Differential effects of phototherapy, adalimumab and betamethasone-calcipotriol on effector and regulatory T cells in psoriasis.

BACKGROUND: Psoriasis is a chronic T-cell-mediated skin disease with marked social and economic burdens. Current treatments are unsatisfactory, with unpredictable remission times and incompletely understood modes of action. Recent advances in our understanding of the pathogenesis of psoriasis have identified the imbalance between CD4+ T effector cells, particularly the T helper (Th)17 subset, and regulatory T cells (Tregs) as key to the development of psoriatic lesions, and therefore a novel therapeutic target. OBJECTIVES: To quantify in patients the effects of three commonly used psoriasis treatment modalities on the Th1, Th2, Th17 and Treg subsets, and to test whether any change correlates with clinical response. METHODS: Flow cytometry was used to enumerate Th1, Th2, Th17 and Treg subsets in blood and skin of patients with psoriasis before and after receiving any of the following treatments: narrowband ultraviolet B (NB-UVB), adalimumab and topical betamethasone-calcipotriol combination (Dovobet® ) RESULTS: All patients responded clinically to the treatments. NB-UVB significantly increased the numbers of circulating and skin Tregs, while, by contrast, adalimumab reduced Th17 cells in these compartments, and Dovobet had dual effects by both increasing Tregs and reducing Th17 cells. CONCLUSIONS: The differential effects reported here for the above-mentioned treatment modalities could be exploited to optimize or design therapeutic strategies to overcome the inflammatory drivers more effectively and restore the Th17-Treg balance in psoriasis.

Community participation for individuals with spinal cord injury living in Queensland, Australia.

STUDY DESIGN: Sequential mixed method design. OBJECTIVES: Determine factors associated with community participation for individuals with spinal cord injury (SCI). SETTING: Queensland, Australia. METHODS: Phase I consisted of a quantitative telephone survey of 270 people who had sustained a SCI within the past 50 years. To verify and interpret survey findings, Phase II involved a qualitative investigation. One focus group, one dyadic and one in-depth interview were conducted with a separate sample of eight people who had sustained a SCI within the past 50 years. RESULTS: In Phase I, employment, paid or unpaid, was the strongest independent factor associated with community participation, whereas time since injury, completeness of injury, secondary conditions and functional independence were also independently associated. In Phase II, participants expressed that survey findings were consistent with their lived experiences. They explained that overall, they needed a strong reason to participate so that benefits outweigh the effort required to participate. Once out in the community, they recognised that other opportunities for participation arise. CONCLUSION: Rehabilitation services need to support individuals with SCI to find meaningful employment and to engage in activities that provide them with a strong reason to participate.

T cell participation in autoreactivity to NC16a epitopes in bullous pemphigoid.

Bullous pemphigoid is a blistering skin disease characterized by autoantibodies against the NC16a domain of bullous pemphigoid 180. This study was performed to characterize and map the fine specificity of T cell responses to NC16a. Peripheral blood mononuclear cells (PBMC) from a total of 28 bullous pemphigoid patients and 14 matched controls were tested for proliferative and cytokine responses to recombinant NC16a and a complete panel of 21 overlapping peptides spanning this region of BP180. Proliferative responses to NC16A and the peptide panel in the patients with active disease were similar in frequency and magnitude to those in healthy donors, and included late responses typical of naive cells in approximately 60% of each group. Interleukin (IL)-4 responses were slightly stronger for six peptides, and significantly stronger for Nc16a, in patients than in controls. Factor analysis identified factors that separate responses to the peptide panel discretely into IL-4, T helper type 2 (Th2) pattern, interferon (IFN)-γ, Th1 pattern and IL-10 or transforming growth factor [TGF-ß, regulatory T cell (Treg )] pattern. Factors segregating IL-10 versus IFN-γ were predicted by active blistering or remission, and TGF-ß or IL-10 versus IFN-γ by age. Finally, we confirmed a significant up-regulation of IgE responses to BP180 in the patients with pemphigoid. This shows the complexity of T cell phenotype and fine autoreactive specificity in responses to NC16A, in patients and in normal controls. Important disease-associated factors determine the balance of cytokine responses. Of these, specific IL-4 and IgE responses show the strongest associations with pemphigoid, pointing to an important contribution by Th2 cytokines to pathogenesis.

