Search

Human faecal waters from 35 healthy non-smoking volunteers (23 from England and 12 from Sweden) consuming their habitual diet were screened for genotoxicity by the single-cell gel electrophoresis (comet) assay using a human colon adenocarcinoma cell line (CACO-2) as the target. Hydrogen peroxide induced DNA damage was categorized as low, intermediate or high for tail moments greater than 5, 17 and 32, respectively: 11 samples were highly genotoxic, four were intermediate, one was low and 19 showed no activity. Endonuclease III treatment significantly increased DNA damage for all except the non-genotoxic faecal waters, suggesting that faecal water genotoxicity may be due, at least in part, to oxidative damage. Faecal water cytotoxicity has previously been attributed to the bile and fatty acid content. In the comet assay no DNA damage was induced by deoxycholate or lithocholate at normal physiological concentrations, suggesting that the genotoxicity of faecal water was due to other substances. Both bile acids induced DNA damage above 300 microM, levels often found in patients with colonic polyps and there was a significant increase in genotoxicity after endonuclease III treatment indicative of oxidative DNA damage. [ABSTRACT FROM PUBLISHER]

Background: Targeting immunometabolism has shown promise in treating autoimmune and inflammatory conditions. Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease involving painful lesions in apocrine gland-bearing skin. Therapeutic options for HS are limited and often ineffective; thus, there is a pressing need for improved treatments. To date, metabolic dysregulation has not been investigated in HS. As HS is highly inflammatory, we hypothesized that energy metabolism is dysregulated in these patients. Metformin, an antidiabetic drug, which is known to impact on cellular metabolic and signalling pathways, has been shown to have anti-inflammatory effects in cancer and arthritis. While metformin is not licensed for use in HS, patients with HS taking metformin show improved clinical symptoms., Objective: To assess the effect and mechanism of action of metformin in HS., Methods: To assess the effect of metformin in vivo, we compared the immune and metabolic profiles of peripheral blood mononuclear cells (PBMCs) of patients with HS taking metformin vs. those not taking metformin. To examine the effect of metformin treatment ex vivo, we employed a skin explant model on skin biopsies from patients with HS not taking metformin, which we cultured with metformin overnight. We used enzyme-linked immunosorbent assays, multiplex cytokine assays and quantitative real-time polymerase chain reaction (RT-PCR) to measure inflammatory markers, and Seahorse flux technology and quantitative RT-PCR to assess glucose metabolism., Results: We showed that metabolic pathways are dysregulated in the PBMCs of patients with HS vs. healthy individuals. In metformin-treated patients, these metabolic pathways were restored and their PBMCs had reduced inflammatory markers following long-term metformin treatment. In the skin explant model, we found that overnight culture with metformin reduced inflammatory cytokines and chemokines and glycolytic genes in lesions and tracts of patients with HS. Using in vitro assays, we found that metformin may induce these changes via the NLR family pyrin domain containing 3 (NLRP3) inflammasome and the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway, which is linked to glycolysis and protein synthesis., Conclusions: Our study provides insight into the mechanisms of action of metformin in HS. The anti-inflammatory effects of metformin support its use as a therapeutic agent in HS, while its effects on immunometabolism suggest that targeting metabolism is a promising therapeutic option in inflammatory diseases, including HS., Competing Interests: Conflicts of interest R.H. has received honoraria from AbbVie, Janssen and UCB; and has acted as a subinvestigator in clinical trials for AbbVie and UCB. N.K. has received honoraria from AbbVie, Janssen, Lilly and UCB; and has acted as a subinvestigator in clinical trials for AbbVie, Moonlake and UCB. B.K. has received grants and/or honoraria from AbbVie, Almirall, Astra Zeneca, Biogen, Bristol Myers Squibb, Celgene, Janssen, Lilly, LEO Pharma, Merck, Moonlake, Novartis, Pfizer and UCB Pharma.​ J.M.F. has received honoraria from Novartis and Moonlake. A.P., C.M.S., E.K.P., J.M.M., A.M.O’R., M.I., P.A.B., J.P.A., C.K., A.Z., S.M. and D.W. declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists.)

