Assessment of the genetic causes of recessive childhood non-syndromic deafness in the UK - implications for genetic testing.

Approximately one in 2000 children is born with a genetic hearing impairment, mostly inherited as a non-syndromic, autosomal recessive trait, for which more than 30 different genes have been identified. Previous studies have shown that one of these genes, connexin 26 (GJB2), accounts for 30-60% of such deafness, but the relative contribution of the many other genes is not known, especially in the outbred UK population. This lack of knowledge hampers the development of diagnostic genetic services for deafness. In an effort to determine the molecular aetiology of deafness in the population, 142 sib pairs with early-onset, non-syndromic hearing impairment were recruited. Those in whom deafness could not be attributed to GJB2 mutations were investigated further for other mapped genes. The genetic basis of 55 cases (38.7%) was established, 33.1% being due to mutations in the GJB2 gene and 3.5% due to mutations in SLC26A4. None of the remaining 26 loci investigated made a significant contribution to deafness in a Caucasian population. We suggest that screening the GJB2 and SLC26A4 genes should form the basis of any genetic testing programme for childhood deafness and highlight a number of important issues for consideration and future work.

A randomized pilot study of SRL172 (Mycobacterium vaccae) in patients with small cell lung cancer (SCLC) treated with chemotherapy.

BACKGROUND: SRL172 is a suspension of heat killed Mycobacterium vaccae, that has been found to be a potent immunological adjuvant when used with autologous cells in animal models. This is a phase II study to test the clinical activity, feasibility and safety of combining SRL172 with chemotherapy to treat patients with small cell lung cancer (SCLC). METHODS: Patients were randomized to receive chemotherapy with (n=14) or without (n=14) SRL172. The chemotherapy was either platinum-based (MVP, n=10) or anthracycline-based (ACE, n=18). SRL172 was given intradermally on day 0, weeks 4, 8 and then 3-6 monthly. RESULTS: The treatment arms were well balanced for disease extent (43% with limited stage in each arm). The toxicity of chemotherapy and overall response at 12-15 weeks (57%) was the same for both treatment regimens. Median survival was 8.6 months and 12.9 for patients treated with chemotherapy alone and with the combination respectively (P=0.10). The survival trend was similar for both disease extent and chemotherapy regimen employed in favour of combination chemotherapy with SRL172. CONCLUSIONS: There is a trend to improved median survival in SCLC with the combination of chemotherapy and SRL172 with no increased toxicity and irrespective of drug regimen. A phase III study examining chemotherapy in combination with SRL172 in SCLC is now underway.

Clinical and immunological assessment of Mycobacterium vaccae (SRL172) with chemotherapy in patients with malignant mesothelioma.

The objectives of this study were to determine the toxicity of intratumoural/intrapleural SRL172 in addition to intradermal SRL172 and standard chemotherapy (mitomycin-C, vinblastine and cisplatin) in patients with malignant mesothelioma. Patients received chemotherapy (mitomycin-C: 8 mg m(-2), vinblastine: 6 mg m(-2), cisplatin 50 mg m(-2)) on a 3-weekly basis for up to six courses. IP SRL172 injections were given 3-weekly prior to chemotherapy and escalated in groups of three patients from 1 microg to 1 mg bacilli in 10-fold increments. Patients were also given ID SRL172 at a dose of 1 mg bacilli 4-weekly. Patients were assessed for toxicity after each course of chemotherapy and for response by CT imaging. Immuno-haematological parameters were analyzed pre-treatment and 1 month after completion of treatment. There was no dose limiting toxicity with IP SRL172 although there was greater toxicity at the highest dose (n=13). There were six out of 16 partial responses (37.5%). Haemato-immunological parameters, measured in seven patients pre and post-therapy, revealed that response rate correlated with a decrease in platelet count and there was an increase in activation of natural killer cells and a decrease in the percentage of IL-4 producing T cells in all tested patients post-treatment. SRL172 can be given safely into tumour deposits and the pleural cavity in patients with malignant mesothelioma and we have established the dose for phase II testing.

Interleukin 10 in Helicobacter pylori associated gastritis: immunohistochemical localisation and in vitro effects on cytokine secretion.

