Karolinska University Hospital Huddinge Research Articles

P-425 Biosilk and ovarioids: recombinant silk as a new tool for establishing a 3D-culture system for human ovarian primary cells

Abstract Study question Can Biosilk be used as scaffold for establishing a 3D-culture system to support attachment and growth of primary cells derived from adult ovarian biopsies? Summary answer The use of recombinant spider silk-based scaffold allowed the formation of 3D-structured ovarioids from both cortex and medulla isolated from 5 different patients. What is known already Fertility in women is adversely affected by several factors, such as age, environmental pollutants and diseases requiring gonadotoxic treatments. Understanding ovarian cell composition and organization may pave the way to novel fertility preservation methods with the ultimate goal of being applied to clinics. So far, there are no clinically established methods for in vitro growth and maturation of human ovarian follicles leading to mature competent oocytes. Therefore, exploring new 3D-culture systems for in vitro reconstruction of ovarian somatic cell niche could lead to development of novel tools to support growth of patient-specific follicles. Study design, size, duration Ovarian tissue was collected from gender reassignment patients (GRP) after informed written consent at Karolinska University Hospital Huddinge from 2019 to 2022. After separation of cortex and medulla, the samples were mechanically and enzymatically dissociated into single-cell suspensions and used to evaluate 3D- and 2D-culture methods. Freshly fixed biopsies from cortex and medulla (3x3x1 mm3) were used as control for the transcriptomic analysis and RNA-FISH assay. Participants/materials, setting, methods Tissue was obtained from five patients aged 23-31 years. Dissociated primary somatic cells seeded on Biosilk-foam scaffolds were kept in culture for 2 weeks, followed by detachment, equal division of the foams and suspension culture for additional 4 weeks (BioSilk-Ovarioids, BSO). BSOs were harvested in PFA and RNAlater (n = 6/patient, respectively) for morphological and transcriptomic analysis. Protein ZO1, and cell type-specific marker genes (AMHR2, PDGFRa, CLDN5, GJA4) were evaluated via immunodetection and RNA-FISH assay, respectively. Main results and the role of chance The sizes of BSOs from both cortex and medulla ranged between 0.5-1 mm at the end of the culture, appearing highly compacted under optical microscope. HE-stained BSO sections revealed that cells were distributed throughout the foams, showing good attachment and distribution. Marker genes were selected for specific cell types [AMHR2-granulosa, PDGFRa-stroma, CLDN5-endothelial, GJA4-perivascular cells (Wagner et al. 2020)] and used to study the representation of different somatic cells in the BSOs. The RNA-FISH analysis confirmed the presence of all cell type-specific marker genes, with predominating presence of stromal cells (PDGFRa). Newly formed ZO1-specific gap junctions were detected in both cortex- and medulla-derived BSOs, appearing mainly in the outer part of the structures. Interestingly, cell layers surrounding the original Biosilk scaffold were observed, especially in medulla-BSOs. Transcriptomic profiling of the samples showed clear separation to three main clusters by principal component analysis: freshly fixed tissue, 2D-cultures, and BSO. Moreover, clustering analysis of differentially expressed genes (DEGs) showed the presence of gene clusters in fresh tissues affected by both 3D/2D-cultures. Further analyses will focus on identification of significantly affected gene ontologies and pathways, which will further guide the optimization of the BSO culture system. Limitations, reasons for caution The tissue was derived from GRPs who always receive androgen treatments prior to surgery. As hormonal treatment may affect the ovarian environment, further trials with untreated patient samples will be needed to generalize the model to fertility preservation patients. Wider implications of the findings The establishment and development of the BSO 3D-culture system may enable the construction of patient-specific clinically significant tools for in vitro folliculogenesis. This would open new avenues for fertility restoration in patients who cannot receive auto-transplants and treatment of infertility in premature ovarian insufficiency if residual follicles remain in tissue. Trial registration number Not applicable

P-424 Effects of in vitro activation vs fragmentation on human ovarian tissue in culture