Human interleukin-27: wide individual variation in plasma levels and complex inter-relationships with interleukin-17A.

Although it is widely believed that interleukin (IL)-27 is anti-inflammatory, its role in controlling human immune responses is not fully established. In particular, its interactions with T helper type 17 (Th)17 cytokines are unclear. Our aims were to establish the relationships between IL-27 and proinflammatory cytokines, including IL-17A, in human sera and cultures of peripheral blood mononuclear cells. Plasma IL-27 levels in 879 healthy humans from 163 families varied widely, but with relatively low heritability (19%). Despite IL-27 including a subunit encoded by Epstein-Barr virus-induced gene 3 (EBI3), there was no correlation of levels with serological evidence of infection with the virus. Although IL-27 has been reported to inhibit IL-17A production, we demonstrated a strong positive correlation in sera, but lower correlations of IL-27 with other proinflammatory cytokines. We verified that IL-27 inhibited IL-17A production by human peripheral blood T cells in vitro, but not that it stimulated IL-10 secretion. Importantly, addition of IL-17A decreased IL-27 production by stimulated T cells but had the opposite effect on resting T cells. Together, these data suggest a model whereby IL-27 and IL-17A exerts complex reciprocal effects to boost inflammatory responses, but restrain resting cells to prevent inappropriate activation.

Indoleamine 2,3 dioxygenase contributes to transferable tolerance in rat red blood cell inducible model of experimental autoimmune haemolytic anaemia.

Autoimmune haemolytic anaemia (AIHA) is caused by autoantibodies against red blood cell (RBC) surface antigens that render RBC susceptible to Fc-mediated phagocytosis and complement-mediated lysis. Experimental AIHA can be induced by injection of rat RBC to naive mice, but a lymphocyte-mediated regulatory mechanism eventually suppresses the production of autoantibodies specific for mouse RBC. Critically, this tolerogenic response can be transferred to naive mice by splenocytes from the rat RBC-immunized mouse. Here we investigate whether indoleamine 2,3 dioxygenase (IDO) or the initiators of IDO cascade, including the cytotoxic T lymphocyte antigen (CTLA)-4 receptor and its soluble isoform, contribute to this tolerogenic mechanism. Splenocytes from experimental AIHA mice were transferred adoptively to naive mice under the cover of anti-CTLA-4, anti-soluble CTLA-4 antibodies or IDO inhibitor 1-methyl tryptophan (1-MT). Recipient mice were immunized with rat RBC and levels of antibody against self-RBC and rat-RBC were monitored. Our results indicate that transfer of tolerance to naive recipients is dependent upon IDO-mediated immunosuppression, as mice receiving previously tolerized splenocytes under the cover of 1-MT were refractory to tolerance and developed haemolytic disease upon further challenge with rat RBC. Initiators of IDO activity, CTLA-4 or soluble CTLA-4 did not mediate this tolerogenic process but, on their blockade, boosted antigen-specific effector immune responses.

Predominance of activated, clonally expanded T helper type 17 cells within the CD4+ T cell population in psoriatic lesions.

Recent evidence points to the T helper type 17 (Th17) subset as key in the pathogenesis of psoriasis, but cells of this type in lesions remain to be fully characterized. Here we isolated, enumerated, functionally tested and clonotyped the CD4(+) Th cell population ex vivo from lesional biopsies and paired peripheral blood samples from psoriasis patients. Th17 cells were over-represented dramatically in lesions from all patients, representing 49-93% of CD4(+) Th cells compared with 3-18% in blood. Most lesional Th17 cells produced interleukin (IL)-17A ex vivo without further stimulation and expressed the CD45RO(+) phenotype characteristic of activated or memory cells. There was no increase in 'natural' [CD25(hi) forkhead box protein 3 (FoxP3(+))] regulatory T cells in lesions versus peripheral blood, but there was enrichment of 'induced' IL-10(+) regulatory T cell numbers in biopsies from some patients. The lesional Th17 cells exhibited a bias in T cell receptor Vß chain usage, suggestive of specific expansion by antigen. The therapeutic challenge is to overcome the dominance of overwhelming numbers of such antigen-specific Th17 cells in psoriatic lesions.

