRTarBase is being utilized to determine a molecular mechanism of non-coding RNAs in cancer progression ; to find obesity . Key mic obesity . A valid obesity . The sec obesity ,60 has b obesity , Mouse G obesity  and Gene obesity  are comp obesity , TarBase obesity , miRCanc obesity , miRWalk obesity , miRGato obesity , miRGate obesity , starBas obesity , HMDD (1 obesity  and otheThis work presents a more comprehensive collection of experimentally validated miRNA-target interactions. The current update has seven times as many MTIs than the miRTarBase 4.5 release, and adds a significant number of MTIs that were collected from high-throughput experimental methods, such as CLIP-seq and PAR-CLIP. This version integrated new features, such as NLP, to recognize miRNA-target interactions between miRNAs and their target genes. Additionally, it provides information on the up- or down-regulation of miRNA and its target gene using TCGA. Abstract annotation data are available for download.The latest release of miRTarBase includes 366 181 curated miRNA-target interactions between 3786 miRNAs and 22 563 target genes, collected from over 4966 articles. The PBA was used to recognize miRNAs in the database, achieving an F-score of 98.7% on a manually curated test data set. The PBA outperformed the traditional rule-based method by 5.2%. The conditional random method was utilized for target gene recognition, achieving an F-score of 70%. For the extraction of MTIs, a set of patterns was automatically generated by the PBA. Each pattern comprises a miRNA, its target gene and other key concept terms, such as those pertaining to unique experimental methods and trigger words. Using a screening mechanism that is based on NLP, the system identified 854 papers that may provide a summary of MTIs. Manual proofreading confirmed that 610 papers mentioned MTIs, indicating that the NLP technique herein had an accuracy of 71%. An automatically updated pipeline was constructed to perform monthly updates that provide the latest MTI information. In summary, miRTarBase 6 serves as a repository of extensive experimental information of value for a wide range of miRNA-related research. miRTarBase will be continuously maintained and updated. The regular update involves manual curation of miRNA-target interactions, and integration of TCGA RNA-seq and miRNA-seq data, providing more comprehensive gene and miRNA expression data across various tumors and samples.


In response to the financial crisis and under the EU/IMF Programme of Financial Support, Ireland committed to make savings on pharmaceutical expenditure, including through a system of generic substitution (GS) and reference pricing (RP) . This inThis study was a retrospective analysis of patient-level national pharmacy claims data from January 2013-October 2014 inclusive. These data were sourced from the publicly-funded Irish community pharmacy drug reimbursement schemes. For each product deemed interchangeable for GS, average prices over the prior 6 months were calculated (‘pre-price’). Pre-prices were then assigned to claims records for the product over the 6 months following RP. Actual ingredient cost expenditure and expected expenditure based on pre-prices were compared and total savings were calculated. Trends in generic versus proprietary product uptake were also monitored.Forty-one product types representing 15 active substances (fifth level ATC) underwent GS and RP between August 2013 and May 2014, resulting in a minimum of 6 months follow-up. For the scheme serving the majority of public patients, GS/RP accounted for an overall combined relative decrease in ingredient cost expenditure of 53% in the 6 months following implementation, amounting to combined 6-month savings of over EUR 35million . Greatest savings were observed for the drug atorvastatin, which incurred a 71% expenditure drop, while lowest relative savings were observed for the drug ramipril, which incurred a 24% expenditure drop. Generic usage rates for the drugs concerned increased on average by 44% with GS/RP introduction.The introduction of GS and RP led to substantial savings which corresponded to pre-policy health service official estimates. Compliance with policies is high with generic drug usage targets now exceeded. Success factors included enactment of supportive legislation in 2013, acceptability of active-substance based GS and the phased nature of the policy introduction. On-going work will identify non-compliance with the policies and will aim to elucidate factors affecting the same. The long-term impact of the policies has yet to be determined.


Although the immune recovery associated with highly active antiretroviral therapy (HAART) in HIV infection has important clinical benefits, this restoration of immunity may result in deterioration, when HAART is initiated in patients with tuberculosis (TB). The immune reconstitution inflammatory syndrome (IRIS) has also been reported in non-HIV persons following anti-TB treatment (ATT). The incidence of IRIS in case-control studies on HIV and non-HIV-infected patients, has been found to be 28-36% and 7-10%, respectively, depending on background tuberculosis prevalence rates.We report two cases illustrating IRIS in treated CNS TB, one in a patient diagnosed with HIV stage C3 and the second in a patient with immunosuppression due to anti-tumor necrosis factor alpha treatment.Mycobacterium tuberculosis susceptible to all anti-TB agents. He initially improved under ATT, but one week after antiretroviral therapy (ART) was started, at 6 weeks after the initiation of ATT, he presented with worsening of his symptoms (left hemiparesis and mixed aphasia), of CSF and MRI changes. He improved after he was started on corticosteroids , but was readmitted with recurrence of the left hemiparesis and worsening aphasia, while reducing the steroid dose to 8 mg of methylprednisolone. Worsening of his neurological status has been reemerging each time we try to stop steroids over a 6-month period.A 40 year-patient, HIV-infected, was diagnosed with tuberculous meningoencephalitis , due to The second case is a 60 year-old patient, with ankylosing spondylitis, treated for 3 years with infliximab, diagnosed with disseminated TB (multiple disseminated tuberculomas and pulmonary TB) histological and bacteriological confirmed. The initiation of ATT has led to neurological improvement, but after 3 weeks of therapy the patient presented with fever and diplopia. These symptoms improved only after corticosteroids administration . At week 18 of ATT the patient was still on steroids.High doses of steroids are usually used to control the IRIS symptoms in TB patients with CNS involvement, but the dosing and duration of corticosteroids should be personalized to each patient. Some patients may require extended courses of corticosteroids.


