Comprehensive flow cytometric reference intervals of leukocyte subsets from six study centers across Europe.

A group of European FOCIS Centers of Excellence adapted panels of the Human Immunophenotyping Consortium (HIPC) for whole blood analysis. Using four core panels [T/regulatory T cell/B/natural killer (T/Treg /B/NK) and myeloid cells] the main leukocyte populations were analyzed in a clinical-diagnostic setting in a harmonized manner across different platforms. As a first step, the consortium presents here the absolute and relative frequencies of the leukocyte subpopulations in the peripheral blood of more than 300 healthy volunteers across six different European centers.

Tissue-resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer.

Tissue-resident memory T (TRM ) cells are CD8+ T lymphocytes that reside in the tissues, including tumours. This T cell subset possesses a magnitude of cytotoxicity, but its epigenetic regulation has not been studied. Here, we investigate the impact of perforin DNA methylation in TRM cells and correlate it with their functional potential. Fifty-three urothelial urinary bladder cancer (UBC) patients were recruited prospectively. The DNA methylation status of the perforin gene (PRF1) locus in TRM cells was investigated by pyrosequencing. Flow cytometry with ViSNE analysis and in-vitro stimulation were used to evaluate TRM cell phenotypes. We discovered that tumour TRM cells have low DNA methylation in the PRF1 locus (32·9% methylation), which corresponds to increased numbers of perforin-expressing TRM cells. Surprisingly, programmed cell death 1 (PD-1) expression is high in tumour TRM cells, suggesting exhaustion. Following interleukin-15 and T cell receptor stimulation, perforin and T-bet expressions are enhanced, indicating that TRM cells from tumours are not terminally exhausted. Moreover, a high number of TRM cells infiltrating the tumours corresponds to lower tumour stage in patients. In conclusion, TRM cells from UBC tumours are epigenetically cytotoxic with signs of exhaustion. This finding identifies TRM cells as potential new targets for cancer immunotherapy.

Peripheral and site-specific CD4(+) CD28(null) T cells from rheumatoid arthritis patients show distinct characteristics.

Proinflammatory CD4(+) CD28(null) T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to identify functional differences between CD4(+) CD28(null) T cells from blood and synovial fluid in comparison with conventional CD28-expressing CD4(+) T cells. Forty-four patients with RA, displaying a distinct CD4(+) CD28(null) T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN-γ, TNF, IL-17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4(+) CD28(null) T cells were significantly more hypomethylated in the CNS-1 region of the IFNG locus than conventional CD4(+) CD28(+) T cells and produced higher levels of both IFN-γ and TNF after TCR cross-linking. CD4(+) CD28(null) T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL-17A production could hardly be detected in CD4(+) CD28(null) cells from the blood, a significant production was observed in CD4(+) CD28(null) T cells from synovial fluid. CD4(+) CD28(null) T cells were not only found to differ from conventional CD4(+) CD28(+) T cells in the circulation, but we could also demonstrate that synovial CD4(+) CD28(null) T cells showed additional effector functions (IL-17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28(null) population.

The effects of chemotherapeutic drugs on human monocyte-derived dendritic cell differentiation and antigen presentation.

Recent studies indicate that chemotherapeutic agents may increase the anti-tumoral immune response. Based on the pivotal role of dendritic cells (DCs) in host tumour-specific immune responses, we investigated the effect of commonly used chemotherapeutic drugs dexamethasone, doxorubicin, cisplatin and irinotecan and glucocorticoids on monocyte-derived DCs (moDCs). Dexamethasone displayed the strongest inhibitory effect on DC differentiation. The effect of cisplatin and irinotecan was moderate, while only weak effects were noticed for doxorubicin. Surprisingly, when the functional consequence of chemotherapy-treated CD14(+) monocytes and their capacity to activate CD4(+) T responders cells were investigated, cisplatin-treated monocytes gave rise to increased T cell proliferation. However, dexamethasone, doxorubicin and irinotecan-pretreated monocytes did not stimulate any increased T cell proliferation. Further investigation of this observation revealed that cisplatin treatment during DC differentiation up-regulated significantly the interferon (IFN)-ß transcript. By contrast, no effect was evident on the expression of interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, IL-6 or IFN-α transcripts. Blocking IFN-ß attenuated the cisplatin-enhanced T cell proliferation significantly. In conclusion, cisplatin treatment enhanced the immune stimulatory ability of human monocytes, a mechanism mediated mainly by the increased production of IFN-ß.

