Increased infiltration and tolerised antigen-specific CD8+ TEM cells in tumor but not peripheral blood have no impact on survival of HCMV+ glioblastoma patients.

Human cytomegalovirus (HCMV) antigens in glioblastoma (GBM) present opportunities for personalised immunotherapy. However, their presence in GBM tissue is still under debate, and evidence of their impact on functional immune responses and prognosis is sparse. Here, we investigated the presence of pp65 (UL83) and immediate early 1 (IE-1) HCMV antigens in a cohort of Norwegian GBM patients (n = 177), using qPCR, immunohistochemistry, and serology. HCMV status was then used to investigate whether viral antigens influenced immune cell phenotype, infiltration, activation and patient survival. Pp65 and IE-1 were detected by qPCR in 23% and 43% of GBM patients, respectively. Furthermore, there was increased seropositivity in GBM patients relative to donors (79% vs. 48%, respectively; Logistic regression, OR = 4.05, 95%CI [1.807-9.114], P = 0.001, also when adjusted for age (OR = 2.84, 95%CI [1.110-7.275], P = 0.029). Tissue IE-1-positivity correlated with increased CD3+CD8+ T-cell infiltration (P < 0.0001), where CD8+ effector memory T (TEM) cells accounted for the majority of CD8+T cells compared with peripheral blood of HCMV+ patients (P < 0.0001), and HCMV+ (P < 0.001) and HCMV- (P < 0.001) donors. HLA-A2/B8-restricted HCMV-specific CD8+ T cells were more frequent in blood and tumor of HCMV+ GBM patients compared with seronegative patients, and donors irrespective of their serostatus. In biopsies, the HCMV-specific CD8+ TEM cells highly expressed CTLA-4 and PD-1 immune checkpoint protein markers compared with populations in peripheral blood (P < 0.001 and P < 0.0001), which expressed 3-fold greater levels of CD28 (P < 0.001 and P < 0.0001). These peripheral blood T cells correspondingly secreted higher levels of IFNγ in response to pp65 and IE-1 peptide stimulation (P < 0.001). Thus, despite apparent increased immunogenicity of HCMV compared with tumor antigens, the T cells were tolerised, and HCMV status did not impact patient survival (Log Rank3.53 HR = 0.85 95%CI [0.564-1.290], P = 0.45). Enhancing immune functionality in the tumor microenvironment thus may improve patient outcome.

Reduced Number of CD8+ Cells in Tonsillar Germinal Centres in Children with the Periodic Fever, Aphthous Stomatitis, Pharyngitis and Cervical Adenitis Syndrome.

The syndrome of periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) is an autoinflammatory disorder of unknown aetiology. Tonsillectomy may cause a prompt resolution of the syndrome. The aim was to study the histologic and immunological aspects of the palatine tonsils in PFAPA, to help understand the pathophysiology of the syndrome. Tonsils from children with PFAPA (n = 11) and children with tonsillar hypertrophy (n = 16) were evaluated histologically after haematoxylin and eosin staining. The number of different cell types was identified immunohistochemically by cluster of differentiation (CD) markers: CD3 (T cells), CD4 (T helper cells), CD8 (cytotoxic T cells), CD15 (neutrophils), CD20 (B cells), CD45 (all leucocytes), CD57 (NK cells) and CD163 (monocytes and macrophages). Tonsils from children with PFAPA showed reactive lymphoid hyperplasia dominated by well-developed germinal centres with many tingible body macrophages. The histologic findings were unspecific, and a similar morphologic appearance was also found in the tonsils from controls. The number of CD8+ cells in germinal centres differed between children with PFAPA [median 9 cells (quartiles: 5, 15)] and controls [18 cells (12, 33) (P = 0.001)] and between children with PFAPA with (median 14 cells; 9, 16) and without (4 cells; 3, 8) aphthous stomatitis (P = 0.015). For the other cell types, no differences in germinal centres were found between children with PFAPA and controls. In conclusion, a lower number of CD8+ cells were found in germinal centres of tonsils in children with PFAPA compared to controls, which may be a feature linked to the aetiology of the syndrome.

Severe human Babesia divergens infection in Norway.

Human babesiosis is a rare but potentially life-threatening parasitic disease transmitted by ixodid ticks, and has not previously been reported in Norway. We report a case of severe babesiosis that occurred in Norway in 2007. The patient had previously undergone a splenectomy. He was frequently exposed to tick bites in an area endemic for bovine babesiosis in the west of Norway. The patient presented with severe haemolysis and multiorgan failure. Giemsa-stained blood smears revealed 30% parasitaemia with Babesia spp. He was treated with quinine in combination with clindamycin, apheresis, and supportive treatment with ventilatory support and haemofiltration, and made a complete recovery. This is the first case reported in Norway; however Babesia divergens seroprevalence in cattle in Norway is high, as is the risk of Ixodes ricinus tick bite in the general population. Babesiosis should be considered in the differential diagnosis of unexplained febrile haemolytic disease.

