Cytokine profiles in high risk injection drug users suggests innate as opposed to adaptive immunity in apparent resistance to hepatitis C virus infection.

A cohort of injection drug users (IDU) have been identified who despite a long history of IDU and sharing of injecting equipment remain seronegative and aviraemic for hepatitis C virus (HCV). They have been termed HCV exposed uninfected (EU). The study of potential innate or adaptive immune mechanisms of resistance to HCV infection in this group is of interest. The aim of this study was to determine the levels of a broad range of cytokines in serum of exposed, uninfected individuals to ascertain whether there is a specific cytokine profile associated with apparent resistance to HCV. Sera from 22 EU individuals were analysed for a range of cytokines and chemokines, and compared to 16 treatment-naive chronic HCV cases (HCV Ab+ RNA+), 16 individuals with spontaneous resolution of HCV (HCV-Ab+ and HCV-RNA-) and 10 healthy unexposed controls. EU subjects had strikingly higher levels of both IL-6 (on average more than 100-fold, P = 0.001) and IL-8 (on average more than 10-fold, P < 0.001) than the comparison groups. Additionally higher levels of tumour necrosis factor-alpha (TNF-α; on average up to threefold, P = 0.02) were seen in EU individuals. The levels of interferon-alpha (IFN-α) were upregulated in all HCV exposed groups in comparison to healthy controls (P = 0.013). Adaptive immune cytokine levels were no different between the groups. Cytokine profiling demonstrated raised levels of pro-inflammatory innate immune cytokines and chemokines in EU IDU, in particular interleukin-6 and interleukin-8. These findings suggest innate immune activation may be the key to prevention of infection in this cohort.

The value of serum neopterin, interferon-gamma levels and interleukin-12B polymorphisms in predicting acute renal allograft rejection.

Acute rejection remains a poor predictor of graft outcome. In this study, we measured serum levels of interferon (IFN)-gamma and neopterin by enzyme-linked immunosorbent assay and a single nucleotide polymorphism (SNP) within the 3' untranslated region of the interleukin (IL)-12 B gene (1188 A/C) to determine whether either of these factors could predict acute rejection in renal transplantation. Significantly higher early post-transplant neopterin levels (days 5-7; 35.7 versus 19.9 nmol/l) were observed in recipients who subsequently rejected their grafts. Post-transplant neopterin levels showed a strong positive correlation with 1-month creatinine levels (Spearman's correlation 0.62, P < 0.001), suggesting macrophage activation early after transplantation. Pretransplant neopterin and IFN-gamma levels and the IL-12B gene SNP did not predict acute rejection in this small retrospective study. The ability to predict acute rejection non-invasively early after transplantation could lead to individual tailoring of immunosuppressive regimens and perhaps lead eventually to longer graft survival.

Effect of stress on basophil function in chronic idiopathic urticaria.

BACKGROUND: Chronic idiopathic urticaria (CIU) is a distressing skin condition involving recurrent itchy hives lasting 6 weeks or longer. The mechanism involves mast cell and basophil degranulation, which releases inflammatory mediators including histamine. In our clinical practice, we have observed that the onset of CIU is often preceded by a major life event. OBJECTIVE: To investigate the role of the hormones of the hypothalamic-pituitary-adrenal (HPA) axis in the link between psychological stress and CIU. METHODS: Thirty people with CIU and 30 normal controls were recruited. A flow cytometric CD63 expression assay was used to quantify basophil activation, and serum cortisol concentrations were measured as an indication of stress. RESULTS: Both corticotrophin releasing factor (CRF) and adrenocorticotrophic hormone (ACTH) were shown to activate basophils. There was no significant difference between numbers of CIU patients and normal controls responding to CFR, ACTH or cortisol. However, the responses in the CIU patients were stronger than those in normal controls. There was also a trend towards higher serum cortisol concentrations in CIU patients. The basophil response to CRF and ACTH correlated with the serum cortisol concentration in normal controls, but not in CIU patients. CONCLUSIONS: Although our data have not supported the hypothesis that stress makes a major contribution to CIU, the heightened basophil response to CFR and ACTH and higher levels of serum cortisol do suggest a derangement of the HPA axis in CIU.

