Bleeding spectrum in children with moderate or severe von Willebrand disease: Relevance of pediatric‐specific bleeding

The bleeding phenotype of children with von Willebrand disease (VWD) needs to be characterized in detail to facilitate diagnosis during childhood and aid in the planning and assessment of treatment strategies. The objective was to evaluate the occurrence, type, and severity of bleeding in a large cohort of children with moderate and severe VWD. We included 113 children (aged 0–16 years) with Type 1 (n = 60), 2 (n = 44), and 3 (n = 9) VWD with von Willebrand factor (VWF) antigen and/or VWF ristocetin cofactor levels ≤ 30 U/dL from a nation‐wide cross‐sectional study (“Willebrand in the Netherlands” study). Bleeding severity and frequency were determined using the International Society on Thrombosis and Hemostasis‐Bleeding Assessment Tool (ISTH‐BAT) with supplementary pediatric‐specific bleeding symptoms (umbilical stump bleeding, cephalohematoma, cheek hematoma, conjunctival bleeding, postcircumcision and postvenipuncture bleeding). We found that all 26 postmenarche girls experienced menorrhagia. Other common bleedings were cutaneous (81%), oropharyngeal (64%), prolonged bleeding from minor wounds (58%), and epistaxis (56%). Pediatric‐specific bleeding symptoms were present in 44% of patients. ISTH‐BAT bleeding score was higher in index cases than in affected family members (median, 12.0 vs. 6.5, P < 0.001), higher in Type 3 VWD than in Type 2 or 1 (17.0 vs. 10.5 or 6.5, P < 0.001) and higher in children with severe (<10 U/dL) than moderate VWD (10–30 U/dL) (11.0 vs. 7.0, P < 0.001). Frequency of any bleeding, epistaxis, and oral cavity was higher in types 2 and 3 than in Type 1 VWD and was associated with VWF levels. We conclude that pediatric‐specific bleeding symptoms occurred in a large proportion of children with moderate or severe VWD and should be included when evaluating children for VWD. Am. J. Hematol. 90:1142–1148, 2015. © 2015 Wiley Periodicals, Inc.     

Adverse childhood experiences and complex health concerns among child welfare-involved children

Adverse childhood experiences (ACEs) contribute to public health concerns, as they have been linked to chronic diseases in adulthood. From the seminal ACEs study in the mid-1990s (Felitti et al., 1998) to today, the Centers for Disease Control (2016) reports well over 50 studies that link ACEs to adult health conditions such as cancer, heart disease, lung disease, and mental illness. This preponderance of evidence has prompted widespread attention to the possibility that preventing and successfully treating ACE-associated traumatic stress would greatly reduce our country’s incidence of chronic disease and the associated public health burden and cost (Danese et al., 2009). To illustrate, one study suggests that child abuse and neglect alone costs the United States $124 billion annually, with individual lifetime costs that are higher than or equal to the economic burden of diabetes and stroke (Fang, Brown, Florence, & Mercy, 2012). That child maltreatment is just one category of 10 total ACEs suggests that the total financial impact of ACEs in the United States is likely much greater and that some populations, such as children involved in child welfare, may be disproportionately affected by the negative effects of adverse childhood experiences.     

Distinct regulation and impact of type 1 T-cell immunity against HPV16 L1, E2 and E6 antigens during HPV16-induced cervical infection and neoplasia

Cervical cancer is the possible outcome of a genital infection with high-risk human papillomavirus type 16 (HPV16) and is preceded by a phase of persistent HPV infection during which the host immune system fails to eliminate the virus. Our previous work showed that failure is reflected by the absence of type 1 T-cell immunity against HPV16 early antigens E2 and E6 in patients with HPV16+ cervical lesions. We now show that a majority of both patients with cervical lesions and healthy subjects display HPV16 L1 peptide-specific type 1 T-cell responses with similar magnitude. The T-cell response in patients was directed at a broad range of peptides within L1, suggesting that during persistent or repeated exposure to HPV16 L1, the immune system maximizes its efforts to counter the viral challenge. Unlike the type 1 T-cell responses against HPV16 early antigens E2 and E6, type 1 T-cell immunity against L1 does not correlate with health or disease. This argues that T-cell responses against early and late HPV16 antigens essentially differ in the manner in which they are induced and regulated, as well as in their impact on the subsequent stages of HPV16-induced cervical disease.     