A population-based investigation of the autoantibody profile in mothers of children with atrioventricular block.

The objective of the study was to investigate the antigen specificity and occurrence of individual autoantibodies in mothers of children diagnosed with atrioventricular (AV) block in a nation-wide setting. Patients with AV block detected before 15 years of age were identified using national quality registries as well as a network of pediatric and adult cardiologists and rheumatologists at the six university hospitals in Sweden. Patients with gross heart malformations, surgically or infectiously induced blocks were excluded. Blood samples were obtained from the mothers and maternal autoantibody profile, including the occurrence of antibodies against Ro52, Ro60, La, SmB, SmD, RNP-70k, RNP-A, RNP-C, CENP-C, Scl-70, Jo-1, ribosomal RNP and histones was investigated in 193 mothers of children with AV block by immunoblotting and ELISA. Autoantibody reactivity was detected in 48% (93/193) of the mothers of children with AV block. In autoantibody-positive mothers, the vast majority, 95% (88/93), had antibodies against Ro52, while 63% (59/93) had autoantibodies to Ro60 and 58% (54/93) had autoantibodies to La. In addition, 13% (12/93) of the autoantibody-positive mothers had antibodies to other investigated antigens besides Ro52, Ro60 and La, and of these anti-histone antibodies were most commonly represented, detected in 8% (7/93) of the mothers. In conclusion, this Swedish population-based study confirms that maternal autoantibodies may associate with heart block in the child. Further, our data demonstrate a dominant role of Ro52 antibodies in association with AV block.

The mode of actions of the Adacolumn therapeutic leucocytapheresis in patients with inflammatory bowel disease: a concise review.

Patients with active inflammatory bowel disease (IBD) have elevated and activated myeloid leucocytes which infiltrate the colonic mucosa in vast numbers. Myeloid leucocytes such as the CD14(+) CD16(+) monocytes are major sources of tumour necrosis factor (TNF)-α, and therefore selective granulocyte/monocyte (GM) adsorption (GMA) should promote remission or enhance efficacy of pharmacological therapy. However, studies in IBD have reported both impressive as well as disappointing efficacy outcomes, indicating that patients' demographic factors might determine responders or non-responders to GMA. Nonetheless, this non-drug intervention has an excellent safety profile, and therapeutic GMA is expected to expand. In this review, attempts have been made to compile an update on the mode of actions (MoA) of the Adacolumn GMA. The MoA of GMA appears to be more than adsorption of excess neutrophils and TNF-producing CD14(+) CD16(+) monocytes per se. Adsorbed GMs release interleukin (IL)-1 receptor antagonist, hepatocyte growth factor and soluble TNF receptors, which are anti-inflammatory. Additionally, a sustained increase in lymphocytes including the regulatory CD4(+) CD25(+) T cells (lymphocyte sparing) is seen post-GMA. The impact of GMA on the immune system is potentially very interesting in the context of treating immune-related diseases. Future studies are expected to add intriguing insights to the MoA of GMA.

Evidence for immunosurveillance in intestinal premalignant lesions.

The immunosurveillance theory argues that the immune system recognizes tumour-specific antigens expressed by transformed cells, which results in the destruction of cancer precursors before they become clinically manifest. As a model for the development of cancer, we set out to study premalignant lesions and immune responses in sentinel lymph nodes from patients with long-standing ulcerative colitis and progression of mucosal dysplasia. Mesenteric lymph nodes draining dysplastic and normal intestinal segments were identified by sentinel node technique during surgery in 13 patients with ulcerative colitis who were subjected to colectomy because of intestinal dysplasia. T cells were extracted from the lymph nodes and analysed by flow cytometry, and lymphocyte proliferation assays were set up in the presence of extracts from dysplastic and normal intestinal mucosa. Increase in CD4/CD8 ratio was observed in sentinel lymph nodes draining dysplastic epithelium compared to normal mucosa. The increase in CD4(+) T cells in relation to CD8(+) T cells correlated with the degree of dysplasia reflected by a significant increase in the ratio against low-grade dysplasia compared to indefinite dysplastic lesions. The T-cell response was specific to antigens from dysplastic epithelial lining as seen in proliferation assays. The observation suggests an important surveillance role for the immune system against premalignant intestinal lesions in patients with long-standing ulcerative colitis.

