Orthodeoxia and postural orthostatic tachycardia in patients with pulmonary arteriovenous malformations: a prospective 8-year series.

Postural changes in 258 patients with pulmonary arteriovenous malformations (PAVMs) reviewed between 2005 and 2013 were evaluated prospectively using validated pulse oximetry methods. Of the 257 completing the test, 75 (29%) demonstrated orthodeoxia with an oxygen saturation fall of at least 2% on standing. None described platypnoea (dyspnoea on standing). The heart rate was consistently higher in the erect posture: 74 (29%) had a postural orthostatic tachycardia of ≥20 min(-1), and in 25 (10%) this exceeded 30 min(-1). Orthostatic tachycardia was more pronounced in PAVM patients than controls without orthodeoxia (age-adjusted coefficient 5.5 (95% CIs 2.6, 8.4) min(-1), p<0.001). For PAVM patients, the age-adjusted pulse rise was 0.79 min(-1) greater for every 1% greater drop in oxygen saturation on standing (p<0.001). In contrast to the postural orthostatic tachycardia syndrome, in this population, there was a trend for more pronounced orthostatic tachycardia to be associated with better exercise tolerance.

Gene vaccination with naked plasmid DNA: mechanism of CTL priming.

The injection of naked plasmid DNA directly into the muscle cells of mice has been shown to induce potent humoral and cellular immune responses. The generation of a cytotoxic T lymphocyte (CTL) response after plasmid DNA injection may involve the presentation of the expressed antigen in the context of the injected myocytes' endogenous major histocompatibility (MHC)-encoded class I molecules or may use the MHC molecules of bone marrow-derived antigen presenting cells (APC) which are capable of providing co-stimulation as well. To resolve which cell type provides the specific restricting element for this method of vaccination we generated parent-->F1 bone marrow chimeras in which H-2bxd recipient mice received bone marrow that expressed only H-2b or H-2d MHC molecules. These mice were injected intramuscularly with naked plasmid DNA that encoded the nucleoprotein from the A/PR/8/34 influenza strain, which as a single antigen has epitopes for both H-2Db and H-2Kd. The resulting CTL responses were restricted to the MHC haplotype of the bone marrow alone and not to the second haplotype expressed by the recipient's myocytes. The role of somatic tissues that express protein from injected plasmids may be to serve as a reservoir for that antigen which is then transferred to the APC. Consequently, our data show that the mechanism of priming in this novel method for vaccination uses the MHC from bone marrow-derived APC, which are efficient at providing all of the necessary signals for priming the T cell.

Human immunoglobulin (IgG) induced deletion of IgM rheumatoid factor B cells in transgenic mice.

The singular ability of immunoglobulin genes to hypermutate their variable regions, while permitting the generation of high-affinity antibodies against foreign antigens, poses a problem in terms of maintenance of immunological self-tolerance. Immunoglobulin gene hypermutation driven by a foreign antigen has the potential to generate antibodies that cross-react with self-components. Consequently, there must exist a mechanism in the periphery for inactivation of mature autoreactive B cell clones. The classical experimental system used to address this problem is the induction of tolerance to soluble, deaggregated human IgG. We have analyzed the mechanism of induction of tolerance to human IgG using transgenic mice that express a human IgM rheumatoid factor (IgM RF) on a large proportion of their B cells. Injection of deaggregated human IgG caused a specific deletion of those B cells that express an intact IgM RF on their cell surface. The degree of RF B cell deletion was proportional to the reduction in the proliferative response of splenocytes to antigen (aggregated human IgG), or to F(ab')2 fragments of anti-human IgM antibodies. Control experiments showed that IgG administration had little effect on the numbers of mouse Ig-bearing cells or their ability to proliferate to a nonspecific mitogen. Thus, the effects of IgG on the human IgM RF B cell are antigen specific and are not due to nonspecific toxic effects of the human IgG preparation. These experiments demonstrate that peripheral exposure to IgG induces deletion of reactive B cells, without any evidence for anergy, and differ from data obtained by other investigators studying tolerance to soluble protein antigens. The results imply that human Igs have distinct properties as soluble antigens, and that peripheral nonresponsiveness to IgG may be due to lymphocyte deletion.

CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.