Recovering is about living my life, as it evolves: perspectives of stroke survivors in remote northwest Queensland.

PURPOSE: Recovering from stroke in remote Australia has rarely been considered, even though rehabilitation services are generally scarce. The primary purpose of this study was to explore stroke recovery, from the perspective of stroke survivors in remote northwest Queensland (NWQ), to explicate the lens through which they view recovering. The secondary purpose was to explore the role of technology to support stroke survivors in remote locations along their recovery journey. METHODS: A qualitative study was undertaken using elements of constructivist grounded theory for data collection and analysis. Semi-structured interviews were conducted with fifteen stroke survivors and two partners living, working or travelling in remote NWQ. RESULTS: From the participants' perspective, recovering in a remote area after stroke is about living my life, as it evolves by endeavouring to recover my way and navigating my recovery in my world. Technology was only considered helpful when it supported participants to recover their way in their world. CONCLUSION: Recovering from stroke from the perspective of stroke survivors in remote NWQ is about living their life, as they want it to be, and as it unfolds within their own context. Technology only has a place when it can support them to recover their way in their world. These findings reinforce the importance of health professionals listening, learning about, and enabling stroke survivors along their recovery journey, within their remote context and support network.Implications for RehabilitationRecovering from the perspective of stroke survivors is about living their life as it evolves.To support stroke survivors from remote areas, health professionals need to listen to and learn from each stroke survivor about what matters to them, what works for them, and about their world; including the challenges (e.g., switching between services) and enablers (e.g., community support) as the stroke survivor perceives them.Finding ways to utilise the strengths within and around them, may improve the recovery process for the stroke survivor in a remote area, ensuring they can access care that meets their needs in their world.Working together with stroke survivors, health professionals need to consider how technology could help them to live their life, while recovering their way and in their world.

Contribution of Candida albicans cell wall components to recognition by and escape from murine macrophages.

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects of C. albicans innate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion of C. albicans by macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4Delta, pmr1Delta, and mnt3 mnt5Delta), whereas O- and N-linked mannan defects (mnt1Delta mnt2Delta and mns1Delta) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1Delta, hwp1Delta, and als3Delta) and yeast-locked mutants (clb2Delta, hgc1Delta, cph1Delta, efg1Delta, and efg1Delta cph1Delta), was similar to that observed for wild-type C. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and process C. albicans.

Immunomodulation against leukemias and lymphomas: a realistic future treatment?

Immunotherapy offers the potential for cure of malignancy without the side effects too commonly seen with conventional chemotherapy. The efficacy of allogenic transplantation and monoclonal antibodies in hematological malignancies illustrate this principle and are now part of routine care. Newer cell based and molecular approaches aimed at stimulating cytotoxic activity against host derived tumor associated antigens are able to 'boost' anti-tumor immunity as judged by immunological assays in vitro. Although clinically meaningful responses were originally less evident, more promising results are now being reported. Our growing understanding of tumor immunology provide rationales for further improvements in the field.

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.

Upper limb recovery after stroke: the stroke survivors' perspective.

PURPOSE: This study investigated stroke survivors' perspective of upper limb recovery after stroke. The aim was to determine factors other than medical diagnosis and co-morbidities that contribute to recovery. The objectives were to explore how stroke survivors define recovery, identify factors they believe influence recovery and determine strategies used to maximize upper limb recovery. METHOD: A qualitative study consisting of three focus groups and two in-depth interviews was conducted with stroke survivors (n = 19) and spouses (n = 9) in metropolitan, regional and rural Queensland, Australia. Data were analysed using principles of grounded theory. RESULTS: Stroke survivors maximize upper limb recovery by 'keeping the door open' a process of continuing to hope for and work towards improvement amidst adjusting to life with stroke. They achieve this by 'hanging in there', 'drawing on support from others', 'getting going and keeping going with exercise', and 'finding out how to keep moving ahead'. CONCLUSIONS: This study provides valuable insight into the personal experience of upper limb recovery after stroke. It highlights the need to develop training strategies that match the needs and aspirations of stroke survivors and that place no time limits on recovery. It reinforces the benefits of stroke support groups and advocates their incorporation into stroke recovery services. These findings can be used to guide both the development and evaluation of stroke survivor centred upper limb training programmes.

