Search

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS‐CoV‐2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS‐CoV‐2 spike protein. Nine antibodies neutralize SARS‐CoV‐2 infection at IC50 values in the subnanomolar range. ELISA‐binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor‐binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N‐terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS‐CoV‐2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS‐CoV‐2‐induced weight loss. The two clusters of potent non‐competing SARS‐CoV‐2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID‐19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives. This article is protected by copyright. All rights reserved

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies., Graphical Abstract, Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions.

The systemic processes involved in the manifestation of life-threatening COVID-19 and in disease recovery are still incompletely understood, despite investigations focusing on the dysregulation of immune responses after SARS-CoV-2 infection. To define hallmarks of severe COVID-19 and disease recovery in convalescent patients, we combined analyses of immune cells and cytokine/chemokine networks with endothelial activation and injury. ICU patients displayed an altered immune signature with prolonged lymphopenia but expansion of granulocytes and plasmablasts along with activated and terminally differentiated T and NK cells and high levels of SARS-CoV-2-specific antibodies. Core signature of seven plasma proteins revealed a highly inflammatory microenvironment in addition to endothelial injury in severe COVID-19. Changes within this signature were associated with either disease progression or recovery. In summary, our data suggest that besides a strong inflammatory response, severe COVID-19 is driven by endothelial activation and barrier disruption, whereby recovery depends on the regeneration of the endothelial integrity.

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly generate human monoclonal antibodies. After immunizing these mice against the spike protein of SARS-CoV-2, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralized SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of neutralizing antibodies binds to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2 induced weight loss. Thus, we report two clusters of potent non-competing SARS-CoV-2 neutralizing antibodies providing potential candidates for therapy and prophylaxis of COVID-19. The study further supports the use of transgenic animals with human immunoglobulin gene repertoires in pandemic preparedness initiatives.

BackgroundCritical thinking is an essential skill that nursing students need to develop. Technological tools have opened new avenues for technology-supported guidance models, but the challenges and facilitators of such guidance models, as well as how they stimulate the development of critical thinking, remain unclear.ObjectiveWe developed a protocol for a mixed methods systematic review to investigate the use of technology-supported guidance models that stimulate the development of critical thinking in nursing education clinical practice.MethodsA convergent integrated design following the Joanna Briggs Institute Manual for Evidence Synthesis will be employed. A pair of authors will select the articles by screening titles and abstracts, and the methodological quality of the articles included in the review will be assessed by a pair of authors according to checklists for specific study designs. The data will be extracted using the standardized Joanna Briggs Institute mixed methods data extraction form and following a convergent integrated approach. The thematic synthesis for data transformation will be used.ResultsDevelopment of a comprehensive systematic search strategy was completed in October 2020. The database searches were performed on October 21, 2020. As of January 2021, analysis and synthesis is ongoing. Completion of this review is expected by January 2021.ConclusionsBy combining evidence from studies with varied methodological approaches, the results should provide broad insight into the use of technology-supported guidance models for clinical practice in nursing education with a focus on the development of nursing students’ critical thinking. The results of this mixed methods systematic review can also be used to develop or improve current technology-supported guidance models for clinical practice in nursing education.International Registered Report Identifier (IRRID)PRR1-10.2196/25126

Long-lived antibody-secreting plasma cells are essential to establish humoral memory against pathogens. While a regulatory transcription factor network has been established in plasma cell differentiation, the regulatory role of miRNAs remains enigmatic. We have recently identified miR-148a as the most abundant miRNA in primary mouse and human plasma cells. To determine whether this plasma cell signature miRNA controls the in vivo development of B cells into long-lived plasma cells, we established mice with genomic, conditional, and inducible deletions of miR-148a. The analysis of miR-148a-deficient mice revealed reduced serum Ig, decreased numbers of newly formed plasmablasts and reduced CD19-negative, CD93-positive long-lived plasma cells. Transcriptome and metabolic analysis revealed an impaired glucose uptake, a reduced oxidative phosphorylation-based energy metabolism, and an altered abundance of homing receptors CXCR3 (increase) and CXCR4 (reduction) in miR-148a-deficient plasma cells. These findings support the role of miR-148a as a positive regulator of the maintenance of long-lived plasma cells.

