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Background: The difference in the immune response to severe acute respiratory syndrome coro-navirus 2 (SARS-CoV-2) in patients with mild versus severe disease remains poorly understood. Recent scientific advances have recognised the vital role of both B cells and T cells; however, many questions remain unanswered, particularly for T cell responses. T cells are essential for helping the generation of SARS-CoV-2 antibody responses but have also been recognised in their own right as a major factor influencing COVID-19 disease outcomes. The examination of T cell receptor (TCR) family differences over a 12-month period in patients with varying COVID-19 disease severity is crucial for understanding T cell responses to SARS-CoV-2., Methods: We applied a machine learning approach to analyse TCR vb family responses in COVID-19 patients ( n = 151) across multiple timepoints and disease severities alongside SARS-CoV-2 infection-naïve (healthy control) individ-uals ( n = 62)., Results: Blood samples from hospital in-patients with moderate, severe, or critical disease could be classified with an accuracy of 94%. Furthermore, we identified significant variances in TCR vb family specificities between disease and control subgroups., Conclusions: Our findings suggest advantageous and disadvantageous TCR repertoire patterns in relation to disease severity. Following validation in larger cohorts, our methodology may be useful in detecting protective immunity and the assessment of long-term outcomes, particularly as we begin to unravel the immunological mechanisms leading to post-COVID complications.

The aim of this study was to explore the proof of concept for using Raman spectroscopy as a diagnostic platform in the setting of systemic lupus erythematosus (SLE). We sought to identify unique Raman signatures in serum blood samples to successfully segregate SLE patients from healthy controls (HC). In addition, a retrospective audit was undertaken to assess the clinical utility of current testing platforms used to detect anti-double stranded DNA (dsDNA) antibodies (n = 600). We examined 234 Raman spectra to investigate key variances between SLE patients (n = 8) and HC (n = 4). Multi-variant analysis and classification model construction was achieved using principal component analysis (PCA), PCA-linear discriminant analysis and partial least squares-discriminant analysis (PLS-DA). We achieved the successful segregation of Raman spectra from SLE patients and healthy controls (p-value < 0.0001). Classification models built using PLS-DA demonstrated outstanding performance characteristics with 99% accuracy, 100% sensitivity and 99% specificity. Twelve statistically significant (p-value < 0.001) wavenumbers were identified as potential diagnostic spectral markers. Molecular assignments related to proteins and DNA demonstrated significant Raman intensity changes between SLE and HC groups. These wavenumbers may serve as future biomarkers and offer further insight into the pathogenesis of SLE. Our audit confirmed previously reported inconsistencies between two key methodologies used to detect anti-dsDNA, highlighting the need for improved laboratory testing for SLE. Raman spectroscopy has demonstrated powerful performance characteristics in this proof-of-concept study, setting the foundations for future translation into the clinical setting.

Biospectroscopy offers the ability to simultaneously identify key biochemical changes in tissue associated with a given pathological state to facilitate biomarker extraction and automated detection of key lesions. Herein, we evaluated the application of machine learning in conjunction with Raman spectroscopy as an innovative low-cost technique for the automated computational detection of disease activity in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis (AAGN). Consecutive patients with active AAGN and those in disease remission were recruited from a single UK centre. In those with active disease, renal biopsy samples were collected together with a paired urine sample. Urine samples were collected immediately prior to biopsy. Amongst those in remission at the time of recruitment, archived renal tissue samples representative of biopsies taken during an active disease period were obtained. In total, twenty-eight tissue samples were included in the analysis. Following supervised classification according to recorded histological data, spectral data from unstained tissue samples were able to discriminate disease activity with a high degree of accuracy on blind predictive modelling: F-score 95% for >25% interstitial fibrosis and tubular atrophy (sensitivity 100%, specificity 90%, area under ROC 0.98), 100% for necrotising glomerular lesions (sensitivity 100%, specificity 100%, area under ROC 1) and 100% for interstitial infiltrate (sensitivity 100%, specificity 100%, area under ROC 0.97). Corresponding spectrochemical changes in paired urine samples were limited. Future larger study is required, inclusive of assigned variables according to novel non-invasive biomarkers as well as the application of forward feature extraction algorithms to predict clinical outcomes based on spectral features.

