UK NEQAS survey of allergen component testing across the United Kingdom and other European countries.

The clinical utility of molecular diagnostic approaches in allergy investigation is being recognized increasingly to play a significant role in the management of allergic patients. Determining the sensitization pattern, which is best achieved through the use of component resolved diagnostics (CRD), allows effective risk stratification, appropriate treatment and patient selection for immunotherapy. In order to assess the diagnostic service provisions for in-vitro allergy testing across Europe, a survey was carried out via the total immunoglobulin (Ig)E and specific IgE external quality assurance schemes run by UK National External Quality Assessment Service (NEQAS) Immunology, Immunochemistry and Allergy. This survey assessed allergy testing, and in particular allergen components offered by the laboratories, and found a wide variability in service provision, particularly between the United Kingdom and other European Union (EU) countries. Furthermore, there was lack of standardization for acquisition of clinical information to aid allergen (and component) selection, gating strategy, testing algorithms and clinical interpretation. Interestingly, a significant proportion of laboratories (the majority from EU) stated that they 'used' the results for peanut components for risk stratification. However, the vast majority of participants were unaware of guidelines relating to the use of allergen component testing, and agreed that further education would assist in reaching a common platform. Hence, this survey has highlighted that although CRD has been adopted into routine diagnostics across Europe, it is potentially compromised by lack of standardized protocols and guidance sources. Consequently, there is a need for local or national standards and education through External Quality Assurance services on the performance and application of CRD into allergy investigation.

Flow cytometric investigation of peri-anaesthetic anaphylaxis using CD63 and CD203c.

The investigation of anaphylactic reactions in the peri-operative period is difficult. Elevation of serum tryptase levels is a good indicator of an anaphylactic event but the ability of subsequent investigations to identify the drug(s) responsible for the reaction is still potentially unreliable. The aim of this study was to examine basophil activation as an investigative tool. We performed flow cytometric analysis of the expression on the cell surface of the basophil activation markers CD63 and CD203c and measured histamine release in 21 patients who were referred with possible peri-operative anaphylaxis. The sensitivity of CD63, CD203c, basophil histamine release and skin prick for the muscle relaxants was found to be 79%, 36%, 36% and 64%, respectively; the specificity was found to be 100%. These results demonstrate the difficulty in investigating the cause of an unexpected clinical event following drug administration, but the higher sensitivity of neo-expression on the cell surface of CD63 suggests that flow cytometric analysis of its neo-expression on basophils in vitro may be a diagnostic aid.

Investigation of alpha nascent polypeptide-associated complex functions in a human CD8(+) T cell ex vivo expansion model using antisense oligonucleotides.

In order to determine molecules involved in the differentiation and proliferation of human CD8(+) cells, two ex vivo expansion models were established: coculture of freshly purified human CD8(+) cells with irradiated autologous feeders (AF) or stimulation with anti-CD3. Two different proliferation kinetics of CD8(+) cells and expression patterns of CD57 were observed between these conditions. Differential display reverse transcriptase-polymerase chain reaction was applied to investigate the differential expression of mRNA species between CD8(+) CD57(+) and CD8(+) CD57(-) populations. A differentially expressed RNA species called alpha nascent polypeptide associated complex (alpha NAC) was found at a higher level in CD8(+) CD57(-) cells than in CD8(+) CD57(+) cells. In the presence of AF, the expression of alpha NAC was reduced on culturing whilst proliferation increased. Similarly, in cultures stimulated with anti-CD3, alpha NAC reverted to its inactive form and differentiation and proliferation increased. Using a phosphorothioate-modified oligodeoxynucleotide antisense directed specifically against alpha NAC mRNA, protein expression was inhibited and increased CD8(+) cell proliferation and CD25 expression were observed irrespective of the culture conditions. This suggests that alpha NAC protein is antiproliferative molecule. This is the first description of the function of the alpha NAC protein in human CD8(+) T cells.

Human CD8+ CTL recognition and in vitro lysis of herpes simplex virus-infected cells by a non-MHC restricted mechanism.

