Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study

We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with ~200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis

Middle Eastern mothers in Sweden, their experiences of the maternal health service and their partner's involvement

<p>Abstract</p> <p>Background</p> <p>Traditional patterns relating to how to handle pregnancy and birth are often challenged due to migration. The purpose of this study was to describe Middle Eastern mothers' experiences of the maternal health care services in Sweden and the involvement of their male partner.</p> <p>Methods</p> <p>Thirteen immigrant mothers from the Middle East who had used the maternal health services in Sweden were interviewed using focus group discussions and individual interviews. These were taped, transcribed and analysed according to Content analysis.</p> <p>Results</p> <p>The four main categories that developed were:</p> <p>• Access to the professional midwife</p> <p>• Useful counselling</p> <p>• Stable motherhood in transition</p> <p>• Being a family living in a different culture</p> <p>Conclusion</p> <p>According to the respondents in this study, understanding the woman's native language or her culture was not vital to develop a good relationship with the midwife. Instead the immigrant woman developed trust in the midwife based on the knowledge and the empathy the midwife imparted.</p> <p>Increasing the amount of first trimester antenatal visits could avoid spontaneous visits to the emergency clinic. There was a greater need for involvement and support by the father during the perinatal period, such as caring for older children and carrying out household chores since the mothers' earlier female network was often lost.</p> <p>Clinical implications</p> <p>There is a need to involve immigrant parents in the available parental education in order to prepare them for parenthood in their new country as well as to explore their altered family situation. Collecting immigrant women and their partner's, experiences of maternal health care services offers a possibility to improve the existing care, both in content, access and availability where the timing of visits and content require further evaluation.</p

The C-Type Lectin of the Aggrecan G3 Domain Activates Complement

Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD) and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP) domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint

Hippocampus Shape Analysis and Late-Life Depression

Major depression in the elderly is associated with brain structural changes and vascular lesions. Changes in the subcortical regions of the limbic system have also been noted. Studies examining hippocampus volumetric differences in depression have shown variable results, possibly due to any volume differences being secondary to local shape changes rather than differences in the overall volume. Shape analysis offers the potential to detect such changes. The present study applied spherical harmonic (SPHARM) shape analysis to the left and right hippocampi of 61 elderly subjects with major depression and 43 non-depressed elderly subjects. Statistical models controlling for age, sex, and total cerebral volume showed a significant reduction in depressed compared with control subjects in the left hippocampus (F1,103 = 5.26; p = 0.0240) but not right hippocampus volume (F1,103 = 0.41; p = 0.5213). Shape analysis showed significant differences in the mid-body of the left (but not the right) hippocampus between depressed and controls. When the depressed group was dichotomized into those whose depression was remitted at time of imaging and those who were unremitted, the shape comparison showed remitted subjects to be indistinguishable from controls (both sides) while the unremitted subjects differed in the midbody and the lateral side near the head. Hippocampal volume showed no difference between controls and remitted subjects but nonremitted subjects had significantly smaller left hippocampal volumes with no significant group differences in the right hippocampus. These findings may provide support to other reports of neurogenic effects of antidepressants and their relation to successful treatment for depressive symptoms

Quantification of Acute Lymphoblastic Leukemia Clonotypes in Leukapheresed Peripheral Blood Progenitor Cells Predicts Relapse Risk Following Autologous Hematopoietic Cell Transplantation