Detection of human papillomavirus type 18 E6 and E7-specific CD4+ T-helper 1 immunity in relation to health versus disease

The most common high-risk human papillomavirus types, HPV16 and 18, differ markedly with respect to their interaction with the host. Clearance of HPV18 infections generally takes longer and HPV18-positive cancers have a poorer prognosis. We therefore evaluated Th1-type immunity against the E6 and E7 oncoproteins of HPV18 in healthy subjects and in patients with HPV18-positive genital cancer, and compared the results to our previously obtained data for HPV16. Approximately 20% of the healthy individuals displayed immunity against HPV18 E6. In contrast, none of the patients showed such responses, despite the presence of HPV18-positive lesions. Several of the patients did respond to HPV18 E7, whereas this immunity is rarely found in healthy subjects. This pattern of immune reactivity is essentially similar to that previously found for HPV16. It is unlikely that this similarity is the result of immunological cross-reactivity between the E6 and E7 antigens of HPV types 16 and 18. Our data confirm the relation between failure of E6-specific Th1 immunity and high-risk HPV-induced cervical neoplasia and argue that parameters other than these determine the differences in pathological impact between HPV types 16 and 18.     

Effective immunotherapy of cancer in MUC1-transgenic mice using clonal cytotoxic T lymphocytes directed against an immunodominant MUC1 epitope

The tumor-associated autoantigen MUCI is intensively studied as a potential target for antigen-specific immunotherapy of cancer. Previous reports concerning experiments in preclinical murine tumor models have provided evidence supporting the feasibility of this approach. However, such studies have not been performed with clonal cytotoxic T lymphocyte populations displaying a highly defined MUC1 specificity. The authors demonstrate that the immunodominant MUC1-specific cytotoxic T lymphocyte response in C57BL/6 mice is directed against an H-2Kb-restricted epitope, MUC1(19-27), which is derived from the N-terminal signal sequence of the MUC1 protein. Processing of this epitope was independent of transporter of antigen presentation and proteasome function. Importantly, successful immunotherapy of MUC1-overexpressing tumors in MUC1-transgenic mice was not accompanied by damage to normal somatic MUC1-positive tissues, even when this involved the infusion of large numbers of clonal cytotoxic T lymphocyte that recognized the immunodominant MUC1 epitope. Although the risk for autoimmune pathology is limited, data indicate that immune tolerance in MUC1-positive subjects restricts the breadth of the MUC1-specific cytotoxic T lymphocyte repertoire that is available for recruitment to immunotherapeutic antitumor responses.     

Frequent display of human papillomavirus type 16 E6-specific memory t-Helper cells in the healthy population as witness of previous viral encounter

Genital human papillomavirus (HPV) infection is common and the majority of infected individuals successfully deal with this virus. Clearance of HPV is presumably mediated by T cells but HPV-16-specific T-cell memory was usually detected in patients with progressive disease and not in healthy subjects, suggesting that HPV-immunity comes too late. We now show the presence of HPV-16 E6-specific memory T-helper (Th) responses in a major fraction (12 of 20) of healthy individuals by application of the IFN-gamma-ELISPOT assay. Although nearly all E6-peptides were recognized, the majority of the responders targeted peptide sequences of the COOH-terminal half (E6(81-158)) of HPV-16 E6. In a direct comparison, the presence of HPV-16 E6-specific T cells coincided with HPV-16 E2-specific T-cell reactivity in healthy individuals, whereas hardly any HPV-16 E7-specific Th immunity was found. This indicates that the induction of T-cell reactivity against HPV-16 E7 is suboptimal during infection when compared with that against HPV-16 E2 and HPV-16 E6. In conclusion, the presence of HPV-16 E6-specific Th memory in the healthy population demonstrates that HPV infection leads to T-cell immunity against immediate early proteins expressed during infection. Because this HPV-16 E6-specific T-cell immunity was frequently detected in healthy subjects, our data suggest that the observed IFN-gamma-producing proliferating T cells circulating in the peripheral blood play a role in protection against persistent HPV infection and associated development of malignancies.     

Vaccinia-expressed human papillomavirus 16 and 18 e6 and e7 as a therapeutic vaccination for vulval and vaginal intraepithelial neoplasia.