A study of cytokine protein secretion, frequencies of cytokine expressing cells and IFN-G gene polymorphisms in normal individuals.

BACKGROUND: Cytokines are major regulators of immune responses, and there is evidence that they play a role in allograft rejection. Before embarking on a detailed study of pretransplant cytokine profiles in renal allograft recipients, we wished to investigate variations in cytokine protein secretion, numbers of cytokine expressing T cells, and cytokine gene polymorphisms in normal volunteers. METHODS: Twenty normal healthy volunteers were studied. Cytokine protein secretion [interleukin- (IL) 2, IL-4, IL-10, and interferon- (IFN) y] and numbers of cytokine expressing CD3+ T cells (IL-2, IL-4, IL-10, and IFN-gamma) were quantified by means of enzyme-linked immunosorbent assay and two-color flow cytometry respectively. IFN-gamma gene polymorphisms were determined by polymerase chain reaction and autoradio graphy. RESULTS: Large interindividual variations in both the quantity of IL-2, IL-4, IL-10, and IFN-gamma cytokine protein secreted and numbers of IL-2 and IFN-gamma expressing T cells were demonstrated. However, numbers of IL-4 and IL-10 expressing cells were found to be below detectable limits by flow cytometry. In the case of IFN-gamma, a bi-modal distribution was seen for the quantity of protein secreted. In addition, correlations were observed between IL-2 protein and frequency of IL-2 expressing T cells. However, no relationship was found between IFN-gamma protein levels, numbers of IFN-gamma expressing cells and IFN-gamma gene polymorphisms. CONCLUSIONS: We have demonstrated large differences in both numbers of T helper 1 cytokine expressing cells and the quantity of T helper 1 and T helper 2 cytokine protein secreted between normal individuals. Although the amount of IL-2 protein secreted appeared to be determined by the frequency of IL-2 expressing cells, this was not the case for IFN-gamma.

In vitro cytokine profiles and their relevance to rejection following renal transplantation.

Graft rejection remains an important cause of renal allograft failure, despite improvements in immunosuppression and HLA typing. Although HLA matching is beneficial, ensuring an exact match it is often impractical. Thus, a reliable in vitro method for quantitating and qualitating alloreactivity is an important goal. In this study, we measured in vitro the cytokine secretion profiles of mononuclear cells from patients prior to renal transplantation by stimulating with anti-CD3 monoclonal antibody and suppressing with cyclosporine. Mononuclear cells from patients who subsequently developed acute cellular rejection secreted higher mean levels of interleukin (IL)-2 and gamma-interferon (IFN-gamma) than those from patients who had no rejection episodes. IFN-gamma secretion was significantly associated with rejection (P = 0.002), whereas IL-2 secretion did not quite reach statistical significance. There was no significant correlation between IL-4 levels and rejection. Although cyclosporine suppressed the secretion of both IL-2 and IFN-gamma, there was no difference in sensitivity to suppression between rejectors and nonrejectors. These results further emphasize the importance of the TH1 lymphocyte subset in renal allograft rejection. The IFN-gamma secretory capacity of alloreactive T cells may influence the outcome of a renal allograft by (1) activating graft infiltrating macrophages and/or (2) up-regulating HLA molecules on the graft.

How important is histocompatibility in bone marrow transplantation?

