Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.

IntroductionDendritic cells (DCs) are the antigen-presenting cells (APCs) capable of inducing adaptive immune responses and tolerance. 1 DCs act as sentinels in peripheral tissues and as APCs in secondary lymphoid organs, filtering their environment and detecting environmental changes that modulate the balance between tolerance and immune response. Following infection and inflammation, DCs induce costimulatory molecules, secrete cytokines, and can change their migration properties. 2,3 Several DC subtypes with unique and overlapping functions have been described. Plasmacytoid DCs (pDCs) are the main interferon (IFN) type I-producing cells, whereas several subtypes of conventional DCs (cDCs) are widely distributed. 4,5 In skin, 2 DC populations, namely Langerhans cells (LCs) and dermal DCs, localize to the epidermis and dermis, respectively. Upon pathogen encounter, they migrate to the draining LN. 6 In skin and spleen, DCs are derived either from blood or from immediate local precursors. [7][8][9][10][11][12][13] DCs are dividing cells rather than terminally differentiated cells. [12][13][14][15] The importance of DC cycling on their homeostasis is currently debated, and DC proliferation has been recently proposed to extend the duration of antigen presentation. 15 The human histiocytic diseases are characterized by abnormal accumulation or proliferation of macrophages or DCs. 16 They are divided into LC or non-LC histiocytosis. In the former category, LC histiocytosis (LCH) is best described. LCH can range from a spontaneously regressing single organ disease to a life-threatening or disabling relapsing multisystem disease. 16 In the latter category, a genetic deficiency in cytotoxic activity, familial hemophagocytic lymphohistiocytosis (FHL), is the principal type implicating macrophages and lymphocytes. In contrast to LCH and FHL, lesions with proliferative DCs with or without cytologic atypia are less well classified and their prevalence remains controversial. 16 Unfortunately, a confusing terminology describing these cases of proliferating DCs persists in the literature as malignant histiocytosis, histiocytic sarcoma, and DC sarcoma.The diagnosis of histiocytic diseases relies on the morphologic, ultrastructural, and immunohistochemical histiocyte properties. The macrophage, DC, or LC lineage markers allow LC to be distinguished from non-LC histiocytosis. For example, diagnostic criteria for LCH include expression of Langerin or detection of Birbeck granules that are pathognomonic, as well as CD1a and S100, which are less specific. According to the International Lymphoma Study Group, histiocytic sarcoma, LC tumor, and LC sarcoma (LCS) are the terms used to separate cases based on the degree of cytologic atypia and on lineage markers. 17 For example, diagnosis of LCS requires the same markers as LCH and the additional presence of cellular and mitotic atypia.While LCS, LC, and DC tumors are clearly neoplastic, it is vigorously debated whether LCH is a reactive or a neoplastic The online version of this article ...

Objective To assess specific, direct, and indirect prenatal ultrasound features in cases of fetal midgut volvulus. Methods Retrospective case series of neonatal volvulus, based on postnatal and prenatal imaging findings that occurred from 2006–2017. Prenatal and postnatal signs including the specific “whirlpool sign” were computed. Postnatal volvulus was confirmed by pathology examination after surgery or neonatal autopsy. Results Thirteen cases of midgut volvulus were identified. Though not a specific sign, a decrease in active fetal movements was reported in eight patients (61.5%). The prenatal whirlpool sign was directly seen in 10 cases, while an indirect but suggestive sign, a fluid‐filled level within the dilated loops, was present in five cases. No intestinal malrotation was observed. Pregnancy outcomes were two terminations of pregnancy, both associated with cystic fibrosis, one early neonatal death, three prenatal spontaneous regressions, and seven favorable outcomes after neonatal surgery with resection of midgut atresia. Conclusions Identification of the whirlpool sign or of a fluid‐filled level within the dilated loops improves the accuracy of ultrasound findings for suspected volvulus. In the absence of total volvulus (in cases of intestinal malrotation) or association with cystic fibrosis, the prognosis appears good.

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.

During thymus development, immature T cells become committed to two distinct lineages based upon expression of αβ or γδ TCR. In the αβ lineage, developing thymocytes progressively extinguish transcription of the TCRγ genes by a poorly understood process known as γ silencing. We show that αβ lineage thymocytes in mice lacking a functional pre-TCR undergo limited proliferation and fail to silence TCRγ genes during development. Stimulation of pre-TCR-deficient immature thymocytes with anti-CD3 Abs does not directly down-regulate TCRγ transcription but restores TCRγ silencing following proliferation. Collectively our data reveal an important role for pre-TCR induced proliferation in activating the TCRγ silencer in αβ lineage thymocytes, a process that may reinforce αβ or γδ lineage commitment.

The ATV is an easy, simple, and accessible 2D method to visualize the FHP with no additional time.

Melan-A specific CD8+ T cells are thought to play an important role against the development of melanoma. Their in vivo expansion is often observed with advanced disease. In recent years, low levels of Melan-A reactive CD8+ T cells have also been found in HLA-A2 healthy donors, but these cells harbor naive characteristics and are thought to be mostly cross-reactive for the Melan-A antigen. Here, we report on a large population of CD8+ T cells reactive for the Melan-A antigen, identified in one donor with no evidence of melanoma. Interestingly, this population is oligoclonal and displays a clear memory phenotype. However, a detailed study of these cells indicated that they are unlikely to be directly specific for melanoma, so that their in vivo expansion may have been driven by an exogenous antigen. Screening of a Melan-A cross-reactive peptide library suggested that these cells may be specific for an epitope derived from a Mycobacterium protein, which would provide a further example of CD8+ T cell cross-reactivity between a pathogen antigen and a tumor antigen. Finally, we discuss potential perspectives regarding the role of such cells in heterologous immunity, by influencing the balance between protective immunity and pathology, e.g. in the case of melanoma development.

What's already known about this topic? Waardenburg syndrome is a form of deafness associated with pigmentation abnormalities, two features that cannot been diagnosed in a fetus. Musculoskeletal abnormalities of the upper limbs are associated in Waardenburg syndrome type III (WS3). What does this study add? We document two cases of WS3 diagnosed at first trimester of pregnancy, because of a homozygous mutation in PAX3. Ultrasound examinations revealed increased nuchal translucency, lack of active movements, bilateral club hands and club feet, and neural abnormalities.