BACKGROUND/AIMS: Interleukin 10 (IL-10) is a counter-inflammatory peptide implicated in the downregulation of human intestinal immune responses. Enhanced secretion of IL-10 has been documented in gastric biopsy organ culture in Helicobacter pylori infection. This study aimed to define the cellular origins of IL-10 in H pylori associated gastritis, and to determine the effects of endogenous IL-10 on proinflammatory cytokine secretion in vitro. METHODS: Endoscopic biopsies were obtained from the gastric antrum at endoscopy from patients with dyspepsia. Two pairs of antral biopsies were cultured in vitro for 24 hours, one pair in the presence of neutralising anti-IL-10 monoclonal antibody, the other pair as controls. The cytokine content of culture supernatants (tumour necrosis factor alpha (TNF-alpha), IL-6, and IL-8) was determined by enzyme linked immunosorbent assay and corrected for biopsy weight. Helicobacter pylori status was established by histology and biopsy urease test, and histopathology graded by the Sydney system. In a subgroup of patients, western blotting was used to establish CagA serological status. Immunohistochemistry for IL-10 was performed on formalin fixed tissues using a combination of microwave antigen retrieval and the indirect avidin-biotin technique. Immunoreactivity was scored semiquantitatively. RESULTS: In vitro culture was performed in 41 patients: 31 with H pylori positive chronic gastritis and 10 H pylori negative. In vitro secretion of TNF-alpha, IL-6, and IL-8 for "control" biopsies was significantly higher in H pylori positive versus negative samples, with values of TNF-alpha and IL-6 correlating with the degree of active and chronic inflammation and being higher in CagA seropositive cases. No evidence for enhanced cytokine secretion was seen in biopsies cocultured in the presence of anti-IL-10 monoclonal antibody. Immunohistochemistry was performed in 29 patients, of whom 13 were H pylori positive. IL-10 immunoreactivity was observed in the surface epithelium in all H pylori positive cases and in 13 of 16 negative cases, especially in areas of surface epithelial degeneration. Lamina propria mononuclear cells (LPMNCs) were positively stained in all H pylori positive cases and in 12 of 16 negative cases, with a significantly greater proportion of positive LPMNCs in the positive group. CONCLUSIONS: This study localised IL-10 protein to the gastric epithelium and LPMNCs. In vitro proinflammatory cytokine secretion was increased in H pylori infection (especially CagA positive infection), but blocking endogenous IL-10 secretion did not significantly increase cytokine secretion. IL-10 is implicated in H pylori infection and might "damp down" local inflammation. The role of gastric IL-10 secretion in determining the clinicopathological outcome of infection merits further study.

Whole blood assay for assessment of the mixed lymphocyte reaction.

When lymphocytes from genetically different individuals are mixed together in tissue culture blast transformation occurs, a reaction known as the mixed lymphocyte reaction (MLR). The MLR is a clinically relevant in vitro assay where lymphocytes from one individual (effector, E) are incubated with the lymphocytes of another individual (stimulator, S) which have been previously rendered incapable of blast transformation by gamma-irradiation. We have standardised a whole blood (WB) MLR assay where the E lymphocytes were provided by 20 microl of WB and the S lymphocytes were provided by irradiated peripheral blood mononuclear cells (PBMCs) either as a mixed pool of 20 donor PBMCs or as single donor PBMC. The optimum number of S lymphocytes needed was comparatively higher than in the standard PBMC MLR: the optimum calculated E:S ratio was 1:20 compared to a E:S ratio of 1:1 or 3:2 in the standard PBMC MLR. In ten normal individuals the WB/PBMC MLR was similar to the standard PBMC/PBMC MLR. As a clinical example, the WB/PBMC MLR proliferative capacity of 13 patients with malignant mesothelioma was no different from the proliferative capacity of their age-sex matched controls. This standardised WB/PBMC MLR assay is a simple and more practical assay than the standard MLR assay and can be incorporated easily in clinical studies with biological end-points.

A randomized phase II study of SRL172 (Mycobacterium vaccae) combined with chemotherapy in patients with advanced inoperable non-small-cell lung cancer and mesothelioma.