Abstract Study question What is the added value of in vitro activation (IVA) protocol compared with fragmentation only in human ovarian tissue culture? Summary answer Although histological assessment shows that the IVA increases follicle survival and growth, IVA and fragmentation stimulate extensive and nearly identical transcriptomic changes in cultured tissues. What is known already Treatments based on activation of the PTEN/PI3K pathway in ovarian tissue in vitro have been administrated to refractory primary ovarian insufficiency (POI) patients and resulted in live births following auto-transplantation of the tissue. However, some studies have shown that mere fragmentation of the tissue produces comparative effects, questioning the added value of chemical stimulation protocol which could potentially activate oncogenic pathways. Study design, size, duration Thirty-three ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section at Karolinska University Hospital Huddinge. The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic) +740Y-P (IVA group) during the first 24 h of culture while half were exposed to vehicle only (fragmentation only group). Subsequently, both groups were cultured for additional 6 days. Media change was performed every other day. Participants/materials, setting, methods Effects on follicles were evaluated by counting and scoring follicles before and after the 7-day culture via HE-stained serial sections. Follicle function was assessed by quantification of steroids by UPLC-MS/MS at the end of the culture. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the 24-h initial culture step. Selected differentially expressed genes (DEGs) were validated by qPCR and immunofluorescence in independent culture experiments. Main results and the role of chance Compared to fragmentation only group, significantly higher follicle survival rate, increased secondary follicle number and sizes were found in IVA group. No significant differences were detected in levels of steroids in culture media on day 7 between the two groups. In our RNA-seq data, when comparing the IVA group to fragmentation only group, only 110 DEGs were found with the relaxed cutoff of FDR<0.1. The signaling pathways affected by IVA involved follicle growth but also inflammation and DNA damage. However, we found that the gene expression was profoundly affected in both IVA and fragmentation only groups compared to the fresh tissue: in total 3,676 and 4,223 DEGs were found (FDR<0.001), respectively. The top enriched gene sets in both groups included several pathways that are known to modulate follicle growth, e.g. PI3K/AKT, MTORC1. We also found that classical steroidogenesis genes (e.g. CYP11A1, CYP17A1) were significantly downregulated in both groups after 24-h culture. Moreover, upregulation of genes related to glycolysis, a process that was shown to promote activation of primordial follicles, and its upstream regulator were shown in the RNA-seq data. This effect was validated by qPCR (ENO1, PKM, LDHA, p < 0.0001). Future validation will be performed using western blot and immunofluorescence. Limitations, reasons for caution The study was performed in an in vitro model where tissue was isolated from the regulation of hypothalamic-pituitary-ovarian (HPO) axis. And thus, further experiments with xeno-transplantation may be needed to explore the effect of IVA in vivo in the future. Wider implications of the findings The impact of 24-h culture on gene expression in ovarian tissue far exceeds the effect of IVA. Yet, follicle growth was stimulated by IVA, which may suggest effects on specific cell populations that are diluted in bulk transcriptomics. Cell type-specific impacts need further studies to conclude about effectiveness of IVA. Trial registration number Not applicable

Students' performance of and perspective on an objective structured practical examination for the assessment of preclinical and practical skills in biomedical laboratory science students in Sweden: a 5-year longitudinal study.

It aims to find students' performance of and perspectives on an objective structured practical examination (OSPE) for assessment of laboratory and preclinical skills in biomedical laboratory science (BLS). It also aims to investigate the perception, acceptability, and usefulness of OSPE from the students' and examiners' point of view. This was a longitudinal study to implement an OSPE in BLS. The student group consisted of 198 BLS students enrolled in semester 4, 2015-2019 at Karolinska University Hospital Huddinge, Sweden. Fourteen teachers evaluated the performance by completing a checklist and global rating scales. A student survey questionnaire was administered to the participants to evaluate the student perspective. To assess quality, 4 independent observers were included to monitor the examiners. Almost 50% of the students passed the initial OSPE. During the repeat OSPE, 73% of the students passed the OSPE. There was a statistically significant difference between the first and the second repeat OSPE (P<0.01) but not between the first and the third attempt (P=0.09). The student survey questionnaire was completed by 99 of the 198 students (50%) and only 63 students responded to the free-text questions (32%). According to these some stations were perceived as more difficult, albeit they considered the assessment to be valid. The observers found the assessment protocols and examiner's instructions assured the objectivity of the examination. The introduction of an OSPE in the education of biomedical laboratory scientists was a reliable, and useful examination of practical skills.

Plasma calprotectin as an indicator of need of transfer to intensive care in patients with suspected sepsis at the emergency department

BackgroundDeciding whether to transfer patients with sepsis from the emergency department (ED) to intensive care units (ICUs) is challenging. We hypothesised that the new biomarker plasma calprotectin (p-calprotectin) could be used to aid the selection of patients for intensive care transfer, since it has been shown to be a promising tool for the determination of sepsis severity in critical care.MethodsThis prospective study was performed on consecutive sepsis alert patients in the ED of Karolinska University Hospital Huddinge. The sepsis alert mandates clinical assessment and decisions regarding treatment, disposition, and level of care by physicians from the ED, the Department of Infectious Diseases, and the ICU. Blood sample analysis for C-reactive protein, procalcitonin, neutrophils, and lymphocytes was routinely performed. P-calprotectin was analysed from frozen plasma samples, using a specific turbidimetric assay.ResultsThree-hundred fifty-one patients who triggered the sepsis alert were available for the study. Among 319 patients who were considered to have an infection, 66 patients (26%) were immediately transferred to the ICU or high-dependency unit (HDU), and 253 patients (74%) were transferred to ordinary wards.Median p-calprotectin was 2.2 mg/L (IQR 1.2–3.9 mg/L) for all patients with infection, it was 3.3 (IQR 1.6–5.2) for those transferred to ICU/HDU and 2.1 (IQR 1.1–3.5) for those transferred to ward units (p = 0.0001).Receiver operating characteristic curve analysis for transfer to the ICU/HDU showed superiority for p-calprotectin compared with procalcitonin and neutrophil–lymphocyte ratio, regarding both all sepsis alert cases and the patients with infection (p < 0.001 for all comparisons). The best p-calprotectin cut-off, 4.0 mg/L, showed a sensitivity of 42.5% and specificity of 83% for transfer to the ICU/HDU among patients with infection.ConclusionsIn sepsis alert patients, p-calprotectin was significantly elevated in patients who were subject to immediate ICU/HDU transfer after assessment by a multidisciplinary team. P-calprotectin was superior to traditional biomarkers in predicting the need for transfer to the ICU/HDU.