A novel cell surface antigen has been identified on a wide range of lymphoid cells and erythrocytes. A mAb YTH 53.1 (CD59) against this antigen enhanced the lysis of human red cells and lymphocytes by homologous complement. Studies of reactive lysis using different species of C56, and of whole serum used as a source of C7-9, indicated that the inhibitory activity of the CD59 antigen is directed towards the homologous membrane attack complex. CD59 antigen was purified from human urine and erythrocyte stroma by affinity chromatography using the mAb YTH 53.1 immobilized on Sepharose, and, following transient expression of a human T cell cDNA library in COS cells, the corresponding cDNA also identified using the antibody. It was found that the CD59 antigen is a small protein (approximately 20 kD as judged by SDS-PAGE, 11.5 kD predicted from the isolated cDNA) sometimes associated with larger components (45 and 80 kD) in urine. The sequence of CD59 antigen is unlike that of other complement components or regulatory proteins, but shows 26% identity with that of the murine LY-6 antigen. CD59 antigen was released from the surface of transfected COS cells by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor; it is therefore likely to be absent from the surface of affected erythrocytes in the disease paroxysmal nocturnal hemoglobinuria.

Function of B cells expressing a human immunoglobulin M rheumatoid factor autoantibody in transgenic mice.

We have generated transgenic mice that express the immunoglobulin (Ig)M heavy chain and kappa light chain genes coding for a human IgM rheumatoid factor (RF), Les. Transgenic B cells expressing human IgM RF show striking similarities to their counterparts in normal humans. They comprise a significant proportion of the adult B cell population, but secrete only low levels of RF into the serum. The RF transgene-expressing B cells localize to primary B cell follicles and the mantle zone regions of secondary follicles in the spleen. Using these mice we have been able to show that one of the central functions of normal RF-expressing B cells may be to act as highly efficient antigen-presenting cells for low concentrations of immune-complexed antigen. High levels of secretion of IgM RF can not be induced under normal circumstances, although RF-expressing B cells proliferate well in vitro to both aggregated human IgG and anti-human IgM antibodies. However, these mice are not intrinsically secretion deficient. By crossing the RF transgenic mice with the autoimmune MRL/lpr background, we find a dramatic increase, > 200-fold, in levels of serum RF. The results strongly suggest that a major function of normal resting RF B cells is unrelated to antibody secretion. Rather, the RF B cells in the follicles may play a role in antigen presentation and regulation of immune responses to antibody-bound nonself-, and possibly self-antigens. This physiologic role of RF B cells may be disrupted in RF-associated autoimmune disease.

Immunostimulatory DNA sequences necessary for effective intradermal gene immunization.

Vaccination with naked DNA elicits cellular and humoral immune responses that have a T helper cell type 1 bias. However, plasmid vectors expressing large amounts of gene product do not necessarily induce immune responses to the encoded antigens. Instead, the immunogenicity of plasmid DNA (pDNA) requires short immunostimulatory DNA sequences (ISS) that contain a CpG dinucleotide in a particular base context. Human monocytes transfected with pDNA or double-stranded oligonucleotides containing the ISS, but not those transfected with ISS-deficient pDNA or oligonucleotides, transcribed large amounts of interferon-alpha, interferon-beta, and interleukin-12. Although ISS are necessary for gene vaccination, they down-regulate gene expression and thus may interfere with gene replacement therapy by inducing proinflammatory cytokines.

Exercise capacity reflects airflow limitation rather than hypoxaemia in patients with pulmonary arteriovenous malformations.

