Prevalence of Human Papillomavirus in 5,072 Consecutive Cervical SurePath Samples Evaluated with the Roche Cobas HPV Real-Time PCR Assay

New commercially available Human Papillomavirus (HPV) assays need to be evaluated in a variety of cervical screening settings. Cobas HPV Test (cobas) is a real-time PCR-based assay allowing for separate detection of HPV genotypes 16 and 18 and a bulk of 12 other high-risk genotypes. The aim of the present study, Horizon, was to assess the prevalence of high-risk HPV infections in an area with a high background risk of cervical cancer, where women aged 23–65 years are targeted for cervical screening. We collected 6,258 consecutive cervical samples from the largest cervical screening laboratory in Denmark serving the whole of Copenhagen. All samples were stored in SurePath media. In total, 5,072 samples were tested with cobas, Hybrid Capture 2 High Risk HPV DNA test (HC2) and liquid-based cytology. Of these, 27% tested positive on cobas. This proportion decreased by age, being 43% in women aged 23–29 years and 10% in women aged 60–65 years. HC2 assay was positive in 20% of samples, and cytology was abnormal (≥ atypical squamous cells of undetermined significance) for 7% samples. When only samples without recent abnormalities were taken into account, 24% tested positive on cobas, 19% on HC2, and 5% had abnormal cytology. The proportion of positive cobas samples was higher than in the ATHENA trial. The age-standardized cobas positivity vs. cytology abnormality was 3.9 in our study and 1.7 in ATHENA. If in Copenhagen the presently used cytology would be replaced by cobas in women above age 30 years, an extra 11% of women would based on historical data be expected to have a positive cobas test without an underlying cervical intraepithelial lesion grade 3 or worse. Countries with a high prevalence of HPV infections should therefore proceed to primary HPV-based cervical screening with caution.     

Secukinumab,a novel anti–IL-17A antibody,shows low immunogenicity potential in human in vitro assays comparable to other marketed biotherapeutics with low clinical immunogenicity

Secukinumab is a human monoclonal antibody that selectively targets interleukin-17A and has been demonstrated to be highly efficacious in the treatment of moderate to severe plaque psoriasis, starting at early time points, with a sustained effect and a favorable safety profile. Biotherapeutics—including monoclonal antibodies (mAbs)—can be immunogenic, leading to formation of anti-drug antibodies (ADAs) that can result in unwanted effects, including hypersensitivity reactions or compromised therapeutic efficacy. To gain insight into possible explanations for the clinically observed low immunogenicity of secukinumab, we evaluated its immunogenicity potential by applying 2 different in vitro assays: T-cell activation and major histocompatibility complex–associated peptide proteomics (MAPPs). For both assays, monocyte-derived dendritic cells (DCs) from healthy donors were exposed in vitro to biotherapeutic proteins. DCs naturally process proteins and present the derived peptides in the context of human leukocyte antigen (HLA)-class II. HLA-DR–associated biotherapeutic-derived peptides, representing potential T–cell epitopes, were identified in the MAPPs assay. In the T-cell assay, autologous CD4⁺ T cells were co-cultured with secukinumab-exposed DCs and T-cell activation was measured by proliferation and interleukin-2 secretion. In the MAPPs analysis and T-cell activation assays, secukinumab consistently showed relatively low numbers of potential T-cell epitopes and low T-cell response rates, respectively, comparable to other biotherapeutics with known low clinical immunogenicity. In contrast, biotherapeutics with elevated clinical immunogenicity rates showed increased numbers of potential T-cell epitopes and increased T-cell response rates in T-cell activation assays, indicating an approximate correlation between in vitro assay results and clinical immunogenicity incidence.     

Expression of the RAI gene is conducive to apoptosis: studies of induction and interference

The RAI gene is also known as iASPP and PPP1R13L. Recent investigations have shown that the region encompassing RAI is important for the development of cancer in young and middle-aged persons. It has been speculated that the RAI product induces apoptosis by blocking NF-kappaB or inhibits apoptosis by blocking p53. Either way the gene could influence the survival of precancerous lesions. Here we report that the expression of RAI mRNA was increased in non-transformed lymphocytes and fibroblasts induced to undergo apoptosis by various means, such as treatment with etoposide, calcium ions, or interleukin-2 and/or serum deprivation. Treatment with etoposide increased the content of RAI protein, too, and caused it to translocate to the nucleus. Inhibition of RAI expression in lymphocytes and fibroblasts with siRNA reduced apoptosis, but treatment with the NF-kappaB-inhibiting substance sulfasalazine relieved this dependence. In the transformed cell line HEK-293 the association between RAI induction and apoptosis seemed broken. Thus, we hypothesize that RAI induction is necessary but not sufficient for apoptosis induction in non-transformed cells. Our results could be explained by a NF-kappaB mediated mechanism.     

