Association of hysterectomy and invasive epithelial ovarian and tubal cancer: a cohort study within UKCTOCS.

OBJECTIVE: To investigate the association between hysterectomy with conservation of one or both adnexa and ovarian and tubal cancer. DESIGN: Prospective cohort study. SETTING: Thirteen NHS Trusts in England, Wales and Northern Ireland. POPULATION: A total of 202 506 postmenopausal women recruited between 2001 and 2005 to the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and followed up until 31 December 2014. METHODS: Multiple sources (questionnaires, hospital notes, Hospital Episodes Statistics, national cancer/death registries, ultrasound reports) were used to obtain accurate data on hysterectomy (with conservation of one or both adnexa) and outcomes censored at bilateral oophorectomy, death, ovarian/tubal cancer diagnosis, loss to follow up or 31 December 2014. Cox proportional hazards regression models were used to assess the association. MAIN OUTCOME MEASURES: Invasive epithelial ovarian and tubal cancer (WHO 2014) on independent outcome review. RESULTS: Hysterectomy with conservation of one or both adnexa was reported in 41 912 (20.7%; 41 912/202 506) women. Median follow up was 11.1 years (interquartile range 9.96-12.04), totalling >2.17 million woman-years. Among women who had undergone hysterectomy, 0.55% (231/41 912) were diagnosed with ovarian/tubal cancer, compared with 0.59% (945/160 594) of those with intact uterus. Multivariable analysis showed no evidence of an association between hysterectomy and invasive epithelial ovarian/tubal cancer (hazard ratio 0.98, 95% CI 0.85-1.13, P = 0.765). CONCLUSIONS: This large cohort study provides further independent validation that hysterectomy is not associated with alteration of invasive epithelial ovarian and tubal cancer risk. These data are important both for clinical counselling and for refining risk prediction models. TWEETABLE ABSTRACT: Hysterectomy does not alter risk of invasive epithelial ovarian and tubal cancer.

Functional and modelling studies of the binding of human monoclonal anti-DNA antibodies to DNA.

The relationships between the antigen-binding specificities of four human monoclonal anti-DNA antibodies and the structural aspects of the combining sites of two of these were examined. Competition ELISAs were used to examine the reactivities of two IgM MAbs (WRI-176 and RT-79) and two IgG mAbs (D5 and B3) to a wide range of polynucleotides. The mAbs WRI-176 and RT-79 were found to bind predominantly ssDNA, with a preference for poly (dT), whilst D5 and B3 bound components of both ss- and dsDNA, and Z-DNA. The mAb B3 also exhibited a preference for A(T) rich nucleotides. Computer models were generated for the Fv regions of WRI-176 and B3. Models for RT-79 and D5 were not generated as the structure of the long CDR-H3 loops in these mAbs could not be predicted. The B3 combining site contains a groove flanked by three arginines at positions CDR-L1-27A, CDR-L2-54 and CDR-H2-53. Using interactive molecular graphics, B-DNA was docked into the B3 antigen combining site along the plane of the VH/VL interface, whilst Z-DNA was best-fitted at approximately 90 degrees to this direction. The models provide a hypothesis to explain the ability of a single autoantibody to bind two different antigens. In addition, aspects of the base specificity of B3 may be explained. The model of the WRI-176 Fv region revealed a relatively flat surface, on which a large number of hydrophobic and aromatic residues were present. Trp-H52, in particular, is prominent on the surface. This may participate in ssDNA binding through base stacking interactions. The models allow identification of potential targets for site-directed mutagenesis.

Anti-cardiolipin/beta-2 glycoprotein activities co-exist on human anti-DNA antibody light chains.

We have recently shown that the human anti-DNA antibodies B3 and 33H11 also bind cardiolipin and that the anti-autoantigen activity resides predominantly on their lambda light chains. We now show that the two auto-antibodies possess strong reactivity to the plasma-protein 2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 with insignificant reactivity to cardiolipin or to beta2-GPI, we now demonstrate that hybrid Fabs with constituent light chain, but not the heavy chain, of B3 or 33H11, exhibit anti-cardiolipin activity. Furthermore, the constructs possessing the auto-antibody-derived light chain also exhibited significant reactivity to beta2-GPI. The results suggest that anti-DNA, anti-cardiolipin and anti-beta2-GPI activities co-exist on the light chains of the antibodies studied and, importantly, these activities could be transferred to antibody constructs by their light chains alone. Computer-generated models of the three-dimensional structures of the auto-antibodies and their hybrids, suggest predominant interaction of their light chains with domain IV of beta2-GPI.