BACKGROUND: Pulmonary arteriovenous malformations (PAVMs) generate a right-to-left shunt. Impaired gas exchange results in hypoxaemia and impaired CO2 clearance. Most patients compensate effectively but some are dyspneic, and these are rarely the most hypoxaemic. AIM: To test degrees of concurrent pathology influencing exercise capacity. DESIGN: Replicate, sequential single centre, prospective studies. METHODS: Cardiopulmonary exercise tests (CPETs) were performed in 26 patients with PAVMs, including individuals with and without known airflow obstruction. To replicate, relationships were tested prospectively in an independent cohort where self-reported exercise capacity evaluated by the Veterans Specific Activity Questionnaire (VSAQ) was used to calculate metabolic equivalents (METs) at peak exercise (n = 71). Additional measurements included oxygen saturation (SpO2), forced expiratory volume in 1 s (FEV1), vital capacity (VC), fractional exhaled nitric oxide (FeNO), haemoglobin and iron indices. RESULTS: By CPET, the peak work rate was only minimally associated with low SpO2 or low arterial oxygen content (calculated as CaO2=1.34 × SpO2 × haemoglobin), but was reduced in patients with low FEV1 or VC. Supranormal work rates were seen in patients with severe right-to-left shunting and SpO2 < 90%, but only if FEV1 was >80% predicted. VSAQ-calculated METS also demonstrated little relationship with SpO2, and in crude and CaO2-adjusted regression, were lower in patients with lower FEV1 or VC. Bronchodilation increased airflow even where spirometry was in the normal range: exhaled nitric oxide measurements were normal in 80% of cases, and unrelated to any PAVM-specific variable. CONCLUSIONS: Exercise capacity is reduced by relatively mild airflow limitation (obstructive or restrictive) in the setting of PAVMs.

Embolisation of pulmonary arteriovenous malformations: no consistent effect on pulmonary artery pressure.

Increasing evidence supports the use of embolisation to treat pulmonary arteriovenous malformations (AVMs). Most pulmonary AVM patients have hereditary haemorrhagic telangiectasia (HHT), a condition that may be associated with pulmonary hypertension. The current authors tested whether pulmonary AVM embolisation increases pulmonary artery pressure (P(pa)) in patients without baseline severe pulmonary hypertension. P(pa) was measured at the time of pulmonary AVM embolisation in 143 individuals, 131 (92%) of whom had underlying HHT. Angiography/embolisation was not performed in four individuals with severe pulmonary hypertension, whose systemic arterial oxygen saturation exceeded levels usually associated with dyspnoea in pulmonary AVM patients. In 143 patients undergoing pulmonary AVM embolisation, P(pa) was significantly correlated with age, with the most significant increase occurring in the upper quartile (aged >58 yrs). In 43 patients with repeated measurements, there was no significant increase in P(pa) as a result of embolisation. In half, embolisation led to a fall in P(pa). The maximum rise in mean P(pa) was 8 mmHg: balloon test occlusion was performed in one of these individuals, and did not predict the subsequent rise in P(pa) following definitive embolisation of the pulmonary AVMs. In the present series of patients, which excluded those with severe pulmonary hypertension, pulmonary artery pressure was not increased significantly by pulmonary arteriovenous malformation embolisation.

Blocking of cytotoxic T cell function by monoclonal antibodies against the CD45 antigen (T200/leukocyte-common antigen).

Cytotoxic T cells are important effectors in graft rejection and antiviral immunity. A full identification of the functional surface molecules used by these cells may help in determining the best strategies for the therapeutic control of rejection responses. Many T cell surface molecules have been implicated in cytotoxic function through the ability of specific monoclonal antibodies to inhibit T cell activity in vitro. Such measures of "inhibition" must be affected by the avidity of interaction of antibody with the particular surface molecule, as well as the avidity of that surface molecule for any physiological ligand. We show that the introduction of an antiglobulin step substantially amplifies the inhibitory effects of certain CD3 and CD8 monoclonal antibodies, presumably by conferring multivalency. In addition the "antiglobulin" approach has permitted, for the first time, the demonstration that monoclonal antibodies to the "common" determinants of the leukocyte-common antigen family (CD45/LCA/T200) prevent cytotoxic T cell function.

Regulation of rheumatoid factor synthesis.