P1443: AUTOLOGOUS NK CELLS AS CONSOLIDATION AFTER FRONT-LINE STEM CELL TRANSPLANTATION IN MULTIPLE MYELOMA: A LONG-TERM FOLLOW-UP

Background: Immunotherapies have shown great anti-tumour effect in a variety of malignancies. In multiple myeloma (MM) antibodies, including bispecific T cell engagers, antibody-drug conjugates, and more recently, CAR T cells have reported exciting clinical results and many of them have been incorporated in the treatment armamentarium. Less is reported for NK cells, in particular autologous NK cells. Initial results from our first-in-human trial ACP-001 with autologous NK cells given as consolidation after front-line autologous stem cell transplantation (ASCT) were recently reported (Nahi et al., 2022, Cell Reports Medicine 3, 100508 February 15, 2022). It showed that the concept was feasible with good safety, including no SAEs, and promising efficacy. We here report the long-term clinical follow-up. Aims: The aim of this report was to investigate long-term safety, disease progression, second-line therapy and survival after autologous NK cell treatment. Methods: This first-in-human trial included six patients with newly diagnosed MM eligible for ASCT at the Karolinska University Hospital Huddinge in Sweden. After standard induction treatment (bortezomib, cyclophosphamide and dexamethasone (VCD) at the time) and ASCT with melphalan 200 mg/m2 (MEL200) the patients received three escalating doses of autologous activated and expanded NK cells. Clinical data on long-term safety, disease progression, second-line therapy and survival were captured from the patients’ electronical charts. Results: No subject were lost to follow-up or had withdrawn from the study. With a median follow-up from study inclusion of 6.9 (6.1-7.2) yrs, and 6.2 (5.4-6.7) yrs from the first NK cell infusion, there are no new reported AE related to the NK cell infusions. All six patients are alive with one subject still in first remission 6.8 yrs after inclusion. Five patients had disease progression 13-51 months after study inclusion and four of them underwent reinduction (VRD (n=2), KRD (n=1), KRD+dara (n=1)) followed by a second ASCT with MEL200. The fifth patient progressed within the first yr after ASCT and received VRD+dara. All five patients responded to second line treatment with PR (n=2), VGPR (n=2) and MRD negative (n=1) but subsequently progressed with a PFS2 ranging from 11.5 to 26.3 months. Summary/Conclusion: This long-term follow-up confirms the safety and feasibility of autologous NK cell therapy as consolidation after front line ASCT in MM. Autologous NK cell treatment in first line did not negatively impact the possibility to administer, or the outcome, of later treatments which is important with all new treatment options for patients with advanced MM.

N-Acetylcysteine Amide Alleviates Cyclophosphamide-Induced Endothelial Damage

Rui He 1,2, Wenyi Zheng 1,2, Terra Slof 3, Eva Wärdell 4, Agneta Månsson-Broberg 4, Svante Norgren 5, Per Ljungman 2, Ying Zhao 1, 2 , Moustapha Hassan 1, 21.Experimental Cancer Medicine, Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, 141 86 Huddinge, Sweden2.Center of Allogeneic Stem Cell Transplantation (CAST) and Clinical Research Center, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden.3.Institute for Life Sciences and Chemistry, HU University of Applied Sciences Utrecht, Oudenoord 330, 3513 EX Utrecht, Netherlands4.Department of Medicine, Division of Cardiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.5.Children´s and Women´s Health Theme, Karolinska University Hospital and Karolinska Institutet, Stockholm, SwedenAimThe current study aimed to investigate the protective role of the novel antioxidant N-acetylcysteine amide (NACA) in cyclophosphamide (Cy)-induced endothelial injury and to explore the underlying mechanism.BackgroundThe alkylating agent Cy is still one of the important corner stones in cancer treatment. Cy is used also as a part of the conditioning regimens prior to hematopoietic cell transplantation (HCT) and as a prophylactic treatment post transplantation in graft-versus-host disease (GVHD). Several studies have shown that high doses of Cy are associated with a number of adverse effects including alopecia, gonadal toxicity, hemorrhagic cystitis, carcinogenesis and late cardiovascular toxicity. Moreover, we have shown that treatment with high-dose Cy caused damage to the arterial endothelium, which might contribute to late cardiovascular disorders. Hitherto, there is no prophylactic strategy for Cy-induced endothelial toxicity. Oxidative stress is one of the main players in the pathogenesis of endothelial disorders and hence is an exploitable trait for targeted treatment.MethodsThe prophylactic efficacy of NACA was evaluated using human aortic endothelial cells (HAECs) and human umbilical endothelial cells (HUVECs). The efficacy of NACA was compared with the clinically approved derivative N-acetylcysteine (NAC). Cy is a prodrug that cannot be activated in the endothelial cells. Therefore, cells were incubated with Cy active metabolite, 4-hydroxycyclophosphamide (4-OH-Cy). In order to reveal the underling mechanism of action, we have investigated and evaluated the following parameters: alteration in cell morphology, cell death pathway, inhibition of DNA damage, variation of redox hemostasis, and neovascularization.ResultsWe found that pre-incubation with 5 mM NACA preserved the normal endothelial cell morphology. However, for the quantification of the obtained results, cell colonies were visualized using crystal violet staining while cell viability was determined using WST-1 assay. Moreover, NACA was found to decrease 4-OH-Cy-induced DNA damage and cell death via caspase-dependent apoptosis in both HAECs and HUVECs. In addition, NACA significantly inhibited 4-OH-Cy-induced oxidative stress by reducing the intracellular levels of reactive oxygen species (ROS) and by improving the cellular antioxidative capacity. Increased activities of catalase and superoxide dismutase (SOD) together with increased levels of reduced glutathione (GSH) were observed. Meanwhile, NACA pre-treatment rebalanced endothelial nitric oxide synthase (eNOS) and arginase I in HAECs, suggesting that NACA may be used as prophylactic strategy to avoid the impaired endothelium-dependent vasodilation caused by 4-OH-Cy. Moreover, data from tube formation assay indicated that NACA preserved the angiogenic capability of endothelial cells that was compromised by 4-OH-Cy through blockage of Notch signaling pathway. Our results showed that in contrast to NAC, its amide derivative NACA showed remarkably superior ability in alleviating the endothelial damage caused by 4-OH-Cy.ConclusionsIn summary, the present investigation showed that the novel antioxidant NACA could be applied to protect against 4-OH-Cy-induced endothelial cell dysfunction and death. Higher bioavailability and better cellular uptake make NACA to a better prophylactic agent compared to NAC. To translate these findings into the clinic, additional in vivo studies are warranted to confirm the protective potential of NACA. DisclosuresLjungman: Takeda: Consultancy, Other: Endpoint committee, speaker; Janssen: Other: Investigator; OctaPharma: Other: DSMB; Enanta: Other: DSMB; Merck: Other: Investigator, speaker; AiCuris: Consultancy.