Toxoplasma gondii causes toxoplasmosis, a worldwide disease. Experimentation withpigs is necessary for the development of new therapeutic approaches to humandiseases. BR-1 mini pigs were intramuscularly infected with T. gondii withtachyzoites (RH strain) or orally infected with cysts (ME-49 strain). Haematology andserum biochemistry were analysed and buffy coat cells were inoculated in mice todetermine tachyzoite circulation. No alterations were observed in erythrocyte andplatelet values; however, band neutrophils increased seven days after infection withME-49. Serology of the mice inoculated with pig blood leucocytes revealed circulatingME-49 or RH strain tachyzoites in the pigs' peripheral blood at two and seven or ninedays post-infection. The tachyzoites were also directly observed in blood smears fromthe infected pigs outside and inside leucocytes for longer periods.Alanine-aminotransferase was high at days 21 and 32 in the RH infected pigs. After 90days, the pigs were euthanised and their tissue samples were processed and inoculatedinto mice. The mice serology revealed the presence of parasites in the hearts, ileumsand mesenteric lymph nodes of the pigs. Additionally, cysts in the mice were onlyobserved after pig heart tissue inoculation. The infected pigs presented similarhuman outcomes with relatively low pathogenicity and the BR-1 mini pig model infectedwith ME-49 is suitable to monitor experimental toxoplasmosis. Toxoplasma gondii causes toxoplasmosis, a worldwide disease that infectsone third of the human population . These animalswere delivered to the Darcy Ribeiro North Fluminense State University (UENF), state ofRio de Janeiro, Brazil, and were maintained in three randomly assigned groups of fourindividuals at a medium size animal facility, with a controlled temperature (25ºC) aswell as food and water ad libitum for two weeks before the experimentalprocedures.T. gondii strains, which carry highand low mortality in mice, respectively. The RH strain was maintained by intraperitonealinjections of tachyzoites and the ME-49 strain was maintained by inoculation of tissuecysts. The cysts were obtained from the brains of the mice three months after infection.The brains were removed, washed with phosphate buffered saline (PBS), macerated andhomogenised. After quantification, the tachyzoites or cysts from the mice were used toinfect the pigs.Swiss Webster mice were bred at the UENF mouse animal facility and were infectedseparately with the RH and ME-49 Ethics - This study was carried out in strict accordance with theBrazilian Law #11794/08. The animal study protocol was reviewed and approved by the UENFCommittee on the Ethics of Animal Experiments (permit 130).Infection, blood collection and fundoscopy - Two groups of four pigseach were infected and a third group was a non-infected control group. The "ME-49" groupwas infected by an oral route with 660 cysts of ME-49 strain and the "RH" group wasinfected intramuscularly with 1 x 107 RH strain tachyzoites, which is anon-cystogenic strain. After infection, the clinical signs of the animals were observeddaily. The pig infections were confirmed by serological testing with the modifiedagglutination test (MAT) (Dubey & Desmonts 1987) before and 30 days after infection.The pigs were sedated with an intramuscular acepromazine (0.22 mg/kg) injection that wascombined with ketamine (20 mg/kg) for blood collection and fundoscopy of all animals.The blood was collected from the auricular vein with ethylenediamine tetracetic acid(EDTA) as an anti-coagulant and purified for serum obtainment. Both procedures wereperformed three times a week in the first 14 days, twice a week for an extra 16 days andweekly up to 90 days. Indirect fundoscopy was performed with an ophthalmoscope .Haematology and serum biochemistry - Blood samples (3-5 mL) with EDTAfrom all of the animals were processed for automatic white blood cell, red blood celland platelets counting (MS4 Melet Schloesing(r)). Haemoglobin concentrations,haematocrit percentages, the mean corpuscular volumes and the mean corpuscularhaemoglobin concentrations were also assayed by a cited automated procedure. Inparallel, blood smears were stained with Giemsa in order to identify and estimate thedifferential leucocytes counts and to detect possible morphological alterations and thepresence of parasites. One hundred leucocytes were differentially counted peranimal.(r)).Serum was collected from 5 mL of blood right after coagulation, stored in individualvials and frozen (-70ºC) until biochemistry determination of the following wasconducted: urea, creatinine, alanine aminotransferase (ALT), aspartate aminotransferaseand alkaline phosphatase. The serum biochemistry was assayed in a semi automaticspectrophotometer  was centrifuged , the buffy coat was collected and washed once with PBSand the cell pellet was resuspended in 1.5 mL of PBS. This suspension was inoculatedintraperitoneally (0.5 mL) in three adult mice. After 40 days, the serum of the micethat survived was submitted to the MAT with a cut-off titre of 1:25 (RH and ME-49groups) and the brains were observed for the presence of cysts (the ME-49 grouponly).Necropsy and histopathology- After 90 days post-infection (dpi), all of the pigs were euthanised,necropsied and the tissues was processed for pathology examination. Tissues were fixedin 10% buffered formalin, routinely processed in alcohol and xylol, embedded in paraffinand 5 μm sections were stained with haematoxylin and eosin. Eleven organs were analysed,including the heart, lung, liver, spleen, kidney, ileum, brain, mesenteric lymph node,skeletal muscle, tongue and retina.Renal stereology - The kidney volumes of all four pigs in each group(infected with the ME-49 or RH strains and non-infected control), cleared of adiposetissue, was measured by the water displacement method. The right kidneys weretransversely sliced in 2 cm sections that were fixed in 4% formaldehyde for latercortical-medullar ratio determination by the point-counting method protocol, with minor modifications, as follows. Each tissue was ground separately withsaline and the homogenates were incubated for 60 min in a shaker at 37ºC with 200 mL ofpre-warmed acidic pepsin solution . The product was filtered through twolayers of gauze and centrifuged at 1,200 g for 10 min. The supernatantwas poured off, the sediment was resuspended in 20 mL of PBS and it was then transferredto a 50 mL centrifuge tube with a conical bottom and neutralised with 15 mL of sodiumbicarbonate . After it was mixed, the solution was centrifuged at 1,200g for 10 min and the sediment was resuspended with 10 mL of PBS thatcontained 1,000 units of penicillin and 100 μg/mL of streptomycin. Blender jars, cuttingboards and other materials were sterilised with soap and hot water (100ºC) betweentissue processing. One millilitre of tissue homogenate was inoculated intraperitoneallyfor each of three adult mice and a control group was inoculated with the same volume ofPBS. Serology and brain cyst searches were performed for parasitaemia determination.Statistical analysis - The numerical data were analysed statically andcompared using the Wilcoxon test and the one-way-ANOVA followed by Dunnett's test. p< 0.05 was considered significant.Clinical data - Two pigs out of four per group became apathetic andlethargic between 2-9 dpi for the RH group and 4-11 dpi for the ME-49 group. One RHgroup pig, which had clinical acute phase signs of the disease, presented increased andabnormal aggressiveness during the chronic phase. No ophthalmic lesion was found in thepigs from both infected groups.The haematological and serum biochemistry parameters - No alterationsin the global number of leucocytes, erythrocytes or platelet values were found among thepig groups, but a significant increase in band neutrophils was seen at 7 dpi in theME-49 group . In our work, one of the RHgroup pigs presented aggressiveness during the chronic phase of the disease, which mayprobably be an indication of neurological alterations caused by the infection.Clinical signs of toxoplasmosis were observed during the acute phase of the disease intwo pigs of each group infected with the RH or the ME-49 strains. These results differfrom oocysts infection (AS-28 strain) in which no major clinical alterations wereobserved in infected animals, except a moderate elevation of rectal temperature . Moreove 3-5 dpi . In contT. gondii control . Nevertheless, thesecells are important because they chemotactically attract dendritic cells that areessential to promote a T-helper 1 response , but with the same dose,parasite form and strain (RH) used in this work. Neutropaenia was observed duringtoxoplasmosis due to neutrophil migration to infected tissues . Theobserved increase in band neutrophil counts was not sufficient to induce leucocytosis.It is known that only extensive tissue damage causes a significant increase of bloodneutrophils that generates leucocytosis , this response may probably be due toileitis that increases recruitment of these cells in mice  and in cresponse . On the ocytosis . Thus, tNeutrophil changes were not seen in the RH group pigs, suggesting a lower immuneresponse in comparison with the ME-49 group. Additionally, the infection venue mayinduce a completely distinct local response with different reflections in the systemicresponse, which is seen as leucocyte fluctuation in the peripheral blood.T. gondiiwas more frequently observed in neutrophils, suggesting that this leucocyte can beimportant for parasitic dissemination in the pigs as demonstrated in mice . Lack of ALT changes in the ME-49 group pigs may suggest that this infection waswell managed by the liver.The ileum, mesenteric lymph nodes and heart were the only ME-49 infected pig tissuesthat induced serum conversion in the mice; however, with the bioassay, cysts were onlyobserved in the brains of mice that were inoculated with heart tissue, suggesting ahigher concentration of parasites in the heart tissue. These results are similar tothose obtained in pigs that were infected with 10 or less VEG strain oocysts, whichshowed many cysts in the tongue, brain and heart .No morphological alteration was detected in the examined infected pig tissues, which wassimilar to results reported by Wingstrand et al. (1997). Kidney transplant may causetoxoplasmosis , indicatT. gondii was ableto infect these pigs with prolonged parasitaemia without clinical signs.The BR-1 mini pigs that were infected with the ME-49 strain used in this work resultedin a suitable model for studying toxoplasmosis because these animals carried theinfection, but survived and presented chronic toxoplasmosis, which is similar to theimmunocompetence observed in humans. Additionally,