Serologic follow-up of children born to mothers with Ro/SSA autoantibodies.

Neonatal lupus erythematosus (NLE) develops in foetuses of mothers with Ro/SSA and La/SSB antibodies and may include foetal atrioventricular block and dermatologic manifestations. In this study, we investigated postnatal Ro and La IgG, IgA and IgM antibody levels up to 1 year of age in 32 children born to Ro/SSA positive mothers. Antibody levels were correlated with NLE manifestations, and the role of breast feeding in transfer of autoantibodies from mother to child was evaluated. Ro52, Ro60 and La IgG antibodies all transferred from the mothers to their foetus in utero and were present in the infant at birth as detected by enzyme-linked immunosorbent assay using recombinant antigens and a synthetic peptide. A significant decrease in Ro52, Ro60 and La IgG autoantibody levels of the infants was observed from birth to 4-5 weeks of age (P < 0.05, P < 0.05 and P < 0.01). Ro- and La-specific IgA and IgM antibodies were detected in the serum from a subset of mothers. However, Ro- and La-specific IgA and IgM antibody levels were low or non-detectable in children raised both with and without breastfeeding. Furthermore, NLE skin lesions developed independently of breastfeeding. Our findings support a role for placental materno-foetal transfer of IgG autoantibodies in the pathogenesis of NLE and indicate that refraining from breastfeeding does not protect from NLE skin involvement.

Antibodies to amino acid 200-239 (p200) of Ro52 as serological markers for the risk of developing congenital heart block.

Maternal autoantibodies to the p200-epitope of Ro52 have been suggested to correlate with development of congenital heart block. The aim of the present study was to evaluate the clinical relevance and predictive value of p200-antibodies in high-risk pregnancies. Sera from 515 Finnish, Swedish and American women were included in the study. Sera originated from 202 mothers with an infant affected by second- or third-degree atrioventricular block (AVB), 177 mothers with rheumatic disease having infants with normal heart rate and female blood donors (n = 136). A novel serological assay for Ro52 p200-antibodies with intra- and inter-assay variability of 3% and 3.8% respectively was developed. Mothers of children affected by AVB II-III had significantly higher p200-antibody levels than mothers with rheumatic disease having children with normal heart rate (P < 0.001). In the Swedish cohort, a distinction between foetuses with normal conduction, AVB I, AVB II and III was possible. A significant difference in anti-p200 levels between AVB I and AVB II-III groups compared with foetuses with normal conduction (P < 0.05 and P < 0.01) was observed. Using p200-antibodies as a second step analysis in Ro52-positive pregnancies increased the positive predictive value for foetal cardiac involvement (AVB I, II or III) from 0.39 (0.27-0.51) to 0.53 (0.37-0.68). In conclusion, Ro52 p200-antibodies may occur in women with unaffected children, but levels are significantly higher in mothers of children with congenital heart block and are suggested as a relevant marker in evaluating the risk for foetal AV block.

Aire deficiency causes increased susceptibility to streptozotocin-induced murine type 1 diabetes.