Background: Treatment for the debilitating disease hidradenitis suppurativa (HS) is inadequate in many patients. Despite an incidence of approximately 1%, HS is often under-recognized and underdiagnosed, and is associated with a high morbidity and poor quality of life., Objectives: To gain a better understanding of the pathogenesis of HS, in order to design new therapeutic strategies., Methods: We employed single-cell RNA sequencing to analyse gene expression in immune cells isolated from involved HS skin vs. healthy skin. Flow cytometry was used to quantify the absolute numbers of the main immune populations. The secretion of inflammatory mediators from skin explant cultures was measured using multiplex and enzyme-linked immunosorbent assays., Results: Single-cell RNA sequencing analysis identified a significant enrichment in the frequency of plasma cells, T helper (Th) 17 cells and dendritic cell subsets in HS skin, and the immune transcriptome was distinct and more heterogeneous than healthy skin. Flow cytometry revealed significantly increased numbers of T cells, B cells, neutrophils, dermal macrophages and dendritic cells in HS skin. Genes and pathways associated with Th17 cells, interleukin (IL)-17, IL-1β and the NLRP3 inflammasome were enhanced in HS skin, particularly in samples with a high inflammatory load. Inflammasome constituent genes principally mapped to Langerhans cells and a subpopulation of dendritic cells. The secretome of HS skin explants contained significantly increased concentrations of inflammatory mediators, including IL-1β and IL-17A, and culture with an NLRP3 inflammasome inhibitor significantly reduced the secretion of these, as well as other, key mediators of inflammation., Conclusions: These data provide a rationale for targeting the NLRP3 inflammasome in HS using small-molecule inhibitors that are currently being tested for other indications., Competing Interests: Conflicts of interest: R.H. has received honoraria from AbbVie, Janssen and UCB, and has acted as a subinvestigator in clinical trials for AbbVie and UCB. K.H.G.M. is the co-founder of and a shareholder in a startup biotechnology company involved in the development of anti-inflammatory therapeutics. B.K. has received grants and/or honoraria from AbbVie, Almirall, AstraZeneca, Biogen, Bristol Myers Squibb, Celgene, Janssen, Lilly, LEO Pharma, Merck, Moonlake, Novartis, Pfizer and UCB Pharma.​ J.M.F. has received honoraria from Novartis and Moonlake. M.R. is a former employee of AbbVie. B.M., C.M.S., A.Z., J.K., R.W.D., K.M.S., J.M., A.P., A.F., M.O’D., K.H., W.J.H. and D.C.W. declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists.)

Hidradenitis suppurativa (HS) is a common cutaneous and systemic inflammatory disease with a significant impact on mental health and quality of life. It is associated with obesity, insulin resistance, metabolic syndrome, cardiovascular (CV) disease, and increased all-cause mortality. Metformin is used frequently in HS treatment and is effective for some patients. The mechanism of action of metformin in HS is unknown. A case-control study of 40 patients with HS (20 on metformin and 20 controls) was conducted to assess differences in metabolic markers, inflammation (C-reactive protein [CRP], serum adipokines, and CV risk biomarkers), and serum immune mediators. Body mass index (BMI), insulin resistance (77%), and metabolic syndrome (44%) were high overall, but not significantly different between the groups. This highlights the need for co-morbidity screening and management. A significant reduction in fasting insulin and a trend towards a reduction in insulin resistance were identified in the metformin group compared with pre-treatment levels. CV risk biomarkers were significantly favourable in the metformin group (lymphocytes, monocyte-lymphocyte ratio, neutrophil-lymphocyte ratio, and platelet-lymphocyte ratio). CRP was lower in the metformin group but was not statistically significant. Adipokines were dysregulated overall but were not different between the two groups. Serum IFN-γ, IL-8, TNF-α, and CXCL1 trended lower in the metformin group but did not reach significance. These results suggest that metformin improves CV risk biomarkers and insulin resistance in patients with HS. When the results of this study are considered alongside other studies in HS and related conditions, it is likely that metformin also has beneficial effects on metabolic markers and systemic inflammation in HS (CRP, serum adipokines, and immune mediators), warranting further research.

