Reactivity of T cells from women with antibodies to the human platelet antigen (HPA)-1a to peptides encompassing the HPA-1 polymorphism.

The human platelet antigen-1a (HPA-1a) is the most common alloantigenic target in fetal and neonatal alloimmune thrombocytopenia (NAIT). Treatment currently depends on the outcome in previous pregnancies. HPA-1 specific T cell responses were determined in 14 HPA-1a alloimmunized women during or after pregnancies affected by NAIT. Peripheral blood mononuclear cells were incubated with peptides encompassing the Leu33Pro polymorphism (residues 20-39 and 24-45 in both Leu33 (HPA-1a) and Pro33 (HPA-1b) forms) or control recall antigens in the presence of autologous sera and T cell proliferation was measured by (3)H-thymidine incorporation. Control antenatal and postpartum sera suppressed T cell proliferation and use of such sera was avoided. Most patients (86%) responded to the HPA-1a peptides with 64% also having weaker T cell proliferation to the HPA-1b peptides; 14% had no activity towards any peptide despite responding to control antigens. Administration of IVIG during pregnancy appeared to reduce T cell reactivity to HPA-1 peptides. Postnatal anti-HPA-1a T cell responses from women who had a severe history of NAIT (an intracranial haemorrhage in a previous fetus) were greater than those from women with a mild history. This assay may have the potential to predict disease severity if performed prior to or early in pregnancy.

Haplotypes of the low-density lipoprotein receptor-related protein 5 (LRP5) gene: are they a risk factor in osteoarthritis?

OBJECTIVE: Several genome-wide scans have revealed an osteoarthritis (OA)-susceptibility locus on chromosome 11q in close proximity to the low-density lipoprotein receptor-related protein 5 (LRP5) gene. The regulation of bone mass is under the control of LRP5 and since increased bone mass is thought to play a role in the pathology of OA we examined LRP5 polymorphisms and haplotypes to determine if variants of this locus may predispose to OA. METHODS: A UK control population of 187 individuals was examined for five commonly occurring polymorphisms against a cohort of 158 DNAs from patients with knee OA. An additional UK cohort was also examined to confirm the findings of the first study; this second group consisted of 110 knee OA patients. Haplotype analysis was also performed on patient and control DNAs. RESULTS: A study of individual polymorphisms revealed no association with disease. However, haplotype analysis of the initial two populations revealed a common haplotype (C-G-C-C-A) that provided a 1.6-fold increased risk of OA (P(c)=0.021). The data obtained from the second cohort confirmed the initial findings, with a 1.6-fold increased risk observed within this cohort for the risk haplotype (P=0.012). CONCLUSIONS: A closer investigation of LRP5 and associated Wnt signalling molecules in OA will help determine disease aetiology and the development of novel treatment strategies that specifically target the bone compartment.

IL-4 and IL-10 modulate autoimmune haemolytic anaemia in NZB mice.

New Zealand Black (NZB) mice spontaneously develop autoimmune haemolytic anaemia (AIHA). Here the effect of injecting NZB mice with plasmids encoding IL-4 (pIL-4) or IL-10 (pIL-10) on NZB disease was tested. Both constructs delayed the development of anaemia as judged by increased haematocrit values as compared with controls, but neither altered the IgG1 to IgG2 red blood cell (RBC) bound autoantibody levels. The increased haematocrit value was associated temporally with increased RBC bound IgG in NZB mice treated with pIL-10, but not pIL-4. By contrast, up-regulation of splenic macrophage FcgammaRIIb2 mRNA was associated temporally with increased haematocrit values in NZB mice given pIL-4. However, no such increase occurred in NZB mice that inhaled a peptide containing a dominant T-cell epitope, although this treatment is known to bias the autoimmune response towards Th2 and to reduce the severity of anaemia. It is considered that IL-4 treatment, in part, ameliorates NZB anaemia by increasing the expression of the inhibitory FcgammaRIIb2 and thereby reducing the capacity of splenic macrophages to phagocytose autoantibody coated RBC, but that this mechanism does not explain the beneficial effects of the inhaled peptide.