The main issue addressed at the Fifth International Workshop on Bone Marrow Transplantation for Leukaemia was the importance of the major histocompatibility complex (MHC) and of minor histocompatibility (minor H) systems in allogeneic bone marrow transplantation (BMT) in humans. It is well established that mismatching for either HLA or minor antigens increases the risks of graft-versus-host disease (GVHD) and graft failure. In HLA-identical sibling transplants minor antigen differences may be responsible for these problems, which might be reduced by matching for minor antigens and improving methods for immunosuppression in vivo. If a suitable family member is not available, then a 'matched' unrelated donor (MUD) may be an alternative. At present MUD transplants have greater risks of GVHD and graft failure and consequently poor survival than HLA-identical sibling transplants; this may be due to unrecognized MHC polymorphisms or to multiple minor H differences. Possible strategies to improve the results of MUD transplants include better HLA matching, new in vitro functional tests for optimizing donor selection and better immunosuppression. Approaches for improving HLA typing include isoelectric focusing for class I and RFLP/allogenotyping or oligonucleotide typing for class II antigens. One obvious disadvantage of exploiting more precise methods for donor matching is the possibility of excluding donors with biologically unimportant histocompatibility differences, which would increase rather than reduce the proportion of patients without donors in a given panel. Functional assays could help in selecting donors on the basis of 'intelligent mismatching' within the HLA system.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxic T lymphocyte precursor (CTL-p) frequency analysis in unrelated donor bone marrow transplantation: two case studies.

HLA 'matched' unrelated donor bone marrow transplantation (BMT) is associated with an increased incidence and severity of acute graft-versus-host disease (GVHD) in comparison with HLA-identical sibling transplants. Using a limiting dilution analysis system for quantitating frequencies of alloreactive cytotoxic T lymphocyte precursors (CTL-p), we previously demonstrated a correlation between CTL-p frequency and HLA disparity between responder and stimulator, and between CTL-p frequency and the incidence of acute GVHD following HLA A, B, DR matched unrelated donor BMT. In this study we assayed CTL-p frequencies in two HLA 'matched' unrelated donor/patient pairs, with single HLA antigenic mismatches detected by allogenotyping or isoelectric focusing but not by HLA serology, and demonstrated that the CTL-ps were specifically directed at the mismatched antigen. Both class I and class II antigens were detected. These data, and our previous work, suggest that high CTL-p frequencies in HLA 'matched' unrelated pairs are indicative of HLA antigenic variants undetected by serology but recognized by molecular typing, and that these are responsible for the value of the assay in predicting acute GVHD after BMT. We propose that this assay system be used in aiding final donor selection before unrelated or mismatched related donor BMT.

Effect of beta propiolactone viral inactivation on alpha1 antitrypsin values.

AIMS: alpha1 Antitrypsin was undetectable in several patient samples treated with 0.5% beta propiolactone, which was used as a virucidal agent. This study was designed to confirm beta propiolactone as the cause and determine why it might have such an effect. METHODS: Volumes of 0, 5, 10, and 20 micro l of beta propiolactone were added to 2 ml aliquots of serum to make final concentrations of 0%, 0.25%, 0.5%, and 1% of beta propiolactone. alpha1 Antitrypsin concentrations and the pH were measured at different time intervals. The effects of adding buffer before the addition of beta propiolactone, NaOH after beta propiolactone, and 6M HCl instead of beta propiolactone were also measured. RESULTS: The addition of beta propiolactone to a volunteer's serum showed a fall in both alpha1 antitrypsin values and pH with increasing time and concentration of beta propiolactone. This effect was also seen when adding HCl, but was partially prevented by buffering the serum or adding NaOH. CONCLUSIONS: These results suggest that it is the acidity of the degradation products of beta propiolactone that is responsible for the fall in alpha1 antitrypsin values. This fall in alpha1 antitrypsin values was dependent on the concentration of beta propiolactone used and the length of time before the test was performed. The effect of beta propiolactone on laboratory tests should be re-evaluated, with attention being paid to sample pH, storage time, and storage temperature.

Cytokine secretion in mixed lymphocyte culture: a prognostic indicator of renal allograft rejection in addition to HLA mismatching.

We have previously demonstrated significant inter-individual variations in cytokine protein secretion between normal individuals and patients prior to renal transplantation. In this study, pre-transplant patient vs. donor mixed lymphocyte cultures (MLC) were set up between 57 renal allograft patient/donor pairs, and secretion of cytokine protein (IL-2, IL-4, IL-6, IL-10 and IFN-gamma) into the culture supernatant measured by ELISA. Significant inter-individual variations in protein secretion in MLC were observed for all cytokines studied. Univariate analysis demonstrated that high levels of IFN-gamma and IL-10 in MLC and spontaneous IL-4, together with female donor sex and a high degree of HLA mismatching (especially HLA-DR) were significantly associated with rejection. However, multivariate analysis revealed the greatest risk of rejection (RR = 25.5, P = 0.003) was associated with a combination of high IL-10 secretion in MLC and mismatching for at least four HLA antigens (HLA-A, -B and -DR). It remains to be determined whether cytokine secretion in MLC is linked to cytokine gene polymorphisms. In future, assays for measuring either cytokine secretion or genetic polymorphisms may prove to be useful in aiding donor selection and tailoring immunosuppressive therapy.