Hidradenitis suppurativa (HS) is a chronic relapsing inflammatory skin disease manifested as painful inflamed lesions including deep-seated nodules, abscesses and sinus tracts. The exact aetiology of HS is unclear. Recent evidence suggests that immune dysregulation plays a crucial role in pathogenesis and disease progression. Innate lymphoid cells (ILC) are a recently identified immune cell subset involved in mediating immunity, however their role in HS has not yet been investigated. Three distinct subsets of ILC- ILC1, ILC2 and ILC3 have been described, and these are involved in skin tissue homeostasis and pathologic inflammation associated with autoimmunity and allergic diseases. In this study, we analysed by multiparameter flow cytometry the frequencies of ILC subsets in skin and peripheral blood mononuclear cells (PBMC) of HS patients and compared these to healthy control subjects and psoriasis patients. The absolute numbers of total ILC and subsets thereof were significantly reduced in the blood of HS patients relative to healthy controls. However, when patients were stratified according to treatment, this reduction was no longer observed in patients undergoing anti-TNF treatment. In HS lesional skin the absolute numbers of ILC were significantly increased relative to control skin. Furthermore, the frequencies of total ILC as well as ILC2 and ILC3 were significantly higher in non-lesional than lesional HS skin. This study analysed for the first time the presence of ILC subsets in the blood and skin of HS patients. Our findings suggest that ILC may participate in HS pathogenesis., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: RHa received support for attendance at meetings from AbbVie and Janssen. OM received support for attendance at meetings from Novartis. BM received support for attendance at meetings from Miltenyi. AM received support for attendance at meeting from AbbVie. BK has received research support/principal investigator (clinical trials) from AbbVie, Alimirall, Janssen, Merck Sharpe Dolme, Moonlake, Novartis,Pfizer and UCB; been a consultant for AbbVie, Amgen, Almirall, Cel-gene, Janssen, Merck Sharpe Dolme, Moonlake, Novartis, Pfizer and UCB; received honoraria from AbbVie, Amgen, Alimrall, Celgene, Janssen, Lilly, Moonlake, Novartis, Pfizer and UCB; and been on scientific advisory boards for AbbVie, Almirall, Celgene, Janssen, Lilly, Moonlake Novartis, Pfizer and UCB. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Petrasca et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Competing Interests: Conflicts of interest: R. Hambly has received honoraria from AbbVie, Janssen and UCB and has acted as a subinvestigator in clinical trials for AbbVie and UCB. B.K. has received research support from or been a principal investigator (clinical trials) for AbbVie, Almirall, Janssen, Merck Sharpe & Dohme, Novartis, Pfizer and UCB; has been a consultant for AbbVie, Almirall, Celgene, Janssen, Merck Sharpe & Dohme, Novartis, Pfizer and UCB; has received honoraria from AbbVie, Almirall, Celgene, Janssen, Lilly, Novartis, Pfizer and UCB; and been on scientific advisory boards for AbbVie, Almirall, Celgene, Janssen, Lilly, Novartis, Pfizer and UCB.