Reduction of human anti-tetanus toxoid antibody in hu-PBL-SCID mice by immunodominant peptides of tetanus toxoid.

Immunotherapy of murine autoimmune and allergic diseases by administration of peptides corresponding to the dominant T cell epitope is a reality. However, problems remain in applying this therapy to reduce antibody responses in humans. To overcome these difficulties, a preclinical system was developed to test the effect of immunodominant peptides from a common antigen, tetanus toxoid (TT), on the long-term human anti-TT response. Individuals whose T cells proliferated against dominant TT peptides were identified. Peripheral blood leucocytes (PBL) from these donors were injected intraperitoneally (i.p.) into mice with severe combined immunodeficiency (SCID) that had been depleted of murine natural killer (NK) cells (hu-PBL-SCID mice). Peptides or PBS were injected i.p. before a further injection of PBL and immunization with TT. The concentration of human IgG and anti-TT in murine plasma was followed for 10 weeks. The total IgG was similar in both groups. By contrast, there was a statistically significant reduction in IgG anti-TT from eight weeks onwards. It is considered that the hu-PBL-SCID model system may provide a means by which the efficacy of peptide immunotherapy for reduction of pathological antibodies in humans can be examined.

Extended haplotypes and linkage disequilibrium in the IL1R1-IL1A-IL1B-IL1RN gene cluster: association with knee osteoarthritis.

The interleukin-1 gene cluster is a key regulator in a number of chronic disease processes. We explored the linkage between nine polymorphic loci in the IL1R1 promoter, eight in the IL1A-IL1B-IL1RN gene complex, and their association with osteoarthritis (OA), a common complex disease associated with low-level inflammation. Using 195 healthy controls, we identified eight novel polymorphisms in the IL1R1 exon 1A region. We found limited LD between IL1R1 and the IL1A-IL1B-IL1RN cluster, although LD within these two individual groups was high. To test association with knee OA, we genotyped 141 patients from Bristol (UK) at the 17 loci. IL1R1 promoter haplotypes showed no association with disease. However, within the IL1A-IL1B-IL1RN complex, we identified a common haplotype conferring a four-fold risk of OA (P=0.00043; Pc=0.0043) and one IL1B-IL1RN haplotype conferring a four-fold reduced risk (P=0.0036; Pc=0.029). To replicate these associations, we subsequently examined 163 knee OA patients from London. Here, the effects of the haplotypes were confirmed: the risk IL1A-IL1B-IL1RN haplotype conferred a two-fold risk of OA (P=0.02), and the protective IL1B-IL1RN haplotype conferred a five-fold reduced risk of OA (P=0.0000008). These results may help to explain the genome-wide scan linkage data and functional observations concerning association between IL-1 and OA.

Dual-energy X-ray absorptiometry applied to the assessment of tibial subchondral bone mineral density in osteoarthritis of the knee.

OBJECTIVE: Plain X-ray is an imprecise tool for monitoring the subchondral bony changes associated with the development of knee osteoarthritis (OA). Our objective was to develop and validate a technique for assessing tibial subchondral bone density (BMD) in knee OA using dual energy X-ray absorptiometry (DXA). DESIGN: Patients with OA of at least one knee underwent DXA scanning of both knees. Regions of interest (ROI) were placed in the lateral and medial compartments of tibial subchondral bone. Weight-bearing plain X-rays and Te (99m) scintiscans of both knees were obtained and scored. RESULTS: One hundred and twelve patients (223 knees) underwent DXA and radiography. Intra-observer CV% was 2.4% and 1.0% for the medial and lateral ROI respectively. Definite OA (Kellgren and Lawrence Grade 2, 3 or 4) was correlated with age-related preservation of subchondral BMD compared to radiographically normal knees. Raised BMD was also associated with subchondral sclerosis, and positive scintigraphy. CONCLUSION: DXA may provide a safe, rapid and reliable means of assessing knee OA. Cross-sectional age-related subchondral tibial BMD loss is attenuated by knee OA.