Drosophila melanogaster derived from two distinct populations (one from sub-Saharan Africa and one from northern Europe). We identified 2308 genes that differed in expression between the sexes and 2474 genes that differed in expression between populations at a false discovery rate of 5%. We also identified more than 1000 genes that showed a sex-by-population interaction in their expression. The genes that differed in expression between sexes showed enrichment for a wide variety of functions, although only 55% of them overlapped with sex-biased genes identified in whole-fly studies. The genes expressed differentially between populations included several that were previously implicated in adaptive regulatory evolution, an excess of cytochrome P450 genes, and many genes that were not detected in previous studies of whole flies. Our results demonstrate that there is abundant intraspecific gene expression variation within in a single, somatic tissue and uncover new candidates for adaptive regulatory evolution between populations.Within species, levels of gene expression typically vary greatly between tissues, sexes, individuals, and populations. To investigate gene expression variation between sexes and populations in a single somatic tissue, we performed a quantitative analysis of the Malpighian tubule transcriptome in adult males and females of  Drosophila melanogaster, most previous studies of sex- or population-biased gene expression have focused on whole flies, body segments, or reproductive organs  have revealed that there are abundant gene expression differences between tissues, developmental stages, sexes, individuals, and populations  and those from derived, non-African habitats. For example, the cytochrome P450 gene Cyp6g1, which shows a large expression difference between African and European flies and confers insecticide resistance when overexpressed , as well as many new genes that were not detected previously. Although most of the differentially expressed genes were consistent between sexes and populations, there were 615 genes that showed sex-biased expression in only one population and 557 genes that showed population-biased expression in only one sex. These findings indicate that there is abundant intraspecific gene expression variation within in a single, somatic tissue.Because 00 years , we examFly strains and rearing conditions were the same as those described in 2. At this age, expression profiles of Malpighian tubules have been shown to be stable in D. melanogaster  after anesthetizing them with COTotal RNA was extracted from dissected tissue using the MasterPure RNA Purification Kit . RNA isolation was performed without DNase I digestion, because this was found to cause partial degradation of the RNA. Purification of mRNA, construction of cDNA libraries, and high-throughput sequencing were carried out by GATC Biotech . In brief, mRNA was enriched by poly(A) selection, sheared, and reverse-transcribed into cDNA using random hexamer primers. Eight individually tagged libraries were pooled and sequenced on one lane of a HiSequation 2500  to produce single-end reads of 50 bp. RNA extraction and library preparation were performed for all samples in parallel to avoid any day or batch effects. The sequence data have been deposited in NCBI’s Gene Expression Omnibus (D. melanogaster transcriptome (including noncoding RNAs) using the annotation of FlyBase release 5.54  and had only minor effects on the downstream analyses, only the results from NextGenMap are presented. Although the reference D. melanogaster genome is derived from a non-African lab strain, a previous simulation study using whole-genome sequences from African and European flies showed that this does not introduce a bias in the number of mapped reads per population  was calculated using the longest transcript of each gene. For the “per transcript” mapping, NextGenMap was run with its most sensitive settings (–kmer-skip 0 -s 0.0) to ensure that only reads with their best match to a single annotated transcript were included. Reads that had equal-quality matches to multiple transcripts were excluded. Because we had only 50-bp single-end reads, this approach greatly reduced the number of uniquely mapped reads and, thus, reduced statistical power to detect differential expression at the transcript level. For this reason, unless otherwise specified, all results refer to the “per-gene” analysis.vs.. Africa) and sex  was used to analyze the eight samples. In this design, the fit of the data to a one-factor model is compared to that to a two-factor model to estimate the effect of each factor (sex and population) on gene expression. To adjust P-values for multiple testing and determine the false discover rate (FDR), Benjamini-Hochberg correction for multiple testing was used  would give an uncorrected P-value of 0.002 and a multiple-test corrected P-value of 0.016. The numbers of analyzed and significant genes for other read-count thresholds are presented in Supporting Information, Table S1. To detect genes that differed in their sex bias between populations (or their population bias between sexes), the aforedescribed analysis was repeated using only data from a single population (or sex) and a one-factor model.Differentially expressed genes were detected using the Bioconductor  as implewas used . For thewas used . Given oP-values were adjusted for multiple testing using the Benjamini-Hochberg correction (Table S2). For this reason, we used the DESeq2 results for further downstream analyses.To assess how DESeq2 performs in comparison to other statistical methods, we also analyzed our data with edgeR (version 3.6.8) . Similarrrection  and an FP-value was below 5%. To account for hierarchical relationships among GO terms, only the most specific terms that were still significant are presented.Differentially expressed genes were functionally annotated using Gene Ontology (GO) terms  and confτ (τ for 14 adult tissues from FlyAtlas (τ range from zero (housekeeping gene) to one (highly tissue-specific gene). In our analyses, genes with τ > 0.7 are considered narrowly expressed . The numbers of genes meeting greater read-count thresholds are presented in Table S1. There were 8231 genes that had an RPKM greater than one when averaged over all libraries. The RPKM values per gene were highly correlated between biological replicates .We generated RNA-seq reads from Malpighian tubules dissected from adult males and females from an African and a European population of nogaster . In totaFile S1). There were significantly more male-biased (1403) than female-biased (905) genes . The male-biased genes also showed a greater degree of sex-bias: the median expression difference between the sexes was 2.13-fold for male-biased genes and 1.25-fold for female biased genes . However, male-biased genes tended to have lower overall expression than female-biased genes, with average RPKM values of 83 and 125, respectively. There was not a strong agreement between a gene’s sex-biased expression in the Malpighian tubules and in the whole fly (In a two-factor (sex and population) analysis of the full data set, 2308 genes were detected as being differentially expressed between females and males at a false discovery rate (FDR) of 5% ; File S1hole fly : 55% of Table S1). For example, when the minimum read-count threshold is increased from 12 to 100, the numbers of female- and male-biased genes are reduced by 12% and 24%, respectively. This indicates that many of the sex-biased genes, especially the male-biased genes, have an overall low expression level. However, even at this higher threshold, there were still significantly more male-biased (1073) than female-biased (798) genes .Increasing the expression threshold for our statistical analysis led to a reduction in the number of sex-biased genes . The genes showing the strongest female-bias in their expression include three yolk protein genes, as well as several genes involved in pheromone metabolism and development . The vast majority of these were cases in which only a single transcript of a gene showed significant differential expression between the sexes (186 transcripts) or multiple transcripts of a gene showed a significant bias toward the same sex (38 transcripts). The former group included genes known to be involved in sex determination, such as Sex lethal and doublesex, as well as seven ribosomal protein genes . There were also three genes  that had one transcript with significantly female-biased expression and another transcript with significantly male-biased expression.In addition to the gene-based analyses, we also investigated sex-biased expression at the level of individual transcripts. For this, we included only