Background: Hidradenitis suppurativa (HS) is a chronic inflammatory skin disorder with significant morbidity. The pathogenesis remains incompletely understood although immune dysregulation plays an important role. It is challenging to treat and approximately 50% of patients respond clinically to adalimumab, the only licensed treatment., Objectives: To examine differences between lesional and nonlesional HS skin at baseline using bulk RNA sequencing, and to compare the transcriptome in the skin before and after 12 weeks of treatment with adalimumab. To examine transcriptomic differences between adalimumab responders and nonresponders using Hidradenitis Suppurativa Clinical Response and the International Hidradenitis Suppurativa Severity Score System (IHS4); and to compare transcriptomic differences based on disease severity (Hurley stage and IHS4)., Methods: We completed bulk RNA sequencing on lesional and nonlesional skin samples of patients before and after 12 weeks of treatment with adalimumab., Results: Baseline differentially expressed genes and pathways between lesional and nonlesional skin highlighted chemokines and antimicrobial peptides produced by keratinocytes; B-cell function; T-cell-receptor, interleukin-17 and nuclear factor-κB signalling; and T-helper-cell differentiation. Transcriptomic differences were identified in lesional skin at baseline, between subsequent responders and nonresponders. Patients with severe HS who did not respond to adalimumab had enriched complement and B-cell activation pathways at baseline. In addition, logistic regression identified CCL28 in baseline lesional HS skin as a potential biomarker of treatment response., Conclusions: This highlights the potential for targeting B-cell and complement pathways in HS treatment and the potential of stratifying patients at baseline to the most suitable treatment based on the skin transcriptome. CCL28 has not previously been identified in HS skin and has potential clinical relevance due to its antimicrobial function and homing of B and T cells at epithelial surfaces. Our results provide data to inform future translational and clinical studies on therapeutics in HS., Competing Interests: Conflicts of interest: R. Hambly has received honoraria from AbbVie, Janssen and UCB and has acted as a subinvestigator in clinical trials for AbbVie and UCB. B.K. has received research support from or been a principal investigator (clinical trials) for AbbVie, Almirall, Janssen, Merck Sharp & Dohme, Moonlake, Novartis, Pfizer and UCB; has been a consultant for AbbVie, Almirall, Celgene, Janssen, Merck Sharp & Dohme, Moonlake, Novartis, Pfizer and UCB; has received honoraria from AbbVie, Almirall, Celgene, Janssen, Lilly, Moonlake, Novartis, Pfizer and UCB; and has been on scientific advisory boards for AbbVie, Almirall, Celgene, Janssen, Lilly, Moonlake, Novartis, Pfizer and UCB., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor licensed for the treatment of type 2 diabetes mellitus (T2DM), has been reported to improve psoriasis., Objective: We compared the effects of sitagliptin treatment, a DPP-4 inhibitor, in combination with narrow-band ultraviolet-B (NB-UVB) phototherapy compared to NB-UVB alone on psoriasis severity, quality of life, cardiovascular disease risk factors and immune parameters in people with moderate psoriasis without T2DM., Methods: In this 39-week, single-centre, randomised controlled trial, people were allocated randomly to receive sitagliptin for 24 weeks with NB-UVB or NB-UVB alone. The primary endpoint was the change in Psoriasis Area and Severity Index (PASI) from baseline to 24 weeks. We estimated that 120 participants would be needed to have 80% power to find a significant difference between the groups., Results: A total of 118 patients were randomised. The median (IQR) baseline PASI was 8.8 (7.5-11.6). At 24 weeks, the mean difference from baseline in PASI (-1.0 [95% CI -2.0 to 0.0]) was significantly larger in the sitagliptin/NB-UVB arm than in the NB-UVB-alone arm (p = 0.044). There were significant differences in the change in Hospital Anxiety and Depression Scale (-2.5 [95% CI -4.0 to -1.0]; p = 0.002) and EuroQol 5-item questionnaire (0.1 [95% CI 0.0-0.1]; p = 0.036) values from baseline to 24 weeks between the sitagliptin/NB-UVB and the NB-UVB-alone arm. There were no treatment-related serious adverse events., Conclusion: Sitagliptin therapy combined with NB-UVB phototherapy significantly improved psoriasis severity, albeit modestly, compared to NB-UVB phototherapy alone in patients with moderate psoriasis without T2DM., (© 2021 S. Karger AG, Basel.)

Background: The burden of illness associated with atopic dermatitis (AD) is significant and multidimensional, especially in those with moderate to severe disease., Objective: Our objective was to evaluate the disease burden of patients with AD in relation to psychological distress, sleep disturbance, and alcohol misuse., Methods: Patients with AD, attending 2 tertiary referral centers in Dublin, Ireland, were recruited. A series of validated questionnaires were used including the Patient-Oriented Eczema Measure, Dermatology Life Quality Index (DLQI), Center for Epidemiologic Studies-Depression Scale, Quality of Life in Atopic Dermatitis Questionnaire, Alcohol Use Disorders Identification Test, and Pittsburgh Sleep Quality Index. The Eczema Area and Severity Index was calculated contemporaneously with the questionnaire completion., Results: One hundred patients completed the questionnaire, of whom 52% were female. Sixty-three percent of patients experienced impaired quality of life as measured by the DLQI. Higher DLQI scores correlated with decreasing age (r = 0.3277, P < 0.0009). Thirty percent were found to be at risk of clinical depression, and higher Center for Epidemiologic Studies-Depression Scale scores correlated with a younger age and eczema severity. Sleep disturbance was greater in those at risk of depression (mean = 10.40 vs 5.79, P < 0.0001). Patients with moderate to severe AD were more likely to score higher on the Alcohol Use Disorders Identification Test, and 25% met the criteria for alcohol use disorder. In relation to sleep, 73% of patients scored higher than 5 on the Pittsburgh Sleep Quality Index, which signifies poor sleep quality., Conclusions: Patients with AD endure a significant burden on health with regard to mental well-being, alcohol use, and sleep quality. Clinicians should consider screening patients for these comorbidities., Competing Interests: The authors have no funding or conflicts of interest to declare., (Copyright © 2020 American Contact Dermatitis Society. All Rights Reserved.)

