Alexandra Rizzitelli

The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement.

A novel dendritic cell (DC)-restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8(+) conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.

Dendritic cells in the thymus contribute to T-regulatory cell induction

Central tolerance is established through negative selection of self-reactive thymocytes and the induction of T-regulatory cells (TRs). The role of thymic dendritic cells (TDCs) in these processes has not been clearly determined. In this study, we demonstrate that in vivo, TDCs not only play a role in negative selection but in the induction of TRs. TDCs include two conventional dendritic cell (DC) subtypes, CD8loSirpαhi/+ (CD8loSirpα+) and CD8hiSirpαlo/− (CD8loSirpα−), which have different origins. We found that the CD8hiSirpα+ DCs represent a conventional DC subset that originates from the blood and migrates into the thymus. Moreover, we show that the CD8loSirpα+ DCs demonstrate a superior capacity to induce TRs in vitro. Finally, using a thymic transplantation system, we demonstrate that the DCs in the periphery can migrate into the thymus, where they efficiently induce TR generation and negative selection.

Signal Regulatory Protein Molecules Are Differentially Expressed by CD8− Dendritic Cells

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein β (Sirpβ1 and Sirpβ4) molecules were identified as differentially expressed in CD8− cDC. Genomic sequence analysis revealed a third Sirpβ member localized in the same gene cluster. These Sirpβ genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpβ1 and β2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpβ genes were expressed by CD8− cDC, but not by CD8+ cDC or plasmacytoid pre-DC. The related Sirpα gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpα and Sirpβ1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpβ1. Cross-linking of Sirpβ1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpβ1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8− cDC.

Incidence and overall survival of malignant ameloblastoma.

Background Malignant ameloblastoma, comprising metastasizing ameloblastoma and ameloblastic carcinoma, represents 1.6–2.2% of all odontogenic tumors. Due to its rare nature, malignant ameloblastoma has only been reported in the literature in small case series or case reports. Using the Surveillance, Epidemiology and End-Results (SEER) database, we have performed a population-based study to determine the incidence rate and the absolute survival of malignant ameloblastoma. Method Using the International Classification of Diseases for Oncology (ICD-O) codes 9310/3 and 9270/3, data from the SEER database were used to calculate the incidence rate and absolute survival rate of population with malignant ameloblastoma. Results The overall incidence rate of malignant ameloblastoma was 1.79 per 10 million person/year. The incidence rate was higher in males than females and also higher in black versus white population. The median overall survival was 17.6 years from the time of diagnosis and increasing age was associated with a statistically significant poorer survival. Conclusions To our best knowledge, we report the largest population-based series of malignant ameloblastoma. The incidence rate was 1.79 per 10 million person/year and the overall survival was 17.6 years.

Switching from a restricted to an effective CD4 T cell response by activating CD8+ murine dendritic cells with a Toll‐like receptor 9 ligand

Freshly isolated quiescent splenic dendritic cell (DC) subtypes differ in their capacity to activate naive CD4 Tu2004cells in culture. The CD8+ DC showed a reduced capacity to stimulate Tu2004cell proliferation compared to either of the CD8– DC subsets, regardless of antigen and DC dose. In contrast to CD8– DC, the quiescent CD8+ DC did not induce IFN‐γ production from CD4 Tu2004cells. The difference between the DC subtypes appeared to be at the level of initial surface molecule interactions, but could not be attributed to differences in expression of MHC classu2004II or B7 family molecules, or to the expression of Fas ligand on DC. However, when activated by inclusion of the Toll‐like receptoru20049 ligand CpG in culture, CD8+ DC became potent stimulators of both CD4 Tu2004cell proliferation and IFN‐γ production. In contrast, similar activation of CD8– DC produced a more modest increase in capacity to stimulate CD4 Tu2004cell proliferation and no increase in capacity to stimulate IFN‐γ production. The difference between a quiescent and an activated state is therefore more extreme for CD8+ than for CD8– DC. The especially tight regulation of the activity of CD8+ DC may be essential for the maintenance of self tolerance.

Serpinb9 (Spi6)-deficient mice are impaired in dendritic cell-mediated antigen cross-presentation

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (GrB) that protects activated cytotoxic lymphocytes from apoptosis. We show here that the CD8+ subset of splenic dendritic cells (DC), specialized in major histocompatibility complex class I (MHC I) presentation of exogenous antigens (cross‐presentation), produce high levels of Sb9. Mice deficient in Sb9 are unable to generate a cytotoxic T‐cell response against cell‐associated antigen by cross‐presentation, but maintain normal MHC‐II presentation to helper T cells. This impaired cross‐priming ability is autonomous to DC and is evident in animals deficient in both Sb9 and GrB, indicating that this role of Sb9 in DC is GrB‐independent. In Sb9‐deficient mice, CD8+ DC develop normally, survive as well as wild‐type DC after antigenic challenge, and exhibit unimpaired capacity to take up antigen. Although the core processing machinery is unaffected, Sb9‐deficient DC appear to process antigen faster. Our results point to a novel, GrB‐independent role for Sb9 in DC cross‐priming.