The current lack of a reliable biomarker of disease activity in anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis poses a significant clinical unmet need when determining relapsing or persisting disease. In this study, we demonstrate for the first time that attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy offers a novel and functional candidate biomarker, distinguishing active from quiescent disease with a high degree of accuracy. Paired blood and urine samples were collected within a single UK centre from patients with active disease, disease remission, disease controls and healthy controls. Three key biofluids were evaluated; plasma, serum and urine, with subsequent chemometric analysis and blind predictive model validation. Spectrochemical interrogation proved plasma to be the most conducive biofluid, with excellent separation between the two categories on PC2 direction (AUC 0.901) and 100% sensitivity (F-score 92.3%) for disease remission and 85.7% specificity (F-score 92.3%) for active disease on blind predictive modelling. This was independent of organ system involvement and current ANCA status, with similar findings observed on comparative analysis following successful remission-induction therapy (AUC > 0.9, 100% sensitivity for disease remission, F-score 75%). This promising technique is clinically translatable and warrants future larger study with longitudinal data, potentially aiding earlier intervention and individualisation of treatment.

Over the past 3 decades, significant advancements in the understanding of the pathophysiology of ANCA-associated vasculitis has led to the development of a multitude of potential candidate biomarkers. Accompanied by the advent of increasingly effective therapeutic strategies, the need for a dependable biomarker to help determine the extent of disease activity and risk of relapse is ever present. Implementation of such a biomarker would enable tailored therapy, optimizing disease control while helping to mitigate unnecessary exposure to therapy and potential treatment-related damage. Although far from perfect, ANCA serology and B-cell population are the two main staple biomarker tools widely used in practice to help supplement clinical assessment. Over recent years, the application and progress of more novel biomarker tools have arisen in both organ-limited and multisystem disease, including genomics, urinary proteins, degradation products of the alternative complement system, cytokines, metabolomics, and biospectroscopy. Validation studies and clinical translation of these tools are required, with serial assessment of disease activity and determination of therapy according to biomarker status correlated with patient outcomes., Competing Interests: F.L. Martin reports having ownership interest in Biocel UK Ltd. All remaining authors have nothing to disclose., (Copyright © 2021 by the American Society of Nephrology.)

Common variable immune deficiency (CVID) is a primary immunodeficiency disease, characterized by hypogammaglobulinemia, recurrent infections and various complications. The clinical heterogeneity of CVID has hindered identification of an underlying immune defect; diagnosis relies on clinical judgement, alongside evidence-based criteria. The lack of pathognomonic clinical or laboratory features leads to average diagnostic delays of 5 years or more from the onset. Vibrational spectroscopic techniques such as Fourier-transform infrared (FTIR) spectroscopy have recently gained increasing clinical importance, being rapid-, non-invasive and inexpensive methods to obtain information on the content of biological samples. This has led us to apply FTIR spectroscopy to the investigation of blood samples from a cohort of CVID patients; revealing spectral features capable of stratifying CVID patients from healthy controls with sensitivities and specificities of 97% and 93%, respectively for serum, and 94% and 95%, respectively for plasma. Furthermore we identified several discriminating spectral biomarkers; wavenumbers in regions indicative of nucleic acids (984 cm -1 , 1053 cm -1 , 1084 cm -1 , 1115 cm -1 , 1528 cm -1 , 1639 cm -1 ), and a collagen-associated biomarker (1528 cm -1 ), which may represent future candidate biomarkers and provide new knowledge on the aetiology of CVID. This proof-of-concept study provides a basis for developing a novel diagnostic tool for CVID.

Robust diagnosis of ovarian cancer is crucial to improve patient outcomes. The lack of a single and accurate diagnostic approach necessitates the advent of novel methods in the field. In the present study, two spectroscopic techniques, Raman and surface-enhanced Raman spectroscopy (SERS) using silver nanoparticles, have been employed to identify signatures linked to cancer in blood. Blood plasma samples were collected from 27 patients with ovarian cancer and 28 with benign gynecological conditions, the majority of which had a prolapse. Early ovarian cancer cases were also included in the cohort (n = 17). The derived information was processed to account for differences between cancerous and healthy individuals and a support vector machine (SVM) algorithm was applied for classification. A subgroup analysis using CA-125 levels was also conducted to rule out that the observed segregation was due to CA-125 differences between patients and controls. Both techniques provided satisfactory diagnostic accuracy for the detection of ovarian cancer, with spontaneous Raman achieving 94% sensitivity and 96% specificity and SERS 87% sensitivity and 89% specificity. For early ovarian cancer, Raman achieved sensitivity and specificity of 93% and 97%, respectively, while SERS had 80% sensitivity and 94% specificity. Five spectral biomarkers were detected by both techniques and could be utilised as a panel of markers indicating carcinogenesis. CA-125 levels did not seem to undermine the high classification accuracies. This minimally invasive test may provide an alternative diagnostic and screening tool for ovarian cancer that is superior to other established blood-based biomarkers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (© 2017 Crown copyright. Clinical and Experimental Immunology, © 2017 British Society for Immunology.)