Abstract Background Although multiple studies have shown superiority of allogeneic hematopoietic cell transplantation (alloHCT) over autologous hematopoietic cell transplantation (autoHCT) for patients with "high-risk" acute lymphoblastic leukemia (ALL), these findings may be explained, in part, by contamination of the peripheral blood progenitor cell (PBPC) leukapheresis product by residual leukemic cells in patients undergoing autoHCT. Methods We retrospectively evaluated minimal residual disease (MRD) via next-generation sequencing (NGS) (Adaptive Biotechnologies, S. San Francisco, CA) in the PBPC leukapheresis products from 32 ALL patients who underwent autoHCT. All patients had "high-risk" ALL, as defined by B-lineage disease with WBC at diagnosis &gt;30,000/uL; high-risk cytogenetics including t(9;22), t(4;11), other 11q23 abnormalities, or monosomy 7; or primary refractory disease. Peripheral hematopoietic cell mobilization consisted of cytarabine 2000mg/m2 IV every 12 hours (16,000mg/m2 cumulative) concurrent with etoposide 40mg/kg cumulative by continuous IV infusion over 4 days. The autoHCT conditioning consisted of 1320cGy total body irradiation (TBI) given in 11 fractions from day -8 to -5, etoposide 60mg/kg IV on day -4, and cyclophosphamide 100mg/kg IV on day -2. Tyrosine kinase inhibitors (TKI) were allowed for patients with Philadelphia chromosome-positive (Ph+) B-ALL. Seven patients (22%) participated in a multi-institutional trial in which the PBPC graft underwent ex vivo complement-mediatedpurging using monoclonal antibodies against CD9/CD10/CD19/CD20 for patients with B-lineage disease, or against CD2/CD3/CD4/CD5/CD8 for patients with T-lineage disease. Kaplan-Meier curves were generated using GraphPad Prism (GraphPad Software, La Jolla, CA) and statistical differences assessed via Log-Rank and Wilcoxon analyses, with a p-value of &lt;0.05 considered significant. Results Twenty-eight patients (88%) had diagnostic bone marrow samples with quantifiable immunoglobulin or T cell receptor (Ig/TCR) gene rearrangements suitable for MRD quantification in the PBPC collections. Twelve (38%) patients had Ph+ B-ALL, 12 (38%) had Ph-neg B-ALL, and 4 (14%) had T-cell ALL. The majority of patients were male, in first complete remission, and autografted between 2000-2009, with a median age at autoHCT of 32 (range 19-55). With a median follow-up of 41 months (range 3-217), median relapse-free survival (RFS) and overall survival (OS) for the entire cohort are 3.2 and 4.2 years, respectively. At 5 years post-autoHCT, 42% of patients remain alive and relapse-free. Of the 28 diagnostic bone marrow specimens, 21 (75%) had rearrangements in more than 1 immunoreceptor locus (Figure 1A). A total of 73 rearrangements were identified across all patients. Clonal Ig heavy chain (IGH) sequences with complete VDJ rearrangement were the most commonly identified rearrangement, present in 17 of 28 patients (61%) and representing 30 of the 73 (41%) total rearrangements. When stratified by graft MRD burden, the median RFS for patients with MRD detectable at a level ≥10-6 (n=13) was 6.5 months, and has not been reached for patients without detectable MRD above this threshold (n=15; p=0.0005; Figure 1B). Ex vivo antibody-based purging failed to eradicate leukemia cells from the PBPC collections of 3 patients with detectable MRD who received a purged graft (Figure 1C), all of whom ultimately relapsed. Of the Ph+ cohort, 6 (50%) had detectable MRD in the PBPC graft. Two (33%) of these patients were not treated with a TKI; 1 relapsed at 4 months post-autoHCT and the other died from non-relapse mortality. Of the 4 Ph+ patients with detectable MRD who received a TKI, 2 (50%) remain long-term relapse-free survivors. Of the 6 patients without MRD who were treated with a post-autoHCT TKI, 5 (83%) remain relapse-free. Conclusion We demonstrate that the NGS-based immunosequencing platform identifies ALL MRD in leukapheresed PBPC collections. The presence of MRD in the autograft strongly predicts relapse, with only 15% RFS in patients harboring ≥10-6 MRD at stem cell collection versus 73% in patients with undetectable MRD. The absence of MRD in PBPC may thus identify a subset of "high-risk" patients likely to achieve long-term remissions without alloHCT. TKI therapy for patients with Ph+ B-ALL may also, in some cases, abrogate the need for alloHCT, even with quantifiable MRD prior to high-dose therapy. Disclosures Damon: Atara: Consultancy; Sunesis: Research Funding; Sigma-Tau: Research Funding; McGraw-Hill: Patents &amp; Royalties: Book Chapter. Andreadis:McGraw Hill: Honoraria; Novartis: Consultancy; Cellerant: Consultancy; Pharmacyclics: Honoraria. Olin:Daiichi-Sankyo: Research Funding. Kong:Adaptive Biotechnologies: Employment, Equity Ownership. Faham:Adaptive Biotechnologies: Employment, Equity Ownership. </jats:sec