Adam P. Croft

Risk of thyroid cancer in survivors of childhood cancer: results from the British Childhood Cancer Survivor Study.

Second primary neoplasms (SPNs) are a recognised late effect of treatment for childhood cancer. Thyroid SPNs can develop after exposure to low‐dose radiation, due to the radio‐sensitivity of the thyroid gland. The British Childhood Cancer Survivor Study (BCCSS) was set up to directly monitor the late effects of treatment, including risk of SPNs, in childhood cancer survivors and includes 17,980 5‐year survivors. We carried out a cohort analysis to determine the risk of thyroid SPNs in the BCCSS, and estimated risk using standardised incidence ratios (SIRs), relative risk (RR) using multivariate Poisson regression and cumulative incidence curves. There were 340,202 person years at risk subsequent to a 5‐year survival, median follow‐up 17.4 years per survivor. We identified 50 thyroid SPNs including 31 (62%) papillary carcinomas, 15 (30%) follicular carcinomas and 4 (8%) other types. 88% of thyroid SPNs developed after exposure to radiotherapy in or around the thyroid gland. SIR overall was 18.0 (95% confidence interval 13.4–23.8). Risk of thyroid cancer was highest after Hodgkins disease: RR 3.3 (1.1–10.1) and Non Hodgkins Lymphoma: RR 3.4 (1.1–10.7) relative to leukaemia (RR 1.0) (p < 0.001). Survivors treated with radiotherapy in childhood had a RR of 4.6 (1.4–15.1) relative to survivors not treated with radiotherapy (RR 1.0), p = 0003. In conclusion, the risk of thyroid cancer in childhood cancer survivors is relatively high in this cohort of childhood cancer survivors. These results will be of use in counselling survivors of childhood cancer exposed to radiation in or around the thyroid area.

Selective increases in regional brain glucocorticoid: A novel effect of chronic alcohol

The hypothalamo-pituitary-adrenal axis shows functional changes in alcoholics, with raised glucocorticoid release during alcohol intake and during the initial phase of alcohol withdrawal. Raised glucocorticoid concentrations are known to cause neuronal damage after withdrawal from chronic alcohol consumption and in other conditions. The hypothesis for these studies was that chronic alcohol treatment would have differential effects on corticosterone concentrations in plasma and in brain regions. Effects of chronic alcohol and withdrawal on regional brain corticosterone concentrations were examined using a range of standard chronic alcohol treatments in two strains of mice and in rats. Corticosterone was measured by radioimmunoassay and the identity of the corticosterone extracted from brain was verified by high performance liquid chromatography and mass spectrometry. Withdrawal from long term (3 weeks to 8 months) alcohol consumption induced prolonged increases in glucocorticoid concentrations in specific regions of rodent brain, while plasma concentrations remained unchanged. This effect was seen after alcohol administration via drinking fluid or by liquid diet, in both mice and rats and in both genders. Shorter alcohol treatments did not show the selective effect on brain glucocorticoid levels. During the alcohol consumption the regional brain corticosterone concentrations paralleled the plasma concentrations. Type II glucocorticoid receptor availability in prefrontal cortex was decreased after withdrawal from chronic alcohol consumption and nuclear localization of glucocorticoid receptors was increased, a pattern that would be predicted from enhanced glucocorticoid type II receptor activation. This novel observation of prolonged selective increases in brain glucocorticoid activity could explain important consequences of long term alcohol consumption, including memory loss, dependence and lack of hypothalamo-pituitary responsiveness. Local changes in brain glucocorticoid levels may also need to be considered in the genesis of other mental disorders and could form a potential new therapeutic target.

Effects of minor laboratory procedures, adrenalectomy, social defeat or acute alcohol on regional brain concentrations of corticosterone

Concentrations of corticosterone in brain areas of TO strain mice were measured by radioimmunoassay. The studies examined the effects of routine laboratory maneuvers, variation during the circadian peak, adrenalectomy, social defeat and acute injections of alcohol on these concentrations. Brief handling of mice increased corticosterone levels in plasma but not in striatum and reduced those in the hippocampus. Single injections of isotonic saline raised the plasma concentrations to a similar extent as the handling, but markedly elevated concentrations in the three brain regions. Five minutes exposure to a novel environment increased hippocampal and cerebral cortical corticosterone levels and striatal concentrations showed a larger rise. However, by 30 min in the novel environment, plasma concentrations rose further while those in striatum and cerebral cortex fell to control levels and hippocampal corticosterone remained elevated. Over the period of the circadian peak the hippocampal and striatal concentrations paralleled the plasma concentrations but cerebral cortical concentrations showed only small changes. Adrenalectomy reduced plasma corticosterone concentrations to below detectable levels after 48 h but corticosterone levels were only partially reduced in the hippocampus and striatum and remained unchanged in the cerebral cortex. Single or repeated social defeat increased both brain and plasma concentrations after 1 h. Acute injections of alcohol raised the regional brain levels in parallel with plasma concentrations. The results show that measurements of plasma concentrations do not necessarily reflect the levels in brain. The data also demonstrate that corticosterone levels can change differentially in specific brain regions. These results, and the residual hormone seen in the brain after adrenalectomy, are suggestive evidence for a local origin of central corticosterone.

