Prophylactic progesterone and preterm birth.

This review summarises the present knowledge of prophylactic progesterone and preterm birth. Preterm birth (less-than 37 weeks) is a leading cause of neonatal mortality and morbidity worldwide. The incidence varies globally but remains low in the Nordic countries (5-6%). Prediction and prevention are complicated due to diverse aetiology, but obstetric history and cervical length can improve prediction. Prophylactic vaginal progesterone initiated between 12 and 24 weeks of gestation is recommended to reduce preterm birth less-than 33-35 weeks in singleton pregnancies with a history of preterm birth or with a short cervix (less-than 25 mm) and can be considered for twin pregnancies with the same risk factors.

A flexible silicone patch for treatment of hemorrhoids after birth: a pilot study of HEMOCIN® Patch.

PURPOSE: To test a non-pharmacological silicone patch for treatment of symptomatic hemorrhoids with focus on usability, safety, and self-reported short-term effect. METHODS: Puerperal women in a Danish maternity ward were cluster randomized to treatment with the HEMOCIN® patch (intervention) or no systematic treatment (control group). On inclusion and after 2 weeks, they completed a questionnaire regarding history and hemorrhoid symptoms scored from 0 to 10. Women in the intervention group also reported on the usability of the patch and any side effects. RESULTS: We included 31 women in the intervention group and 33 in the control group. Twenty-eight (90.3%) women in the intervention group and 27 (81.8%) women in the control group responded to follow-up. Except from a difference in the severity of swelling at inclusion, there were no differences between the two groups for any symptoms, neither at inclusion, nor at follow-up, or in the change of symptoms during the two weeks (p > 0.05). Twenty-three women (85.2%) in the control group used medical treatment vs. one woman in the intervention group. The patch was used on an average of 9.3 days, 15.5 h/day and for 7.1 h before changing the patch. No severe side effects were reported. CONCLUSION: This pilot study finds that the HEMOCIN® patch is a safe and feasible treatment option for hemorrhoids. However, we did not detect any significant effect on hemorrhoid symptoms. The patch could be an option for people who seek non-pharmacological treatment for symptomatic hemorrhoids or need long-term treatment without steroid side effects.

Outcomes of monoamniotic twin pregnancies managed primarily in outpatient care-a Danish multicenter study.

INTRODUCTION: Monoamniotic twin pregnancies are high-risk pregnancies, and management by inpatient or frequent outpatient care is recommended. We report the outcomes of a national cohort of monoamniotic twin pregnancies managed primarily as outpatients. MATERIAL AND METHODS: We prospectively analyzed the recorded data from the Danish Fetal Medicine Database, local databases, and medical records of all monoamniotic twin pregnancies diagnosed at the first trimester scan or later, and managed at the six major fetal medicine centers in Denmark over a 10-year period. RESULTS: Sixty-one monoamniotic twin pregnancies were included. Thirteen pregnancies were terminated early. Of the remaining 48 pregnancies with a normal first trimester scan, there were 36 fetal losses (25 spontaneous miscarriages <22+0  weeks, 3 late terminations and 8 intrauterine deaths >22 weeks) and 60 liveborn children (62.5%), all of whom were delivered by cesarean delivery at a median gestational age of 33+0  weeks. Three children had minor malformations and there was 1 pregnancy with twin-to-twin transfusion syndrome. After 26+0  weeks, 78.8% were managed as outpatients. Intrauterine death occurred in 3.8% of outpatients and in 28.6% of inpatients (admitted due to complications). At weeks 32, 33 and 34, the prospective risk of intrauterine death was 6.9%, 4.2% and 5.9%, respectively. CONCLUSION: In this nationwide, unselected population, only 62.5% of fetuses with a normal first trimester scan were born alive. In contrast, the mortality was 3.8% after 26 weeks among the 78.8% of the cohort that was managed as outpatients. More knowledge is still needed to predict which pregnancies are at the highest risk of intrauterine death.

[Establishing a screening programme in Denmark for foetal and neonatal alloimmune thrombocytopenia].

Foetal and neonatal alloimmune thrombocytopenia (FNAIT) can cause cerebral haemorrhage in newborns. FNAIT occurs in women, who do not have the thrombocyte type human platelet antigen (HPA)-1a and are carrying an HPA-1a positive foetus. Maternal antibodies can cause thrombocytopenia in the foetus or newborn. Antenatal screening for FNAIT can easily be integrated in the already existing national screening programme for rhesus immunisation. Prophylactic treatment with immunoglobulines for pregnancies at risk can prevent neonatal complications. We argue, that the WHO criteria for a screening programme for FNAIT are met.

Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications.

Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

Glycan elongation beyond the mucin associated Tn antigen protects tumor cells from immune-mediated killing.

Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr), STn (NeuAcα2-6GalNAc-Ser/Thr), T (Galß1-3GalNAc-Ser/Thr), and ST (NeuAcα2-6Galß1-3GalNAc-Ser/Thr) antigens are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing only the shortest possible mucin-like glycans (Tn and STn). Glyco-engineering was performed by zinc finger nuclease (ZFN) knockout (KO) of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast and pancreatic cancer cell lines T47D and Capan-1 increases sensitivity to both NK cell mediated antibody-dependent cellular-cytotoxicity (ADCC) and cytotoxic T lymphocyte (CTL)-mediated killing. In addition, we investigated the association between total cell surface expression of MUC1/MUC16 and NK or CTL mediated killing, and observed an inverse correlation between MUC16/MUC1 expression and the sensitivity to ADCC and CTL-mediated killing. Together, these data suggest that up-regulation of membrane bound mucins protects cells from immune mediated killing, and that particular glycosylation steps, as demonstrated for glycan elongation beyond Tn and STn, can be important for fine tuning of the immune escape mechanisms in cancer cells.