High-Throughput Functional Ex-Vivo Drug Testing and Multi-Omics Profiling in Patients with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common leukemia in adults and the prognosis is dismal. The heterogeneity of driver mutations in AML from one patient to another is significant. Although new treatment targets and modalities have recently been introduced, a lack of therapies to cover the entire spectrum of molecularly distinct AML classes remains a challenge. Furthermore, often only a fraction of the patients carrying mutations actually respond to the drugs predicted to target such genomic subtypes in AML. Our question was if outcome can be improved by optimizing existing and emerging therapies? To address this, we applied direct functional drug testing ex vivo in patients with newly diagnosed AML, along with deep molecular profiling to identify new therapeutic and diagnostic opportunities for specific genetic subclasses. To date, 122 patient samples have been included in the study. We have collected AML patient mononuclear cells isolated from fresh bone marrow aspirates from the Karolinska University Hospital Huddinge and Uppsala University Hospital, or applied biobanked samples from the Swedish acute leukemia biobank. The cells from the patient are tested for 72h with up to 525 conventional and investigative oncology drugs in a 5-point concentration range to determine optimal treatment options. The test is based on bulk cell viability and the cells are cultured in bone marrow stroma-conditioned media. The data is compared to healthy bone marrow controls to filter out generally toxic drugs and combinations. Excess patient material is applied for multi-omics analysis, including genome sequencing, deep proteomic profiling, concentrations of soluble factors, bulk/single-cell RNA sequencing and mass cytometry. Our results indicate significant heterogeneity in functional drug responses across individual patients, even among those with the same founder mutations. We could identify potential targeted treatments for most patients based on the exvivo testing. For instance, patients that show resistance to the Bcl-2 inhibitor Venetoclax can be associated with a distinct drug sensitivity response pattern that includes drugs with different mechanisms of actions. Based on the clustering of drug response data in all clinical samples, we could identify groups of patients with similar global drug responses, and drugs with similar patterns of efficacy across all patients. Disclosures Lehmann: Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbive: Membership on an entity's Board of Directors or advisory committees; TEVA: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kallioniemi:Medisapiens: Other: Co-founder and stockholder; Sartar Therapeutics: Other: Co-founder and stockholder; Astra-Zeneca: Other: Joint Grant; Pelago: Other: Joint Grant; Takara: Other: Joint Grant; Abbot: Other: Licensing income.

Reduced bleeding with DRY CUT® transurethral resection of the prostate (TURP) compared to standard TURP

Objective: To compare bleeding and transfusion rate between patients who undergo standard transurethral resection of the prostate (TURP) and patients who undergo DRY CUT® TURP.Materials and methods: A retrospective comparison was made of 626 patients who underwent a standard monopolar TURP during 2004–2007 at the Karolinska University Hospital Huddinge with 620 patients who underwent monopolar DRY CUT® TURP during 2011–2014 at the same clinic. Transfusion rate, perioperative bleeding, prostate volume, resection weight, use of anticoagulation therapy, presence of prostate cancer, whether the operation was performed by a specialist doctor in urology or a resident and length of hospital stay were evaluated.Results: The median bleeding was 300 ml (IQR = 100–645 ml) in the group of patients who underwent standard TURP compared to 75 ml (IQR 30–268 ml) in the DRY CUT® TURP group. The bleeding quotient for standard TURP was 2.3-times the perioperative bleeding for DRY CUT® TURP. In a logistic regression model the patients who underwent standard TURP were more likely to undergo blood transfusion compared to DRY CUT® TURP (OR = 3.18, 95% CI = 1.72–5.88). The results were not affected by adjustment for patient age, presence of prostate cancer, anticoagulation therapy or operation performed by a specialist in urology. However, the resection weight did influence the bleeding quotient.Conclusions: The shift from standard TURP to DRY CUT® TURP has decreased the perioperative bleeding and need for blood transfusion at our hospital.