First-trimester diagnosis and management of pregnancies implanted into the lower uterine segment Cesarean section scar.

OBJECTIVE: To describe first-trimester ultrasound diagnosis and management of pregnancies implanted into uterine Cesarean section scars. METHODS: All women referred for an ultrasound scan because of suspected early pregnancy complications were screened for pregnancies implanted into a previous Cesarean section scar. The management of Cesarean section scar pregnancies included transvaginal surgical evacuation, medical treatment with local injection of 25 mg methotrexate into the exocelomic cavity and expectant management. RESULTS: Eighteen Cesarean section scar pregnancies were diagnosed in a 4-year period. The prevalence in the local population was 1 : 1800 pregnancies. Surgical treatment was used in eight women and it was successful in all cases. The respective success rates of medical treatment and expectant management were 5/7 (71%) and 1/3 (33%). Five women (28%) required blood transfusion and one woman (6%) had a hysterectomy. CONCLUSIONS: Cesarean section scar pregnancies are more common than previously thought. When the diagnosis is made in the first trimester the prognosis is good and the risk of hysterectomy is relatively low.

T-cell specificity in murine autoimmune haemolytic anaemia induced by rat red blood cells.

Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of rat and murine RBC antigens recognized by T-cells from mice with this disease. Splenic T-cells from mice with AIHA proliferated in response to multiple murine RBC membrane components, each of which is recognized by rat RBC induced autoantibodies. Thus, there were responses to murine autoantigen fractions that correspond in apparent molecular mass with the anion channel Band 3, with spectrin from the membrane skeleton and with the high and low molecular mass glycophorins, and the equivalent fractions from rat RBC also stimulated proliferation by T-cells. It was confirmed that purified Band 3 from murine and rat RBC also elicited responses. In contrast with the results in AIHA, T-cells from healthy control mice failed to respond to the antigens from either species, with the exception of proliferation induced by murine spectrin in one experiment and weak responses elicited by rat Band 3. It is suggested that T-cells activated by multiple cross-reactions between rat and murine RBC proteins, and by epitope spreading, are necessary to drive autoantibody production in this model of AIHA.

The effects of pressure on chondrocyte tumour necrosis factor receptor expression.

This work was performed to determine whether one aspect of load, pressure, could alter tumour necrosis factor (TNF) receptor type I (RI) expression on chondrocytes. Encapsulated tsT/AC62, osteoarthritic (OA) or non-arthritic (NA) chondrocytes were centrifuged at speeds representing 5 or 20 MPa, incubated for specific periods, released from alginate and TNFRI and II (TNFRII) expression determined by flow cytometry. Significant (p<0.05, n=4) changes in tsT/AC62 chondrocyte TNFRI expression were apparent 24 hours after application of 20 MPa. Five or 20 MPa increased OA chondrocyte TNFRI expression; chondrocytes from some OA patients were markedly sensitive to 20 MPa. NA chondrocyte TNFRI expression usually decreased in response to 5 and 20 MPa. Significant pressure-induced differences in TNFRI expression between NA and OA groups were apparent at 5, but not 20 MPa. Pressure did not significantly alter TNRFII expression on tsT/AC62, NA or OA chondrocytes. These results suggest a mechanism whereby sensitivity of chondrocytes to the effects of TNFalpha may be increased, in susceptible individuals, in regions of the joint that experience peak loading.

Susceptibility to physiological concentrations of IL-1beta varies in cartilage at different anatomical locations on human osteoarthritic knee joints.