Pandemic influenza vaccination elicits influenza-specific CD4+ Th1-cell responses in hypogammaglobulinaemic patients: four case reports.

In these case reports, we investigated pandemic influenza 2009 vaccination of primary hypogammaglobulinaemic patients. Three combined variable immunodeficiency (CVID) patients and one X-linked agammaglobulinaemia (XLA) patient were vaccinated with the pandemic vaccine A/California/7/2009 (H1N1)-like split virus (X179a) adjuvanted with the oil-in-water emulsion AS03. Subsequently, serum and peripheral blood mononuclear cells were sampled and used to measure the haemagglutination inhibition (HI) and antibody-secreting cell (ASC) responses. In addition, the IFN-γ, IL-2 and TNF-α producing CD4(+) Th1-cell response was determined as these cytokines are important indicators of cell-mediated immunity. Two of the CVID patients responded to vaccination as determined by a >4-fold rise in HI antibodies. These subjects also had influenza-specific ASC numbers, which, albeit low, were higher than prevaccination levels. In addition, vaccination induced CD4(+) Th1-cell responses in both the XLA patient and the CVID patients, although the frequency of influenza-responsive cells varied amongst the patients. These results suggest that hypogammaglobulinaemia patients can mount a CD4(+) Th1 cell-mediated response to influenza vaccination and, additionally, that influenza vaccination of some hypogammaglobulinaemia patients can produce an influenza-specific humoral immune response. The findings should be confirmed in larger clinical studies.

Quantification of S100A12 (EN-RAGE) in blood varies with sampling method, calcium and heparin.

S100A12 is a calcium-binding protein predominantly found in neutrophil granulocytes and monocytes. Its usefulness in monitoring inflammatory disease states depends on documentation that assay results are reliable. This study aimed at defining guidelines for blood sampling, selection of optimal material handling and reference intervals in healthy controls while taking into account the basic features of S100A12. An enzyme linked immunosorbent assay was developed based upon antibodies induced in rabbits by injection of recombinant S100A12. Our studies confirm that oligomers of S100A12 are generated in the presence of calcium. Structural changes in S100A12 mediated by calcium influence the interaction with antibody. This is proposed as the background for our very low readings of S100A12 in Ethylene Diamine Tetraacetic Acid (EDTA) plasma. Individual S100A12 levels did not change substantially over a 5-week sampling period. Based upon testing of 150 blood donors we suggest reference intervals of S100A12 in serum to be 49-1340 microg/l for women and 27-1750 microg/l for men. The estimated mean concentrations were 234 microg/l in serum samples (range 12-15791), 114 microg/l (range 3-17282) in re-calcified EDTA plasma and 48 microg/l (range 2-14843) in heparin plasma. Without adding calcium to EDTA plasma before running the assay, concentrations were around 2 microg/l (16 persons). S100A12 quantification is assumed to become relevant for diagnostic use in many disease states. The importance of the handling and analysing conditions for a reliable result was examined. We recommend serum collected in gel-containing tubes as the preferred sample material and have suggested reference intervals for healthy individuals.

Placental expression of glycophosphatidylinositol (GPI)-anchored proteins in paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal stem cell disorder in which a defect of glycophosphatidylinositol (GPI)-anchored proteins leads to higher morbidity and mortality because of intravascular haemolysis, haemoglobinuria, pancytopenia and an increased frequency of thrombotic events. We report here the clinical features of a pregnant woman with PNH and present an immunhistochemical analysis of complement regulators, leukocyte activation markers and placental alkaline phosphatase (PALP) on syncytiotrophoblasts and inflammatory cells in her placenta. Placental tissue from normal deliveries served as controls. The patient had severe PNH with haemolysis, thrombosis episodes and signs of bone marrow failure. Placental syncytiotrophoblasts and villous cells of fetal origin in both normal placentas and the placenta from the PNH patient expressed PALP and the complement regulators CD46, CD55 and CD59. Additionally, CD11b-positive leukocytes of presumed maternal origin were negative for CD15 in the PNH placenta, while they stained positive within the villous space and in normal placentas. These findings show that fetally derived cells in the PNH placenta expressed GPI-linked molecules that are known to be of importance for a successful pregnancy outcome.