RNA-seq reads that could be mapped unambiguously to a specific transcript of a gene with multiple annotated transcripts. In total, we were able to map 66.5 million reads to 13,482 transcripts of 6594 multiple-transcript genes. Although the lower number of mapped reads reduced our statistical power to detect differential expression in comparison to the gene-based analysis, we were still able to detect 230 transcripts from 207 genes that differed in expression between females and males at a FDR of 5% (File S1). There was no significant difference between the number of Africa-biased (1230) and Europe-biased (1244) genes . However, the degree of population bias was slightly higher for Europe-biased (1.42-fold) than for Africa-biased (1.28-fold) genes . Increasing the expression threshold for our statistical analysis led to a slight reduction in the number of population-biased genes (Table S1). For example, when the minimum read-count threshold is increased from 12 to 100, the numbers of Africa- and Europe-biased genes are reduced by 3.9% and 3.5%, respectively. This indicates that the vast majority of the population-biased genes have a relatively high overall level of expression.A total of 2474 genes showed consistent, significant differences in their level of Malpighian tubule expression between the African and European populations in both sexes ; File S1P = 0.0002). Of these genes, there were similar numbers showing overexpression in Africa (22 genes) and Europe (18 genes). The GO categories overrepresented among the genes with significantly greater expression in Africa indicate that these genes, in addition to playing roles in transport and energy metabolism that are expected for the Malpighian tubules, are also involved in male courtship behavior, morphogenesis, and growth (Table S5). Genes with functions in stress response and centrosome organization were more highly expressed in the Malpighian tubules of European flies than of African flies (Table S6). The genes with the greatest overexpression in African flies included those with annotated functions in the nervous system and perception, whereas the genes with the greatest overexpression in European flies included mainly those involved in metabolism . The vast majority of these were cases in which only a single transcript of a gene showed significant differential expression between the populations (430 transcripts) or multiple transcripts of a gene showed a significant bias toward the same population (65 transcripts). There were also seven genes , mostly of unknown function, that had one transcript with significantly Africa-biased expression and another transcript with significantly Europe-biased expression.At the transcript level, we detected 511 transcripts from 468 genes that differed in expression between the African and European populations at a FDR of 5% (P < 0.001). Interestingly, nine of the 10 genes with the greatest male bias also differed in expression between populations, whereas none of the 10 genes with the greatest female bias differed in expression between populations . This pattern held for larger sets of genes: 45 of the top 100 male-biased genes showed population-biased expression, whereas only 10 of the top 100 female-biased genes showed population-biased expression . These observations indicate that male-biased genes, especially those with strong male bias, are more likely to differ in expression between populations than female-biased genes. Of the 2474 genes that showed population-biased expression in our two-factor analysis, 716 (29%) also showed sex-biased expression (P = 0.024).Of the 2308 genes that showed sex-biased expression in our two-factor analysis, 716 (31%) also showed population-biased expression . A greatpression . A slighP = 0.0019) and the molecular function “structural constituent of ribosome” . There were also 417 genes with sex-biased expression only in the European population  and the molecular function “endopeptidase inhibitor activity” . We also found three genes of unknown function  that showed conflicting sex bias between the populations .In addition to the 2308 genes detected as sex-biased in the joint analysis of the African and European populations, we found 198 genes with sex-biased expression only in the African population  and “response to chemical” . We also found 307 genes with population-biased expression that was limited to males  and five were Europe-biased in females, but Africa-biased in males .There were 2474 genes that differed in expression between the populations in the combined analysis of both sexes. In addition to these, we found 250 genes with population-biased expression that was limited to females  to one (highly tissue-specific gene) (P < 0.0001 in both cases) . However, such genes represented only a small minority (1–2%) of all genes with sex- or population-biased expression  . Howeverh cases) . In termpression .Materials and Methods). This led to an increase in the proportion of genes detected as sex-biased in the brain (2.4%), but it was still much lower than in the Malpighian tubules. There were also more genes with highly sex-biased expression in the Malpighian tubules: 848 genes showed greater than a twofold expression difference between the sexes in the Malpighian tubules , whereas only 50 genes showed greater than a twofold expression difference between the sexes in the brain . Thus, despite the conspicuous behavioral differences between females and males , there appears to be much less sexually dimorphic gene expression in the brain than in other tissues.Although the Malpighian tubule is a somatic tissue shared by females and males, it displays a considerable number of genes that are expressed differentially between the sexes. Of the genes with sufficient expression for statistical analysis, 18.4% differed in expression between the sexes. In contrast, a previous study using the same experimental design found much less sex-biased expression in the brain, where only 0.8% of the genes differed in expression between the sexes (vs. RNA-seq) or experimental designs  that were employed. Consistent with the latter, we found 615 genes for which sex-biased expression was observed in only one of our two populations. This finding suggests that the differences between the studies may reflect natural intraspecific variation in gene expression. Further differences may be attributable to the increased sensitivity that oligonucleotide-based microarrays have to detect isoform-specific expression of transcripts, which was limited in our RNA-seq analysis.In terms of sex-biased expression, our results are generally concordant with those of a previous microarray study that examined Malpighian tubule expression in males and females of a single laboratory strain (Cyp6g1 (2.1-fold overexpression in Europe), CG10560 (2.4-fold overexpression in Africa), and CG9509 (1.9-fold overexpression in Europe), all of which show functional and population genetic evidence for recent adaptive evolution (Cyp6g1, which confers insecticide resistance when overexpressed, is a well-documented example of such adaptation (Overall, we found that 19.7% of genes differed in their Malpighian tubule expression between a population from the ancestral species range in sub-Saharan Africa and one from the derived species range in Europe. This finding suggests that gene regulation has changed considerably in response to local environmental conditions. Furthermore, because we used a “common garden” experimental design in which flies from both populations were raised under identical conditions for multiple generations prior to RNA extraction, we can assume that the differences in expression have an underlying genetic basis and are not the result of phenotypic plasticity. Several of the genes that were previously identified as showing expression divergence between populations in whole-fly studies were also detected as differing in expression between populations in the Malpighian tubule. These include It is also noteworthy that genes involved in the innate immune response are enriched among those showing population-specific differences in sex-biased expression, with there being an excess of antimicrobial genes with female-biased expression in the African population. The Malpighian tubules are known to play an important role in immune defense (