The role of VH determinants in systemic lupus erythematosus.

The V-regions of anti-DNA antibodies contain determinants which can drive the autoimmune in SLE. Most of the evidence comes from murine studies where VH-derived epitopes accelerate the disease process in lupus prone-mice and can elicit mild inflammatory changes reminiscent of lupus in healthy animals. T helper cells reactive with VH peptides arise spontaneously during the disease and are thought to assist production of both anti-peptide antibodies and the generation of autoantibodies that deposit in the glomeruli. In mice stimulatory epitopes may be unique to autoantibodies. As tolerogens VH peptides may delay or diminish the autoimmune response by altering the production of cytokines. An artificial VH peptide, (pCONCENSUS) has been derived and this inhibits responses to VH and other autoantigens but leaves the murine immune system intact and able to generate reponses to external antigens. Limited number of studies of V-region determinants of human anti-DNA MAbs indicate prior sensitization of lupus T cells to VH determinants and that V-region reactive T cells are not deleted in periphery of healthy individuals.

Suppressive effects of a novel antioxidant compound on human T cell functions in vitro.

The use of antioxidant compounds with differing modes of action has clearly demonstrated involvement of oxidative processes in the activation of T lymphocytes. In this paper, we show that a novel antioxidant (lazaroid U75412E, a free radical scavenger) suppressed mitogen-induced T cell proliferation in vitro. Similar results were obtained with diphenylene iodonium (DPI), a known inhibitor of NADPH oxidase. The lazaroid was further shown to inhibit IL 2 production but to be less potent in suppressing IL 2 receptor expression. Thus, scavenger-type antioxidants act on T cells primarily by blocking a signal necessary for the induction of IL 2 synthesis such as the activation of NF kappa B. Furthermore, the potent inhibition of lymphocyte responses caused by the specific enzyme inhibitor DPI provides direct proof of the source of the oxidants involved in these processes.

Immunization with a peptide of Sm B/B' results in limited epitope spreading but not autoimmune disease.

An experimental model of systemic lupus erythematosus has recently been described in normal animals. We sought to confirm and extend this model, which involved immunization of normal rabbits and mice with a peptide of Sm B/B', PPPGMRPP. This peptide is an early target of the immune response in anti-Sm-positive patients with lupus. The peptide was used in a multiple Ag peptide format, with multiple copies of PPPGMRPP bound to an inert lysine backbone. New Zealand White rabbits and A/J and C57BL/10ScSn mouse strains were immunized with PPPGMRPP-MAP. Pepscan assays were used to determine the epitope spreading of the anti-PPPGMRPP-MAP response to other octamers of SmB/B' following immunization. We obtained high titer anti-PPPGMRPP-MAP IgG responses in the New Zealand White rabbits and A/J mice. The rabbits immunized with PPPGMRPP-MAP showed varying degrees of epitope spreading, while the A/J mice showed no spreading. We observed no autoantibodies to dsDNA or other anti-nuclear autoantibodies in our animals by ELISA or immunofluorescence, although anti-nuclear autoantibodies were found by Western blotting in some of the rabbits. No evidence of clinical disease was seen in our normal animals. These data underline the difficulties often associated with the reproduction of animal models in different laboratories.

Molecular expression systems for anti-DNA antibodies--2.

Antibodies to double-stranded DNA are the best-known serological markers of systemic lupus erythematosus, and are closely associated with its renal pathogenesis. How these antibodies recognize DNA is not fully understood. An understanding of the relationship between the functional attributes of an antibody with the three-dimensional structure of its antigen-combining site would allow an insight into the rules that dictate auto-antibody-nucleic acid interaction and consequent pathogenicity of the autoantibody. Data from such studies could assist the development of novel drugs as an approach to specific therapies that can inhibit or disrupt protein-nucleic acid interactions. A full understanding of the binding specificities can be achieved only by experimental determination of detailed three-dimensional structure of these antibodies alone, and of their complexes with specific DNA antigens. A prerequisite of such a study is the ability to produce multimilligram quantities of the antibody protein. However, these antibodies are particularly difficult to express, probably due to their DNA-binding activity. This review attempts to focus on the recent developments on the over-expression of anti-DNA antibody fragments in heterologous cell expression systems and their purification to homogeneity that would in turn allow their structural studies via crystallization.

Peptides from antibodies to DNA elicit cytokine release from peripheral blood mononuclear cells of patients with systemic lupus erythematosus: relation of cytokine pattern to disease duration.