Rheumatoid arthritis is associated with high titers of IgM and IgG autoantibodies against IgG (rheumatoid factors, RF) and is prevalent in individuals with the HLA haplotypes Dw4, Dw14 and DR1. Our investigations have aimed to determine the molecular genetic basis for RF autoantibody synthesis in people, in an effort to understand eventually how RF production may become improperly regulated in rheumatoid patients. The results have defined several cross-reactive idiotypes on the heavy and light chains of RFs that are serologic markers for specific immunoglobulin variable region genes. These autoantibody associated genes are highly conserved in human populations and are preferentially rearranged and expressed during early B cell development, and in certain lymphoproliferative diseases. They may be associated with a B cell subpopulation that is important for the processing and presentation to T cells of protein antigens trapped in immune complexes. These RF-associated idiotypes are eventually lost during the T cell dependent antibody diversification that accompanies rheumatoid arthritis. The stimuli for the diversification have not been clearly established. However, the rheumatoid arthritis disease susceptibility determinant on the beta-1 chain of individuals with the HLA Dw4, Dw14 or DR1 haplotypes is reproduced by the gp110 protein of the Epstein-Barr virus, and is a potent stimulus for T cell proliferation. Moreover, anti-gp110 antibodies are abundant in rheumatoid arthritis patients. Thus, it is possible that their continual binding and processing of gp110-IgG immune complexes by RF precursor B cells in the joints and other extravascular sites may lead to the emergence of self-reactive T cells that can trigger anti-IgG autoantibody synthesis in the absence of an external antigen.

Probing of the rheumatoid factor (RF) V gene repertoire in rheumatoid arthritis (RA) by hybridoma clones.

Twenty human monoclonal antibodies with rheumatoid factor (RF) specificity were produced from fusions using B lymphocytes derived from the synovial tissue of two patients with rheumatoid arthritis (RA) and one with the polyarticular form of juvenile rheumatoid arthritis (JRA) (1). All the 20 monoclonal antibodies were IgM. Fourteen of these were classical RFs with specificity restricted to IgG, and included 12 kappa and 2 lambda proteins. When the fine specificity for IgG Fc determinants were investigated most of them showed the Ga specificity. In addition, 5 lambda and 1 kappa monoclonal RF antibodies showed polyreactivity and also reacted with various other antigens than IgG (1). The 14 monoreactive RFs were further studied for the expression of RF-related cross-reactive idiotypes (CRI) and variable heavy (VH) and light chain (VL) subgroups. Only four of the twelve kappa RFs expressed the V kappa III subgroup. Three of them belong to the V kappa IIIb sub-subgroup and expressed the CRI 17.109. One of these 3 clones in addition expressed the VH I associated CRI G6. Five other monoreactive RFs expressed either or both of the VH III associated CRI B6 and D12 (2). Using staphylococcal protein A (SPA) binding as well as Northern blotting techniques (2), studies indicated that 10 out of the 12 RFs studied expressed the VH III regions and 2 expressed the VH I region. These data, both for the heavy and light chains, indicated a different V gene usage by the RF derived from RA patients than by the RF M-components derived from patients with mixed cryoglobulinemia and Waldenström's macroglobulinemia but without RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic immunization for allergy immunotherapy.

Allergic disorders are characterized by the prevalence of immunoglobulin (Ig) isotype E antibodies, and are considered to result from enhanced T helper type-2 (Th2) responses to allergens. A Th2 response is characterized by enhanced humoral responses and the production of IL-4 and IL-5 by CD4(+) T cells (Th2) in response to antigen (1,2). These "Th2 cytokines" enhance the allergic response by inducing B cell isotype switching to IgE (3-5), by inducing undifferentiated Th0 cells to further differentiate into Th2 cells, and by inducing eosinophil growth, and differentiation (5). In addition, the Th2 cytokines inhibit Th0 differentiation into Th1 cells, thereby reducing the recruitment of interferon gamma (IFN-γ) producing Th1 cells that could then down-regulate or modulate the Th2 responses (3,5). Consequently, the ability of a Th2 response to allergens to exert a positive feedback effect leads to a vicious cycle and may explain the exacerbation of allergic responses that follows continued exposure to allergens in atopic humans (2,6).

Inhibition of allergic inflammation in the lung by plasmid DNA allergen immunization.