Encephalitis is parenchymal brain inflammation due to infectious or immune-mediated processes. However, in 15–60% the cause remains unknown. This study aimed to determine if the cytokine/chemokine-mediated host response can distinguish infectious from immune-mediated cases, and whether this may give a clue to aetiology in those of unknown cause.We measured 38 mediators in serum and cerebrospinal fluid (CSF) of patients from the Health Protection Agency Encephalitis Study. Of serum from 78 patients, 38 had infectious, 20 immune-mediated, and 20 unknown aetiology. Of CSF from 37 patients, 20 had infectious, nine immune-mediated and eight unknown aetiology.Heat-map analysis of CSF mediator interactions was different for infectious and immune-mediated cases, and that of the unknown aetiology group was similar to the infectious pattern. Higher myeloperoxidase (MPO) concentrations were found in infectious than immune-mediated cases, in serum and CSF (p = 0.01 and p = 0.006). Serum MPO was also higher in unknown than immune-mediated cases (p = 0.03). Multivariate analysis selected serum MPO; classifying 31 (91%) as infectious (p = 0.008) and 17 (85%) as unknown (p = 0.009) as opposed to immune-mediated. CSF data also selected MPO classifying 11 (85%) as infectious as opposed to immune-mediated (p = 0.036). CSF neutrophils were detected in eight (62%) infective and one (14%) immune-mediated cases (p = 0.004); CSF MPO correlated with neutrophils (p<0.0001).Mediator profiles of infectious aetiology differed from immune-mediated encephalitis; and those of unknown cause were similar to infectious cases, raising the hypothesis of a possible undiagnosed infectious cause. Particularly, neutrophils and MPO merit further investigation. Encephalitis is a neurological emergency, which presents as headache, cognitive and behavioural disturbance, sometimes with focal neurological signs; many cases progress to seizures, coma and death . The mosThere is mounting evidence that the host inflammatory response, driven by cytokines, chemokines and associated mediators, may play a pivotal role in the pathogenesis of encephalitis –14, and Earlier work has demonstrated the importance of considering the significance of any identified mediators in the pathogenesis of disease in the context of leucocyte subsets within the CNS . The earTherefore, we conducted a study to examine the mediator profiles in the cerebrospinal fluid (CSF) and serum of patients with acute encephalitis to address two questions. Firstly, does the mediator profile differ between those of infectious and immune-mediated aetiologies; and secondly might characteristic mediator profiles suggest whether those of unknown aetiology are more likely to be infectious or immune-mediated.We analysed samples available from adult patients in a previous prospective clinical and diagnostic study- the Health Protection Agency  Aetiological Study of Encephalitis in England; this recruited 203 patients from 24 centres which each recruited over 2 years (2005–2008); methods have been described in detail [Patients provided written informed consent, the procedure for which was approved in the HPA study by the North and East Devon Multicentre Research Ethics Committee (Reference: 05/Q2102/22). This sub-study was approved by the HPA Encephalitis Study Steering Group and also by the Pan-Manchester Research and Development Group for the University of ManchesterSerum and CSF samples were collected at recruitment and stored at -80°C. Thirty mediators  were assessed using cytometric bead array . A further panel for eight matrix metalloproteinases was performed using the same technique where additional volumes were available. Mediators were identified from previous literature and chosen to reflect, in part:Leucocyte proliferation and differentiation ;Leucocyte chemotaxis ;Adhesion molecules ;Markers of BBB permeability ;Antiviral and related peptides ;Broadly pro-inflammatory interleukins ; andBroadly anti-inflammatory interleukins .Fluorescence intensity was determined using a Bio-Rad platform . Standards and samples were analysed in duplicate and mean value used. Standard curves were adjusted at the points of fluorescence intensity saturation, generating a sigmoid curve with 6–8 fluorescence points, as described . To avoiFreeze/thaw cycles were minimised, typically to 2–3, in the central laboratory. Nevertheless, to minimise any potential impact of any possible variation in sample storage, concentrations were median-centred and normalised for each patient, using established methodology ,17. TherThe Mann-Whitney U test was used for categorical variables and Kendall’s rank for continuous variables (SPSS 2011). Step-wise discriminant function analysis was used to develop a multivariate model, which underwent a leave-one-out cross-validation to protect against type 1 error; the power of each mediator is expressed using Wilk’s Lambda coefficient, from perfect discriminatory power (0.0) to none (1.0). This assisted by generating a model based not just on absolute concentrations of selected mediator(s), but also with a numerical weighting applied to selected mediator(s). This was generated using data from the majority of patients and tested on a random subset in a split-reliability analysis. Significance was defined as p<0.05.A total of 95 patients were identified, 78 of whom had serum samples of sufficient volume available; these comprised 38 with an infectious aetiology , 1 influenza A, 1 measles, 1 HIV, 1 toxoplasmosis); 20 immune-mediated cases , and 20 of unknown aetiology. For 37 patients CSF samples of sufficient volume were available, 19 with an infectious aetiology , 9 immune-mediated , and 8 of unknown aetiology. Additional samples were available to assess the MMP panel for 62 serum and 18 CSF samples.The following mediators were not identified in >80% of the cohort and were therefore removed from further analysis: TNFR1 and 2, eSelectin, CXCL9, IL-17α, and VEGFα.The mediators that tended to be found at higher concentrations in either the CSF or the serum were similar across all the aetiological groups . SignifiUnivariate analysis of mediator concentrations showed many similarities but also several differences between the aetiological groups Tables  and 4. TIn particular, higher concentrations of MPO were identified in both the serum and CSF of patients with infectious than immune-mediated aetiologies (p = 0.01 and p = 0.006 respectively) Figs  and 3. IHierarchical cluster analysis of mediators from all patients identified two broad clusters of mediators, which were used in the heat-maps. The first cluster of serum mediators contained IL-1α, IL-1β and IL-10, in addition to other mediators; in CSF these same mediators were clustered together with the addition of IL-1RA. The second cluster in serum contained several mediators including IFNα, IFNβ, IFNγ and IFNο; these same mediators clustered together in CSF. Heat-maps derived from serum mediator concentrations showed strong positive correlations between mediators in the first cluster and strong negative correlation in between mediators in the second cluster in all aetiological groups and this did not appear to be substantially different between groups . HoweverTo see which specific mediators might be used to distinguish between aetiological groups we used discriminant function analysis for pairwise comparisons of the groups. For discriminant function analysis of serum data this generated a model that used serum MPO alone to distinguish 31 (89%) of 35 samples as infective and five (31%) of 16 as immune-mediated . This technique also generated a model using serum MPO alone to distinguish 17 (85%) of 20 samples of unknown aetiology and six (38%) of 16 immune-mediated samples ; both were confirmed on the leave-one-out cross validation. No serum variables qualified as distinguishing samples of infective versus unknown aetiology. A serum MPO concentration cut-off of >0.8pg/mL correctly identified 27 (77%) infective, 17 (85%) as unknown, and 8 (50%) immune-mediated cases; to assess the usefulness of this cut off value for MPO this was then applied to samples from 7 patients (3 infective and 4 immune-mediated) that had not been included in the initial analysis. In this split-reliability analysis all three cases were identified as infective but none of the four were identified as immune-mediated.Discriminant function analysis of CSF data generated a model which again used MPO alone to correctly classify 11 (85%) of 13 cases as infective and four (67%) of six cases as immune-mediated ; this was confirmed on the leave-one-out cross validation. A CSF MPO concentration cut-off of 1.0pg/mL correctly classified nine (69%) infective cases and four (67%) immune-mediated cases. The proportion of cases correctly classified was the same when applied to the raw data using a cut-off of 1000pg/mL. To assess the usefulness of this cut off value we assessed this in 9 patients (6 infective and 3 immune mediated) not included in the original analysis. In this split-reliability analysis three of six and one of three cases were classified as infective or immune-mediated respectively.As MPO is released during degranulation of neutrophils, we assessed whether these findings may reflect the relative neutrophil concentration between the different aetiological groups. Eight (62%) of the infective group had detectable neutrophils in the CSF, as opposed to only one (14%) of the immune-mediated group (p = 0.004). Moreover, across the whole cohort there was a strong positive correlation between CSF neutrophil count and CSF MPO concentration  . In the This study has shown that the cytokine and associated mediator profiles in UK patients with infectious encephalitis differ from those with immune-mediated aetiologies, particularly in the CSF. For patients with unknown aetiology patterns in CSF most closely resembled infectious cases, suggesting they may have undiagnosed infection. MPO was the mediator of greatest significance, with higher concentrations in patients with infection, and CSF MPO concentration was strongly correlated with CSF neutrophil count.There are an estimated 0.07–12.6 cases of encephalitis per 100,000/year . The mosTherefore, we analysed CSF and serum samples from 95 patients with encephalitis recruited prospectively in the HPA Encephalitis Study . We founDiscriminant analysis of both CSF and serum identified MPO as the mediator that most strongly discriminated cases of an infective or unknown aetiology from immune-mediated cases. CSF concentrations of MPO correlated with CSF neutrophil count, suggesting that CSF neutrophils may be the primary source of MPO identified. A higher percentage of patients had detectable neutrophils in the CSF in the infectious as opposed to immune-mediated group, although this was not as strong an association as the MPO concentration, perhaps suggesting that the MPO in the CSF may also reflect neutrophil extracellular traps ; these aNeutrophils are known to be an important part of the host response early in viral encephalitis, before lymphocytes predominate; in bacterial central nervous system infections