Modest work has been performed to improve the sensitivity of residual disease detection or investigate the contribution that the immune system makes in controlling metastatic tumor growth, in particular, the frequency and biological actions of peptide-specific CD8+ T lymphocytes in limiting metastatic disease and/or maintaining remission. Fifty-three peripheral blood samples from 32 prostate cancer (PC) patients were investigated for the presence of circulating prostate-specific antigen (PSA)-expressing cells (CPECs) using a highly sensitive and specific assay combining immunomagnetic epithelial cell enrichment with nested RT-PCR of PSA mRNA. Using HLA-A2 tetramer complexes, frequency of CD8+ T cells specific for PSA-derived peptides was determined. Additionally, serum concentrations of PSA and testosterone were measured. CPECs were detected in 26% of peripheral blood samples from PC patients. CD8+ T cells specific for PSA-derived peptides were detected at low frequency in HLA-A2-positive PC patients. The correlation between these PSA-specific CD8+ T cells and residual prostate tumor cells and clinical measures was investigated. Our data suggest that frequency of PSA-specific CD8+ T cells is correlated to CPECs, but not to serum PSA level.

Induction of CTL responses specific for prostate-specific antigen (PSA)-derived peptides in healthy individuals and patients with prostate cancer (PC) was investigated. Eight PSA-derived peptides that have the potential to bind HLA-A2 molecules were examined. Peripheral blood lymphocytes isolated from HLA-A2-positive volunteers were expanded using autologous mature, PSA-derived peptide-pulsed dendritic cells. The expansion of IFN-gamma-secreting CD8+ T cells specific for three of the eight PSA-derived peptides (PSA-2(108-117), PSA-4(141-150) and PSA-6(146-154)) was detected in healthy individuals, but not in patients with PC. Using HLA-A2/peptide tetramers, the PSA-specific CD8+ T cells were detectable at low frequency both in healthy individuals and patients with PC. Using flow cytometric cytotoxicity assays, the expanded effectors from healthy individuals were able to kill the PSA-expressing epithelial cell line LNCaP and the peptide-pulsed T2 cells in a MHC class I-restricted manner without involving NK activity. However, such killing by effectors expanded from prostatectomized patients involved a complete or a significant NK activity. Specific recognition of PSA-derived peptides in healthy individuals may occur by an adaptive CTL immune response, while such recognition in PC patients may additionally or alternatively be mediated by an innate NK immune response. In conclusion, our work indicates that the PSA-specific CD8+ T cells exist in both healthy individuals and PC patients, but they have impaired function in patients as they failed to release IFN-gamma and to kill targets without involving NK activity.

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.

There is growing interest in the in vitro generation of dendritic cells (DC) from peripheral blood monocytes, but the effect of the method chosen to isolate CD14+ monocytes for subsequent DC generation is poorly documented. The method used to isolate monocytes may have an impact on the subsequent function of DC by affecting their ability to express costimulatory molecules (CD80/86), maturation marker (CD83) and/or to produce important immunomodulatory cytokines. In this study, we show that the positive selection of monocytes by anti-CD14-coated microbeads inhibits the lipopolysaccharide (LPS)-induced production of interleukin (IL)-12, IL-10 and tumour necrosis factor-alpha (TNF-alpha) from human DC. However, when DC were grown from monocytes isolated by plastic adherence, LPS induced the production of much higher levels of these cytokines. DC derived from adherence-isolated monocytes induced the development of potent cytotoxic T lymphocytes of the Tc1 subset specific for influenza matrix protein, as confirmed by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), cytotoxicity assay, major histocompatibility complex (MHC)-peptide tetrameric complexes and T helper 1/T helper 2 (Th1/Th2) cytokine production assays.

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.

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 (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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.