PURPOSE: Anogenital intraepithelial neoplasia is a chronic disorder associated with infection by high-risk human papillomavirus (HPV) types. It is frequently multifocal and recurrence after conventional treatment is high. Boosting HPV-specific cell-mediated immune responses may reduce progression to carcinoma and could lead to disease clearance. We have tested the safety, immunogenicity, and efficacy of a recombinant vaccinia candidate vaccine (TA-HPV) in women with anogenital intraepithelial neoplasia. EXPERIMENTAL DESIGN: Twelve women, aged 42-54 years with high-grade HPV-positive vulval or vaginal intraepithelial neoplasia of up to 15 years duration, completed a Phase II study of TA-HPV, a live recombinant vaccinia virus, expressing modified versions of the E6 and E7 open reading frames from HPV-16 and HPV-18. RESULTS: The vaccine was well tolerated. Five of 12 (42%) patients showed at least a 50% reduction in total lesion diameter over 24 weeks with 1 patient showing complete regression of her lesion. Overall, 83% of women showed some improvement with an average decrease in lesion size of 40%. All cases showed an increased IgG titer and T-cell response to the vaccinia virus. An IFN-gamma enzyme-linked immunospot assay using pooled 22-mer peptides spanning HPV-16 E6 and E7 showed an increased specific T-cell response after vaccination in 6 of the 10 cases available for testing. There was no increase in specific cytotoxic response to selected individual HLA class I-restricted HPV-16 E6/7 peptides. CONCLUSIONS: The results suggest that the vaccine may have an effect on HPV-positive vulval intraepithelial neoplasia/vaginal intraepithelial neoplasia and that additional studies are warranted to develop an effective therapeutic vaccine.     

Cell‐derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII–dependent mechanism

Objective

To determine the cellular origin of synovial microparticles, their procoagulant properties, and their relationship to local hypercoagulation.

Methods

Microparticles in synovial fluid and plasma from patients with rheumatoid arthritis (RA; n = 10) and patients with other forms of arthritis (non‐RA; n = 10) and in plasma from healthy subjects (n = 20) were isolated by centrifugation. Microparticles were identified by flow cytometry. The ability of microparticles to support coagulation was determined in normal plasma. Concentrations of prothrombin fragment F₁₊₂ (by enzyme‐linked immunosorbent assay [ELISA]) and thrombin–antithrombin (TAT) complexes (by ELISA) were determined as estimates of the coagulation activation status in vivo.

Results

Plasma from patients and healthy controls contained comparable numbers of microparticles, which originated from platelets and erythrocytes. Synovial microparticles from RA patients and non‐RA patients originated mainly from monocytes and granulocytes; few originated from platelets and erythrocytes. Synovial microparticles bound less annexin V (which binds to negatively charged phospholipids) than did plasma microparticles, exposed tissue factor, and supported thrombin generation via factor VII. F₁₊₂ (median 66 nM) and TAT complex (median 710 μg/liter) concentrations were elevated in synovial fluid compared with plasma from the patients (1.6 nM and 7.0 μg/liter, respectively) as well as the controls (1.0 nM and 2.9 μg/liter, respectively).

Conclusion

Synovial fluid contains high numbers of microparticles derived from leukocytes that are strongly coagulant via the factor VII–dependent pathway. We propose that these microparticles contribute to the local hypercoagulation and fibrin deposition in inflamed joints of patients with RA and other arthritic disorders.

     

Prolongation of skin graft survival by modulation of the alloimmune response with alternatively activated dendritic cells

BACKGROUND: Activation of immature dendritic cells (DC) in the presence of the glucocorticoid hormone dexamethasone (DEX) results in alternatively matured DC that present antigen in the absence of a proper co-stimulatory context. This maturation process is irreversible, making these cells an attractive potential tool for the induction of antigen-specific T-cell tolerance in vivo. The authors explored the possibility of using these DC for the induction of transplantation tolerance in a fully allogeneic setting in mice. METHODS: Immature dendritic cells (D1, an immature splenic DC line derived from B6 mice) were pretreated with DEX for 24 hr, after which lipopolysaccharide or nothing was added to the culture for another 48 hr. These cells were analyzed for their in vitro and in vivo stimulating or tolerizing capacities. RESULTS: In line with their phenotype, including decreased interleukin (IL)-12 production, in vitro co-culture of alternatively matured D1 (B6 origin; H-2b) with completely allogeneic T cells of BALB/c origin led to a significant decrease in the alloreactive T-cell response. A single injection of 1 x 10(6) alternatively matured H-2b DC into BALB/c mice induced a different alloimmune response compared with mature DC. The responding T cells showed a lower proliferation rate and a lower interferon-gamma production, whereas a significantly higher proportion of the cells produced IL-10 as measured ex vivo by enzyme-linked immunospot assay. Furthermore, injection with alternatively matured DC, followed by transplantation of fully mismatched skin grafts (C57BL/6), led to a significantly prolonged survival compared with that of mature DC-pretreated mice or untreated mice. The immunomodulatory effect was antigen specific, as third-party reactive alloresponses were not affected. CONCLUSIONS: The authors' data constitute the first direct demonstration that DC alternatively matured in the presence of glucocorticoid hormones can be exploited for the specific suppression of the alloreactive Th1 response, resulting in a delayed skin graft rejection in a complete major histocompatibility complex-incompatible strain combination.