Cytotoxic T lymphocytes (CTL) are important for the recognition and lysis of virally infected cells, but their effectiveness can be limited by viral immune evasion mechanisms. We investigated the immunophenotype and function of human CD8+ T cells raised in response to herpes simplex virus (HSV). The expanded population contained cells of an activated and mature phenotype, as determined by the expressions of CD25, CD45RO, CD57, CD95 and HLA-DR. Cultured cells also expressed CD45RA. These cells lysed autologous and allogeneic HSV-infected lymphoblastoid cell line (LCL) targets via a non-major histocompatibility complex (MHC) restricted recognition pathway. Inhibition assays showed the mechanism of cytotoxicity to be calcium-dependent, granule exocytosis pathway, rather than the internal disintegration pathway. Cold target competition assays indicated that a common CTL population contributed to the recognition of autologous and allogeneic-infected targets. These effectors showed recognition of infected targets which was distinct from that of K562 cells. Non-MHC restricted lysis-associated molecule 2B4 (CD244) was upregulated on culturing and made a significant contribution to lysis of FcgammaR-bearing targets in a redirected killing assay. These findings suggest that CTL can recognize virally infected cells through a combination of non-MHC restricted mechanisms and may result in more efficient lysis than classical CD8+ T cells.

An optimised biphasic culture system for the generation of functional dendritic cells from patients with acute lymphoblastic leukaemia at presentation and in clinical remission.

We have tested the hypothesis that functional dendritic cells (DC) may be generated from patients with acute lymphoblastic leukaemia (ALL). We evaluated the production of DC from blast cells taken at presentation from nine children with ALL. Blast cells were expanded in serum-free medium supplemented with Flt3L, G-CSF, GM-CSF, IL-3, IL-6 and SCF for 7 days and subsequently stimulated with Flt3L, GM-CSF and TGF-beta for a further 14 days, with the addition of TNF-alpha for the final 48 h of culture. Cultured cells had the morphological appearance of DC and expressed the DC-associated antigens CD1A (range 2-87%) and CD83 (15-44%). Expression of the co-stimulatory molecules CD80 and CD86 was increased and the majority of these cells retained their expression of CD34 (73+/-4%) and HLA-DR (79+/-5%). Seven of the nine ALL had a leukaemia-specific abnormality and DC generated from five of these seven cases were derived from the leukaemic clone. Leukaemic DC derived from four HLA-A*02-positive ALL pulsed with CMV-associated peptides could induce significant proliferation of peptide-specific CD8+ T cells. This specificity was verified using tetrameric complexes of HLA class l/antigenic peptide. DC could also be generated from cells taken at times of complete remission of ALL and from normal controls using these culture conditions. These findings show that functional DC can be generated both from ALL blasts and from patients in remission; these might be utilised in future for immunotherapeutic strategies in the treatment of ALL.

Functional analysis of the CD8+CD57+ cell population in normal healthy individuals and matched unrelated T-cell-depleted bone marrow transplant recipients.

The biological activities of CD8+ that co-express CD57 remain poorly defined. It is unclear whether all CD8+ cells have the potential to become CD57+ or whether they represent a unique subset with distinct functions. Several studies have reported the association between elevated numbers of CD8+CD57+ and a wide range of clinical disorders such as viral reactivation of human cytomegalovirus (HCMV). In this study, we have investigated the relationship between viral reactivation and the effect of diminished interleukin (IL)-2 production. Using CD8+ cells isolated from patients at various times after allogeneic transplants and in vitro models of HCMV infection, we determined their combined effect on CD8+CD57+. Our results show that high numbers of CD8+CD57+ correlated with diminished killing of HCMV-infected targets. In addition, we showed a synergistic effect between IL-2 and HCMV in the expansion of CD8+CD57+ cells. Furthermore, these cells after anti-CD3 stimulation did not produce tumour necrosis factor (TNF)-alpha or interferon (IFN)-gamma. Interestingly, IL-10 production was elevated in several patients which appeared to be associated with the time from transplant.

Interleukin-10-induced CD8 cell proliferation.

Interleukin (IL)-10, a product of T helper 2 (Th2) lymphocytes, has been shown to be an important regulator of lymphoid and myeloid cells, inhibiting mitogen, peptide and alloantigen-induced T-cell proliferation and IL-2 production. The microenvironment at the time of cell activation, notably the presence or absence of cytokines such as IL-10, interferon-gamma (IFN-gamma) and IL-2, is believed to determine the lineage and magnitude of cell-mediated responses. In this study, we show that recombinant human IL-10 (rhIL-10) exerts a dose-dependent inhibitory effect on human peripheral blood mononuclear cells stimulated in vitro, when these cells have not previously been exposed to rhIL-10. Furthermore, incubation of these cells with high doses of rhIL-10, either before or at the time of activation, results in inhibition which is followed several days later by the emergence of a population of CD8 positive cells. This rhIL-10-responsive CD8, positive cell population still emerges even when the cells are washed following incubation with rhIL-10 prior to cell activation. Using purified CD8 populations this was shown to be a direct action of rhIL-10 on CD8 cells and not via CD4 positive cells and monocytes. This finding was only observed when cells were activated with a cross-linking anti-CD3 antibody and not when activated with phorbol-12-mystrate-13-acetate (PMA) and calcium ionophore (CaIon), suggesting that the effect is mediated through cell-surface receptors. Analysis of CD8 positive clones reveal production of Tc2 patterns of cytokines and reduced cell cytotoxicity to allogeneic, natural killer and lymphokine activated cell targets.