OBJECTIVES: To determine whether cartilage biopsies from specific regions of osteoarthritic knee joints differ in susceptibility to the degradative effects of the amounts of interleukin-1 beta (IL-1 beta; 1-10 pg/ml) found in osteoarthritic joints. To establish whether biopsies are sensitive to the effects of either IL-1 beta or TNFalpha or both catabolic cytokines. METHODS: Cartilage from specified regions of 22 osteoarthritic knee joints was examined. Biopsies were incubated for 14 days without or with IL-1 beta or TNFalpha at physiological concentrations and GAG release into supernatants assessed. RESULTS: Variation was observed in susceptibility to the effects of 1-10 pg/ml IL-1 beta in biopsies from different sites within the same joints and the same site in different joints. The number of regions responding to the cytokine increased significantly (P< 0.0063, Chi square test) with concentration: only 10% (2/21) of all regions tested were susceptible to the effects of 1 pg/ml IL-1 beta, whereas 45% (9/20) were susceptible to the effects of 5 pg/ml and 56% (10/18) to the effects of 10 pg/ml IL-1 beta. Significantly fewer regions (4%, 2/47) responded to both IL-1 beta and TNFalpha (P< 0.047, Chi square test); biopsies from some patients responded to neither cytokine. CONCLUSIONS: IL-1 beta, at the low concentrations detected in joints, can degrade cartilage from susceptible locations. Susceptibility of some cartilage biopsies to the effects of either IL-1 beta and TNFalpha, but not both, suggests the signalling receptors for the two major catabolic cytokines are not usually expressed concurrently. The fact that some biopsies respond to neither cytokine suggests that in some patients the local concentration of inhibitors may be high or that other catabolic stimuli predominate. These results could have important implications for pharmacological intervention strategies.

Mechanism of anti-D-mediated immune suppression--a paradox awaiting resolution?

During pregnancy, women can be immunized by fetal red blood cells (RBCs) of an incompatible blood group. Subsequent transplacental passage of the antibodies can result in fetal morbidity or mortality due to RBC destruction. The administration of anti-D antibodies to D(-) women after delivery of a D(+) infant, and subsequent prevention of Rhesus (Rh) D haemolytic disease of the fetus and newborn, is the most successful clinical use of antibody-mediated immune suppression. The passive IgG anti-D might prevent immunization to D(+) RBCs by an IgG Fcgamma receptor (Fcgamma R)-dependent mechanism such as crosslinking the D-specific B-cell receptor and inhibitory FcgammaRIIb. However, recent murine studies demonstrate that the suppressive effects of antibodies to heterologous RBCs can be Fcgamma R-independent, suggesting other mechanisms might contribute.

Helper T cells in antibody-mediated, organ-specific autoimmunity.

The production of pathogenic autoantibodies in organ-specific autoimmune diseases is largely T cell dependent. For many of these diseases, the precise specificities and cytokine profiles of the T cells that respond to the corresponding autoantigens have now been identified. This knowledge has been exploited to treat some models of antibody-mediated autoimmunity using peptides corresponding to the dominant helper epitopes, giving impetus to the development of a similar approach in the equivalent human diseases.

Tumor necrosis factor alpha can contribute to focal loss of cartilage in osteoarthritis.

UNLABELLED: OBJECTIVE; To evaluate the potential for tumor necrosis factor alpha (TNFalpha)-induced focal loss of cartilage in osteoarthritic (OA) knee joints. DESIGN: Fresh cartilage from specified regions of OA joints was immunostained for TNF-receptor (R) bearing chondrocytes. Cartilage explants from the same regions were cultured with or without small amounts of TNFalpha and cumulative GAG release into supernatants measured. Concentrations of TNFalpha, p55 and p75 soluble (s) TNF-R in supernatants from cultured OA and non-arthritic (NA) synovium were measured by ELISA. RESULTS: TNF-R bearing chondrocytes were identified in OA cartilage; more specimens contained p55 TNF-R- than p75 TNF-R-bearing chondrocytes and differences in TNF-R distribution were apparent in cartilage from different regions of the same knees. TNFalpha at 5, 1, 0.5 and 0.25 ng/ml (but not 0.1 ng/ml) significantly increased glycosaminoglycans (GAG) release from cartilage explants in a dose-dependent manner. Variation in susceptibility to TNFalpha was observed in explants from different sites. TNFalpha and p75 sTNF-R, but not p55 sTNF-R, concentrations were significantly higher in OA, as compared with NA, supernatants. A significant correlation between TNFalpha and p75 sTNF-R measurements was apparent only in NA supernatants. CONCLUSIONS: Variations in chondrocyte TNF-R expression occur in OA cartilage in vivo. TNFalpha at concentrations produced by OA synovium in vitro, can degrade cartilage matrix. In most OA supernatants sTNF-R concentrations were insufficient to abrogate the effects of TNFalpha. Thus conditions exist in some OA knees for TNFalpha to contribute to focal loss of cartilage.

Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat-shock protein 60.

The hypothesis that T-cell responses to the 60 000 MW family of heat-shock proteins (hsp) may be related to the severity of rheumatoid arthritis (RA) was examined. Peripheral blood mononuclear cells (PBMC) from most normal individuals and both early and established RA patients proliferated in vitro in response to human hsp 60 and mycobacterial hsp 65 as well as tetanus toxoid (TT) and mycobacterial purified protein derivative (PPD). PBMC from some patients with established RA gave responses to hsp 60 that were above the normal range and/or peaked earlier than PBMC from normal individuals. The responses of PBMC from established RA to hsp 65, but not PPD or TT, were also higher than those from normal individuals, but the peak responses to all three antigens appeared delayed. Thus a selective increase in responsiveness to hsp 60 develops with disease duration in many RA patients. Six assessments of disease activity and severity were made but apart from rheumatoid factor titre, they were unrelated to the proliferative response. Similarly, disease activity and severity did not differ between those RA patients whose hsp 60 stimulated cells produced interferon-gamma and those who did not, although patients whose hsp 60-stimulated T cells produced interleukin-4 (IL-4) and/or IL-10, appeared to have less disease activity and severity than those who did not. Significant negative correlations were found between IL-10 production by hsp 60-stimulated cells and disease assessments. It is considered that RA is less severe in those patients whose hsp 60-stimulated cells produce T-helper 2 type cytokines.

Increased serum C reactive protein may reflect events that precede radiographic progression in osteoarthritis of the knee.

OBJECTIVE: Raised serum C reactive protein (CRP) and hyaluronate (HA) concentrations predict the progression of knee osteoarthritis (OA) in the long term but the consistency of these relations with time is unknown. The purpose of this work was therefore to determine if raised CRP and HA at entry and three years before entry (-3 years) predict radiological progression of knee OA in a group of patients between entry and five years. METHODS: Knee radiographs from 90 patients with knee OA at entry and five years follow up were assessed for progression of disease over five years. The concentrations of serum CRP and HA were measured at entry (n=90) and also in 40 serum samples available from -3 years. Odds ratios (OR) for predicting progression were calculated by logistic regression. RESULTS: Serum CRP at entry was not predictive of progression between entry and five years (OR 1.12, 95% CI 0.81, 1.55) but serum CRP at -3 years was predictive of progression (OR 1.90, 95% CI 1.01, 3.28). Serum HA concentration at entry predicted progression between entry and five years (OR 2.32, 95% CI 1.16, 4.66). CONCLUSION: These results are consistent with previous reports relating to HA, and suggest that raised serum CRP reflects events that precede a period of later radiographic progression in knee OA. However, because of the large overlap between groups, the serum CRP or HA concentration are not good predictors of individual patient progression and have a poor sensitivity and specificity.

Ability of T cells from patients with rheumatoid arthritis to respond to immunoglobulin G.