the neutrophil counts predominate, and are typically much higher. Interestingly, neutrophils have been identified recently in murine models of HSV encephalitis, and may be important for reducing viral load, albeit whilst also being associated with the production of, potentially neurotoxic, reactive oxygen species –35. HistOur study suggests that MPO in the CSF could be explored as a biomarker for a potential infectious aetiology in patients with encephalitis. Also serum MPO was also associated with an infective aetiology so may represent a potential biomarker and both require validation in prospective studies. For example interesting developments have been made in point-of-care tests for protein biomarkers, such as the lateral flow assays developed for CSF lactate to distinguish bacterial from viral meningitis . In addiIn CSF there were many more differences in the concentrations of mediators between the aetiological groups. Specifically, higher concentrations of IL-8 were identified in both the infectious and unknown group as opposed to immune-mediated cases, which would be consistent with the key finding of the role of neutrophils in our study, given the important role of IL-8 in neutrophil activation –49. In aInterestingly no significant differences were found in the concentration of any of the mediators between the infectious and unknown groups in either the serum or the CSF. Only one other study has assessed profiles between encephalitis aetiologies and identified a higher concentration of IFNγ and TNFR1 in 13 patients with HSV than 15 with ‘non-herpetic limbic encephalitis’ . We did Pooling the patients with different infectious aetiologies together, allowed analysis of factors which were common between them, and which distinguished them from immune-mediated encephalitis. It also allowed a comparison with those whose aetiology was unknown., This analysis makes the assumption that the pathology is similar in infectious cases. Whilst most is known about HSV infection, which is associated with necrosis, neutrophil invasion, haemorrhagic change and secondary autoimmunity in some, fewer histopathological data are available for other aetiologies ,52,53. IAlthough the patients for whom no aetiology could be identified had a mediator profile suggestive of infection, they had all undergone an extensive work up for infectious causes, through the HPA study ,54. TherIn summary, whilst there were many similarities identified, the cytokine and chemokine profiles in the CSF of patients with encephalitis of an infectious aetiology differed from those with an immune-mediated aetiology; those in whom no cause could be identified best reflected those of an infectious aetiology. MPO may represent a potential biomarker to distinguish encephalitis due to infection versus immune-mediated causes, which may in part relate to neutrophils in the CSF.BDM is an NIHR Academic Clinical Lecturer and this work received support as part of an NIHR Doctoral Research Fellowship (NIHR-DRF-2010-03-97). TS received support from the NIHR Health Protection Research Unit in Emerging and Zoonotic Infections at Liverpool. TS was also supported by the MRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by the laboratory team at the Vaccine Evaluation Unit, Public Health England, Manchester, UK. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health or Public Health England. The samples and data from these patients have been used in associated studies arising from the original Health Protection Agency  Aetiological Study of Encephalitis in England.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS) offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research.Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for  Unfortunately, promising activities observed in two-dimensional (2-D) monolayer culture could not always be satisfactorily confirmed in animal studies or in clinical trials, because of the inability to replicate the extracellular microenvironment where cells reside in tumor tissues , relative to 0.46 μg/ml (95% CI 0.28–0.75) for the monolayer culture, which was consistent with previous reports [50 and MCRI values were summarized in 50 values determined with spheroids were approximately 50.3-fold, 60.0-fold, and 83.3-fold higher than that for monolayer cells. The data demonstrated that 3-D spheroid culture of MCF-7 cells were less susceptible to DOX than their 2-D counterpart. It was also notable that the spheroids of larger sizes exhibited higher drug resistance. This was consistent with the result we observed in the above penetration studies. The results indicated that this newly generated in vitro spheroid assay could be suitable and effective for drug screening.AlamarBlue Cell Viability assays were performed to determine drug cytotoxicity to monolayer culture and spheroid culture. Spheroids seeded with 2000, 4000, and 8000 cells per micro-well had diameters close to 300, 400, and 500 μm on day 3 , respect reports , 33. ToxCell cycle analysis by flow cytometry was shown in Apoptosis of MCF-7 cells in spheroid culture and monolayer culture were performed by an AnnexinV-FITC Apoptosis Assay, and the results were listed in The complex spatial and physical structure of spheroids provides a physiological context that more closely mimics the tumor microenvironment. Multicellular drug resistance has been associated with cell contact inhibition and adverse microenvironmental conditions like hypoxia, acidic extracellular pH and nutritional depletion, 36. Manin vivo. Compared with monolayer cultures on conventional 2-D plastic surfaces, spheroid culture models provide a physiological context that more closely mimics the native tumor microenvironment[in vitro microtumor model was more predictive and more precise in mimicking in vivo drug efficacy.Three-dimensional spheroid culture models are increasingly becoming essential tools in cancer research as they allow cellular responses that more closely mimic events occurring vironment–44, and The model has many advantages. The prefabricated micropore scaffold allowed for the rapid cellular assembly to form spheroids without any external forces, and enabled quantitative and qualitative analysis of an individual spheroid or a single cell. This model also offered a high yield of spheroids with excellent control over sizes. Standardization of spheroid size is critical for the reproducible evaluation in drug assays as cell biology and diffusion limitations vary with the size of MCTS. Furthermore, the transparent agarose scaffolds allowed for monitoring of spheroid formation by traditional optical microscopy, and the generated spheroids with compact and rigid structure could be recovered from scaffolds easily without affecting the physical characteristics. Wan Yong Ho et al. reported a spheroid culture model for drug screening using the MTT Assay, in which a non-adherent agarose surface was pre-coated in 96-well plate for producing spheroids with uniform sizes . HoweverIn our study, agarose has been used because of its high water content and biocompatibility. Because this agarose surface strongly resists cell adhesion, the cells settle from suspension into discrete populations of equivalent numbers for cultures of uniform-sized spheroids. Using MCF-7 cell lines derived from breast carcinoma, we demonstrated the ability to accurately control the size of multicellular tumor spheroids based on cell seeding number. MCF-7 spheroids were prepared ranging from 200 to 600 μm in diameter based on initial cell seeding number . Additio50 value was 23.8 μg/ml, approximately 50-fold higher than that of monolayer culture  exhibit a heterogeneous architecture with an inner necrotic core, intermediate quiescent region, and a peripheral proliferating region –9. The gThe complex spatial and physical structure of spheroids affects the signal transduction pathways, and ultimately changes the cellular phenotypic expression and cell behavior. CD 44 is a cell membrane proteoglycan that mediates cell-cell and cell-matrix adhesion , 53. CD 50 values of three culture systems were approximately 50-fold, 60-fold, and 80-fold, respectively, higher than that of monolayer cells.Herein, we examined a scalable method to prepare uniformly sized spheroids, and demonstrated applications in drug screening. Using homogeneous spheroids with three different sizes, we investigated the dependence of drug cytotoxicity with spheroid size , and compared the results with traditional 2-D cultures. To our knowledge, this is the first study to show the relationships between spheroid sizes and drug resistance. As shown in The drug permeability, cell cycle distribution, and hypoxia state are all factors that associated with distinctive chemoresistance. The penetration capability of anticancer drugs can be influenced by MCTS size. In in vitro drug testing and basic cancer cell research.In summary, a new microwell-based spheroid culture model can provide massive uniform spheroids with a narrow size distribution. Cells grown as MCTS developed multicellular chemoresistance similar to the tumor with decreased drug penetration, cell cycle arrest, hypoxia, and altered gene expressions. Drug resistance was demonstrated to be strongly dependent on the size of the MCTS. This study will contribute to the development of 3-D MCTS models, which can provide valuable tools for