Background: Hidradenitis suppurativa (HS) is a chronic debilitating inflammatory disease, associated with metabolic syndrome, obesity and insulin resistance. Metformin, an oral hypoglycaemic agent, may play an important role in delaying or preventing the onset of diabetes and metabolic syndrome. Metformin has been reported as having efficacy in HS. It may have a role in the treatment of HS and its associated co-morbidities. Objective: To evaluate metformin use, response and tolerability in a HS population. Methods: A retrospective chart review of patients attending a specialist Dermatology HS clinic over 12 months. All patients treated with metformin were included. Results: Fifty-three HS patients received metformin; 85% female; mean age was 37 years and mean weight was 102 kg. The mean duration of metformin was 11.3 months and mean dose was 1.5 g/days. The 6- and 12-month drug survival were 61% and 39%, respectively. Metformin was well tolerated. Gastrointestinal side effects were experienced by 11%. Subjective clinical response was seen in 68% ( n  = 36) with 19% (7/36) of these having quiescent disease with metformin monotherapy. 25% had no improvement. Insulin resistance was seen in 75%. Its presence did not predict clinical response to metformin. Conclusion: Metformin is an effective, well tolerated and inexpensive treatment that represents a viable treatment option for HS. Key message: Metformin is an effective; well tolerated and inexpensive treatment in the management of HS.

Psoriasis is a chronic autoimmune disease mediated by dysregulated immune responses in dendritic cells (DC) and T cells. The stress-response enzyme heme oxygenase-1 (HO-1) has been described as protective in animal models of psoriasis, however, implementation of HO-1-based therapies is hindered by the lack of clinically-suitable HO-1 inducers. The plant-derived polyphenols, carnosol and curcumin, have been identified as candidate HO-1 inducers however there has been little investigation into their effects on human immune cells. We demonstrate that treatment of human DC with these polyphenols limits DC maturation, reduces pro-inflammatory cytokine production, and prevents induction of allospecific T cell responses, in a manner partially dependent on carbon monoxide (CO). We also characterised their effects in ex-vivo psoriasis PBMC and report that curcumin, but not carnosol, strongly reduces T cell proliferation and cytokine poly-functionality, with reduced expression of psoriatic cytokines IFNγ, IL-17, GM-CSF and IL-22. This study therefore supports reports highlighting the therapeutic potential of curcumin in psoriasis by providing insight into its immunological effects on healthy human DC and psoriasis PBMC. We also demonstrate, for the first time, the anti-inflammatory effects of carnosol in human immune cells.

Implantable ventricular assist devices (VADs) have proven efficient in advanced heart failure patients as a bridge-to-transplant or destination therapy. However, VAD usage often leads to infection, bleeding, and thrombosis, side effects attributable to the damage to blood cells and plasma proteins. Measuring hemolysis alone does not provide sufficient information to understand total blood damage, and research exploring the impact of currently available pumps on a wider range of blood cell types and plasma proteins such as von Willebrand factor (vWF) is required to further our understanding of safer pump design. The extracorporeal CentriMag (Thoratec Corporation, Pleasanton, CA, USA) has a hemolysis profile within published standards of normalized index of hemolysis levels of less than 0.01 g/100 L at 100 mm Hg but the effect on leukocytes, vWF multimers, and platelets is unknown. Here, the CentriMag was tested using bovine blood (n = 15) under constant hemodynamic conditions in comparison with a static control for total blood cell counts, hemolysis, leukocyte death, vWF multimers, microparticles, platelet activation, and apoptosis. The CentriMag decreased the levels of healthy leukocytes (P < 0.006), induced leukocyte microparticles (P < 10(-5) ), and the level of high molecular weight of vWF multimers was significantly reduced in the CentriMag (P < 10(-5) ) all compared with the static treatment after 6 h in vitro testing. Despite the leukocyte damage, microparticle formation, and cleavage of vWF multimers, these results show that the CentriMag is a hemocompatible pump which could be used as a standard in blood damage assays to inform the design of new implantable blood pumps., (© 2014 The Authors. Artificial Organs published by Wiley Periodicals, Inc. on behalf of International Center for Artificial Organs and Transplantation (ICAOT).)