On the importance of albumin binding for the flux of 7α-hydroxy-3-oxo-4-cholestenoic acid in the brain

We confirmed previous findings by a Japanese group that there is an accumulation of 7α-hydroxy-3-oxo-4-cholestenoic acid (7-Hoca) in human subdural hematomas. The accumulation correlated with the time from the bleeding to the sample collection. We present evidence that these accumulations are likely to be caused by the strong affinity of 7-Hoca to albumin and the marked difference between plasma and brain with respect to levels of albumin. In the circulation, 80-90% of 7-Hoca is bound to albumin with a ratio between the steroid acid and albumin of ∼1.4 ng/mg. In cerebrospinal fluid (CSF), the ratio between 7-Hoca and albumin is ∼30 ng/mg. When albumin or hemolyzed blood in a dialysis bag was exposed to CSF, there was a flux of 7-Hoca from CSF to the albumin. We suggest that the major explanation for accumulation of 7-Hoca in subdural hematoma is a flux from the brain into the hematoma due to the high affinity to albumin and the high capacity of 7-Hoca to pass biomembranes. We discuss the possibility that the markedly different ratios between 7-Hoca and albumin in circulation and brain can explain the flux of 7-Hoca from the brain into circulation against a concentration gradient.

In tribute to Singh Chhatwal.

Environmental Microbiology ReportsVolume 8, Issue 5 p. 555-555 Tribute In tribute to Singh Chhatwal Anna Norrby-Teglund, Corresponding Author Anna Norrby-Teglund [email protected] Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, S-14186 Stockholm, SwedenFor correspondence. E-mail: [email protected]. Tel. +46 8 585 83297; Fax +46 8 746 7637.Search for more papers by this author Anna Norrby-Teglund, Corresponding Author Anna Norrby-Teglund [email protected] Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, S-14186 Stockholm, SwedenFor correspondence. E-mail: [email protected]. Tel. +46 8 585 83297; Fax +46 8 746 7637.Search for more papers by this author First published: 23 October 2016 https://doi.org/10.1111/1758-2229.12463Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume8, Issue5Special Issue: Special Issue on Pathogen and Antibiotic Resistance EcologyOctober 2016Pages 555-555 RelatedInformation

Neonatal adaptation in infants prenatally exposed to antidepressants--clinical monitoring using Neonatal Abstinence Score.

BackgroundIntrauterine exposure to antidepressants may lead to neonatal symptoms from the central nervous system, respiratory system and gastrointestinal system. Finnegan score (Neonatal Abstinence Score, NAS) has routinely been used to assess infants exposed to antidepressants in utero.AimThe purpose was to study neonatal maladaptation syndrome in infants exposed to selective serotonin reuptake inhibitors (SSRI) or serotonin-norepinephrine reuptake inhibitors (SNRI) in utero.MethodRetrospective cohort study of women using antidepressants during pregnancy and their infants. Patients were identified from the electronic health record system at Karolinska University Hospital Huddinge containing pre-, peri- and postnatal information. Information was collected on maternal and infant health, social factors and pregnancy. NAS sheets were scrutinized.Results220 women with reported 3rd trimester exposure to SSRIs or SNRIs and who gave birth between January 2007 and June 2009 were included. Seventy seven women (35%) used citalopram, 76 used (35%) sertraline, 34 (15%) fluoxetine and 33 (15%) other SSRI/SNRI. Twenty-nine infants (13%) were admitted to the neonatal ward, 19 were born prematurely. NAS was analyzed in 205 patients. Severe abstinence was defined as eight points or higher on at least two occasions (on a scale with maximum 40 points), mild abstinence as 4 points or higher on at least two occasions. Seven infants expressed signs of severe abstinence and 46 (22%) had mild abstinence symptoms. Hypoglycemia (plasma glucose <2.6 mmol/L) was found in 42 infants (19%).ConclusionSevere abstinence in infants prenatally exposed to antidepressants was found to be rare (3%) in this study population, a slightly lower prevalence than reported in previous studies. Neonatal hypoglycemia in infants prenatally exposed to antidepressant may however be more common than previously described.

PO-0679 Neonatal Adaptation In Infants Prenatally Exposed To Antidepressants; Clinical Monitoring Using Neonatal Abstinence Score

Background Intrauterine exposure to antidepressants may lead to neonatal symptoms from the central nervous, respiratory and gastrointestinal system. Neonatal Abstinence Score, NAS, has routinely been used to assess infants exposed to antidepressants in utero . Aim To study neonatal maladaptation syndrome in infants exposed to selective serotonin reuptake inhibitors (SSRI) or serotonin norepinephrine reuptake inhibitors (SNRI) in utero . Method Retrospective cohort study of women delivering at Karolinska University Hospital Huddinge between January 2007 and June 2009, who were using antidepressants during pregnancy, and their infants. Information was collected on maternal and infant health, social factors and pregnancy. NAS sheets were scrutinised. Results Two hundred and twenty women with reported 3rd trimester exposure to SSRI/SNRI were included. Seventy-seven women (35%) used citalopram, 76 (35%) sertraline, 34 (15%) fluoxetine, 33 (15%) other SSRI/SNRI. Twenty nine infants (13%) were admitted to the neonatal ward, 19 were born prematurely. Hypoglycemia (plasma glucose Conclusions Severe abstinence was rare in this cohort of infants exposed to SSRI/SNRI and the majority of all scored infants had no signs of neonatal maladaptation. Hypoglycemia was detected in one out of five infants and might be overrepresentated in SSRI/SNRI exposed infants.