Tetanus immunity among pregnant women attending antenatal care in Dar es Salaam, Tanzania.

This study was conducted to investigate immunity to tetanus among pregnant women with verbal histories or documentation of having been vaccinated under the current five-dose tetanus toxoid (TT) schedule. It examined sera from 176 pregnant women attending antenatal care at Muhimbili Medical Centre in Dar es Salaam, Tanzania. Tetanus antitoxin level of 0.1 IU/ml was considered protective. Our findings show that 94.9% of women had tetanus antitoxin > or = 0.1 IU/ml. Multivariate analysis revealed that time after last vaccination, TT doses received and TT vaccination status explained 7.5%, 5.7% and 2.3% of variations in tetanus antitoxin levels respectively. Pregnant women with non-protective levels of tetanus antitoxin (5.1%) pose great risks of neonatal tetanus to their newborns and are also susceptible to maternal tetanus. Proper keeping of TT vaccination records is vitally important to avoid hyper-immunisation.

Immunity to tetanus in male adults in Dar es Salaam, Tanzania.

OBJECTIVE: To determine immunity to tetanus in male blood donors with previous diphtheria-pertussis-tetanus (DPT)/tetanus toxoid (TT) vaccination. DESIGN: A cross sectional study, conducted in September 1999. SETTING: Blood bank, Muhimbili Medical Centre, Dar es Salaam, Tanzania. METHODS: Using an antigen competition ELISA technique, serum tetanus anti-toxin levels in two hundred male blood donors were determined. RESULTS: Vaccination history was absent in 43 (21.5%) blood donors, whereas 60 (30%) and 97 (48.5%) reported childhood DPT and TT vaccination, respectively. Tetanus anti-toxin was undetectable in 47 (23.5%) blood donors and the levels were below that considered protective (> or = 0.1 IU/ml) in 25 (12.5%). Among those with undetectable level, 43 (91.5%) had no vaccination history. Time after last DPT/TT vaccination correlated significantly with tetanus anti-toxin levels (r2=-0.331, p=0.001). In multivariate analysis, TT doses received and time after last vaccination explained 4.8% and 29.4%, respectively, of the variations in tetanus anti-toxin levels. CONCLUSION: Seventy two (36%) male blood donors were susceptible to tetanus and the susceptibility was highest from 48 years. A regular TT booster dose at 10 yearly intervals is recommended to provide adequate and long lasting immunity in male adults. Proper keeping of vaccination records is emphasised.

Antibodies to tetanus toxoid in women of childbearing age in Dar es Salaam and Bagamoyo, Tanzania.

Our aim was to determine tetanus immunity in women of childbearing age (15-44 years) with histories and/or documentation of having been vaccinated with Tetanus Toxoid (TT) under the Expanded Programme on Immunization in Dar es Salaam and Bagamoyo, Tanzania. Using an ELISA technique, serum levels of TT antibody, antibody avidity and distribution of TT IgG subclass antibodies were determined in 207 apparently healthy women. A TT antibody level of 0.1 IU/ml was considered protective. 99% and 100% of women in Dar es Salaam and Bagamoyo, respectively, had a TT antibody level > or = 0.1 IU/ml. Anti-toxin binding avidity was found to be high in most of the women. In addition to TT IgG3 subclass antibody, TT IgG1 subclass antibody was the most dominant subclass type. A substantial number of women also had TT IgG2 and TT IgG4 subclass antibody responses. A better recording system on TT immunization is recommended to avoid hyper-immunization of women and to optimize the cost-effectiveness of the immunization programme.

[High dose chemotherapy with autologous stem cell support in cancer patients]. / Høydosebehandling med autolog stamcellestøtte hos kreftpasienter.

INTRODUCTION: In 1996 it was decided that high-dose chemotherapy with peripheral stem cell support should be offered by all five university hospitals in Norway. MATERIAL AND METHODS: We report on the first 49 patients from the western part of Norway treated with this modality at Haukeland University Hospital in the 1996-98 period. RESULTS: All patients had a total of > 2-10(6) CD34 positive cells/kg collected before high-dose chemotherapy. To achieve this critical stem cell dose, five patients had to have three or more stem cell collections; four of them had to be mobilised several times. Poor stem cell mobilisation was mostly marked in patients with soft tissue sarcoma and testicular cancer, but was also observed in a few heavily pre-treated patients with non-Hodgkins lymphoma. With the exception of one lymphoma patient who developed a rapid bone marrow relapse, all patients had satisfactory sign of bone marrow regeneration after reinfusion of the stem cells. This also applied to the poor mobilisors. No treatment-related deaths have occurred. Four to 38 months after high-dose therapy, 33% of patients with multiple myeloma and 52% of patients with malignant lymphoma were alive and in complete remission. Three of the four patients with soft tissue sarcoma relapsed 3-7 months after high-dose chemotherapy.

