Z.N. Berneman

Immunosuppression induced by immature dendritic cells is mediated by TGF‐β/IL‐10 double‐positive CD4+ regulatory T cells

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (iDC) is caused by the absence of inflammatory signals in steady‐state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)‐β and interleukin (IL)‐10 double‐positive CD4+ T cells within 1 week of autologous DC/T cell co‐cultures. In iDC/T cell cultures, where antigen‐specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF‐β and IL‐10 secreted by CD4+CD25−FOXP3− T cells. In addition, the suppressive capacity of CD4+ T cells conditioned by iDC was transferable to already primed antigen‐specific CD8+ T cell cultures. In contrast, addition of CD4+ T cells conditioned by mDC to primed antigen‐specific CD8+ T cells resulted in enhanced CD8+ T cell responses, notwithstanding the presence of TGF‐β+/IL‐10+ T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine‐secreting regulatory T cells. We show that iDC‐conditioned CD4+ T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4+ T cells. Furthermore, TGF‐β+/IL‐10+ T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.

Laboratory Diagnosis and Molecular Classification of von Willebrand Disease

A complete set of laboratory investigations, including bleeding time, PFA-100 closure times, factor VIII (FVIII) coagulant activity (FVIII:C), von Willebrand factor (VWF) ristocetin cofactor (VWF:RCo), collagen binding (VWF:CB), antigen (VWF:Ag) and propeptide (VWFpp), ristocetin-induced platelet aggregation (RIPA), multimeric analysis of VWF and the response of FVIII:C and VWF parameters to desmopressin (DDAVP), is necessary to fully diagnose all variants of von Willebrand disease (VWD) and to discriminate between type 1 and type 2 and between severe VWD type 1 and type 3. The response to DDAVP of VWF parameters is normal in pseudo VWD (mild VWF deficiency due to blood group O), in mild VWD type 1 and in carriers of recessive severe VWD type 1 and 3. The response to DDAVP is rather good but restricted followed by increased clearance in dominant type 1/2E, good but transient in mild type 2A group II, good for VWF:CB, with only poor response for VWF:RCo in 2M and 2U, poor in 2A group I, 2B, 2C and 2D, and very poor or non-responsive in severe recessive VWD type 1 and 3. Homozygosity or double heterozygosity for nonsense (null) mutations in the VWF gene result in recessive VWD type 3. The combination of a nonsense and missense mutation or of two missense mutations (homozygous or double heterozygous) may cause recessive severe VWD type 1. Recessive VWD type 2A subtype IIC (2C) is caused by homozygous or double heterozygous gene defects in the D1–D2 domain. Homozygosity or double heterozygosity for a FVIII binding defect of the VWF is the cause of recessive VWD type 2N (Normandy) characterized by low FVIII:C, mild or moderate VWF deficiency and normal VWF multimers. Dominant VWD type 1/2E is a mixed quantitative and qualitative multimerization defect caused by a heterozygous cysteine mutation in the D3 domain resulting in abnormal multimerization with a secretion and clearance defect of VWF not due to increased proteolysis. Dominant VWD type 1 Vicenza is a qualitative defect with normal secretion but rapid clearance with equally low levels of FVIII:C, VWF:Ag, VWF:RCo, VWF:CB and the presence of unusually large VWF multimers in plasma due to a specific mutation (R1205H) in the D3 domain. Dominant VWD type 2M and 2U are caused by loss-of-function mutations in the A1 domain resulting in quantitative/qualitative deficiencies with a selectively decreased platelet-dependent function with decreased VWF:RCo but normal VWF:CB, a relative decrease in large VWF multimers and the presence but relative loss of large VWF multimers. VWD type 2A and 2B show loss of large VWF multimers due to increased proteolysis. Dominant type 2A is caused by heterozygous missense mutations in the A2 domain. VWD type 2B is due to gain-of-function mutations in the A1 domain and differs from 2A by a normal VWF multimeric pattern in platelets and increased RIPA. DDAVP response curves and VWFpp/Ag ratios contribute to the diagnostic differentiation of VWD type 1 and 2. Rapid clearance of VWF after DDAVP with increased VWFpp/Ag ratios >10 appears to be diagnostic for VWD Vicenza. VWD type 1/2E due to the mutations in the D3 domain uniformly show increased VWFpp/Ag ratios ranging from 3.2 to 4.69 indicating clearance of the VWF/FVIII complex. Normal VWFpp/Ag ratios in mild VWD type 1 with mutations in the D1-D2 and the D4-B-C domains reflect a synthesis/secretion defect.