Background: Methadone maintenance treatment in primary care is cost-effective and improves outcomes for opiate-dependent patients. A more developed understanding of the evolving needs of this important cohort will facilitate further improvements in their integrated care within the community., Objectives: The aim of this study was to compare the burden of chronic disease, multi-morbidity and intensity of health-service use between methadone-maintained patients (MMPs) and matched controls in primary care., Methods: This is a retrospective matched case-control design. Data on chronic disease and health service use was collected in 13 computerized GP surgeries on 414 patients (207 MMPs and 207 controls). Twelve months of records were examined. MMPs were compared with controls matched by gender, age, socio-economic status (SES) and GP surgery., Results: MMPs suffered more chronic disease (OR = 9.1, 95% CI: 5.4-15.1, P < 0.001) and multi-morbidity (OR = 6.6, 95% CI: 4.3-10.2, P < 0.001). They had higher rates of respiratory, psychiatric and infectious disease. MMPs of lower SES had more chronic disease than their peers (OR = 7.2, 95% CI: 2.4-22.0, P < 0.001). MMPs attended the doctor more often with medical problems (OR = 15.4, 95% CI: 8.2-28.7, P < 0.001), with a frequent requirement to have medical issues addressed during methadone-management visits. Their care generated more telephone calls (OR = 4.4, 95% CI: 2.8-6.8, P < 0.001), investigations (OR = 1.8, 95% CI: 1.2-2.7, P = 0.003), referrals (2.6, 95% CI: 1.7-4.0, P < 0.001), emergency department visits (2.1, 95% CI: 1.3-3.6, P = 0.004), outpatient attendances (2.3, 95% CI: 1.51-1.43, P < 0.001) and hospital admissions (3.6, 95% CI: 1.6-8.1, P = 0.001)., Conclusion: Correcting for routine methadone care and drug-related illnesses, MMPs had a higher burden of chronic disease and used both primary and secondary health services more intensively than matched controls.

Although there is much epidemiological evidence for an interaction between diet and colorectal cancer risk, the mechanisms by which diet might protect against colorectal cancer are still unclear. Here we report the significant up-regulation of carcinogen-induced apoptosis in the colon of rats fed a diet containing low-risk factors for colon cancer, namely low fat content, high calcium and high non-digestible carbohydrate. The dose-dependent induction of apoptosis in colonic crypts by the carcinogen 1,2-dimethylhydrazine (DMH) was significantly greater in rats receiving the low-risk compared with a high-risk (high fat, low calcium, low non-digestible carbohydrate) diet (P<0.001). There were also significant interactions of colon region with DMH dose and region by diet, with the greatest increases in apoptosis occurring in the mid and distal regions of the colon compared with the proximal region. Since we have previously shown the low-risk diet to be non-toxic, these new results suggest a diet-induced up-regulation of apoptosis, which may represent a mechanism of protection against the early stages of carcinogenesis in the colon.