Regeneration of dendritic cells in aged mice

Age-related thymic involution causes a decreased output of thymocytes from the thymus, thereby resulting in impairment of T cell-mediated immunity. While alterations in the T cell and non-haematopoietic stromal compartments have been described, the effects of thymic involution on thymic dendritic cells (DC) are not clearly known. Thymic DC play an essential role in shaping T cell-mediated immune responses by deleting self-reactive thymocytes to establish central tolerance and by inducing regulatory T-cell (Treg) development. It is therefore important to assess the prevalence of and alterations to thymic DC with age, as this may impact on their function. We assessed the numbers and proportions of the three distinct subsets of thymic DC in ageing mice, and showed that these subsets are differentially regulated. This is expected as thymic DC subsets have different origins of development. We further assessed the responses of thymic DC in a regenerative environment, such as that induced by sex-steroid ablation (SSA), and clearly showed that, consistent with global thymus regrowth, all three DC populations increased in numbers and regained their relative proportions to thymocytes after an initial lag period. These findings are important for the clinical translation of thymic regenerative approaches, and indicate that SSA facilitates the maintenance of critical processes such as negative selection and Treg induction through promoting thymic DC regeneration.

A randomized ‘N-of-1’ single blinded clinical trial of barbed dermal sutures vs. smooth sutures in elective plastic surgery shows differences in scar appearance two-years post-operatively

BACKGROUNDnBarbed sutures have unidirectional circumferential shallow barbs, which distribute tension throughout the wound and close wound securely without the need to tie knots.nnnOBJECTIVESnWe compare two different methods of wound closure in elective plastic surgical cases: barbed 3/0xa0V-Loc™180 suture and smooth 3/0xa0Maxon™ sutures, both polyglyconate monofilament synthetic absorbable sutures. We assessed the aesthetic long-term results with a minimum two year follow up.nnnMETHODSnThis is a prospective, randomized controlled study with internal control. A single surgeon performed all cases. Patients who underwent elective operations that involved long wound closure were enrolled in the study. Each patient acted as their own internal control with half their wound being sutured with 3/0xa0V-Loc™180 barbed suture and the other half with smooth 3/0xa0Maxon™ deep dermal sutures and then a subcuticular skin closure. In both groups, the superficial fascial system was closed with 1 Vicryl interrupted sutures on both sides. Long-term cosmesis was evaluated using the modified Hollander cosmesis score by review of standardized postoperative photographs by 9 blinded plastic surgeons and specialist registrars.nnnRESULTSnThe study reports on 33 female patients. The time taken for wound closure was significantly reduced using the barbed suture (pxa0<xa00.001). There was no difference in the complication ratio in either group. Two-year aesthetic outcome was significantly superior when using the barbed suture (pxa0=xa00.0075).nnnCONCLUSIONnBarbed sutures closure of long wounds is faster and produces a better long-term aesthetic outcome than smooth sutures.

A surgical view on the treatment of Madelung's disease

Benign symmetrical lipomatosis (Madelungs disease) is a rare condition of unclear aetiology characterized by numerous, unencapsulated lipomatous deposits. The only effective treatment is by surgical intervention; however, there is no consensus in the optimal approach. We present the case of a patient who required staged, multi‐modality treatment to achieve disease quiescence. The case highlights the usefulness of magnetic resonance imaging as a tool for assessment as well as preoperative planning.

Serpinb9 is a marker of antigen cross-presenting dendritic cells

HighlightsSerpinb9 expression is highest in DCs capable of cross‐presentation.Serpinb9 expression defines two different populations of CD8+ DCs.High expression of Serpinb9 identifies CD8+ DCs capable of cross‐presentation. ABSTRACT Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (grB) that protects cytotoxic lymphocytes from grB‐mediated death. In addition, Sb9 is also expressed in accessory immune cells, including dendritic cells (DCs), although its role is debated. Recently, we have demonstrated that Sb9 plays a grB‐independent role in cross‐presentation of antigens by CD8+ DCs. Here, using a mouse line expressing green fluorescent protein knocked in under the control of the Sb9 promoter, we demonstrate that Sb9 expression is highest in those tissue‐resident and migratory DC subsets capable of cross‐presentation. Further, we show that CD8+ DCs can be divided into two subsets based on Sb9 expression, and that only the subset expressing higher levels of Sb9 is capable of cross‐presentation. These findings add support for role for Sb9 cross‐presentation, and indicate that high Sb9 expression is a novel marker of cross‐presentation capable DCs.