Long-term epidemiology of bacterial susceptibility profiles in adults suffering from febrile neutropenia with hematologic malignancy after antibiotic change

Objective: The aim of this study was to investigate the epidemiology and antibiotic susceptibility profiles of isolated bacterial organisms in relation to empiric treatment of neutropenic fever over a 15-year period. Methods: All patients with or at risk for febrile neutropenia and treated in the hematology ward of the Antwerp University Hospital during 1994–2008 were prospectively included. Skin, blood, and urine cultures were taken. Oral quinolone prophylaxis was started in patients with neutropenia without fever. Empiric starting therapy consisted of amikacin in combination with cefepime. Results: A total of 3624 bacteria were isolated. The most common pathogens were coagulase-negative Staphylococci (46%), followed by Escherichia coli (25%), Enterobacteriaceae (15.6%), Staphylococcus aureus (7.2%), and Pseudomonas aeruginosa (3.8%). The balance between Gram-positive and Gram-negative bacteria remained stable, with a majority of Gram-positive bacteria. A shift from oxacillin-sensitive to oxacillin-resistant coagulase-negative Staphylococci was observed. Regarding susceptibility patterns, no vancomycin resistance was detected in coagulase-negative Staphylococci or in S. aureus. The E. coli susceptibility rates remained stable. However, 66% of bloodstream infections were ciprofloxacin-resistant. A reduced susceptibility of P. aeruginosa strains to meropenem was noticed. Conclusions: Improvement in antibiotic susceptibility of inducible Enterobacteriaceae following a switch of empiric antibiotic therapy was maintained 15 years after starting the latter treatment. Further improvement in antibiotic susceptibility of these bacteria to ceftazidime was observed, but continuous vigilance is warranted.

Folate and Vitamin B12 deficiency presenting as pancytopenia in pregnancy: a case report and review of the literature

We present a case of extreme pancytopenia in a 27-year-old pregnant woman. The initial picture was compatible with a severe hematological problem in the category of aplastic anemia, paroxysmal nocturnal hemoglobinuria or even acute leukemia. The further biochemical investigations revealed, however, a folate deficiency. Nowadays this is a very rare cause of pancytopenia. Next to this she also had a Vitamin B(12) deficiency due to intrinsic factor failure. The recent literature is discussed.

Characterization of Interleukin-15-Transpresenting Dendritic Cells for Clinical Use

Personalized dendritic cell- (DC-) based vaccination has proven to be safe and effective as second-line therapy against various cancer types. In terms of overall survival, there is still room for improvement of DC-based therapies, including the development of more immunostimulatory DC vaccines. In this context, we redesigned our currently clinically used DC vaccine generation protocol to enable transpresentation of interleukin- (IL-) 15 to IL-15Rβγ-expressing cells aiming at boosting the antitumor immune response. In this study, we demonstrate that upon electroporation with both IL-15 and IL-15Rα-encoding messenger RNA, mature DC become highly positive for surface IL-15, without influencing the expression of prototypic mature DC markers and with preservation of their cytokine-producing capacity and their migratory profile. Functionally, we show that IL-15-transpresenting DC are equal if not better inducers of T-cell proliferation and are superior in tumor antigen-specific T-cell activation compared with DC without IL-15 conditioning. In view of the clinical use of DC vaccines, we evidence with a time- and cost-effective manner that clinical grade DC can be safely engineered to transpresent IL-15, hereby gaining the ability to transfer the immune-stimulating IL-15 signal towards antitumor immune effector cells.