Effects of the glucocorticoid antagonist, mifepristone, on the consequences of withdrawal from long term alcohol consumption

BACKGROUND Studies were carried out to test the hypothesis that administration of a glucocorticoid Type II receptor antagonist, mifepristone (RU38486), just prior to withdrawal from chronic alcohol treatment, would prevent the consequences of the alcohol consumption and withdrawal in mice. MATERIALS AND METHODS The effects of administration of a single intraperitoneal dose of mifepristone were examined on alcohol withdrawal hyperexcitability. Memory deficits during the abstinence phase were measured using repeat exposure to the elevated plus maze, the object recognition test, and the odor habituation/discrimination test. Neurotoxicity in the hippocampus and prefrontal cortex was examined using NeuN staining. RESULTS Mifepristone reduced, though did not prevent, the behavioral hyperexcitability seen in TO strain mice during the acute phase of alcohol withdrawal (4 hours to 8 hours after cessation of alcohol consumption) following chronic alcohol treatment via liquid diet. There were no alterations in anxiety-related behavior in these mice at 1 week into withdrawal, as measured using the elevated plus maze. However, changes in behavior during a second exposure to the elevated plus maze 1 week later were significantly reduced by the administration of mifepristone prior to withdrawal, indicating a reduction in the memory deficits caused by the chronic alcohol treatment and withdrawal. The object recognition test and the odor habituation and discrimination test were then used to measure memory deficits in more detail, at between 1 and 2 weeks after alcohol withdrawal in C57/BL10 strain mice given alcohol chronically via the drinking fluid. A single dose of mifepristone given at the time of alcohol withdrawal significantly reduced the memory deficits in both tests. NeuN staining showed no evidence of neuronal loss in either prefrontal cortex or hippocampus after withdrawal from chronic alcohol treatment. CONCLUSIONS The results suggest mifepristone may be of value in the treatment of alcoholics to reduce their cognitive deficits.

CD248+ stromal cells are associated with progressive chronic kidney disease.

Stromal fibroblasts are the primary cells of the kidney that produce fibrotic matrix. CD248 is a stromal marker expressed on fibroblasts and pericytes within the human kidney. Here, we tested whether CD248 expression in the kidney colocalizes with fibrosis and if it is associated with known determinants of chronic kidney disease (CKD). CD248 expression was located and quantified in situ by immunohistochemistry in kidney biopsies from 93 patients with IgA nephropathy and compared with 22 archived biopsies encompassing normal kidney tissue as control. In normal kidney tissue, CD248 was expressed by resident pericytes, stromal fibroblasts, and was upregulated in human CKD. The expression was linked to known determinants of renal progression. This relationship was maintained in a multivariate analysis with CD248 expression linked to renal survival. CD248 was expressed by a population of α-smooth muscle actin (SMA)(+) myofibroblasts and α-SMA(-) stromal cells but not expressed on CD45(+) leukocytes. Thus, CD248 defines a subset of stromal cells, including but not limited to some myofibroblasts, linked to albuminuria and tubulointerstitial damage during tissue remodeling in CKD.

The mesenchymal stem cell marker CD248 (endosialin) is a negative regulator of bone formation in mice

OBJECTIVE CD248 (tumor endothelial marker 1/endosialin) is found on stromal cells and is highly expressed during malignancy and inflammation. Studies have shown a reduction in inflammatory arthritis in CD248-knockout (CD248(-/-) ) mice. The aim of the present study was to investigate the functional effect of genetic deletion of CD248 on bone mass. METHODS Western blotting, polymerase chain reaction, and immunofluorescence were used to investigate the expression of CD248 in humans and mice. Micro-computed tomography and the 3-point bending test were used to measure bone parameters and mechanical properties of the tibiae of 10-week-old wild-type (WT) or CD248(-/-) mice. Human and mouse primary osteoblasts were cultured in medium containing 10 mM β-glycerophosphate and 50 μg/ml ascorbic acid to induce mineralization, and then treated with platelet-derived growth factor BB (PDGF-BB). The mineral apposition rate in vivo was calculated by identifying newly formed bone via calcein labeling. RESULTS Expression of CD248 was seen in human and mouse osteoblasts, but not osteoclasts. CD248(-/-) mouse tibiae had higher bone mass and superior mechanical properties (increased load required to cause fracture) compared to WT mice. Primary osteoblasts from CD248(-/-) mice induced increased mineralization in vitro and produced increased bone over 7 days in vivo. There was no decrease in bone mineralization and no increase in proliferation of osteoblasts in response to stimulation with PDGF-BB, which could be attributed to a defect in PDGF signal transduction in the CD248(-/-) mice. CONCLUSION There is an unmet clinical need to address rheumatoid arthritis-associated bone loss. Genetic deletion of CD248 in mice results in high bone mass due to increased osteoblast-mediated bone formation, suggesting that targeting CD248 in rheumatoid arthritis may have the effect of increasing bone mass in addition to the previously reported effect of reducing inflammation.

CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism

Introduction CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. Objective To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. Design CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248−/− and wild-type controls with carbon tetrachloride (CCl4) treatment. Results Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248−/− mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248−/− mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-β at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248−/− HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248−/− HSC was confirmed by significantly reduced c-fos expression in CD248−/− HSC compared with wild-type HSC. Conclusions Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury.

A Differential Role for CD248 (Endosialin) in PDGF-Mediated Skeletal Muscle Angiogenesis

CD248 (Endosialin) is a type 1 membrane protein involved in developmental and pathological angiogenesis through its expression on pericytes and regulation of PDGFRβ signalling. Here we explore the function of CD248 in skeletal muscle angiogenesis. Two distinct forms of capillary growth (splitting and sprouting) can be induced separately by increasing microcirculatory shear stress (chronic vasodilator treatment) or by inducing functional overload (extirpation of a synergistic muscle). We show that CD248 is present on pericytes in muscle and that CD248-/- mice have a specific defect in capillary sprouting. In contrast, splitting angiogenesis is independent of CD248 expression. Endothelial cells respond to pro-sprouting angiogenic stimulus by up-regulating gene expression for HIF1α, angiopoietin 2 and its receptor TEK, PDGF-B and its receptor PDGFRβ; this response did not occur following a pro-splitting angiogenic stimulus. In wildtype mice, defective sprouting angiogenesis could be mimicked by blocking PDGFRβ signalling using the tyrosine kinase inhibitor Imatinib mesylate. We conclude that CD248 is required for PDGFRβ-dependant capillary sprouting but not splitting angiogenesis, and identify a new role for CD248 expressed on pericytes in the early stages of physiological angiogenesis during muscle remodelling.

Refractory multisystem sarcoidosis responding to infliximab therapy

Chronic progressive multisystem granulomatous disease is seen in 10–30% of patients with sarcoidosis and can result in end organ damage. Corticosteroids are the mainstay of treatment with the addition of cytotoxic agents in severe cases. Some patients are refractory to such treatment and, therefore, management is a challenge. There is currently limited evidence for biological agents such as infliximab, a monoclonal anti-tumor necrosis factor-α antibody in the treatment of multisystem sarcoidosis. We report outcomes of three patients with extensive multisystem sarcoidosis refractory to conventional treatment and treated at our center. Clinical assessment and radiographic imaging were used to assess the response to infliximab treatment. Infliximab therapy induced clinical remission in all three patients, and this clinical response correlated with radiographic evidence of the resolution of granulomatous disease. Serum ACE level was reduced in all cases, and daily steroid dosage was reduced. We propose that infliximab can be an effective treatment in patients with multisystem complex sarcoidosis refractory to conventional drug therapy and can result in sustained clinical remission. Our experience supports the urgent need for randomized controlled clinical trials of anti-TNF therapy in refractory systemic sarcoidosis.

Nimodipine prior to alcohol withdrawal prevents memory deficits during the abstinence phase.

Effects of the dihydropyridine, nimodipine, an antagonist at L-type calcium channels, on the memory loss in rats caused by long term alcohol consumption were examined. Either a single dose of nimodipine or 2 weeks of repeated administration was given prior to withdrawal from 8 months of alcohol consumption. Memory was measured by the object recognition test and the T maze. Both nimodipine treatments prevented the memory deficits when these were measured between 1 and 2 months after alcohol withdrawal. At the end of the memory testing, 2 months after cessation of chronic alcohol consumption, glucocorticoid concentrations were increased in specific regions of rat brain without changes in plasma concentrations. Both nimodipine treatment schedules substantially reduced these rises in brain glucocorticoid. The data indicate that blockade of L-type calcium channels prior to alcohol withdrawal protects against the memory deficits caused by prolonged alcohol intake. This shows that specific drug treatments, such as nimodipine, given over the acute withdrawal phase, can prevented the neuronal changes responsible for subsequent adverse effects of long term consumption of alcohol. The results also suggest the possibility that regional brain glucocorticoid increases may be involved in the adverse effects of long term alcohol intake on memory. Such local changes in brain glucocorticoid levels would have major effects on neuronal function. The studies indicate that L-type calcium channels and brain glucocorticoid levels could form new targets for the treatment of cognitive deficits in alcoholics.