Background The Public Health Intervention Wheel (PHIW) is a population-based practice model that encompasses three levels of practice (community, systems, and individual/family) and 17 public health interventions. Each intervention and practice level contributes to improving population health. Public health nurses (PHNs) provide care at the three levels of practice. Prevention of falls is a public health issue and the majority of falls happen at home. Therefore, prevention and management of falls in the community could benefit from a public health systems approach by PHNs underpinned by the PHIW. Case presentation A hypothetical case is presented of a 78-year-old gentleman who had a fall which resulted in a fractured right acetabulum and surgery before being discharged home. Methods The aim of this paper was to use a case summary to illustrate PHN practice in the context of the PHIW as applied to falls management and prevention. This paper focuses on fall incidence and PHN response in Ireland and Norway. The PHIW is described and relevant interventions from the PHIW are applied to PHN practice in managing the case. Conclusions The PHIW model provides insight into the potential scope of public health nursing in falls, articulating PHN practice in the community.

Purpose To present an emerging innovative care model that supports participation and thriving by older adults in residential care, by introduction to new technology and mobilizing volunteer services. Design Qualitative, exploratory study, introducing tablet computers to 15 older adults in two municipalities. Methods The intervention encompassed weekly workshops over the course of 1 year with volunteer adolescents as personal tutors. Observations of workshops, interviews with nurses, and repeated semistructured interviews with older adult participants eliciting their perspective on use, experiences, perceived usefulness, and overall evaluation of the intervention. Findings A model of four components is suggested to support participation and thriving by older adults in residential care: (a) simplified tools: iPad—technology relatively easy to use; (b) person-centered process: one-to-one tutoring following each individual's own pace; (c) young volunteers to teach technology, establishing an intergenerational arena; and (d) being mindful of driving forces that encourage use and learning. We found that all kinds of use and all levels of mastery generated a sense of pride that supported thriving and enjoyment. Conclusions These findings support the use of new technology and use of volunteer services for sustaining thriving in older adults. The person-centered approach stimulates use of the tablet, and participants showed enjoyment, more social participation, and reported subjective experiences of thriving. Clinical Relevance Innovative models of care that prevent (or postpone) functional decline and support thriving in older adults are highly sought after in health care. A model that systematically involves volunteer services comes with potentials to alleviate nurses’ workload, and then the intervention is seen as a manageable and low-cost initiative in residential care.

The study explored how 125 older patients and 92 next of kin experienced and assessed continuity in long-term home health care. Data were collected by means of structured interviews. A majority of the patients indicated that having a high number of health care personnel involved in their care was not problematic. For patients, informed and skilled health personnel along with knowing the visiting personnel may compensate for a high number of personnel. For next of kin, accepting a high number of personnel was related to the carers being informed about the patient’s situation. This study indicates that, in terms of patient satisfaction, the overall quality of care is more important than the number of people providing the care.

We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice.

In 2013, cancer immunotherapy was selected as the breakthrough of the year by Science’s editors and monoclonal antibody therapy is considered one of its most successful tools. When castration-resistant prostate cancer (PCa) disease progresses and despite androgen deprivation treatment, it typically metastasizes, leading to poor patients’ prognosis and survival. In this case, immunotherapy represents the only chance of treatment. In a previous study carried out by our team on the role of cytoplasmic PML1 in the context of epithelial to mesenchymal transition (EMT) in PCa2, we identified a new promising PCa target for antibody-based therapy. Our target, of which we cannot disclose the name for patenting reasons and that we will further refer to as Protein-X, is a type 1 transmembrane protein that is specifically expressed by PCa cell. Transcriptome profiling obtained using Nanostring technology with a cancer progression panel showed that the knockdown of Protein-X leads to an upregulation of the epithelial EMT marker E-cadherin in PCa cells, suggesting its role in the process. Furthermore, the clinical value of Protein-X as a diagnostic and/or prognostic biomarker of invasive and aggressive PCa, is also being investigated by a histopathologist using immunohistochemistry analysis of Protein-X’s expression on tissue microarrays (TMAs). To develop a monoclonal antibody-based therapy against Protein-X, mouse monoclonal antibodies (mAbs) were generated using hybridoma technology. MAbs-producing hybridomas were sequenced and purified mAbs were tested for their specificity by flow cytometry, immunofluorescence, ELISA and immunoblotting assays. Using in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay, we show that the antibodies mediate cell cytotoxicity. This result will be further investigated in vivo using a xenograft mouse model. Future experiments will focus on designing and generating chimeric antigen receptor (CARs) modified adoptive T-cell targeting of Protein-X-expressing tumor cells and bispecific antibodies. In summary, we aim to develop an anti-Protein-X antibody-based therapy against invasive prostate cancer, which will potentially represent a new tool to target aggressive PCa responsible for cancer metastasis and poor prognosis in PCs patients. Citation Format: Anna Di Biase, Tarik Regad, Jayakumar Vadakekolathu, Desmond Powe, Hans-Martin Jäck, Edith Roth. Generation of monoclonal antibodies against a newly identified target for invasive prostate cancer and their use in the development of an antibody-based targeting therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B152.