The ability of T cells from rheumatoid factor (RF)-positive patients with rheumatoid arthritis (RA) to respond to immunoglobulin G (IgG) was assessed. Peripheral blood mononuclear cells (PBMC) from RA patients and normal individuals were cultured with and without human IgG or Mycobacterium tuberculosis-purified protein derivative (PPD) for 7 days and their proliferative response measured at intervals by their ability to take up tritiated thymidine. PBMC from 14/26 RA patients proliferated in response to IgG (taking a stimulation index of 3 or above as positive). The peak response varied between individuals but usually occurred on day 5, the same day, or 1 day later than the peak response to PPD. By contrast, PBMC from a significantly lower proportion (1/9) of normal individuals and patients with other arthritides (0/6) responded to IgG, although all responded to PPD. PBMC from 9/14 RA patients responded to Fab fragments of IgG but only 3/9 to the Fc fragment. Higher proliferative responses from RA PBMC were elicited by IgG aggregates than the original IgG preparation, but PMBC from 5/5 normal individuals and 5/6 patients with other arthritides failed to respond to the aggregates. The response to IgG was human leucocyte antigen (HLA)-DR restricted and mediated by CD4+ T cells. It is considered that these results advance the hypothesis that IgG-reactive T cells contribute to the initiation or perpetuation of RA.

Galactosylation of serum IgG and autoantibodies in murine models of autoimmune haemolytic anaemia.

A number of systemic autoimmune diseases are associated with increased levels of the agalactosyl (G0) IgG isoforms that lack a terminal galactose from the CH2 domain oligosaccharide. The current aim was to determine whether the galactosylation of serum IgG is also reduced in a classic antibody-mediated, organ-specific autoimmune condition, and whether the pathogenic autoantibodies are preferentially G0. In two murine forms of autoimmune haemolytic anaemia (AIHA), sera and autoantibodies eluted from erythrocytes were obtained, and the levels of G0 measured using a lectin-binding assay. Serum IgG galactosylation was unaffected following the induction of AIHA in CBA/Igb mice by immunization with rat erythrocytes, but in all animals with the disease the IgG autoantibodies generated were more G0 than the sera. The anti-rat erythrocyte antibodies were similar to the autoantibodies in being preferentially G0, and when CBA/Igb mice were immunized with canine erythrocytes as a control foreign antigen, there was again a bias towards the production of G0 IgG antibodies. In NZB mice with chronic, spontaneous AIHA, the concentration and galactosylation of both serum IgG and autoantibodies were lower than in the induced model, and the ratio of G0 IgG in the serum and erythrocyte eluates varied markedly between different individuals. Our interpretation of these results is that changes in serum IgG or autoantibody galactosylation are not consistent in different models of AIHA, and that production of low galactosyl antibodies can be a feature of a normal immune response.

Splenic but not thymic autoreactive T cells from New Zealand Black mice respond to a dominant erythrocyte Band 3 peptide.

Previous work from our laboratory suggested that erythrocyte Band 3 peptide 861-874 is the dominant epitope recognized by splenic T cells from adult New Zealand Black (NZB) mice that are developing autoimmune haemolytic anaemia (AIHA). Here, it is shown that splenic T cells from 6-week-old NZB mice mount a vigorous in vitro proliferative response to peptide 861-874 and some other selected Band 3 peptides. As the donors grow older, splenic T cells respond to an increasing number of Band 3 peptides and the magnitude of their response also becomes greater. Splenic T cells from 3-week-old NZB mice still responded vigorously to peptide 861-874 and Band 3. By contrast, neither thymocytes nor single-positive CD4-enriched thymus cells from NZB mice responded to peptide 861-874 or Band 3, although they responded to concanavalin A (Con A). However, thymocytes from mice expressing a transgenic T-cell receptor (TCR)-specific for myelin basic protein (MBP) peptide Ac 1-9 responded vigorously to Ac 1-9. It is considered that the T-cell response of NZB mice to Band 3 is initially focused on peptide 861-874 and later spreads to other Band 3 peptides as the disease progresses and that peptide 861-874-reactive T cells are primed in the periphery rather than the thymus.