Levels and avidity of antibodies to tetanus toxoid in children aged 1-15 years in Dar es Salaam and Bagamoyo, Tanzania.

A study was undertaken to determine the serological response in children (aged 1-15 years) immunized with diphtheria-pertussis-tetanus vaccine (DPT) alone or with a tetanus toxoid (TT) booster dose under the Expanded Programme on Immunization in Dar es Salaam and Bagamoyo, Tanzania. Using an ELISA technique, serum levels of anti-TT antibody, antibody avidity and anti-TT IgG subclasses were determined in 138 apparently healthy children. Our findings revealed that 94.7% and 98% of children aged 1-5 years in Dar es Salaam and Bagamoyo, respectively, had anti-TT antibody levels above that considered protective (> or = 0.1 IU/ml). Among 6-15-year-old children, 53.3% in Dar es Salaam and 55% in Bagamoyo had anti-TT antibody levels > or = 0.1 IU/ml. The avidity index of anti-TT antibodies was high in most of the younger children, 84.2% in Dar es Salaam and 92% in Bagamoyo. Significantly fewer older children in Dar es Salaam and Bagamoyo (53.3% and 50%, respectively) had high avidity index antibodies. The predominant anti-TT IgG subclasses were IgG1 and IgG3. It is concluded that the current DPT immunization schedule provides adequate tetanus immunity for children under 5. However, about half of the older children had no protection against tetanus.

Expression of annexin II in glioma cell lines and in brain tumor biopsies.

Annexin II is a calcium and phospholipid binding protein and a substrate for protein-tyrosine kinases. Increased levels of annexin II are observed in various cancer cells and tissues, and the molecule has been proposed as a marker of malignancy in vivo. Annexin II was expressed in four glioma cell lines (D-54MG, D-37MG, U251MG and GaMG), as determined by Western blot analyses, immunofluorescence staining and flow cytometric measurements. In addition, annexin II expression was also found in cryostat sections obtained from 15 consecutive brain tumor biopsies: Ten were histologically classified as glioblastomas, one as an astrocytoma, two as meningiomas and two as brain metastases. Cultured spheroids from the glioma cell lines and from three of the glioblastoma biopsies showed lower levels of annexin II, than found in the monolayers of the cell lines and in the freshly cut biopsies. The annexin II expression of the cell lines were not found to be related to their proliferative, migratory or invasive properties. These findings indicate that although annexin II may serve as a marker of malignancy in vivo, its expression can be reduced in vitro, and appear unrelated to malignant features of glioma cell lines.

Suppressive effect on lymphoproliferation in vitro by soluble annexin II released from isolated placental membranes.

PROBLEM: Syncytiotrophoblast microvillous plasma membranes (StMPM) are potent suppressors of lymphoproliferation in vitro. We have previously shown that soluble annexin II (AII) is present at higher levels in retroplacental serum (RPS) than in peripheral serum, and that soluble AII has an immunosuppressive effect. The aims of this study were to determine whether AII can be released from StMPM and whether soluble AII from StMPM exerts any immunosuppressive effect. METHOD OF STUDY: Isolated StMPM were incubated in growth medium for 18 hr and supernatants were prepared by ultracentrifugation. Soluble AII was detected by immunoblotting. StMPM, StMPM supernatant, and affinity-purified AII were analysed in a lymphoproliferation assay for immunomodulating activity. RESULTS: AII heavy chain and its p11 light chain were detected both in StMPM supernatant and in RPS after removal of StMPM particles by ultracentrifugation. StMPM, StMPM supernatant, and purified AII suppressed lymphoproliferation in a dose-dependent manner. Absorption of AII from StMPM supernatant reduced the suppressive activity. The suppressive effect of StMPM supernatant and purified AII was completely reversed by heating at 100 degrees C for 30 min or by adding recombinant interleukin-2 at 100 units/ml. Although StMPM and affinity-purified AII suppressed the proliferation of lymphocytes from all donors tested, StMPM supernatant suppressed the proliferation of lymphocytes from 12 of 23 donors. Six of eight female non-suppressed donors were multiparae, whereas five of five female suppressed donors were nulliparae. CONCLUSIONS: Annexin II is released by isolated placental membranes in vitro and is present in RPS, indicating in vivo release of AII at the fetomaternal interface, probably as AII heterotetramer. AII has immunosuppressive activity and may be important in fetal allograft survival.