Autologous stem cell transplant (ASCT) is the standard of care in transplant-eligible multiple myeloma patients and is associated with significant improvement in progression-free survival (PFS), complete remission rates (CR), and overall survival (OS). However, majority of patients eventually relapse, with a median PFS of around 36 months. Relapses are harder to treat and prognosis declines with each relapse. Achieving and maintaining “best response” to initial therapy is the ultimate goal of first-line treatment and sustained CR is a powerful surrogate for extended survival especially in high-risk multiple myeloma. ASCT is often followed by consolidation/maintenance phase to deepen and/or maintain the response achieved by induction and ASCT. Novel agents like thalidomide, lenalidomide, and bortezomib have been used as single agents or in combination. Thalidomide use has been associated with a meaningful improvement in PFS and EFS, however, with substantial side effects. Data with lenalidomide maintenance after-ASCT is favorable, but the optimal duration of lenalidomide maintenance is still unclear. Bortezomib use has been associated with superior outcomes, predominantly in high-risk myeloma patients. Combination regimens utilizing a proteasome inhibitor  with an immunomodulatory drug  have provided the best outcomes. This review article serves as a review of the best available evidence in post-ASCT approaches in multiple myeloma. Autologous stem cell transplant (ASCT) is the standard of care in transplant-eligible patients, based on several phase III trials and meta-analyses in the mid-1990s showing significant improvement in progression-free survival (PFS) and complete remission rates (CR) as well as overall survival (OS) . In thei deepen and maintain the response achieved by HDC-ASCT in myeloma patients. The benefit of maintenance therapy is thought to be due to suppression of clonal proliferation of myeloma cancer cells thereby delaying or preventing relapse.The treatment algorithm for newly diagnosed multiple myeloma (MM) has evolved over the last two decades with the incorporation of novel agents in myeloma induction regimens prior to ASCT , 6. ImprIn this review article, we will briefly review the significance of the depth of response to induction chemotherapy and will focus on how to deepen or maintain response by means of a consolidation approach, maintenance, or both.Multiple studies have suggested a survival advantage from attaining a deeper response to induction chemotherapy and HDC-ASCT, and sustained CR was shown to be a surrogate for OS in myeloma population –14 see . In a meFurthermore, achieving stringent CR (sCR), as defined by IMWG criteria, is associated with even better outcomes compared to CR, with median time-to-progression of 50 months with sCR versus 20 months with CR , 12. The Controversies in the Assessment of Minimal Residual Disease in MM, Corradini et al. reviewed the clinical significance of MRD negativity using highly sensitive techniques and concluded that, at the present time, the goal of assessing MRD is for risk stratification and to evaluate response to novel agents particularly in the context of clinical trials [The techniques for assessing disease burden in MM have evolved over time. Sensitive methods to detect MRD have been used to evaluate either immunophenotypic response or molecular response , 18, 19.l trials .P = 0.005) and high-risk cytogenetics by FISH  were the only independent factors that predicted unsustained CR and shortened OS [Immunophenotypic response assessment by multiparameter flow cytometry (MFC) was evaluated after ASCT in a subset of patients enrolled in Myeloma Research Council (MRC) IX, the Spanish GEM 2000, and GEM 2005 trials. Absence of MRD at D + 100 was associated with statistically significant improvement in both PFS and OS in patients with favorable and adverse cytogenetics , 21, 22.Molecular response can be assessed by either PCR-based techniques  or next generation sequencing (NGS). MRD assessment by PCR was evaluated in several trials and homogenously showed that lower levels of MRD were associated with markedly better survival and longer disease-free periods , 23–25.It should be noted, though, that these results were based on subsets of patients enrolled in the cited trials where MRD assessment was done only in those who had a bone marrow sample at D + 100 (in those assessed by MFC), thus raising concern for selection bias. Additionally, variety of technical factors can affect the sensitivity, specificity, and applicability of MFC such as time of sampling with respect to treatment, number of markers, number of cells counted, and marrow cellularity. On the other hand, PCR and NGS, despite being very sensitive for MRD detection, are limited by the technical complexity and the lack of validated threshold to separate high from low MRD to better allow establishment of prognostic variables –26.In conclusion, all methods reliably differentiated between MRD-positive versus -negative cases and successfully correlated MRD negativity with improved outcomes. MFC appears to have the greatest applicability, whereas PCR and NGS demonstrated higher sensitivity. Other techniques for MRD assessment such as detection of circulating tumor cells and whole-body PET-CT/MRI seem to have potential to add sensitivity for MRD detection, especially in extramedullary disease and/or oligo- or nonsecretory MM . HoweverDespite the remarkable improvement in the outcome of MM treatment over the last two decades with the use of immunomodulatory and novel agents, MM remains an incurable disease. Multiple studies over the last few years have examined the role of maintenance, consolidation, or both to eliminate residual disease after HDC-ASCT in MM. Consolidation is typically a short course, more intensive, with the main goal of deepening the response achieved by induction chemotherapy and HDC-ASCT. Maintenance is given for a prolonged period of time with the goal of preventing/delaying disease progression. Both approaches showed favorable outcomes in terms of delaying relapse and need for second line intensive therapy. However, it remains unclear whether there is a significant survival advantage to justify the cost and toxicity of continued treatment. The ideal regimen should be easy to deliver, convenient to use, cost-effective, have modest toxicity and lead to improved PFS and ideally OS over retreatment at relapse , 28.n = 125). Significant improvements were noted in both PFS  and OS  favoring those receiving the 50 mg dose [Historically, interferons (IFN) and glucocorticoids were the first agents studied in the maintenance setting after induction. Berenson et al. compared alternate-day oral prednisone at 2-dose levels (10 mg versus 50 mg) in a subset of patients achieving at least 25% tumor reduction following induction therapy during their enrollment in SWOG 9210 trial . However, OS rate at 5 years from diagnosis was similar in both arms.Two major phase III trials evaluated the role of lenalidomide use after HDC-ASCT in MM and showed improvement in PFS and TTP –49. In tP < 0.001).In comparison, CALGB-100104 trial that evaluated lenalidomide maintenance after ASCT did not include a consolidation phase and it permitted crossover . 460 patβ-2 microglobulin levels, or patients who achieved a CR prior to the start of maintenance therapy or placebo.A subgroup analysis showed that patients treated with lenalidomide induction therapy had significantly longer survival if they received lenalidomide maintenance, compared to those who received a placebo. The same analysis also showed that lenalidomide maintenance did not provide a survival advantage for patients who were treated with thalidomide induction therapy, patients who had elevated P = 0.008). Interestingly, OS analysis of patients crossing over from placebo to lenalidomide within 6–12 months of randomization showed significant benefit from lenalidomide maintenance [Based on these results, the study was unblinded and 82 out of 128 patients in the placebo arm crossed over to the lenalidomide maintenance arm. Dr. McCarthy updated the results of CALGB-100104 trial in 2013 at the International Myeloma Workshop in Japan. Intention-to-treat analysis conducted at a median follow-up of 48 months after crossover showed median TTP of 50 months in the lenalidomide maintenance arm versus 27 months in the placebo arm. OS benefit was maintained at 48 months . This study also compared lenalidomide maintenance to no maintenance after high-dose melphalan or MPR consolidation. In comparison to no maintenance, lenalidomide maintenance significantly reduced the risk of progression independently of previous induction/consolidation regimen  with a trend towards improved 3-year OS rates in the lenalidomide maintenance arm . Response rates improved during maintenance therapy in both high-dose melphalan and MPR arms. Interestingly, despite a similar CR rate, PFS improved in those who received high-dose melphalan in comparison to MPR chemotherapy. One possible explanation stated by authors is that response was assessed with standard laboratory tests rather than MRD detection by immunophenotypic or molecular techniques which may have revealed subtle differences in response between the two groups [Most recently, Palumbo et al. reported results of the open-label, randomized, phase III study comparing melphalan 200 mg/mo groups . The cono groups .P = 0.034) [Enthusiasm for lenalidomide maintenance after HDC-ASCT in MM was subdued by the concern for increased risk of SPMs reported in both IFM and CALGB trials. Interim analysis of IFM trial showed increased risk of SPMs (3.1 versus 1.2 per 100 patients per year) in the lenalidomide maintenance arm . Based o= 0.034) . However= 0.034) .The incidence of SPMs following lenalidomide exposure was reviewed in a recently published meta-analysis of 7 randomized trials including 2620 patients treated with lenalidomide versus 598 patients treated with no lenalidomide exposure . Risk ofOn the other hand, acute myeloid leukemia (AML) and myelodysplasia (MDS) were reported in high frequency in untreated patients with monoclonal gammopathy of undetermined significance (MGUS) . This suIn conclusion, data with lenalidomide maintenance after ASCT is favorable. However, the optimal duration of lenalidomide maintenance is still unclear. Lenalidomide provides the most benefit in those who fail to achieve CR or very good partial response (VGPR), by IMWG criteria, after ASCT.P = 0.007); however, no OS benefit was seen. This approach seemed to be most beneficial for patients achieving less than a VGPR after induction and ASCT [The use of bortezomib in the post-ASCT setting in myeloma was evaluated in multiple randomized trials as consolidation, maintenance and in combination with other agents , 54, 55.P = 0.006). There was a trend towards improvement in 5-year OS survival rates (61% with bortezomib regimens versus 55% in nonbortezomib arm) but this did not reach statistical significance (P = 0.07). Bortezomib maintenance significantly improved nCR/CR rate from 31% to 49%, which was found, in a landmark analysis, to be associated with better PFS and OS at 12 months. However, in this trial, no random assignment for maintenance therapy was performed; and, therefore, the effect of that cannot be independently assessed. Furthermore, the major part of the difference in nCR/CR rates between the two groups was observed after induction and ASCT favoring the bortezomib arm. Equivalent upgrade of response was noted with either bortezomib or thalidomide maintenance. Nonetheless, maintenance treatment with bortezomib was much better tolerated than thalidomide maintenance, with fewer patients stopping treatment prematurely. Subgroup analysis showed that the superior outcomes with bortezomib were predominantly accomplished in patients with high-risk disease and myeloma-related renal failure or those with del(17p) and del(13q) [P = 0.004) and OS  were significantly improved in the bortezomib-containing arm compared to those who did not receive bortezomib. In patients with normal serum creatinine, PFS remained superior in the bortezomib arm, whereas OS was similar in both groups. Patients with abnormal FISH results for del(13q14), t, and del(17p13) were compared with patients without the abnormality. PFS was worse in all patients with del(13q14) regardless of the treatment arm whereas OS in patient with del(13q14) was similar to those who did not carry the deletion. Of note, OS in patients with the deletion was significantly better in the bortezomib arm in comparison to the nonbortezomib arm. In patients with del(17p13), both PFS  and OS  were significantly better in the bortezomib arm. The presence of t was associated with worse outcomes compared to patients without this translocation. There was a trend towards better outcomes in patients with t who received bortezomib-containing regimen; however, this did not reach statistical significance. This leads us to the conclusion that the use of bortezomib in the post-ASCT setting can potentially overcome the adverse effects of abnormal cytogenetics, a subgroup that has always been associated with inferior outcomes.HOVON-65 trial evaluated bortezomib given during induction and during maintenance in 827 newly diagnosed myeloma patients . Patientdel(13q) . In patiTrials in the recent years focused on combination regimens for consolidation/maintenance after ASCT. The Spanish PETHEMA study was a 3-arm randomized trial that evaluated bortezomib/thalidomide maintenance after ASCT versus single-agent IFN or single-agent thalidomide for a period of 3 years . After aCavo et al. evaluated the combination bortezomib/thalidomide/dexamethasone (VTD) versus thalidomide/dexamethasone (TD) as consolidation therapy after ASCT in 474 newly diagnosed myeloma patients . After cTotal therapy (TT) trials are series of studies conducted by the Arkansas Myeloma Group utilizing all active antimyeloma agents upfront to achieve a maximal tumor cytoreduction and thereby increase the frequency and duration of CR, with the goal of extending PFS and OS see .P = 0.038). Despite more adverse features in TT-3B and overall higher-risk population, all outcomes  were similar in the 2 protocols suggesting that 3-year maintenance with a bortezomib-containing regimen is superior to shorter maintenance, along with the reportedly more effective immunomodulatory effect of lenalidomide compared to thalidomide used in TT-3A [The introduction of bortezomib in the post-ASCT setting started with TT-3A (VTD-PACE for 2 cycles as consolidation followed by either VTD for 1 year or TD for 2 years). The upfront addition of bortezomib resulted in improved outcomes, compared with TT-2 trials that randomized patients upfront to receive/not receive thalidomide as part of induction, consolidation, and maintenance. To validate these findings along with bortezomib pharmacogenomic data, a successor trial TT-3B enrolled another 177 patients. TT-3A and TT-3B were identical in design, except for the fact that the maintenance phase of TT-3B included VRD  and was continued for 3 years rather than 1 year in TT-3A . Compariin TT-3A –58. Furtin TT-3A .Most recently, Nooka et al. reported results of a single-arm trial evaluating consolidation and maintenance therapy with lenalidomide, bortezomib, and dexamethasone (RVD) in patients with high-risk cytogenetics, defined as del TP53, del(1p), t, or t. Patients received different induction regimens followed by ASCT and subsequently RVD maintenance for 3 years followed by lenalidomide until disease progression. Following initiation of RVD maintenance, 51% of patients achieved sCR with 96% achieving at least VGPR as best response. Median PFS for all patients was 32 months and 3-year OS of 93% was reported .Furthermore, in their recent phase II IFM study, Roussel et al. reported high-quality response and favorable tolerability. This phase II trial included 31 newly diagnosed MM patients who were eligible for ASCT. Overall, 27% of patients were classified as having high-risk chromosomal abnormalities based on either having a del(17p) or t abnormality, determined by FISH. Patients received three cycles of RVD and then proceeded to ASCT. Two months after recovery of blood counts, patients who had not progressed received consolidation therapy consisting of two cycles of RVD. Patient subsequently received maintenance therapy with continuous lenalidomide (Revlimid) for one year. Responses deepened significantly after ASCT and consolidation therapy in comparison to responses at the end of induction: 40% reaching sCR versus 10%; 58% of patients were MRD-negative versus 16%. Responses also improved further for some patients during maintenance therapy. After all treatment sequences, 68% of patients had achieved MRD negativity. At a median follow-up of 39 months, the estimated 3-year PFS and OS were 77% and 100%, respectively . None ofDendritic cell vaccination, an example of this approach, works by induction of idiotype-specific T- and B-cell response that can stimulate the body's own immune system to fight and eradicate myeloma cells following administration of idiotype-protein pulsed dendritic cells. Although this seems to be a safe strategy, it has not shown any effects on survival and, to date, remains experimental –64.Other forms of immunotherapy include the monoclonal antibody Elotuzumab (anti-CS1) and Daratumumab (anti-CD38) that have activity both as single agents and in combination with other novel therapeutics. Novel proteasome inhibitors such as Carfilzomib, Marizomib, Ixazomib, and Oprozomib may provide better outcomes in the future. Antibodies to various myeloma cell markers, such as CD40, CD56, CD74, IL-6, TRAIL, and RANKL, combined with proteasome inhibitors, conventional chemotherapy, and/or immunomodulatory agents, may serve as maintenance targets for future research with an ultimate goal of significantly improving OS or potentially curing multiple myeloma , 65.Over the last decade, the trials mentioned above have led to markedly improved outcomes in myeloma and answered multiple questions regarding optimal induction regimens as well as highlighting the importance of consolidation/maintenance after ASCT in the era of novel agents. While considering consolidation/maintenance strategies after HDC-ASCT, one needs to take into careful consideration the cost and toxicity involved with the different strategies in order to improve outcomes. The use of sensitive techniques to detect MRD provides a stratification tool to guide further treatment following ASCT.Patients with low-risk disease and normal cytogenetics, who achieve CR or sCR (per IMWG criteria) after ASCT, especially if this excellent response can be confirmed by MRD negativity, may forgo further therapy after ASCT. These patients will need to be closely followed for evidence of relapse. Patients with high-risk cytogenetics and those with low-/standard-risk cytogenetics who achieve less than a CR should be considered for maintenance treatment. Average time to start consolidation/maintenance is typically 60–100 days after ASCT.Nooka et al. have reported excellent outcomes for patients with high-risk cytogenetics using combination RVD followed by lenalidomide maintenance. However, this needs further validation in a phase II/III trial design to confirm these superior results. Roussel regimen  is promising particularly in the setting of less than a CR with HDC-ASCT.Our institutional approach for high-risk cytogenetics and low-/standard-risk cytogenetic patients who are still MRD positive after HDC-ASCT is an approach similar to Roussel regimen's with continuation of RVD beyond 2 cycles until achievement of MRD negativity followed by lenalidomide maintenance as long as tolerated or until progression.