Aire-deficient mice are a model of the human monogenic disorder autoimmune polyendocrine syndrome type I (APS I) characterized by a progressive autoimmune destruction of multiple endocrine glands such as the adrenal cortex, the parathyroids and the beta-cells of the pancreas. The disease is caused by mutations in the autoimmune regulator (AIRE) gene, a putative transcription factor expressed in thymic medullary epithelial cells and in antigen-presenting cells of the myeloid lineage in peripheral lymphoid organs. As Aire(-/-) mice do not spontaneously develop endocrinopathies, we wanted to evaluate the autoimmune multiple low-dose streptozotocin (MLDSTZ) diabetes model in Aire(-/-) mice. Surprisingly, Aire heterozygote mice were most susceptible to MLDSTZ-induced diabetes, whereas Aire(-/-) mice displayed an intermediate sensitivity to diabetes. Furthermore, Aire(-/-) macrophages produced higher levels of TNF-alpha and lower levels of IL-10 following streptozotocin stimulation, and Aire(-/-) mice developed a higher frequency of islet cells autoantibodies as a sign of increased activation. However, the number of islet infiltrating F4/80(+) Aire(-/-) macrophages was significantly decreased which was attributed to an increased susceptibility to streptozotocin cytotoxicity of Aire(-/-) macrophages. In conclusion, Aire(-/-) macrophages display an increased activation after STZ stimuli, but suffer from increased susceptibility to STZ cytotoxicity. These results support an important function of Aire in the control of peripheral tolerance through myeloid antigen-presenting cells.

Sentinel node lymphocytes: tumour reactive lymphocytes identified intraoperatively for the use in immunotherapy of colon cancer.

The sentinel node is the first lymph node to receive lymphatic drainage from a tumour and is usually the first site of metastases. Today, the sentinel node is used for tumour staging. Here, we focus on its immunological role and investigate lymphocytic function in sentinel nodes, identified intraoperatively by peritumoural dye injection, from 15 patients with colon cancer. Tumour infiltrating lymphocytes, sentinel and nonsentinel lymph node cells and peripheral blood leukocytes were studied by flow cytometry, proliferation assays and interferon-gamma secretion after activation with autologous tumour homogenate. Whereas tumour-infiltrating lymphocytes were nonresponsive in the proliferation assays, lymphocytes from sentinel nodes proliferated dose dependently and secreted interferon-gamma upon stimulation with tumour homogenate. The responses were of varying magnitude and tended to be weaker in metastatic sentinel nodes. Sentinel node lymphocytes represents an enriched source of tumour reactive lymphocytes, and may be useful in future trials of adoptive immunotherapy.

Eosinophil granulocytes are activated during the remission phase of ulcerative colitis.

AIM: The aim of this study was to establish a method of investigating intestinal eosinophil and neutrophil granulocytes by flow cytometry, and to compare the distribution and activity of these cells in different stages of ulcerative colitis (UC). METHODS: Biopsy samples were taken from six locations of the entire colon and from the terminal ileum in 10 patients with active total UC, 10 patients with inactive total UC, eight patients with active distal UC, and 11 control subjects. Cell suspensions from biopsies and from peripheral blood were incubated with fluorophore conjugated monoclonal antibodies. The use of scatter plot-gating and specific antibodies was established in a flow cytometry assay. RESULTS: Eosinophils were more numerous and more active in patients with active UC than in controls. Interestingly, during inactive UC, the number of activated eosinophils was even larger. Eosinophil activity was high in the rectum of patients with distal colitis but was also slightly elevated in the proximal colon. Neutrophils were increased in number and activity during active but not inactive UC. In patients with distal colitis, activated neutrophils were only found in the sigmoid colon and rectum. CONCLUSION: With this method, we confirm that neutrophils participate in the inflammatory process during active UC, and that they express a resting phenotype during remission. The finding of activated eosinophils in inflamed intestine strengthens the view of these cells as proinflammatory and tissue damaging. Nevertheless, our new finding of high eosinophil activation during inactive UC suggests that eosinophils play a role in repair of injured epithelium.

Identification of sentinel nodes in patients with colon cancer.

AIMS: Lymphatic mapping and sentinel node biopsy entails better staging in malignant melanoma and breast cancer and we used this technique in patients with colon cancer to possibly improve detection of lymphatic spread. METHODS: Thirty patients with invasive adenocarcinomas were investigated. The tumour status in identified sentinel node(s) was compared with the status in all other harvested regional nodes for each patient. Patients were followed at regular visits for more than 30 months. RESULTS: Sentinel nodes were identified in all cases, either per-operatively (28 cases) or at dissection of the formalin-fixed specimen (2 cases). The sentinel nodes were diagnostic for the entire lymphatic field in 28 patients and the false negative rate was 2/12. In four cases, the sentinel nodes were the only metastatic nodes. After at minimum 30 months, three patients had died of colon cancer metastases. CONCLUSION: This method improved the identification of patients with lymph-node metastases, especially those with only few metastatic nodes. Patients dying from metastatic disease had lymph-node metastases at diagnosis.