The use of Teflon cell culture bags to expand functionally active CD8+ cytotoxic T lymphocytes.

We have investigated the ability of Teflon cell culture (TCC) bags, compared to conventional tissue culture flasks and plates, to support the expansion of human CD8+ T cells in response to an allogeneic stimulus. TCC bags, which are compatible with good manufacturing practice (GMP), facilitated CD8+ T cell growth as well as conventional culture vessels and resulted in cytotoxic T cells which were able to kill allogeneic targets. Growth characteristics were compared by investigating the number, immunophenotype and cell cycle properties of the cells generated. The kinetics of cell growth were not significantly different over the first 14 days of culture in each vessel type, with the cell counts being highest at day 10 in all cases. However, the TCC bags resulted in a significantly higher proportion of cells with the morphology of typical lymphocytes than tissue culture flasks after 14 and 18 days in culture. There were no significant differences in the percentage of typical lymphocytes expanded in TCC bags compared to those expanded in plates. Expanded CD8+ cells maintained their initial level of expression of CD3, CD11a, CD18 and T cell receptor (alphabeta heterodimer, TCR (alphabeta)) but increased expression of CD45RO, CD95 and of activation markers HLA-DR and CD25 in each culture vessel. Studies of cell cycle parameters showed that each vessel supported CD8+ T cell stimulation, as demonstrated by significantly higher levels of S phase than fresh PBMN cells. The cells generated in TCC bags were able to kill allogeneic targets and also possessed natural killer (NK) cell activity. Thus, TCC bags are able to support the expansion of CD8+ T lymphocytes as well as flasks or tissue culture plates and are applicable to lymphocyte expansion for use in immunotherapy.

Minimal residual disease status before allogeneic bone marrow transplantation is an important determinant of successful outcome for children and adolescents with acute lymphoblastic leukemia.

The efficacy of allografting in acute lymphoblastic leukemia (ALL) is heavily influenced by remission status at the time of transplant. Using polymerase chain reaction (PCR)-based minimal residual disease (MRD) analysis, we have investigated retrospectively the impact of submicroscopic leukemia on outcome in 64 patients receiving allogeneic bone marrow transplantation (BMT) for childhood ALL. Remission BM specimens were taken 6 to 81 days (median, 23) before transplant. All patients received similar conditioning therapy; 50 received grafts from unrelated donors and 14 from related donors. Nineteen patients were transplanted in first complete remission (CR1) and 45 in second or subsequent CR. MRD was analyzed by PCR of Ig or T-cell receptor delta or gamma rearrangements, electrophoresis, and allele-specific oligoprobing. Samples were rated high-level positive (clonal band evident after electrophoresis; sensitivity 10(-2) to 10(-3)), low-level positive (MRD detected only after oligoprobing; sensitivity 10(-3) to 10(-5)), or negative. Excluding 8 patients transplanted in CR2 for isolated extramedullary relapse (all MRD-), MRD was detected at high level in 12 patients, low level in 11, and was undetectable in 33. Two-year event-free survival for these groups was 0%, 36%, and 73%, respectively (P <.001). Follow-up in patients remaining in continuing remission is 20 to 96 months (median, 35). These results suggest that MRD analysis could be used routinely in this setting. This would allow identification of patients with resistant leukemia (who may benefit from innovative BMT protocols) and of those with more responsive disease (who may be candidates for randomized trials of BMT versus modern intensive relapse chemotherapy).

Minimal residual disease status as a predictor of relapse after allogeneic bone marrow transplantation for children with acute lymphoblastic leukaemia.