Thalassaemia is a group of genetic diseases where haemoglobin synthesis is impaired. This chronic anaemia leads to increased dietary iron absorption, which develops into iron overload pathology. Treatment through regular transfusions increases oxygen capacity but also provides iron through the red cells' haemoglobin. An essential treatment, in parallel with transfusions, is the use of chelating agents to remove the excess iron deposited in tissues. These deposits are found in the liver, spleen, heart, and pancreas and are associated with cardiac failure and diabetes. The deposits in these tissues of patients have been isolated as haemosiderin. Thalassaemia patients are particularly at risk of free radical induced damage. Thus, the present study has investigated, as a model system, human cells in vitro in the Comet assay in the presence of free radicals. This assay measures DNA damage, particularly DNA strand breakage. The effects of iron overload on cells oxidatively stressed with hydrogen peroxide (H(2)O(2)) have been determined as well as the effect of the chelating agent, deferoxamine. Iron overload was simulated with ferric (FeCl(3)) and ferrous chloride (FeCl(2)), ferrous sulphate (FeSO(4)) and haemosiderins. Both human lymphocytes from a male and a female donor and human adenocarcinoma colonic cells showed an increase in DNA damage in the Comet assay after treatment with H(2)O(2). Ferric chloride produced an increase in DNA damage in human colonic cells, but little or no damage in human lymphocytes. Ferrous chloride also produced weak DNA damage in human lymphocytes, but ferrous sulphate produced a dose-related response. Deferoxamine produced no DNA damage. When H(2)O(2) was combined with FeCl(3), FeCl(2), or FeSO(4), the DNA damage produced was as least as great as or slightly greater than with H(2)O(2) alone. When deferoxamine was combined with H(2)O(2) and FeSO(4) there was a consistent decrease in response. There was little or no decrease in response when deferoxamine was combined with H(2)O(2) and FeCl(3) or FeCl(2), but at high (100-300microm) doses there were changes in the appearance of cellular DNA from Comet tails to dense centres surrounded by a diffuse area. This was probably as a consequence of chelation processes. Haemosiderin produced no damage. The three fractions of haemosiderin examined were of three different densities and from a Thai patient where the oxyhydroxide phase is the ferrihydrite. The colour change was similar to that for FeCl(3), but the level of the ferric ion in the haemosiderin was possibly too low in the sample to produce a response. The next stage is to examine peripheral lymphocytes from thalassaemic patients, with and without chelation therapy, whose cells may be more sensitive to simulated iron overload and to lower levels of haemosiderin. Teratogenesis Carcinog. Mutagen. 20:11-26, 2000., (Copyright 2000 Wiley-Liss, Inc.)

In previous studies, N-(N'-acetyl-L-propyl)-N-nitrosoglycine (APNG) has been shown to be a potent mutagen in a variety of genotoxicity assays and a carcinogen in a limited cancer study. APNG decomposes to a carboxymethyldiazonium ion, which can also be generated from potassium diazoacetate (KDA). KDA is particularly interesting because it is a stable nitrosated derivative of glycine, one of the most common dietary amino acids. KDA has been shown to produce more O6 carboxymethyl- and O6 methyl-adducts than APNG, so it was anticipated that it might also be a potent genotoxic agent. Thus in the present study KDA has been investigated in the single cell gel electrophoresis (Comet) assay, which primarily measures DNA strand breakage. Since KDA has been shown to be formed in the gut, the genotoxic effects of KDA were investigated in vitro in human adenocarcinoma colon Caco-2 cells, and in rat primary colon cells and compared to responses from human peripheral lymphocytes. KDA induced DNA damage in the three cell types, confirming that KDA is genotoxic in a range of mammalian cells.

The single cell gel electrophoresis assay (Comet assay) was used to measure DNA damage in peripheral lymphocytes from a group of individuals from The Gambia in order to determine whether such damage could be associated with increased exposure to aflatoxin in this population. Responses obtained were correlated to responses previously obtained [1] in a cross-sectional study in the same individuals of various cytogenetic alterations [chromosomal aberrations, micronuclei (crest positive and negative staining), and sister chromatid exchanges], and aflatoxin-albumin adducts. Analysis of variance methods were used to assess the effects of smoking, GSTM1 genotype, sex, age, and smoking status. A comparison was also made between The Gambian individuals and a group of healthy, non-smoking volunteers in the United Kingdom where aflatoxin exposure would be expected to be low. From the earlier study [1], it was determined that the levels of the sister chromatid exchanges and micronuclei were higher in The Gambian group than in a European group where aflatoxin exposure was lower, but that there were no correlations between the adduct levels and the cytogenetic abnormalities at the individual level. In the present study, DNA damage as measured in the Comet assay was not significantly higher than in the healthy United Kingdom volunteers. In addition, there were no associations between cytogenetic damage, GSTM1 genotype, age, sex, lifestyle factors (smoking and aflatoxin exposure), and Comet response at the individual level. Comet response was higher in females than males in The Gambia if one outlier was excluded from analysis and not taking into account other sources of variability. It would appear that DNA damage as measured in the Comet assay in peripheral blood lymphocytes is not a sensitive genotoxic marker of aflatoxin exposure in this population.