A study of cytokine gene polymorphisms and protein secretion in renal transplantation.

Although there is evidence that cytokine gene polymorphisms are associated with varying quantities of cytokine protein production, the exact role of these polymorphisms in allograft rejection remains unclear. In a previous study, we demonstrated a significant association between high IL-10 secretion in mixed lymphocyte culture (MLC), together with HLA mismatching for at least 4-6 antigens, with the occurrence of acute rejection following renal transplantation. We, therefore, wished to ascertain whether cytokine gene polymorphisms are associated with varying levels of protein secretion and/or allograft rejection in the same group of patients. Cytokine protein secretion in MLC for IL-4, IL-6, IL-10 and IFN-gamma was measured by ELISA in 49 patient-donor pairs. Protein secretion for the above cytokines was also measured in phytohaemagglutinin (PHA) stimulated cultures in 30 normal controls. In both patient and control groups, single nucleotide polymorphism analysis for IL-4 G(-590)T, IL-6 G(-174)C, IL-10 G(-1082)A, IL-10 C(-819)T, IL-10 C(-592)A, TNF-alpha G(-308)A and microsatellite analysis for IFNG (CA repeat) was performed. No correlation was found between cytokine gene polymorphisms and cytokine protein secretion in either mitogen stimulated cultures (control group) or MLC (patient group). In addition, no correlation was demonstrated between cytokine gene polymorphisms and renal allograft rejection.

Dendritic cells and their potential therapeutic role in haematological malignancy.

The generation of an effective immune response is dependent on the efficient capture and presentation of antigen by antigen-presenting cells. The most potent antigen-presenting cells are dendritic cells (DC). These cells have the capability of activating naive helper and cytotoxic T cells. In recent years it has been demonstrated that in vivo responses to a number of solid tumours can be generated by DC pulsed with either purified tumour antigen or whole tumour cell lysate. In addition, a number of in vivo studies using DC have also been attempted in solid tumours, with some encouraging results. In haematological malignancies, there is now strong evidence that previous T cell anergy can be reversed and significant anti-tumour immune responses generated, in vitro, against the majority of leukaemias. As far as in vivo studies in haematological malignancies are concerned, although T cell responses have been demonstrated in the majority of cases and some dramatic early clinical responses reported, overall results appear disappointing. However, considering the fact that many of these studies were performed in patients with advanced disease and that such therapeutic strategies are still in their infancy, the overall results are actually quite encouraging. Although there is a real potential for DC immunotherapy in the future, it is important to be realistic about the limitations and obstacles to its development. It is highly unlikely that any form of immunotherapy is going to be effective in advanced disease due to the physical bulk of tumour, the immunosuppressive effects of tumours themselves and to any secondary immunosuppression following standard cancer therapy. The potential for immunotherapy is likely to lie either in adjunctive therapy or for treating minimal residual disease. Even in those situations, one of the major obstacles to be overcome is the state of immunological anergy or tolerance that many tumours seem able to induce. Indeed, there is evidence that, under certain circumstances, DC themselves can present antigen in such a way as to produce this state of anergy. Although, in vitro manipulation of DC and T cells can generate tumour-specific T cells from previously "anergic" cells, once reintroduced in vivo, these cells will be re-exposed to the tumour environment with the risk of being rendered anergic again.

Abnormal T-cell function in B-cell chronic lymphocytic leukaemia.