Aim To explore continuity of care from a manager’s perspective: How do managers understand the concept of continuity of care and how do they assess continuity of care in home health-care? How do managers work to ensure continuity of care when managing care provision for patients in their homes? Background Continuity of care is considered a prerequisite for service quality. Managers can influence continuity of care through managing day-to-day work. Methods Semi-structured interviews of a purposive sample of 16 managers from 12 municipal units. Theory-driven and data-driven analysis was applied. Results Ideally, to promote continuity of care, the number of carers should be limited. Yet, in practice, discontinuity of care was accepted as a working compromise. The managers faced two paradoxes: the continuity ideal vs. the practicalities of home health-care, and caring for patients vs. caring for staff. Conclusions The managers were forced to prioritize, in conflict with ideals or professional standards, patients’ wellbeing or the wellbeing of the staff. Ensuring continuity of care for all patients did not seem feasible. Implications for nursing management The study highlights management paradoxes: possible unintended consequences of prioritizing needs for continuity of care should be objects of reflection in care management.

Krüppel-like factor 2 (KLF2) controls T lymphocyte egress from lymphoid organs by regulating sphingosin-1 phosphate receptor 1 (S1Pr1). Here we show that this is not the case for B cells. Instead, KLF2 controls homeostasis of B cells in peripheral lymphatic organs and homing of plasma cells to the bone marrow, presumably by controlling the expression of β 7 -integrin. In mice with a B cell-specific deletion of KLF2, S1Pr1 expression on B cells was only slightly affected. Accordingly, all splenic B cell subsets including B1 cells were present, but their numbers were increased with a clear bias for marginal zone (MZ) B cells. In contrast, fewer peyers patches harboring fewer B cells were found, and fewer B1 cells in the peritoneal cavity as well as recirculating B cells in the bone marrow were detected. Upon thymus-dependent immunization, IgG titers were diminished, and antigen-specific plasma cells were absent in the bone marrow, although numbers of antigen-specific splenic plasmablasts were normal. KLF2 plays also a role in determining the identity of follicular B cells, as KLF2-deficient follicular B cells showed calcium responses similar to those of MZ B cells and failed to down-regulate MZ B cell signature genes, such as CD21 and CXCR7.

Objective. To explore the first-line leaders’ role in quality work in long-term care in Norway, in order to determine how that work is related to such success characteristics as leadership, staff, patients, performance, information and information technology. Design. Cross-sectional telephone survey. The text was analysed using content analysis. Setting. Thirty-two Norwegian municipalities stratified according to region and population size. Participants. Sixty-four first-line leaders in nursing homes and home-based care. Main outcome measure. The clinical microsystem approach is used as a framework by defining and designing measureable variables. Results. Thirty-six leaders described how they initiated and motivated employees to be active in quality work; the remaining leaders indicated that they played a passive role. The first-line leaders played a key role in implementing national quality policies and regulations. The quantity of other success characteristics was low. Conclusions. The municipalities delegated the responsibility of implanting national policies to the first-line leaders. Missing were key quality success criteria such as macro- and meso-perspectives for the municipality as a whole and co-operation with other leaders in the organization and fostering of relevant learning. Quality work was fragmented rather than comprehensive and systematic.