Rapid divergence in the course of Drosophila evolution reveals structural important domains of the Notch antagonist Hairless

 Hairless is a member of the Notch signalling pathway, where it acts as antagonist by binding to Suppressor of Hairless [Su(H)], thereby inhibiting Notch target gene activation. The pathway and its members are highly conserved in metazoans from worms to humans. However, a Hairless orthologue from another species has not yet been identified. The identification of Hairless in largely diverged species by cross-hybridization has failed so far probably due to a low degree of conservation. Therefore, we turned to D. hydei where a Hairless mutation has been described before. The D. hydei Hairless orthologue is reasonably well conserved with regard to gene structure and expression. The prospective Hairless protein orthologues share several highly conserved regions which are separated by quite diverged stretches. As to be expected, the largest region of high conservation corresponds to the Su(H) binding domain. This region is also functionally conserved, since this D. hydei protein domain binds very strongly to the D. melanogaster Su(H) protein. The other conserved regions support our earlier structure-function analysis since they nicely correspond to previously defined, functionally important protein domains. Most notably, the very C-terminal domain which is very sensitive to structural alterations, is nearly identical between the two species. In summary, this evolutionary study improves the knowledge on functionally significant domains of the Hairless protein, and may be helpful for the future identification of homologues in other animals, especially in vertebrates. Received: 26 August 1998 / Accepted: 9 November 1998     

Biotin deficiency decreases life span and fertility but increases stress resistance in Drosophila melanogaster

Biotin deficiency is associated with fetal malformations and activation of cell survival pathways in mammals. In this study we determined whether biotin status affects life span, stress resistance, and fertility in the fruit fly Drosophila melanogaster. Male and female flies of the Canton-S strain had free access to diets containing 6.0 (control), 4.8, 2.5, or 0 pmol biotin/100 mg. Biotin concentrations in diets correlated with activities of biotin-dependent propionyl-CoA carboxylase and biotin concentrations in fly homogenates, but not with biotinylation of histones (DNA-binding proteins). Propionyl-CoA carboxylase activities and biotin concentrations were lower in males than in females fed diets low in biotin. The life span of biotin-deficient males and females was up to 30% shorter compared to biotin-sufficient controls. Exposure to oxidative stress reversed the effects of biotin status on survival in male flies: survival times increased by 40% in biotin-deficient males compared to biotin-sufficient controls. Biotin status did not affect survival of females exposed to oxidative stress. Exposure of flies to cold, heat, and oxidative stress was associated with mobilization of biotin from yet unknown sources. Biotin deficiency decreased fertility of flies. When biotin-deficient males and females were mated, the hatching rate (larvae hatched per egg) decreased by about 28% compared to biotin-sufficient controls. These findings are consistent with the hypothesis that biotin affects life span, stress resistance, and fertility in fruit flies.     

Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.     

Association with membrane protrusions makes ErbB2 an internalization-resistant receptor

In contrast to the epidermal growth factor (EGF) receptor, ErbB2 is known to remain at the plasma membrane after ligand binding and dimerization. However, why ErbB2 is not efficiently down-regulated has remained elusive. Basically, two possibilities exist: ErbB2 is internalization resistant or it is efficiently recycled after internalization. By a combination of confocal microscopy, immunogold labeling electron microscopy, and biochemical techniques we show that ErbB2 is preferentially associated with membrane protrusions. Moreover, it is efficiently excluded from clathrin-coated pits and is not seen in transferrin receptor-containing endosomes. This pattern is not changed after binding of EGF, heregulin, or herceptin. The exclusion from coated pits is so pronounced that it cannot just be explained by lack of an internalization signal. Although ErbB2 is a raft-associated protein, the localization of ErbB2 to protrusions is not a result of raft binding. Also, an intact actin cytoskeleton is not required for keeping ErbB2 away from coated pits. However, after efficient cross-linking, ErbB2 is removed from protrusions to occur on the bulk membrane, in coated pits, and in endosomes. These data show that ErbB2 is a remarkably internalization-resistant receptor and suggest that the mechanism underlying the firm association of ErbB2 with protrusions also is the reason for this resistance.     

TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.     

Role for IL-10 in suppression mediated by peptide-induced regulatory T cells in vivo

Regulatory CD4(+) T cells were induced in the Tg4 TCR transgenic mouse specific for the N-terminal peptide (Ac1-9) of myelin basic protein by intranasal administration of a high-affinity MHC-binding analog (Ac1-9[4Y]). Peptide-induced tolerant cells (PItol) were anergic, failed to produce IL-2, but responded to Ag by secretion of IL-10. PItol cells were predominantly CD25(-) and CTLA-4(+) and their anergic state was reversed by addition of IL-2 in vitro. PItol cells suppressed the response of naive Tg4 cells both in vitro and in vivo. The in vitro suppression mediated by these cells was not reversed by cytokine neutralization and was cell-cell contact-dependent. However, suppression of proliferation and IL-2 production by PItol cells in vivo was abrogated by neutralization of IL-10. These results emphasize an important role for IL-10 in the function of peptide-induced regulatory T cells in vivo and highlight the caution required in extrapolating mechanisms of T regulatory cell function from in vitro studies.