There is increasing evidence of T cell dysfunction in B cell chronic lymphocytic leukaemia (B-CLL) which may contribute to the aetiology and progress of the disease. An absolute CD8+ lymphocytosis correlates with disease progression and low expression of CD4 and CD8 (as found in autoimmune disease) is seen with abnormal expression of other surface molecules. Although the expression of T cell surface activation markers, CD25 and CD152, may be increased on culture in B-CLL serum, response to the common mitogens, PHA and PWM, is reduced. This and the excess of CD8 cells may explain partly the variable cooperation of T cells with B cell production of immunoglobulin in B-CLL. In the context of T cell cross-talk with antigen presenting cells, B-CLL B cells are poor antigen presenters. But the T cells themselves have significant abnormalities of expression of the many antigens and ligands necessary for this process. In particular, they exhibit variable expression of the low affinity and non-specific adhesion molecules LFA-1 and ICAM-1, variable, clonally restricted and skewed expression of the TCR repertoire (implying repeated antigenic stimulation possibly by CLL antigens), reduced CD28 and CD152 expression (implying impairment of ability to start or stop an immune response) and reduced IL2 and CD25 (IL2 R) expression (critical for positive feed-back in maintenance and expansion of the T cell response to antigen presentation). Although the production of IL2 and other cytokines by the T cell in B-CLL may be impaired, production of the anti-apoptotic cytokine IL4 is not and there may be a unique and expanded subset of CD8/CD30 cells capable of releasing IL4. The relationship of this T cell subset to the malignant B cell in vivo is unknown. However, T cells which are CD4+/CD152+/CCR4+ migrate selectively in vitro in response to the chemokine CCL22 (specific for the receptor CCR4) produced by the malignant B cells and are always seen amongst the malignant cells in bone marrow and lymph nodes from B-CLL patients. Other abnormalities of cytokine secretion are described. These findings suggest that the T cell in B-CLL may be unable to start, maintain and complete an immune response to the malignant B cell and other antigens and may be involved directly in sustaining the tumour. However, autologous tumour specific cytotoxicity has been shown in vitro and T cells which recognise tumour-derived heavy chain fragments circulate in vivo. If adoptive immunotherapy of any nature is to succeed in B-CLL, manipulation to optimise these CTL responses is needed to overcome the profound and variable T cell dysfunction in this disease.

Pre-incubation with interleukin-4 mediates a direct protective effect against the loss of pancreatic beta-cell viability induced by proinflammatory cytokines.

Loss of pancreatic beta-cells in type I diabetes is associated with an increase in T helper 1 (Th1) proinflammatory cytokines in the islet milieu, with a concomitant reduction in Th2 anti-inflammatory cytokines. In animal models, manoeuvres designed to polarize Th1 responses towards Th2, particularly involving interleukin (IL)-4, have been shown to protect against insulitis and diabetes. The aim of this study was to determine whether IL-4 can exert a direct effect on beta-cell viability. The rat pancreatic beta-cell line, BRIN-BD11, was used. IL-4R mRNA expression was assayed by reverse transcription-polymerase chain reaction and DNA sequencing and protein expression measured using anti-IL-4R antibodies and confocal microscopy. Cells were pretreated in vitro with IL-4, incubated with IL-1beta and interferon (IFN)-gamma and DNA fragmentation and nitrite production analysed by flow cytometry and Griess assay, respectively. Expression of type I (IL-4R alpha and common gamma-chain) and type II (IL-4R alpha, IL-13R alpha-1) IL-4R mRNA transcripts, together with cell surface expression of IL-4R, was demonstrated. Pre-incubation with IL-4 reduced significantly cell death induced by IL-1beta alone or by a combination of IL-1beta and IFN-gamma, although this was not accompanied by a reduced production of nitrite. The protective effect of IL-4 was not seen when all three cytokines were added simultaneously. These results demonstrate, for the first time, expression of IL-4 receptor components on rat pancreatic beta-cells and reveal a direct protective effect on the loss of viability mediated by proinflammatory cytokines when beta-cells are pre-incubated with IL-4.

In vitro dendritic cell-induced T cell responses to B cell chronic lymphocytic leukaemia enhanced by IL-15 and dendritic cell-B-CLL electrofusion hybrids.