Mycobacterial preparations have been used with limited success against cancer apart from superficial bladder cancer. Recently, a therapeutic vaccine derived from Mycobacterium vaccae has been given to patients with prostate cancer and melanoma indicating a possible beneficial effect on disease activity in such patients. We have recently initiated a series of randomized studies to test the feasibility and toxicity of combining a preparation of heat-killed Mycobacterium vaccae (designated SRL172) with a multidrug chemotherapy regimen to treat patients with inoperable non-small cell lung cancer (NSCLC) and mesothelioma. 28 evaluable patients with previously untreated symptomatic NSCLC and mesothelioma were randomized to receive either 3 weekly intravenous combination chemotherapy alone, or chemotherapy given with monthly intra-dermal injections of SRL172. Safety and tolerability were scored by common toxicity criteria and efficacy was evaluated by survival of patients and by tumour response assessed by CT scanning. The toxicity of chemotherapy was similar in the two groups. SRL172 caused mild inflammation at the injection site. In the group of patients randomized to receive chemotherapy combined with SRL172, there was a trend towards improved response rate (54% vs. 33%) with more patients in the combined arm receiving radical surgery and radiotherapy, improved median survival (9.7 months vs. 7.5 months) and improved 1 year survival (42% vs. 18%). SRL172 appeared to improve sleep (P = 0.08) and improved appetite (P = 0.01). There was no detectable change in serum cytokine levels for gamma-interferon and TNF-alpha before and after treatment. In patients with NSCLC and mesothelioma, there may be a beneficial interaction when chemotherapy is administered in combination with SRL172. Confirmation of this effect and further investigation is underway in a randomized phase III trial and in laboratory models.

Flow cytometric visualisation of cytokine production by CD3-CD56+ NK cells and CD3+CD56+ NK-T cells in whole blood.

BACKGROUND: Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring. METHODS: With a three-colour fluorescent labelling technique, specific cytokine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK population or on the CD3+/CD56+ NK-T-cell population. RESULTS: Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and ionomycin stimulation. CONCLUSIONS: This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3+CD56+ NK-T lymphocytes in patients with relevant diseases.

A highly sensitive whole blood natural killer cell assay.

The assessment of natural killer cell activity at baseline and the monitoring of this activity during treatment is important in many diseases especially in patients with cancer and AIDS. An optimised and standardised whole blood chromium release assay is described using K562 cells, the standard target erythroleukaemic cell line. The tumour cell lysis observed using whole blood is comparable to that obtained with the standard 4 h lysis assay using peripheral blood mononuclear cells as effector cells. Results with the whole blood assay are reproducible when the incubation with K562 cells is performed over a period of 18 h. The assay necessitates only 0.6 ml of blood collected in 10 IU/ml of sodium heparin as the anticoagulant. In this report, depletion experiments, also standardised using whole blood, show that the effector cells in the whole blood assay are contained within the CD56 + cell population. This assay will be of interest where the immunological status of patients with different diseases need to be frequently monitored.

Lack of T cell response to cardiac myosin and a reduced response to PPD in patients with idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDCM) is the main cause of cardiac transplantation in young adults in the 20-40 years age group in the Western world. Recent evidence supports a possible role for autoimmune pathogenesis in IDCM and it has been suggested that T cells could mediate the disease. Cardiac myosin is one of the putative autoantigens recognized by antibodies from patients with IDCM, but T cell responses to cardiac myosin have not previously been assessed. Proliferation to cardiac myosin by peripheral blood mononuclear cells (PBMC) from patients, their relatives and controls was assessed in a lymphoproliferation assay specifically designed to measure low frequency T cell precursor responses. The study group consisted of 23 patients with IDCM and 29 relatives. The control groups consisted of 10 patients with heart failure secondary to ischaemic heart disease (IHD) and 22 healthy laboratory controls. A response to myosin was observed in 16.7% of the subjects studied. However, these responses were all of low precursor frequency and no dose response for antigen-specific proliferation could be observed. More importantly, there was no correlation between myosin-specific T cell responses and IDCM, as only one IDCM patient and four IDCM relatives (three out of the four with left ventricular enlargement (LVE)) were among the 14 subjects whose PBMC exhibited a proliferative response. However, proliferation of PBMC to purified protein derivative of Mycobacterium bovis (PPD) was significantly suppressed in IDCM patients when compared with the laboratory control group (P<0.05). PPD response data suggested that the PPD suppression correlated with disease progression. The results of our present study indicate an absence, or lack, of cardiac myosin-specific peripheral blood T cells in IDCM patients, along with the possibility of underlying impaired cell mediated immunity, reflected in the suppressed responses to PPD. Future studies looking at T cell immune mechanisms in IDCM should concentrate on the analysis of T cells from the heart itself, or look at other potential cardiac antigens from normal and diseased heart tissue.