Aims. The present study explores how community nurses addressed patient care in the EPR and the comprehensiveness of their documentation. Background. The need for comprehensive nursing documentation in home health care is considerable and quality is regarded as a prerequisite for continuity of care. Documentation according to the nursing process is considered to be of good quality due to its logical structure. Nurses in home health care face different challenges than nurses in institutionalised care because of long-term patient situations and a focus on chronic illness rather than acute disease. Design. Retrospective study. Method. The study was performed on a sample of 91 patient records. Data were analysed in three phases: (1) systematising the unstructured text, (2) structuring the text according to the nursing process and (3) assessing the comprehensiveness using a validated instrument. Results. The home care nurses documented patient care chronologically along a time axis rather than using a logical structure according to the nursing process. The documentation reflected today’s overall emphasis on patient participation, as more than 70% of the notes on nursing status were connected to subjective nursing status. Paradoxically, the nurses showed a lack of attention to the patients’ ability to communicate. Only two of 264 documented nursing diagnoses were connected to communication. The comprehensiveness of the documentation, however, was incomplete. Conclusions. Home health care nurses are attentive to patient participation but fail to address patients’ needs with regard to communication. The documentation is incomplete when assessed according to the steps of the nursing process. A question that arises is whether the nursing process may be a limitation for the quality of the nursing documentation. Relevance to clinical practice. The study contributes to identifying areas of improvement in documentation by nurses in home health care.

Progressive loss of hip containment attributable to dysplasia epiphysealis hemimelica of the right proximal femur and macrodactyly of the right second toe was diagnosed and monitored by radiographs and magnetic resonance imaging in a 7-year-old boy. The patient had early surgical correction including shortening osteotomy of the hypertrophic toe and partial resection of the involved superolateral femoral head-neck junction combined with a Pemberton-like acetabuloplasty. This treatment restored containment and function. At 4 years followup, the femoral head remained contained and the patient was participating fully in the activities of daily life for his age-group. A review of the literature suggests monitoring the hip with magnetic resonance imaging may allow early identification of a hip at risk for loss of containment. This treatment may save the hip from severe deformity and early secondary osteoarthritis.

Pre-B cell receptor (pre-BCR) signals are essential for pro-B cells to mature efficiently into pre-B cells. The pre-BCR is an Ig-like transmembrane complex that is assembled from two μH chains (μHC) and two surrogate L chains consisting of the non-covalently associated polypeptides VpreB and λ5. In λ5−/− mice, pro-B cell maturation is impaired, but not completely blocked, implying that a μHC induces differentiation signals in the absence of λ5. Using a mouse model, in which transgenic μHC expression can be controlled by tetracycline, we show that in the absence of λ5, the transgenic μHC promotes in vivo differentiation of pro-B cells, induces IL-7-dependent cell growth, and is expressed on the surface of pre-B cells. Our findings not only show that an incomplete pre-BCR can initiate signals, but also challenge the paradigm that an IgHC must associate with an IgLC or a SLC to gain transport and signaling competency.

Operationsziel Stabiles, belastungsfahiges und bewegliches Huftgelenk. Indikationen Unbehandelte luxierte Huftgelenke bei uber sechs Monaten alten Sauglingen. Persistierend instabiles Gelenk bei junger als sechs Monaten alten Sauglingen nach achtwochigem Repositions- und Retentionsversuch. Kontraindikationen Beidseitige Luxation bei Arthrogryposis multiplex. Hohe Huftluxation mit Inkongruenz der Gelenkpartner bei Jugendlichen und Erwachsenen. Operationstechnik Anterolateraler Zugang und Darstellung des Intertrochantergebiets. Fenestrierung der Gelenkkapsel. Resektion des Ligamentum teres und des Pulvinars, partielle Resektion des Ligamentum transversum und der Iliopsoassehne, Reposition unter Innenrotation und Abduktion. Fixierung des Huftkopfes in Repositionsstellung mit einem durch den Schenkelhals in das Azetabulum gebohrten Kirschner-Draht. Intertrochantare Osteotomie und Fixierung der Fragmente in Neutralstellung der Gliedmase mit zwei Kirschner-Drahten. Becken-Bein-Gipsverband mit Fusteil fur sechs Wochen in Neutralstellung des Beins. Nach sechs Wochen Entfernung des Gipsverbandes und aller Kirschner-Drahte. Keine Orthese. Bei persistierender Pfannendysplasie Azetabuloplastuk empfohlen. Ergebnisse 48 Huftgelenke wurden zwischen 1981 und 1995 operiert; durchschnittliche Nachuntersuchungszeit 8,3 Jahre (sechs Monate bis 18 Jahre). Die Nachuntersuchungsergebnisse aller Patienten wurden nach Barrett et al. bewertet. Es kam zu keiner Reluxation. Bei 32 Patienten fanden sich ausgezeichnete und gute, bei sieben mittelmasige und bei weiteren sieben schlechte Ergebnisse. Bei Operationen bis zum sechsten Lebensmonat traten keine, bei Eingriffen zwischen dem sechsten und zwolften Lebensmonat zwei Huftkopfnekrosen auf; bei nach dem zwolften Lebensmonat operierten Kindern kam es zu vier Huftkopfnekrosen. Die besten klinischen und radiologischen Gesamtergebnisse waren bei Eingriffen bis zum sechsten Lebensmonat zu verzeichnen. Die klinischen und radiologischen Gesamtergebnisse verschlechterten sich kontinuierlich bei Operationen nach dem sechsten Lebensmonat.