Inhibition of phytohaemagglutinin-induced lymphoproliferation by soluble annexin II in sera from patients with renal cell carcinoma.

Annexin II (AII) is a member of a family of glycoproteins which bind negatively charged phospholipids in a calcium-dependent manner. Annexins are membrane-associated proteins, expressed both in normal and malignant cells, but have also been detected as soluble molecules in serum and other body fluids. Because of their adhesive properties, it has been suggested that annexins play a role in the metastatic process. An ELISA was established for quantification of soluble AII. Within-run variation was 5.2-10.4% and run-to-run variation 12.4-15.6%. Soluble AII was detected in all sera studies. A strongly positive serum was arbitrarily given the value 100 AII units and used as reference serum. The mean level in sera from 20 normal blood donors was 49 (SE 5.6) AII units. Sera from peripheral blood of five patients with renal cell carcinoma and sera from blood obtained from the renal vein of the same patients contained 47 (SE 20) and 83 (SE 28) AII units, respectively. In two patients, AII levels were increased in renal vein serum as compared with peripheral blood serum. Interestingly, in both cases, and in none of the three remaining cases, phytohaemagglutinin-stimulated lymphoproliferation was suppressed by renal vein serum as compared with peripheral blood serum. Affinity absorption of AII from the renal vein sera with increased AII levels strongly reduced their immunosuppressive activity. Addition of affinity-purified AII to cell cultures suppressed lymphoproliferation. These data show that the level of AII is markedly increased in renal vein sera from some patients with renal cell carcinoma, suggesting that AII may be locally released in vivo. The study also demonstrates an immunosuppressive effect of soluble AII in vitro. We speculate that soluble AII released by the tumour has immunosuppressive properties. This study identifies soluble AII as a novel immunosuppressive factor in sera from patients with renal cell carcinoma. A further study including a larger number of patients is currently in progress, in order to investigate the pathological significance of this finding.

Resistance of ovarian teratocarcinoma cell spheroids to complement-mediated lysis.

We have shown previously that it is possible to target complement-mediated killing against cultured ovarian tumour cells in vitro. As malignant ovarian cells usually grow in solid nodules in vivo, we have in the present study examined the effectiveness of complement killing against ovarian teratocarcinoma cells (PA-1) growing in three-dimensional tumour microspheroids (TMSs). Our study shows that PA-1 cells growing in TMSs are less susceptible to complement-mediated killing than cells growing in monolayer cultures, even after neutralization of protectin (CD59), the main inhibitor of complement lysis. Cells in suspension and cells growing in TMSs showed a similar expression of membrane co-factor protein (MCP, CD46) and CD59. Decay-accelerating factor (DAF, CD55) was not detected on the surface of cells in suspension, but appeared focally on the outermost cell layers of the TMSs. Complement-activating antibodies bound to all PA-1 cells in suspension but only to the most peripherally located cells in TMSs, even though the target antigens were similarly expressed in the two systems. Antibody-induced complement activation on PA-1 cells in suspension led to C3 and C5b-9 deposition on most cells, while C3 and C5b-9 were only found on the outermost layers of the TMSs. The increased complement resistance of tumour cells growing in three-dimensional spheroids is partly because of an insufficient penetration of antibodies and complement into the TMSs. TMSs are a useful model for the development of more efficient ways to kill malignant cells in micrometastases with monoclonal antibodies and complement.

Surface annexin II on placental membranes of the fetomaternal interface.

PROBLEM: The phospholipidbinding membrane protein annexin II has been demonstrated to possess FcR activity for IgG and has been localized to the outer part of the syncytiotrophoblast cell layer. The question has arisen whether annexin II is exposed on the surface of syncytiotrophoblast cells thus enabling it to take part in the transport of IgG across the maternal barrier. METHOD: Syncytiotrophoblast microvillous plasma membranes were analyzed by flow cytometry for annexin II as well as established surface molecules. Fresh, fixed placental tissue was preincubated with antibodies to annexin II or known trophoblast surface molecules, and analyzed by confocal laser scanning microscopy. RESULTS: Annexin II and its subunit p11 were expressed on the surface of the syncytiotrophoblast microvillous plasma membranes as were other established surface proteins (CD46, CD59, placental alkaline phosphatase), using both flow cytometry and confocal microscopy. Annexin was not detected on the surface of viable cultured trophoblast cells. CONCLUSION: Annexin II is exposed on the surface of syncytiotrophoblast cells as a heterotetramer together with its light chain p11. It is exposed to maternal blood and may be instrumental in IgG transport across the placental barrier by binding.