HLA class II-restricted proliferative and cytotoxic T cell (CTL) responses to B cell chronic lymphocytic leukaemia (B-CLL) can be generated using autologous dendritic cells (DCs) pulsed with tumour cell lysate. In this study a number of different approaches were used to optimize further the in vitro system. First, the effects of a variety of maturation agents were studied. The addition of TNF-alpha, polyriboinosinic polyribocytidylic acid (Poly(I:C)) and LPS to autologous DCs resulted in the emergence of only a small percentage of CD83+ DCs, IFN-alpha having no demonstrable effect. Only the addition of Poly(I:C) to DCs resulted in modestly increased specific cytotoxicity to B-CLL targets, IFN-alpha and LPS having no effect. Secondly, T cells were pretreated with IL-15, prior to culturing with lysate-pulsed autologous DCs. A significant increase in T cell activation (P = 0.038), IFN-gamma secretion (P = 0.030) and specific cytotoxicity to B-CLL targets (P = 0.006) was demonstrated compared to untreated T cells. Thirdly, monocyte derived DCs electrofused with B-CLL B cells were compared with lysate-pulsed DCs. T cells stimulated by fused DCs generated higher levels of specific cytotoxicity to autologous B-CLL B cell targets than those stimulated by lysate pulsed DCs (P = 0.013). Blocking studies demonstrated inhibition of this cytotoxicity by both anti-CD4 (P = 0.062) and anti-CD8 monoclonal antibodies (P = 0.018), suggesting the generation of both HLA class I- and HLA class II-restricted CTL responses. In summary, in vitro B-CLL-specific T cell responses can be enhanced further by preincubating T cells with IL-15 and using autologous fused DC-B-CLL hybrids instead of autologous lysate-pulsed DCs. These preliminary data require confirmation with larger numbers of patients. Such an approach, however, may eventually provide effective immunotherapy for treatment of B-CLL.

Generation in vitro of B-cell chronic lymphocytic leukaemia-proliferative and specific HLA class-II-restricted cytotoxic T-cell responses using autologous dendritic cells pulsed with tumour cell lysate.

Immunotherapy using dendritic cells has shown encouraging results in both haematological and non-haematological malignancies. In this study, monocyte-derived dendritic cells from patients with B-CLL were cultured for 6 days in the presence of IL-4 and GM-CSF. Autologous B-CLL T-cells were cultured alone or with B-CLL lysate-pulsed and unpulsed autologous dendritic cells. IFN-gamma secretion was assessed using ELISA. Cytotoxicity was assessed, after 21 days in culture and re-stimulation, using flow cytometry with and without blockade by anti-HLA class I, anti-HLA class II, anti-CD4, anti-CD8 and anti-TCRalphabeta monoclonal antibodies. B-CLL T cells stimulated with B-CLL lysate-pulsed autologous dendritic cells showed a significant (P = 0.0004) increase in IFN-gamma secretion and a significant (P = 0.0008) increase in specific cytotoxicity to autologous B-cell targets, but none to autologous T cell or B cell targets from healthy individuals. B-CLL T cells cultured with (non-B-CLL) B-cell lysate-pulsed B-CLL dendritic cells showed no significant response. Pulsing dendritic cells from healthy volunteers with an autologous (non-B-CLL) B-cell lysate did not stimulate proliferation, cytokine production or cytotoxicity by autologous T cells. Pulsing B-CLL dendritic cells with allogeneic B-CLL lysates and culturing with autologous T-cells elicited cytotoxicity against autologous B-CLL targets in some cases, but not in others. Cytotoxicity was significantly reduced by blocking with anti-HLA class II (P = 0.001), anti-TCRalphabeta (P = 0.03) and anti-CD4 (P = 0.046) antibodies. Phenotyping of the responding T-cell population demonstrated the majority to be CD4 positive. Our data demonstrate that HLA class II-restricted proliferative and cytotoxic T-cell responses to B-CLL can be generated using autologous dendritic cells pulsed with tumour cell lysate.

Humoral immunity and bronchiectasis.