We have analysed the behaviour of minimal residual disease (MRD) after allogeneic bone marrow transplantation (allo-BMT) in 71 children with acute lymphoblastic leukaemia (ALL). The method relied on PCR of IgH, TCRdelta and/or TCRgamma gene rearrangements followed by electrophoretic size resolution and allele-specific oligoprobing. Patients were similarly conditioned; 55 received marrow from unrelated donors and 16 from related donors. MRD was assessed at various time-points up to 24 months after BMT. Three children were not evaluable due to transplant-related mortality. MRD was detected in 28/32 patients (88%) who relapsed post-BMT; 16 were positive at all times and 12 were initially negative but became positive at a median of 3 months (range 1.5-11) prior to relapse. In contrast, only eight of 36 (22%) patients who remained in continuing complete remission (CCR) (median follow-up 43 months, range 20-94) showed MRD at any time after BMT (P<0.0001). In these eight patients MRD was found up to 9 months after transplant and at low levels (0.01-0.001%). All eight (median follow-up 39 months, range 24-87) had at least two MRD-negative samples tested subsequently and five of the eight had evidence of grade I-II acute graft-versus-host disease (GvHD), raising the possibility of a graft-versus-leukaemia effect. In general, any evidence of MRD after allo-BMT is a poor prognostic sign. However, if immunotherapy were to be targeted towards patients with evidence of persisting MRD after BMT, the method described would expose only a small proportion of patients to unnecessary additional toxicity.

Cytomegalovirus (CMV) infection in AIDS patients is associated with a CD3 receptor-mediated T cell hyporesponsiveness.

HIV+ individuals with human CMV (HCMV) reactivation have a CD3 receptor-mediated T cell hyporesponsiveness when compared with CD4-matched HIV+ and HCMV- control groups. The impairment of proliferation was not reversed by exogenous IL-2. A typical increase in NFkappaB expression was observed following cross-linking of the CD3 receptor, but did not lead to increased CD25 cell surface expression or cell proliferation. The HCMV-induced non-responsiveness was not observed when cells were stimulated with phorbol esters. Lymphocytes cultured with media collected from cell cultures infected with HCMV showed a dose-dependent inhibition in the total T cell population even though cells staining dually for CD8/57 increased in number. The altered growth factor requirements of CD8/57+ cells may therefore account for their presence in AIDS and patients following bone marrow transplantation.

Faecal tumour necrosis factor-alpha in individuals with HIV-related diarrhoea.

OBJECTIVE: HIV-related gastrointestinal infection is associated with diarrhoea, weight loss, mucosal inflammation and increased intestinal permeability. As tumour necrosis factor (TNF)-alpha may mediate these features this cytokine was measured in the faeces of HIV-seropositive individuals with diarrhoea to assess its role in the pathogenesis of HIV-related gastrointestinal disease and the association with specific intestinal pathogens. DESIGN: Prospective study. METHODS: Two hundred and four HIV-seropositive individuals provided stool samples that were analysed for faecal TNF-alpha (FTNF-alpha) using a standard sandwich enzyme-linked immunosorbent assay. RESULTS: Stool from patients with bacterial, cytomegalovirus (CMV) and microsporidial diarrhoea had significantly elevated FTNF-alpha compared with those who had pathogen-negative diarrhoea (P < 0.05). FTNF-alpha was not raised in cryptosporidiosis, pathogen-negative or solid stool. In subjects with diarrhoea of more than 2 weeks duration and three stool samples negative for enteric pathogens, FTNF-alpha greater than 15 U/ml has a sensitivity of 88% and a specificity of 66% for the diagnosis of diarrhoea-related CMV enteritis. CONCLUSION: TNF-alpha production may have a role in the pathogenesis of bacterial, microsporidial and CMV-related diarrhoea in HIV-seropositive individuals. Thus, anti-TNF-alpha agents may have a therapeutic role in the management of these conditions. FTNF-alpha greater than 15 U/ml in apparently pathogen-negative diarrhoea may suggest endoscopic gastrointestinal biopsy to diagnose CMV enteritis.

Detection of cytoplasmic IL-1 beta in peripheral blood mononuclear cells from intensive care unit (ICU) patients.

Cytokines including IL-1 beta have been implicated in the pathophysiology of sepsis and the systemic inflammatory response. It is believed that certain critically ill patients may be 'primed' with respect to cytokine production, and that subsequent 'triggers' may cause exaggerated cytokine production in these patients with exacerbation of their clinical condition; however, no means of identifying 'primed' patients has been described. The presence of cytoplasmic IL-1 beta within peripheral blood mononuclear cells (PBMC) from patients in the ICU was investigated as a means of identifying 'primed' patients, using fluorescent antibody labelling and flow cytometry. The study revealed that PBMC from ICU patients had a different staining pattern for IL-1 beta than those from healthy subjects, and that PBMC from certain ICU patients did indeed stain strongly for IL-1 beta; however, the presence of these strongly staining cells was not associated with clinical condition or outcome. It is concluded that whilst it might be possible to identify 'primed' patients in the ICU using this technique, this is of no clinical value as a predictor of clinical course.

Monitoring cytokine production in peripheral blood during acute graft-versus-host disease following allogeneic bone marrow transplantation.