The nature of the immune response (Th1/Th2) in mice to protein antigens or allergens was compared to that of immunization with pDNA encoding the same antigens. pDNA immunization induced a Th1 response and no IgE antibodies whereas the proteins induced a Th2 response and IgE antibodies. Furthermore, the pDNA induced Th1 response dominated over the protein elicited Th2 response in a secondary immune response. In particular, a preexisting Th2 response (as is the case in allergic patients) did not prevent a new Th1 response to an allergen-pDNA booster injection. The major reason why pDNA immunization induced a Th1 response to allergens was the presence of immunostimulatory non-coding DNA sequences (ISS) in the plasmid constructs having a CpG motif. These ISS caused antigen presenting cells to secrete INF-alpha, INF-beta and IL-12, all cytokines that induce naive T cells to differentiate into CD4+ Th1 cells and CD8+ Tc1 cells. Passive transfer of both Th1 and Tc1 cells from pDNA immunized mice into naive mice inhibited a Th2 response and IgE antibody formation to a subsequent injection of allergen in alum. pDNA immunization or ISS-oligonucleotide injection prior to allergen challenge reduced both immediate type airway sensitivity and late phase allergen induced eosinophil filtration of the lung. Allergen-pDNA immunization may provide a novel type of immunotherapy for the treatment of allergic diseases in man. Since only small amounts of allergen are secreted by the allergen-pDNA transformed cells, allergen-pDNA immunotherapy will unlikely carry the risk of the anaphylactic reactions that are associated with classical allergen injection immunotherapy.

Group B streptococcal disease in infants: a case control study.

OBJECTIVES: To describe and quantify the maternal and neonatal factors associated with Group B streptococcus (GBS) disease in infants <90 days of age. SETTING: Neonatal Units in London, Oxford, Portsmouth and Bristol. PATIENTS: Cases were infants <90 days of age with invasive GBS disease diagnosed between 2000 and 2003, and controls were healthy infants born in the same hospital and in the same birth weight category. MAIN OUTCOME MEASURES: Demographic and clinical data on the mother, baby, birth and neonatal stay. RESULTS: 138 cases and 305 controls were recruited. The majority of cases (74%) presented in the first week of life (early onset, EO); most on day 1 (89%). 65% of EO cases had one or more clinical risk factors (prematurity, prolonged rupture of membranes (PROM), known maternal GBS carriage, fever during labour). A multivariable logistic regression analysis found that the strongest independent associations with GBS disease were known maternal carriage of GBS (odds ratio (OR) 6.9), maternal infection in the peripartum period (OR 4.2) and maximum temperature in labour (OR 2.2 per degrees C). GBS disease was associated with twice the likelihood of PROM and fetal tachycardia (p = 0.05 and 0.07 respectively). EO cases had lower Apgar scores and were more likely to have respiratory distress and convulsions, and to require tube feeding than controls. They spent longer in hospital than controls, requiring longer stays at all levels of care. CONCLUSIONS: Independent of birth weight, a number of maternal, birth and neonatal factors are significantly associated with GBS disease. The management of babies with GBS disease results in an appreciable use of hospital resources.

Generation and characterization of two monoclonal self-associating IgG rheumatoid factors from a rheumatoid synovium.

OBJECTIVE: To study IgG rheumatoid factor (RF) from rheumatoid synovium. METHODS: We fused the K6H6/B5 human-mouse heterohybridoma with unstimulated rheumatoid synovial B cells to generate IgG-RF-secreting hybridomas. RESULTS: The RFs from 2 such hybridomas bound specifically to the Fc fragment of human IgG and self-associated to form immune complexes. Such immune complexes are a major characteristic of the pathogenic IgG-RFs in rheumatoid synovium. CONCLUSION: IgG-RF-secreting hybridomas have been obtained. Analyses may reveal the underlying mechanisms of the induction of IgG-RF.

CD4 monoclonal antibody pairs for immunosuppression and tolerance induction.

A pair of rat anti-mouse CD4 monoclonal antibodies (mAb) have been selected which bind to different epitopes of the molecule. Both the mAb are rat IgG2b and show clear synergistic activity in complement lysis in vitro. When injected together in vivo, they exhibit an improved immunosuppressive effect, compared to each antibody alone, on allogeneic graft rejection, humoral responses and on tolerance induction. Limiting dilution analysis indicates that the in vivo depletion of interleukin 2-producing cells is improved using both mAb by 2-3-fold over that obtained with the individual antibodies. As little as 60 ng per mouse of the CD4 antibody pair was sufficient to allow the induction of tolerance to human gamma-globulin, even without elimination of the CD4+ cells. The results suggest that appropriate antibody pairs may be good candidates for effective immunosuppressive serotherapy in man.