THU0578-HPR Wide-Spread Pain and Pain Intensity in Patients with Early Rheumatoid Arthritis. A Comparison between Smokers and Non- Smokers. A Cross-Sectional Study

BackgroundPain is a frequent symptom in patients with rheumatoid arthritis (RA). In patients with established RA about 34% had wide-spread pain more than three months and pain was more likely...

Postoperative pancreatic fistula formation according to ISGPF criteria after D2 gastrectomy in Western patients

Limited information is available on the incidence of postoperative pancreatic fistula (POPF) after D2 gastrectomy with the strict use of the International Study Group of Pancreatic Fistula (ISGPF) criteria, particularly so in Western patients. All patients who underwent gastrectomy for adenocarcinoma at the Karolinska University Hospital Huddinge from 2006 until June 2012 were identified via hospital records and reviewed for type of surgical procedure, postoperative morbidity, incidence, and risk factors for POPF. Ninety-two of 107 cases had a D2 gastrectomy eligible for evaluation of POPF, of which 83 (90%) also underwent bursectomy. Seven patients fulfilled the criteria for POPF grade A (7.6%), 5 met the criteria for POPF grade B (5.4%), and 6 the criteria for POPF grade C (6.5%). The incidence of POPF grade B or C was 4.9% among the 82 patients for whom no pancreatic resection was performed and 70% among 10 cases with concomitant pancreatic resection. The latter (OR 156.2, 95% CI 8.00-3046.93) and age (OR 1.2, 95% CI 1.02-1.35) were found to be the only risk factors for POPF after gastrectomy upon a multivariate analysis. In this series of Western patients, POPF grade B or C according to the ISGPF criteria was uncommon after D2 gastrectomy without pancreatic resection. Bursectomy was not a risk factor for POPF.

In search of the molecular consequences of 8p21 deletion in multiple myeloma: commentary on Gmidéne et al

We have read with great interest but also astonishment, the publication by Gmidene et al. regarding the deletion of chromosomal region 8p21.3 in multiple myeloma (MM) patients [1]. While this study claims to be the first to present the deletion of this region in MM patients, we would like to draw attention to previous work in the field published by us and others. In the January 2009 issue of the British Journal of Haematology, we have presented our observations on the deletion of the 8p21 region and the prognostic significance it presents [2]. Using a commercially available FISH probe located on the 8p21 region, we have identified that 14/58 patients tested (24 %) carried this deletion at the time of diagnosis. In 2010, a larger study by Neben et al. has evaluated 236 MM patients and reported a 19 % frequency for this cytogenetic abnormality [3]. Although using a smaller group of patients, the study by Gmidene et al. describes a similar frequency of the deletion: 21.6 % (8/37 patients) that falls in line with the two previously published observations. Although there are minor differences in the FISH probes used for analysis in these three studies, the similarity of frequencies reported strongly suggests that a similar group of patients are identified with the different approaches. The study by Gmidene et al. confirms our previous report and taken altogether with Neben et al., these data convincingly demonstrate that del(8)(p21) is seen in about 1/5 of MM patients. We have also reported that del(8)(p21) stands out as a significant factor for poor prognosis in MM patients receiving autologous stem cell transplantation [2]. Survival analysis has revealed that loss of8p21 wassignificantlyassociated with poor progression-free survival (PFS: 12 vs. 27 months in patients without the deletion, p \0.05) and overall survival (OS: 24 vs. 67 months in patients without the deletion, p\ 0.005). Moreover, in this group of patients, multivariate analysis of prognostic factors has also identified del(8)(p21) as an independent prognosticfactorforbothPFS(p = 0.033)andOS(p = 0.029). Similarly, Neben et al. have also identified del(8)(p21) as a statistically significant prognostic factor for both PFS (p = 0.011) and OS (p = 0.040), while this significance was lost after p values were adjusted for ISS criteria[3]. In multivariate analysis, del(8)(p21) did not present as an independent factor, possibly due to the use of a larger number of variables compared to our study as well as differences in treatment regimens. Unfortunately, the current study by Gmidene et al. does not present any survival data on the reported patients. Regarding the correlation of the occurrence of different chromosomalaberrations, thehierarchicalclusteringanalysis in our study has presented a very close link between del(8)(p21) and t(4;14), while the correlations with del(17)(p13) and del(13)(q14)weresomewhatweaker.Nebenetal.havereported a significant correlation with del(13)(q14) (p = 0.04) and the absence of correlation with the occurrence of amp1(q)(21) (p = 0.06). Confirming the previous work, Gmidene et al. also suggest a link of del(8)(p21) with del(13)(q14) and t(4;14). In our 2009 paper as well as a review on chromosomal aberrations in MM that was published in 2010 [4], we have drawn attention to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor gene cluster located in this locus and hypothesized that the downregulation of TRAIL receptor expression due to del(8)(p21) might cause decreased sensitivity of tumor cells to TRAIL-mediated apoptosis [5], providing an advantage for immune escape of the malignant clone [6]. In a similarly structured discussion, Gmidene et al. also speculate about the possibility of such a phenomenon resulting from the deletion of TRAIL receptor T. Sutlu E. Alici G. Gahrton (&) H. Nahi Department of Medicine, M54, Karolinska Institutet, Center for Hematology and Regenerative Medicine, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden e-mail: gosta.gahrton@ki.se