Objective. To investigate whether histone-specific T helper (Th) cells that are able to induce anti-doublestranded DNA (anti-dsDNA) antibodies can be isolated from patients with systemic lupus erythematosus (SLE) and to characterize the cytokine secretion pattern of such Th clones. Methods. Peripheral blood mononuclear cells from SLE patients and healthy donors were stimulated with autologous apoptotic cell material or purified histones, expanded with interleukin-2 (IL-2), and cloned by limiting dilution. Histone reactivity of clones was examined by histone-specific proliferation and cytokine release. Cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and CTLL-2 bioassay. Induction of anti-dsDNA antibodies was measured in cocultures of autologous B cells and Th clones by ELISA. Results. Numerous histone-specific T cell receptor (TCR) α/β+ Th clones were established from 2 of 3 patients with active SLE and from 1 of 2 healthy individuals. Most Th clones secreted IL-2, interferon-γ (IFNγ), and IL-4, whereas some produced predominantly IL-2 and lFNγ. Th clones that could stimulate the production of anti-dsDNA antibodies were derived from SLE patients and from a healthy individual. Conclusion. Th cells specific for histones may play an important role in the pathogenesis of SLE by inducing autoantibodies to dsDNA. Both Th1 and Th2 cytokines may be involved in the pathogenesis of SLE. The presence of histone-specific Th cells in a healthy individual indicates the importance of peripheral tolerance for preventing autoimmunity to nuclear antigens.

Aim To provide a method for assessing the degree of interpersonal continuity adapted to context and to measure and assess the degree of interpersonal continuity for long-term recipients dependent on daily home health care. Background Interpersonal continuity is important to the quality of care for long-term dependents. In high-frequency home healthcare services where patients receive daily care from many nurses or other health personnel over time, interpersonal continuity may be difficult to attain. Design A cross-sectional study with a descriptive design. Methods Information concerning 79 patients receiving long-term frequent care was collected during four weeks in a maximum variation sample of Norwegian municipalities, from January 2009–May 2010. We measured interpersonal continuity objectively using indices of dispersion and the next-day sequence of health personnel. For each measure, we computed the highest feasible level of continuity that could be attained in this home healthcare context given a standard shift plan. This level was then used as benchmark against which the actual level of continuity was assessed. Results Patients received on average 51 visits from a mean of 17 different carers during four weeks. The results revealed a low degree of interpersonal continuity in practice, far below what was feasible according to the benchmarks. Conclusion High-frequency home health care was characterized by interpersonal discontinuity, but with potential for improvement. Objective measures of interpersonal continuity, when the benchmark is adapted to the context, are useful tools for planning and surveying continuity of care.

Precursor BCR (pre-BCR) signaling governs proliferation and differentiation of pre-B cells during B lymphocyte development. However, it is controversial as to which parts of the pre-BCR, which is composed of Igμ H chain, surrogate L chain (SLC), and Igα-Igβ, are important for signal initiation. Here, we show in transgenic mice that the N-terminal non-Ig-like (unique) tail of the surrogate L chain component λ5 is critical for enhancing pre-BCR-induced proliferation signals. Pre-BCRs with a mutated λ5 unique tail are still transported to the cell surface, but they deliver only basal signals that trigger survival and differentiation of pre-B cells. Further, we demonstrate that the positively charged residues of the λ5 unique tail, which are required for pre-BCR self-oligomerization, can also mediate binding to stroma cell-associated self-Ags, such as heparan sulfate. These findings establish the λ5 unique tail as a pre-BCR-specific autoreactive signaling motif that could increase the size of the primary Ab repertoire by selectively expanding pre-B cells with functional Igμ H chains.