Plasma concentrations and peripheral blood cells containing cytoplasmic cytokines were monitored during the post-transplant period in 10 patients who had received allogeneic bone marrow transplants (BMT) for the correction of inherited genetic disorders. The presence of CD14-positive cells containing cytoplasmic interleukin-1 alpha and beta in the peripheral blood was indicative of acute graft-versus-host disease (GVHD). Plasma concentrations of IL-1 alpha, IL-1 beta and TNF-alpha were significantly raised in the GVHD group when compared with the uneventful days. There was, however, poor temporal correlation between the plasma concentrations and clinical manifestations of acute GVHD. Cells containing cytoplasmic IL-6 were present in the peripheral blood when patients had clinically suspected and/or microbiologically confirmed infection. The results from this study demonstrate that analysis of peripheral blood cells for cytoplasmic IL-1 alpha and IL-1 beta are better markers of acute GVHD than is monitoring plasma concentrations of these cytokines.

Cytokine gene expression in skin and lymphoid organs in graft versus host disease.

AIM: To determine if human graft versus host disease (GvHD) is associated with any detectable change in cytokine gene expression in the skin and lymphoid organs. METHODS: Reverse transcriptase and the polymerase chain reaction were used to amplify mRNA for interleukins-1 (IL-1), -2 (IL-2), -4 (IL-4) and -6 (IL-6), IL-2 receptor (IL-2R), tumour necrosis factors alpha (TNF-alpha) and beta (TNF-beta), gamma interferon (IFN gamma) and granulocyte macrophage colony stimulating factor (GM-CSF) in frozen punch biopsy specimens of skin and necropsy samples of skin, lymph node, and spleen. RESULTS: No cytokine mRNA was detected in the punch biopsy specimens except weak signals for IL-6 and IL-1 and GM-CSF in two normal donors and IL2-R in one patient with GvHD. In samples of skin taken at necropsy, however, significant quantities of mRNA for TNF-alpha, TNF-beta, and IL-4 were detected in patients who had or had had GvHD in contrast to those without the disease whose skin lacked mRNA for these products but contained detectable quantities of IL-1, IL2-R, IL-6 and GM-CSF. There seemed to be a reciprocal relation between TNF-alpha and IL-4. In necropsy samples of lymph node and spleen a pattern of cytokine production similar to that in the skin was observed with a preponderance of TNF-alpha, TNF-beta and IL-4 in patients with GvHD and GM-CSF and IL-6 in those without the disease. CONCLUSIONS: The local synthesis of these molecules would explain many of the morphological and immunohistological features of GvHD. The failure to detect TNF-alpha, TNF-beta, and IL-4 in skin biopsy specimens exhibiting GvHD is probably due to their small size but further investigations are required.

Influence of collection and separation of blood samples on plasma IL-1, IL-6 and TNF-alpha concentrations.

The anticoagulant used for the collection of blood was found to influence in vitro cytokine production in whole blood. Lithium heparin in certain collection tubes was found to contain endotoxin and induced cytokine synthesis in a time-dependent manner whereas endotoxin-free lithium heparin did not. No induction of cytokine occurred in the presence of EDTA which was also able to inhibit endotoxin-induced cytokine synthesis. Synthesis or absence of cytokine correlated with the induction of messenger RNA. Investigation of the kinetics of cytokine induction in whole blood revealed that tumour necrosis factor alpha (TNF) was detectable after 2 h of incubation at 37 degrees C and interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) after 3 h. In certain samples IL-1 and IL-6 were detectable in plasma separated immediately from blood collected into endotoxin-free lithium heparin, presumably reflecting in vivo synthesis, and similar concentrations were detected after 3 h of incubation of whole blood at 37 degrees C. These data indicate that as long as blood is collected into endotoxin-free anticoagulant then cytokine measurements will reflect the in vivo status.

Combined detection of phenotype and Y chromosome by immunoenzyme labelling and in situ hybridisation on peripheral lymphocytes.

A novel staining method for simultaneously determining the immunophenotype and sex of peripheral lymphocytes is described. Cell surface markers on lymphocytes are identified using specific monoclonal antibodies located by a peroxidase anti-peroxidase staining technique (PAP). Lymphocytes are subsequently hybridised with a biotinylated Y chromosome-specific sequence probe and this is located by avidin-biotin-alkaline phosphatase staining. Following the two staining steps the preparation is examined and in the same field lymphocytes show brown peroxidase staining of cell surface markers and red staining of the Y chromosome. The application of this combined staining technique provides a convenient method for studying the development of different cell lineages following sex-mismatched bone-marrow transplantation and for the identification of chimeric situations. The method has been shown to be sensitive and reproducible.