Human breast cancer cell lines are required as models for use in the understanding of breast carcinoma, and for improving the ability of cell screens to detect appropriate anti-cancer agents. Four human breast cancer cell lines (MT-1, MaTu. MT-3 and MC4000) were established from human tumour xenografts grown in nude mice. All the lines were shown to be of human origin by karyotype analysis, were epithelial in morphology by both light and electron microscopy, were positive for cytokeratin 18, and were free from mycoplasma, bacterial, yeast and fungal contamination. All of the new lines were shown to be ER and PgR negative, while using the same procedures (i.e. radioligand binding and immunohistochemical staining) the positive control cell line MCF-7 was shown to be positive. MaTu had been previously reported as ER and PgR positive in vivo and it may be that this characteristic had been lost due to in vitro selection pressures. The growth rates of all the new breast cancer cell lines were similar and within the limits required for incorporation into a panel for screening anti-cancer drugs by a microtetrazolium based, colorimetric growth inhibition assay. Three of the lines (MT-1. MaTu and MC4000) were also able to grow into macroscopic colonies for use in a non-agar clonogenic assay. In addition, both MT-1 and MaTu formed spheroids and were clonogenic in soft-agar. The new lines demonstrated a wide range of sensitivities to anticancer agents commonly used in the treatment of breast cancer, and together with their corresponding xenografts are providing additional systems for the evaluation of new compounds.

Formulated diets associated with a high risk (HR) or low risk (LR) for colon cancer were used to assess the effect of diet on putative metabolic biomarkers in human flora-associated rats: The HR diet was high in fat and sucrose and low in calcium and fiber; the LR diet was low in fat and high in starch, calcium, and fiber. The nutrient-to-energy ratio and energy intake were the same for both diets. Body and liver weights were significantly higher in animals fed the HR diet, possibly due to greater energy availability from fat. Cecal weights were significantly higher in animals fed the LR diet, presumably due to a bulking effect of the fiber and increased bacterial biomass. The HR diet significantly altered cecal bacterial enzyme activity: beta-glucuronidase activity increased 2.5-fold, and beta-glucosidase activity was halved. Ammonia production and the bacterial metabolism of 2-amino-3-methyl-7H-imidazo[4,5-f] quinoline (IQ) to 7-hydroxy-IQ (7OHIQ) were significantly higher in animals fed the HR diet. The HR diet, which contained factors common to diets consumed throughout the Western world, increased beta-glucuronidase activity, elevated cecal ammonia concentrations, and enhanced the genotoxic risk from 7OHIQ formation, three putative metabolic biomarkers of colorectal cancer. The significance of the reduction in beta-glucosidase is unclear.

Clinical and coronary arteriographic findings were evaluated in patients with angina pectoris who were considered not to have diabetes mellitus or to have chemical or clinical diabetes. Each of the three groups consisted of 100 consecutive referred patients. Neither the age of the patients nor duration of symptoms differed significantly among the groups. Hypertension, gout, and peripheral vascular disease were more frequent in the patients with clinical diabetes. There was no difference in serum cholesterol concentration among the groups, but plasma triglyceride levels and the frequency of type 4 hyperlipoproteinemia were significantly higher (p less than 0.01) in the chemical and clinical diabetic groups than in the nondiabetic patients. Coronary arteriographic observations indicated that the severity of the coronary arterial disease was greater in both diabetic groups than in nondiabetic patients. The difference in the coronary scores among the three groups of patients interacts to some extent with the triglyceride level, since a high score in the diabetic groups was noted only in the presence of an elevated tryglyceride concentration. The results indicate that the increased severity of coronary arterial disease in diabetic patients is not attributable to age, duration of symptoms, hypertension, type -4 hyperlipoproteinemia, or apparent severity of the glucose intolerance.