Bronchiectasis is a common complication of primary antibody deficiency but the incidence of antibody deficiency as an underlying cause of bronchiectasis is largely undefined. In this study the humoral immune status of a cohort of bronchiectatic patients was investigated to detect the frequency of significant antibody deficiency and to determine the extent of immunological investigation which is appropriate for routine assessment of bronchiectasis patients. Fifty-six out-patients (with a mean age of 59.6 years) had serum immunoglobulins, IgG subclasses and specific antibodies to capsular polysaccharides of Haemophilus influenzae and Streptococcus pneumoniae measured. Where specific antibody -levels were low, where possible, appropriate immunization with pneumococcal or conjugated Haemophilus polysaccharide vaccines was offered and the responses quantified. Three of 56 patients had low total serum IgG levels. Thirteen of 56 had deficiencies of either a single IgG subclass or combinations of two or more subclasses, with IgG4 being most frequently implicated (9/56). Twenty-nine of 56 had low basal specific polysaccharide antibody levels. Test immunization, where performed, produced satisfactory responses in all cases except one, where a specific defect of responsiveness to pneumococcal polysaccharide was identified. This study indicates that antibody deficiency is an uncommon aetiological/underlying factor in the causation of bronchiectasis beyond the fourth decade and that detailed investigation of humoral immune status as a routine in bronchiectasis patients, at least at this age, is not generally justified.

Analysis of the expression of critical activation/interaction markers on peripheral blood T cells in B-cell chronic lymphocytic leukaemia: evidence of immune dysregulation.

B-cell chronic lymphocytic leukaemia (B-CLL) is characterized by an accumulation of clonal malignant B cells. The intrinsic characteristics that permit this accumulation have been extensively studied and described. However, it is possible that proliferation and survival of this malignant clone is facilitated by a disruption in the interaction between B and T cells that normally regulate the immune system. In this study, using flow cytometry and cell culture techniques, marked abnormalities of the expression of certain key activation and interaction molecules on the peripheral blood T cells of patients with B-CLL were demonstrated. In particular, on comparison with normal controls, there was a marked reduction in the number of circulating T cells expressing CD25 (interleukin 2 receptor) (P = 0.007), CD28 (P = 0.01) and CD152 (CTLA-4) (P = 0.001). There was also a reduction in the number of circulating T cells expressing CD4 (P = 0.03), CD5 (P = 0.05) and CD11a (P = 0.01). There was no difference in the number expressing T-cell receptor alphabeta (P = 0.1), CD8 (P = 0.4), CD54 (P = 0.4) and CD154 (P = 0.5), and the only marker expressed on a greater number of circulating T cells in B-CLL patients was HLA-DR (P = 0.05). These results suggest that there is a profound T-cell dysregulation that may contribute to the survival of the malignant B cells in patients with B-CLL and to the related autoimmune phenomena of the disease.

Pretransfused patients with severe aplastic anaemia exhibit high numbers of cytotoxic T lymphocyte precursors probably directed at non-HLA antigens.

Patients with severe aplastic anaemia (SAA) have a relatively high risk of graft rejection after transplantation of bone marrow from HLA-identical siblings compared with patients transplanted for leukaemia. This is presumed to be due to pretransplant blood transfusions which sensitize the recipient's immune system to non-HLA antigens expressed on donor marrow cells. To test this hypothesis, we estimated in the peripheral blood of 18 SAA patients, the frequencies of alloreactive cytotoxic T lymphocyte precursors (CTL-p) directed against mononuclear cells from syngeneic twins, HLA-identical siblings or HLA-matched unrelated individuals. The results were compared with the frequencies of CTL-p in 13 healthy subjects or leukaemia patients who had not been transfused. Pretransfused patients with SAA had significantly higher frequencies of CTL-p directed against HLA-identical siblings (P = 0.002), and against HLA-matched unrelated individuals (P = 0.004), than did untransfused individuals. In contrast, the frequency of CTL-p in two pretransfused patients against syngeneic mononuclear cells was very low. We propose that the increased numbers of CTL-p in the blood of pretransfused SAA patients are directed against non-HLA antigens on target cells and may be the result of prior blood transfusion.