Potential for novel MUC1 glycopeptide-specific antibody in passive cancer immunotherapy.

MUC1 is an important target for antibodies in passive cancer immunotherapy. Antibodies against mucin glycans or mucin peptide backbone alone may give rise to cross reactivity with normal tissues. Therefore, attempts to identify antibodies against cancer-specific MUC1 glycopeptide epitopes havebeen made. We recently demonstrated that a monoclonal antibody against the immunodominant Tn-MUC1 (GalNAc-α-MUC1) antigen induced ADCC in breast cancer cell lines, suggesting the feasibility of targeting combined glycopeptide epitopes in future passive cancer immunotherapy.

The number of regulatory T cells in transbronchial lung allograft biopsies is related to FoxP3 mRNA levels in bronchoalveolar lavage fluid and to the degree of acute cellular rejection.

BACKGROUND: The transcription factor Forkhead Box P3 (FoxP3) is a marker of regulatory T cells (Tregs) - a subset of T cells known to suppress a wide range of immune responses. These cells are considered to be pivotal for the induction of tolerance to donor antigens in human allografts. We aimed to correlate the number of lymphocytes expressing FoxP3 in transbronchial biopsies from lung allografts with the FoxP3 expression in bronchoalveolar lavage fluid (BALF). In addition, we aimed to correlate the number of FoxP3+ cells in transbronchial biopsies with the degree of acute cellular rejection in lung allografts. MATERIALS AND METHODS: The expression of FoxP3 was evaluated using immunohistochemical staining in 40 lung allograft biopsies obtained from 23 patients. The number of Tregs was related to the FoxP3 mRNA levels as determined using qRT-PCR in corresponding BALF samples from the same patients. Furthermore, the number of Tregs was related to the degree of acute allograft rejection (according to ISHLT criteria, A0-A4). RESULTS: Regression analysis showed a significant concordance between the number of Tregs in lung tissue and the level of FoxP3 mRNA relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels in BALF (n=40, p=0.0001). In addition, we found a significant increase in the number of Tregs during acute allograft rejections of grades A2 and higher (median: 32.6Tregs/mm(2)) when compared to those of grades A1 and A0 (median: 4.9Tregs/mm(2)) (p=0.0002). DISCUSSION AND CONCLUSION: The association between the distribution of Tregs in transbronchial biopsies and the level of FoxP3 mRNA in BALF indicates that assessment of FoxP3 mRNA in BALF is a reliable non-invasive method for evaluating the number of Tregs in lung tissue. Furthermore, the association between the number of Tregs in lung tissue and the degree of acute cellular rejection shows that Tregs are recruited to the site of inflammation and may be involved in the regulation of acute rejection. Thus, Tregs may play a role in the cellular processes that affect lung allograft outcome.

Glycan-mediated modification of the immune response.

Aberrantly glycosylated tumor antigens represent promising targets for the development of anti-cancer vaccines, yet how glycans influence immune responses is poorly understood. Recent studies have demonstrated that GalNAc-glycosylation enhances antigen uptake by dendritic cells as well as CD4+ T-cell and humoral responses, but prevents CD8+ T-cell activation. Here, we briefly discuss the relevance of glycans as candidate targets for anti-cancer vaccines.

Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity.

Protein glycosylation often changes during cancer development, resulting in the expression of cancer-associated carbohydrate antigens. In particular mucins such as MUC1 are subject to these changes. We previously identified an immunodominant Tn-MUC1 (GalNAc-α-MUC1) cancer-specific epitope not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis demonstrated high affinity binding (KD = 1.7 nM) of mAb 5E5 to its target, Tn-MUC1. Immunolabelling with mAb 5E5 revealed surface expression of the Tn-MUC1 epitope in breast cancer tissue and cell lines, and mAb 5E5 induced ADCC in two human breast cancer cell lines, MCF7 and T47D. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies.

Cancer associated aberrant protein O-glycosylation can modify antigen processing and immune response.

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.

Elevated mRNA levels of CTLA-4, FoxP3, and granzyme B in BAL, but not in blood, during acute rejection of lung allografts.

INTRODUCTION: Regulatory T cells (Tregs) have been related to acute rejection as have the cytotoxic T cells, their immunological counterpart. High expression of cytotoxic markers has been related to acute rejection incidents following both kidney and intestine transplantation, while the correlation between FoxP3 expression and acute rejection is still being debated. Some studies have been performed on blood samples from lung-transplanted patients, while others have investigated the local immune response in the lungs by analysing broncho-alveolar-lavage (BAL) fluids or biopsies. Biopsies are considered the gold standard in diagnosis of acute rejection. AIM: The aim was to measure the expression of both Treg (FoxP3, CD25 and CTLA-4) and cytotoxic (granzyme B, granulysin, and perforin) markers in BAL and blood samples from lung-transplanted patients to investigate the possible relation of expression and acute rejection incidents in order to develop a non-invasive diagnostic method for acute rejection. MATERIALS AND METHODS: 24 lung-transplanted patients were included in this 6-month cross-section study. BAL and blood samples were analysed for FoxP3, CD25, CTLA-4, granzyme B, granulysin, perforin, CD4 and CD8 mRNA by QRT-PCR with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as primary reference. RESULTS: We demonstrate that the mRNA levels in BAL relative to GAPDH of nearly all markers are elevated during acute rejection; CTLA-4, FoxP3, and granzyme B significantly, while a strong tendency is seen among the others. No significant differences were detected in blood. CONCLUSION: CTLA-4, FoxP3 and Granzyme B mRNA levels are elevated during acute rejection in BAL, but not in blood, following lung transplantation, indicating that evaluation of Treg and cytotoxic marker expression in BAL can be used in the assessment of allograft rejection state.