Measurement of T-helper cytokines secreted by cord blood mononuclear cells in response to allergens.

It has been proposed that in utero factors may predispose towards the development of childhood atopy. To test this hypothesis, it will be necessary to measure T-helper cell (Th) cytokines secreted by human cord blood mononuclear cells (CBMC) stimulated by allergens. However, to date, it has proven impossible to measure allergen-specific CBMC secretion of the key Th cytokine interleukin-4 (IL-4) using conventional sandwich ELISA techniques. We report for the first time the successful measurement of IL-4 secreted by CBMC stimulated by the allergens timothy grass pollen and house dust mite extract. The method is an adaptation of a novel cell-based ELISA (celELISA), which demonstrated an increased (up to 20-fold) sensitivity to detect IL-4. The method is simple, precise, is no more costly than a conventional ELISA, and can identify individuals in a general population whose CBMC exhibit different cytokine biases in response to allergens. The frequency distribution of IL-4 and interferon-gamma (IFN-gamma) CBMC responses to allergens in the general population approximates to a log-normal distribution, which will permit the application of linear regression techniques in the identification of in utero factors which influence Th bias.

Serial changes in the galactosylation of autoantibodies and serum IgG in 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 C(H)2 domain oligosaccharide. The aims were to determine whether there are also persistently high levels of G0 autoantibodies or serum IgG in autoimmune haemolytic anaemia (AIHA), and whether any changes in galactosylation over time are related to the course of disease. Autoantibodies eluted from red blood cells, and serum IgG, were obtained from a patient with chronic AIHA over a 21 month period, and the degree of galactosylation measured using a lectin-binding assay. There were wide fluctuations in the galactosylation of autoantibody and serum IgG, but these changes were unrelated to the severity of the anaemia. The galactosylation of autoantibody and serum IgG varied independently, and the autoantibodies were preferentially G0 in comparison with serum IgG in only half of the serial samples. We conclude that AIHA differs from other, systemic autoimmune conditions in that high levels of G0 autoantibodies or serum IgG are not persistent, and that changes in galactosylation do not parallel the course of disease.

Quantitation of erythrocyte-bound IgG subclass autoantibodies in murine autoimmune haemolytic anaemia.

A quantitative and sensitive cellular enzyme-linked immunosorbent assay was developed for determining the number of molecules of IgG of each subclass bound to the surface of murine red blood cells (RBC). To develop standard titration curves, RBC from normal mice were treated with tannic acid and coated with a known concentration of purified myeloma of each IgG subclass. The quantity of each subclass bound to the surface of erythrocytes was determined by calculating the protein concentration of the bound IgG, which was then converted into number of molecules of IgG/RBC. The assay was used to quantify the number of autoantibodies of all four IgG subclass bound to the erythrocytes of mice injected with rat RBC. Twenty one days after the first immunisation, a mean number of 84,000 molecules of IgG1/RBC were detected, which increased to 114,500 molecules/RBC on day 28. On days 56 and 96 the mean concentration of IgG1 remained high, however by day 110 the mean level of IgG1 had decreased slighty to 69,500 molecules/RBC. By contrast, the mean concentration of IgG2a autoantibodies was considerably lower throughout the experiment, starting at 40,200 molecules/RBC on day 21 and dropping to 2,500 molecules/RBC by day 110. The mean quantities of IgG2b and IgG3 autoantibodies were similar to each other, and intermediate between the levels of IgG1 and IgG2a autoantibodies.