Plasma Amino Acids as Predictors for Outcome in Patients at the Intense Care Unit

Amino acids (AA) are important metabolites in blood plasma. In critically ill patients muscle wasting and successive organ failure affect recovery and are one of the most common causes of death in the intense care unit (ICU). Disturbances in the plasma metabolites such as AA could report on complications and act as predictor for outcome.Here, we present the analysis of 22 amino acids concentrations in plasma of successively admitted critically ill patients (N= 174) in the ICU at the Karolinska University Hospital Huddinge [1]. The admission scoring of APACHE II, SOFA and the mortality up to 6 months were registered. Arterial blood samples were taken within 24 hours after admission and analysed by HPLC with pre‐column derivatization and an internal standard of norvaline [2].Patients were grouped into six‐months survivors and non‐survivors. Statistical analysis of AA concentrations was accomplished using R, version 2.15.1. A Wilcoxon rank sum test showed that phenylalanine (p= 0.03), alanine (p=0.03), tyrosine (p= 0.002), 3‐methylhistidine (p=0.006), carnitine (p=0.03) and ornithine (p= 0.08) were differed between the two groups with higher plasma concentrations in patients that died. The plasma concentrations of branched chain amino acids were not significantly different. Three patients showed highly elevated concentrations of two AA (Ala, Gln) possibly indicating a subgroup of patients with specific clinical characteristics such as single organ failure.In conclusion, those patients that died showed higher plasma concentration of several AA. Some of the amino acids (Phe, Tyr, 3‐methylhistidine) may represent a negative protein turnover i.e. increased muscle protein loss.This work was supported by a grant from the Swedish Research Council and Karolinska University Hospital Huddinge.

Umbilical cord blood T cells cultured with IL-7 in addition to IL-2 exhibit a higher degree of polyfunctionality and superior proliferation potential

Event Abstract Back to Event Umbilical cord blood T cells cultured with IL-7 in addition to IL-2 exhibit a higher degree of polyfunctionality and superior proliferation potential Sofia Berglund1, 2*, Jens Gertow1, Isabelle Magalhaes1, Michael Uhlin1, 2 and Jonas Mattsson1, 2 1 Karolinska Institute, Dept of Laboratory Medicine, Sweden 2 Karolinska University Hospital Huddinge, Centre for Allogeneic Stem cell Transplantation (CAST), Sweden BACKGROUND: Umbilical cord blood transplantation (UCBT) is an increasingly used stem cell source for allogeneic stem cell transplantation. One disadvantage to UCBT is that treatment with donor lymphocyte infusion (DLI) is not possible. We wanted to explore if cultured T cells from the UCB graft could offer an alternative to DLI in this setting. METHODS: T cells were positively selected with CD3+/CD28+ magnetic beads and cultured with IL-2 at escalating concentrations. In addition, IL-7 (20 ng/ml) was added to half of the cultures. After 7 days of expansion the T cells were analyzed by flow cytometry to determine phenotype, and cytokine production. RESULTS: There was significantly greater proliferation in cultures to which IL-7 had been added. Phenotypical differeces such as a higher CD4+/CD8+, a lower percentage of central memory T cells (CD45RO+ CCR7+) and a higher percentage of effector memory T cells (CD45RO+ CCR7-) was seen with IL-7. We assessed the intracellular production of IL-2, TNF-alpha, IFN-gamma and CD107a, and found that a higher percentage of polyfunctional T cells (positive for three or four factors) was seen in cells cultured with IL-7 in CD3+ and CD3+CD8+ cells, with a trend towards the same association also in CD3+CD4+ cells. CONCLUSION: There seem to be a proliferation advantage in adding IL-7 to a lower dose of IL-2. Addition of IL-7 also seem to have an impact on the differentiation status of the cells. T cells cultured in IL-7 had more polyfunctional traits, possibly increasing the activity of a putative future DLI. Acknowledgements The authors would like to thank the maternity ward, in particular Boel Niklasson, at Karolinska University hospital Huddinge, Stockholm, Sweden, for their kind assistance in obtaining the umbilical cord blood units. This study was supported by grants from Swedish Research Council (K2012-64X-22020-01-3) and The Swedish Childhood Cancer Foundation (PROJ10/052) and Swedish society for medical research. Keywords: umbilical cord blood, IL-7, T cell expansion, DLI, Cell Culture Techniques Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Berglund S, Gertow J, Magalhaes I, Uhlin M and Mattsson J (2013). Umbilical cord blood T cells cultured with IL-7 in addition to IL-2 exhibit a higher degree of polyfunctionality and superior proliferation potential. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00697 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Sofia Berglund, Karolinska Institute, Dept of Laboratory Medicine, Stockholm, 14186, Sweden, sofia.berglund@ki.se Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sofia Berglund Jens Gertow Isabelle Magalhaes Michael Uhlin Jonas Mattsson Google Sofia Berglund Jens Gertow Isabelle Magalhaes Michael Uhlin Jonas Mattsson Google Scholar Sofia Berglund Jens Gertow Isabelle Magalhaes Michael Uhlin Jonas Mattsson PubMed Sofia Berglund Jens Gertow Isabelle Magalhaes Michael Uhlin Jonas Mattsson Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Human Polyomaviruses Were Not Detected in Cerebrospinal Fluid of Patients with Neurological Complications After Hematopoetic Stem Cell Transplantation