Autoantibodies to alpha-fodrin in primary Sjögren's syndrome and SLE detected by an in vitro transcription and translation assay.

OBJECTIVE: To investigate the prevalence of alpha-fodrin autoantibodies in primary Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) with and without secondary SS, using an in vitro transcription and translation assay (ITT). METHODS: cDNA encoding JS-1, the amino-terminal portion of alpha-fodrin, was used for ITT. Immunoprecipitation was performed with sera from 56 primary SS patients and 67 SLE patients, 14 with and 53 without secondary SS. Correlations to RF, ANA, anti-dsDNA, anti-SS-A and anti-SS-B antibodies, hypergammaglobulinemia, labial salivary gland biopsy grade, extraglandular manifestations and a modified SLE disease activity index (mSLEDAI) were made. RESULTS: Autoantibodies against alpha-fodrin were detected in 16/56 (29%) of primary SS patients and in 25/53 (47%) of sera from SLE patients without secondary SS. In SLE patients with secondary SS the prevalence was 3/14 (21%). None of the blood donors showed alpha-fodrin reactivity. Correlations were found to RF, ANA, anti-dsDNA antibodies and a positive mSLEDAI score. CONCLUSION: The frequency of alpha-fodrin autoantibodies detected by this method is similar in sera from primary SS patients and SLE patients with or without secondary SS. The presence of alpha-fodrin autoantibodies seems to reflect non-organ-specific autoimmunity in primary SS and SLE and to be of limited discriminating value.

Autoimmune adrenal insufficiency: recognition and management.

The main cause of Addison's disease is an autoimmune organ-specific destruction of the cells in the adrenal cortex by an autoreactive process of activated immune cells directed against the steroid-synthesising enzyme 21-hydroxylase. The diagnosis of Addison's disease is suspected in a patient presenting with symptoms of fatigue, bodyweight loss, anorexia, salt craving, and signs of low blood pressure and hyperpigmentation of the skin. Laboratory findings include electrolyte disturbances, and typically an elevated serum potassium level and sometimes a low serum sodium level is found together with low plasma levels of basal and corticotropin-stimulated hydrocortisone (cortisol). An aetiological diagnosis can rapidly be made using commercially available assays demonstrating the presence of autoantibodies directed against 21-hydroxylase. Determination of 21-hydroxylase autoantibodies also permits early diagnosis before a complete adrenocortical destruction has occurred. Thus, a window of opportunity for an early immunomodulatory intervention therapy may exist. Patients presenting with an acute adrenocortical crisis should be treated with 100mg of hydrocortisone and saline intravenously without awaiting laboratory results. Maintenance therapy includes substitution of glucocorticoid and mineralocorticoid steroids, using divided and lower total dosages of glucocorticoids than previously used.

Impaired immunoproteasome assembly and immune responses in PA28-/- mice.

In vitro PA28 binds and activates proteasomes. It is shown here that mice with a disrupted PA28b gene lack PA28a and PA28b polypeptides, demonstrating that PA28 functions as a hetero-oligomer in vivo. Processing of antigenic epitopes derived from exogenous or endogenous antigens is altered in PA28-/- mice. Cytotoxic T lymphocyte responses are impaired, and assembly of immunoproteasomes is greatly inhibited in mice lacking PA28. These results show that PA28 is necessary for immunoproteasome assembly and is required for efficient antigen processing, thus demonstrating the importance of PA28-mediated proteasome function in immune responses.

Intercellular adhesion molecule-1 inhibits interleukin 4 production by naive T cells.