Differential effects of immunisation with mycobacterial 65 kD heat shock protein on two models of autoimmunity.

The effects of preimmunisation with the 65 kD mycobacterial heat shock protein (hsp65) on 2 murine models of autoimmunity were compared. Experimental autoimmune haemolytic anaemia (AIHA) can be provoked in mice by repeated injection with rat red blood cells (RBC). In this model, preimmunisation with hsp65 10 days before induction of disease resulted in a partial, but significant, reduction in RBC-bound autoantibody levels measured by Coombs' test. However, preimmunisation with human IgG (hIgG) was associated with a similar suppressive effect. Administration of neither hsp65 nor hIgG affected the direct or indirect anti-rat agglutinin titres of mice subsequently injected with rat RBC. Injection of hsp65 or hIgG prior to induction of AIHA elicited the production of IgG antibodies against the respective immunogen, as judged by enzyme-linked immunosorbent assays. In contrast to the results in experimental AIHA, pristane-induced arthritis (PIA) was effectively prevented by preimmunisation with hsp65, but not with hIgG. It is considered that, whilst hsp65 injection may slightly reduce subsequent anti-RBC autoantibody production in AIHA by antigenic competition, such a mechanism cannot account for the substantial protection against PIA afforded by hsp65 preimmunisation. We suggest that the high, sustained production of anti-hsp65 antibodies observed in mice given hsp65 and pristane may play a role in specifically suppressing arthritogenic immune responses in PIA.

Red cell-reactive non-specific immunoglobulins and autoantibodies in the sera of normal and anaemic dogs.

The levels of red blood cell (RBC) membrane-reactive IgG were measured in sera from normal and anaemic dogs using an enzyme-linked immunosorbent assay with erythrocyte ghosts as the target antigen (g-ELISA). The anaemic dogs were classified as cases of primary autoimmune haemolytic anaemia (AIHA), other anaemias with elevated levels of RBC-bound immunoglobulin detected by a direct enzyme-linked antiglobulin test (DELAT), or DELAT-negative anaemias. The g-ELISA detected IgG capable of binding RBC membranes in all the serum samples tested, and the levels were significantly higher (P < 0.05) in dogs with AIHA than in cases of DELAT-negative anaemia or in healthy animals. When sera were preabsorbed with RBC ghosts, g-ELISA readings were reduced in two of three AIHA cases that were tested, but not in dogs from any other group. There was no correlation between g-ELISA results and serum IgG levels in the cases of AIHA, but there was a significant relationship (rs = 0.74, P < 0.001) between these parameters in the dogs with other DELAT-positive anaemias. When spectrin(s) from the internal RBC membrane skeleton was used as the target antigen in the s-ELISA, sera from all groups of anaemic dogs yielded a wide range of values. Although the levels of spectrin-reactive IgG were significantly higher (P < 0.05) in AIHA cases than normal dogs, suggesting that haemolysis provoked production of anti-membrane skeleton antibodies, the highest reading was recorded in a healthy animal. It is concluded that all canine sera contain IgG non-specifically reactive with RBC membranes and that serum antibodies against the erythrocyte surface can also be detected in some dogs with primary AIHA. Distinct spectrin-reactive serum IgG antibodies can also be demonstrated in normal and AIHA positive dogs.

Red blood cell glycophorins as B and T-cell antigens in canine autoimmune haemolytic anaemia.