AbstractAbstract 4503Neurological complications after allogenetic hematopoetic stem cell transplantation (HSCT) are associated with increased mortality. Reactivation of JC-virus (JCV), a well-known human polyomavirus (HPyV) can be associated with progressive multifocal leukoencephalopathy (PML) after HSCT. Knowledge of whether reactivation of the newly discovered HPyVs KIPyV, WUPyV, Merkel cell PyV (MCV), HPyV 6, HPyV7, trichodysplasia spinulosa PyV (TSV) and HPyV9 is associated to neurological complications after HSCT is however limited. Cerebrospinal fluid (CSF) from 32 HSCT patients with neurological symptoms was therefore analyzed for presence of the above HPyVs including BK-virus (BKV), JCV, and primate PyVs lymphotropic PyV (LPV) and Simian Virus 40 (SV40), but found negative by PCR for all these PyVs. IntroductionThe incidence of neurological complications after HSCT is up to 15% and related to increased mortality. Risk factors are e.g. busulfan (Bu), electrolyte disturbances, low platelet count, high blood pressure, ciklosporin toxicity and viral reactivation, of which HPyV JCV is one.2007–2008, three new HPyVs were described: KIPyV, WUPyV, both found in nasopharyngeal aspirates and Merkel cell PyV (MCV), detected in Merkel Cell Carcinoma, a skin cancer detected in elderly persons or in immunocompromized persons. We earlier examined for these three HPyVs in CSFs of 20 patients with neurological complications after HSCT, but they were all negative by PCR. From 2009 and on, another five HPyVs (HPV6, HPV7, TSV and HPyV9) were detected, and to possibly find whether these PyVs were responsible for neurological conditions in patients having undergone HSCT, we have extended our study and analyzed CSF from 32 patients who presented with neurological complications after HSCT for all known HPyVs.Patients, Material and Methods PatientsFrom January 1st 2000 to April 20th 2011 843 patients underwent HSCT at the Centre for Allogenic Stemcell Transplantation, Karolinska University Hospital Huddinge, Sweden. Of these patients, 65 suffered from neurological symptoms and 32 had sufficient amounts of CSF for further analysis. Mean age was 38 years (1–63). The female/male ratio was 13/19. 15 patients received stem cell from a matched unrelated donor, 3 from cord blood and 6 from siblings. 20/32 patients had Bu in the conditioning therapy, 7 received TBI and 5 patients got other conditioning therapy. Multiplex PCR analysisA PCR detecting the presently known 9 HPyVs, including, BKV, JCV, KIPyV, WUPyV, MCV, HPyV6, 7, TSV and HPyV9 as well as the primate LPV and SV40 was developed and used for the analysis. The PCR was initiated with denaturation of CSFs (10 μl) for 9 min at 94°. DNA was extracted from 5 μl CSF/sample using 2×Qiagen Multiplex PCR Master Mix and a PCR protocol containing 15 min in 94°C for denaturation 94°C 15 min followed by 40 cycles with 94°C 20 sec, 50°C, 1 min 30 sec, 71°C 1 min 20 sec and ended with 71°C for 4 min. PyVs amplicons were identified using bead based multiplex analysis with a MagPix system from Luminex. The assay could detect all 11 viruses with a sensitivity of <10 copies/sample. Results and DiscussionAmong the 32 HSCT patients with neurological symptoms, the most frequent were headache and fever, (41%), followed by seizures (25%) and confusions/hallucinations (16%). At the end of the study period 16/32 patients with a mean follow up time of 3.5 years had survived.Because HyPV reactivation is increased in immunocompromised patients, neurological signs due to reactivation of these viruses are more likely to occur. Despite neurological symptoms, no DNA from the known 9 HPyVs or from LPV or SV40 was detected by the Luminex multiplex based assay in any of the 32 CSF samples.The detection assay used, with detection limits of between 5–10 copies of the respective viruses, should be sensitive enough to detect viral DNA if the corresponding virus was responsible for neurological disease.Many episodes of neurological complications after HSCT are still not given a diagnosis. Our negative finding emphasizes the need for a broader search for causes, especially when new treatment options are becoming available. Disclosures:No relevant conflicts of interest to declare.

Prevention of ovarian cancer – let's do something

Prevention of ovarian cancer – let's do something

Activated Apoptotic Cells Induce Dendritic Cell Maturation via Engagement of Toll-like Receptor 4 (TLR4), Dendritic Cell-specific Intercellular Adhesion Molecule 3 (ICAM-3)-grabbing Nonintegrin (DC-SIGN), and β2 Integrins

Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-α (TNF-α). Cell-cell contact-dependent signaling involved β2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4(-/-) mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFκB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.