The type of cytokines produced during T cell responses determines susceptibility or resistance to many pathogens and influences the development of autoimmunity and allergy. To define the role of individual accessory molecules in cytokine production during primary immune responses, Drosophila cell lines expressing murine major histocompatibility complex class II molecules with defined combinations of accessory molecules were used to present peptide antigen to naive T cell receptor transgenic T cells. Significantly, expression of B7.1 or B7.2 without additional accessory molecules led to very high production of interleukin (IL)-4, which contrasted with minimal IL-4 production elicited by conventional antigen presenting cells (APC). However, coexpression of ICAM-1 and B7 on Drosophila APC induced little IL-4, suggesting an inhibitory role for intercellular adhesion molecule-1 (ICAM-1). In support of this idea, stimulation of T cell receptor transgenic T cells with peptide presented by splenic APC devoid of ICAM-1 (from ICAM-1-deficient mice) led to high IL-4 production. Thus, the level of IL-4 production by naive CD4(+) T cells during typical primary responses appears to be controlled, at least in part, by T-APC interactions involving ICAM-1.

The role of H2-O and HLA-DO in major histocompatibility complex class II-restricted antigen processing and presentation.

The function of major histocompatibility complex (MHC) class II molecules is to sample exogenous antigens for presentation to CD4+ T helper cells. After synthesis in the endoplasmic reticulum, class II molecules are directed into the endosomal system by association with the invariant chain (Ii), which is sequentially cleaved, generating class II dimers loaded with Ii-derived peptides (CLIP). These class II-peptide complexes are physiological substrates for H2-M/HLA-DM, a resident of the endosomal/lysosomal system which facilitates the removal of CLIP from newly synthesised class II alpha beta dimers. Exchange of CLIP for antigenic class II-binding peptides is also promoted by the action of H2-M/HLA-DM, resulting in stable peptide-class II complexes that are transported to the cell surface for presentation to CD4+ T cells. Recent evidence suggests that this H2-M/HLA-DM-mediated 'peptide editing' is influenced by another MHC class II-encoded molecule, H2-O/HLA-DO. This non-polymorphic alpha beta heterodimer is associated with H2-M/HLA-DM during intracellular transport and within the endosomal system of B cells. H2-O/HLA-DO alters the peptide exchange function of H2-M/HLA-DM in a pH-dependent manner, so that H2-M/HLA-DM activity is limited to more acidic conditions, corresponding to lysosomal compartments. Indeed, H2-O/HLA-DO may serve to limit the presentation of antigens after fluid phase uptake by B cells, while augmenting presentation of antigens internalised via membrane Ig receptors. Such a mechanism may maintain the fidelity of the B-cell-CD4+ T-cell interaction, counteracting self reactivity arising from less stringent lymphocyte activation. Here, data evaluating the role of H2-O/HLA-DO shall be reviewed and its putative function discussed.

Altered antigen presentation in mice lacking H2-O.

HLA-DM catalyzes the release of MHC class II-associated invariant chain-derived peptides (CLIP) from class II molecules. Recent evidence has suggested that HLA-DO is a negative regulator of HLA-DM in B cells, but the physiological function of HLA-DO remains unclear. Analysis of antigen presentation by B cells from mice lacking H2-O (the mouse equivalent of HLA-DO), together with biochemical analysis using purified HLA-DO and HLA-DM molecules, suggests that HLA-DO/H2-O influences the peptide loading of class II molecules by limiting the pH range in which HLA-DM is active. This effect may serve to decrease the presentation of antigens internalized by fluid-phase endocytosis, thus concentrating the B cell-mediated antigen presentation to antigens internalized by membrane immunoglobulin.

A family with hereditary extra-adrenal paragangliomas without evidence for mutations in the von Hippel-Lindau disease or ret genes.

OBJECTIVES: To characterize a family with hereditary paraganglioma, and to search for germline mutations in the von Hippel-Lindau disease (VHL) tumour suppressor gene and the ret proto-oncogene. DESIGN: Patient records and histopathological reports were reviewed. Available tumour samples were reinvestigated using immunohistochemical techniques. The VHL gene was investigated by single strand conformational polymorphism analysis of PCR products amplified from exons 1, 2 and 3 and the 3' untranslated region. The ret gene was analysed by amplifying and sequencing exons 10, 11 and 16. PATIENTS: A family with paragangliomas in three consecutive generations was investigated. RESULTS: The affected individuals were found to have multiple extra-adrenal paragangliomas. All three affected individuals had retroperitoneal tumours, and two also had paraganglioma in the neck. No mutations of the VHL or ret genes were detected. CONCLUSIONS: The described family may represent a novel dominantly inherited neuroendocrine tumour syndrome.