Pathogenic autoantibodies from two dogs with autoimmune haemolytic anaemia (AIHA) were shown to react with glycophorin from the canine red blood cell (RBC) membrane. Autoantibodies in both cases bound to purified glycophorin in enzyme-linked immunosorbent assays (ELISAs), and the major autoantigen immunoprecipitated by the antibodies corresponded in apparent molecular mass with glycophorin. Furthermore, neuraminidase treatment of the precipitated antigen, or of canine glycophorin, resulted in identical changes in apparent molecular mass in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Such removal of sialic acid from glycophorins was demonstrated to cause shifts in SDS-PAGE migration that are unique among RBC membrane proteins. In two further cases of AIHA, where autoantibodies did not immunoprecipitate the glycophorin pattern, ELISAs revealed that RBC-reactive IgG was present in serum and RBC elutes, but that these antibodies failed to bind to canine glycophorin. Thus, we consider that autoantibodies specific for glycophorin are present in some, but not all, dogs with AIHA. T-cells from a case of AIHA proliferated in vitro in response to autologous RBC, or to multiple RBC membrane components fractionated by SDS-PAGE. Three fractions, corresponding to major glycophorins, to the RBC anion channel band 3, and to spectrin from the membrane skeleton, were stimulatory. In contrast, T-cells from healthy dogs failed to respond to RBC, or to any blot fractions with the exception, in one animal, of the fraction bearing spectrin. It is suggested that activation of autoreactive T-cells with multiple specificities may be necessary to provide sufficient help for pathogenic autoantibody production.

Autoimmune haemolysis in the dog: relationship between anaemia and the levels of red blood cell bound immunoglobulins and complement measured by an enzyme-linked antiglobulin test.

A direct enzyme-linked antiglobulin test (DELAT) was used to measure the levels of red blood cell (RBC) bound IgG, IgM, IgA and C3 in dogs with autoimmune haemolytic anaemia (AIHA). At presentation, one or more DELAT parameters was raised in each AIHA case, and the RBC were typically coated with immunoglobulin of more than one class, together with C3. There was no relationship between the levels of RBC-bound IgG, IgM or IgA and the severity of the anaemia, although a significant negative correlation (rs = -0.66, P < 0.02) was found between bound C3 and blood haemoglobin concentration. These results indicate that the level of sensitisation of erythrocytes with IgG alone is not a reliable predictor of the severity of haemolysis in different cases, and that the pathogenesis of AIHA can be complex, involving multiple immunoglobulin classes and complement in the destruction of RBC. A significant relationship (rs = 0.63, P < 0.02) was found between serum IgG concentration and haemoglobin levels, and it is suggested that this may be due to free IgG inhibiting the interaction of IgG-sensitised RBC with macrophages. Serial measurements from individual AIHA cases during treatment revealed that the levels of RBC-bound immunoglobulins fell simultaneously with improvements in anaemia. In one dog, a relapse was associated with increases in bound IgG and IgM. Transient relative reticulocytopenia at presentation was common, but was not related to the severity of the anaemia. However, in other cases there was a persistent failure to increase RBC production, which was associated with slower recovery.

Proliferative responses of peripheral blood mononuclear cells from normal dogs and dogs with autoimmune haemolytic anaemia to red blood cell antigens.

Autoimmune haemolytic anaemia (AIHA), one of the most common autoimmune diseases of the dog, is characterised by binding of autoantibody to erythrocyte membrane antigens leading to a decreased red blood cell (RBC) life-span. Failure of self-tolerance with activation of autoreactive T-lymphocytes is thought to play a key role in the initiation of such autoimmune events. Peripheral blood mononuclear cells (PBMC) were obtained from 11 clinically normal dogs, six clinically normal relatives of two littermate dogs which died from AIHA, and four dogs which had recovered from primary AIHA. Cells were stimulated in vitro with a panel of canine RBC-derived antigens (RBC membranes, glycophorin, spectrin, five 15-mer glycophorin peptides), the non-recall antigen keyhole limpet haemocyanin (KLH), and the mitogen concanavalin A (Con A). The kinetics of the proliferative responses to specific antigens were assessed by serially sampling the cultures from days 4 to 10. PBMC from all dogs responded strongly to Con A (day 2) and to KLH (maximal response on days 7 to 10) under appropriate culture conditions. Two of 11 normal dogs responded weakly to RBC membranes (mean stimulation index = 4.25). In contrast, PBMC from all dogs recovered from AIHA responded to RBC membranes (mean SI = 9.2 +/- 2.5) and occasionally to other erythrocyte antigens. Similar responses were recorded with PBMC from dogs related to AIHA cases. It is considered that although normal individuals harbour erythrocyte-reactive lymphocytes, such cells are primed in dogs with AIHA or a genetic susceptibility to this disease.