Peptides from VH regions of antibodies to DNA drive immune responses in systemic lupus erythematosus (SLE). We studied peptide-induced cytokine release by peripheral blood mononuclear cells (PBMC) of patients, the influence of peptide concentration, disease characteristics and HLA-D haplotypes. Cells secreting cytokines (IFNgamma, IL-2, IL-4 and IL-10) were measured by ELISPOT in PBMC from 31 patients with SLE and 20 matched healthy controls in response to seven peptides (A-G) from the CDR1/FR2 to CDR2/FR3 VH regions of human anti-DNA MAbs. Disease activity was assessed by SELENA-SLEDAI. HLA-DR and -DQ alleles were determined by molecular typing techniques. PBMC from significantly higher proportions of SLE patients than controls responded to VH peptides by generating IFNgamma and IL-10. Type of cytokines released in response to at least one peptide (D) depended on antigen concentration. Cytokine release was not associated with clinical features of SLE except for disease duration. A shift occurred from IFNgamma, IL-4 and IL-10 production in early disease to IL-4 and IL-10 in late disease (suggesting increasing TH2-like responses over time). Three peptides (B, D, G) were more stimulatory in the SLE patients than controls. Although none of the peptides was restricted by any particular MHC class II allele, among responders there was increased prevalence of HLA- DQB1*0201 and/or DRB1*0301, alleles known to predispose to SLE. Thus, responses to some VH peptides are more frequent in SLE and vary with disease duration. Increased responses in individuals with HLA class II genotypes that predispose to SLE suggest that peptide presentation by those molecules permits brisker peripheral blood cell responses to autoantibody peptides, thus increasing risk for disease.

Analysis of three new idiotypes on human monoclonal autoantibodies.

We have identified and characterised three new idiotypes on human IgM McAbs generated from the splenocytes of a SLE patient with active disease. RT-6, which binds H1 and Sm/RNP, expresses essentially a private Id. Its expression is limited to a small number of human McAbs and the sera from patients with infectious diseases. In contrast RT-72Id and RT-84Id, expressed on McAbs which are polyreactive for two or more antigens, have a public distribution. RT-72Id and RT-84Id are found on McAbs from murine and human adult, and foetal tissues. In sera, significant numbers of SLE, RA and patients with other autoimmune diseases are positive for both Ids. RT-84Id is also elevated in SLE relatives and spouses, and in patients with Klebsiella infection. No correlation with disease activity, IgM or IgG levels was observed with either Id. However, RT-72Id was significantly associated with anti-ssDNA antibodies and RhF. RT-6Id and RT-72Id are located on the framework regions of the mu heavy chain, whereas RT-84Id is present on the kappa light chain, within the binding site. The McAbs are encoded by mainly germline genes: heavy chains of RT-6, RT-72 and RT-84 are encoded by the genes VH26, VH4.22 and VH4.21, respectively, and the light chain sequences of RT-6 and RT-72 are derived from DPL11 and HK102. Immunofluorescent staining revealed the presence of RT-72Id and RT-84Id positive immunoglobulin deposits in 18% and 45%, respectively, of the lupus renal sections compared with none in the disease control group, suggesting that these Ids may contribute to the pathology of the disease.

Interplay of four idiotypes and interaction with autoantibodies in lupus patients, their relatives and their spouses.

Our aim was to investigate links between systemic lupus erythematosus (SLE)-associated autoantibodies, idiotypes (Id) and genetic predisposition to their development. We studied four public Ids (16/6, WRI 176 beta, RT72 and RT84), identified the Km and Gm phenotypes and sought six selected autoantibodies in 32 SLE patients, 174 of their relatives and 15 spouses. Though anti-double-stranded DNA antibody was uncommon in the relatives (9%), the range of antinuclear reactivities was as broad in the relatives as in the probands. Antibodies to the synthetic peptide U1-RNP-A 35-38 were found in 56% of the patients, 28% of their relatives and 20% of the spouses, whereas antibodies to the Golgi apparatus was present in 7% of the patients, 26% of their relatives and 33% of the spouses. However, most of these family members were unaffected. RT84 Id was positively associated with antibodies to Sm-D peptide 1-20 and to Ro/SSA 60 kD peptide 304-324, but negatively associated with anti-dsDNA activity. The median of age was significantly lower in the RT84 Id-positive than in the RT84 Id-negative individuals. These data suggest that genetic as well as environmental factors are involved in the aetiology of SLE. In addition, RT84-carrying immunoglobulins (Ab2) might be directed to one of many cross-reactive Ids of dsDNA-binding antibodies (Ab1), perhaps down-regulating their production.