Transgenic expression of B- and T-cell receptors (BCRs and TCRs, respectively) has been a standard tool to study lymphocyte development and function in vivo. The generation of transgenic mice is time-consuming and, therefore, a faster method to study the biology of defined lymphocyte receptors in vivo would be highly welcome. Using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells, TCRs can be expressed rapidly in mice of any background. We aimed at adopting this retrogenic technology to the in vivo expression of BCRs. Using a well characterised BCR specific for hen egg lysozyme (HEL), we achieved surface expression of the retrogenically encoded BCR in a Rag-deficient pro B-cell line in vitro. In vivo, retrogenic BCRs were detectable only intracellularly but not on the surface of B cells from wild type or Rag2-deficient mice. This data, together with the fact that no BCR retrogenic mouse model has been published in the 7 years since the method was originally published for TCRs, strongly suggests that achieving BCR-expression in vivo with retrogenic technology is highly challenging if not impossible.

Apoptotic cell death is important in the development and homeostasis of multicellular organisms1 and is a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing an inflammatory response or tissue damage1,2. We now show that the presence of apoptotic cells during monocyte activation increases their secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10) and decreases secretion of the proinflammatory cytokines tumour necrosis factor-α (TNF-α), IL-1 and IL-12. This may inhibit inflammation and contribute to impaired cell-mediated immunity in conditions associated with increased apoptosis, such as viral infections, pregnancy, cancer and exposure to radiation.

Background and Objectives Our long-term goal is to understand how one class of interfering non-coding RNAs, the so-called micro-RNAs (miRNAs), regulates and fine-tunes the differentiation of mature B cells into effector cells, i.e., memory B cells and antibody-secreting plasma cells. miRNAs control the expression of specific target genes at the post-transcriptional level by binding to target sequences e.g., in the 3’-untranslated region of mRNAs, which, depending on the degree of the binding, results either in a block of translation or an accelerated degradation of the respective target mRNA. Lineage-specific deletion of the miRNA-processing DICER protein as well as of individual miRNAs revealed the importance of miRNA pathway in early steps of central B cell maturation. However, the mechanisms by which miRNA-dependent circuits control the antigen-induced phase of B cell activation of mature naive B cells and their subsequent differentiation into effector cells remain largely elusive. Methods To change this situation we established a transgenic knock-in mouse line with a floxed allele of DGCR8, an essential subunit of the nuclear miRNA processing complex. B cell-specific deletion of DGCR8 resulted in a complete maturation block at the pro-B stage, indicating that miRNA processing is essential for central B cell maturation. We have also established a culture system that allows us to delete a floxed DGCR8 gene in freshly isolated B cells using retrovirally transduced Cre. Using this system, we found that the miRNA pathway is required for mitogen-induced in vitro proliferation of mature B cells. Results We have also obtained genome-wide miRNA expression profiles of all major mouse B cell subsets, including long-lived plasma cells, and found a profound upregulation of one miRNA in plasma cells. Ectopic expression of this plasma cell signature-miRNA in primary mouse B cells accelerated the differentiation into antibody-secreting plasmablast, as indicated by upregulation of CD138 and enhanced IgM secretion. We also verified several targets of this miRNA, e.g., Bach2 and MiTF, that are part of the transcriptional circuit that controls germinal centre reactions and plasma cell differentiation. Conclusions These studies will provide new molecular insights into regulatory circuits that control the production of antibodies and could potentially lead to new avenues for diagnosing or treating diseases associated with aberrant plasma cell development, e.g., primary antibody deficiencies, plasma cell malignancies, and autoimmune disorders.

Two spectrophotometric methods for cholinesterase determination which utilize thiocholine chloride esters as substrates were developed. The disappearance of the C-S bond at 229 mμ during the enzymic hydrolysis of the thiocholine chloride esters, permitted the measurement of the rate and activity of the enzyme samples. The reduction of indophenol by the thiocholine liberated during the enzymatic hydrolysis made the evaluation of enzyme activity feasible in the visible spectrum at 600 mμ. These methods are simple, rapid, and accurate in analyzing cholinesterase activity or inhibition. They also serve to measure the cholinesterase activity of as little as 0.01–0.001 ml samples of blood, serum, or tissue extracts.