Maria Grazia Ennas

Genetic variation in TNF and IL10 and risk of non-Hodgkin lymphoma: a report from the InterLymph Consortium

BACKGROUND Common genetic variants in immune and inflammatory response genes can affect the risk of developing non-Hodgkin lymphoma. We aimed to test this hypothesis using previously unpublished data from eight European, Canadian, and US case-control studies of the International Lymphoma Epidemiology Consortium (InterLymph). METHODS We selected 12 single-nucleotide polymorphisms for analysis, on the basis of previous functional or association data, in nine genes that have important roles in lymphoid development, Th1/Th2 balance, and proinflammatory or anti-inflammatory pathways (IL1A, IL1RN, IL1B, IL2, IL6, IL10, TNF, LTA, and CARD15). Genotype data for one or more single-nucleotide polymorphisms were available for 3586 cases of non-Hodgkin lymphoma and for 4018 controls, and were assessed in a pooled analysis by use of a random-effects logistic regression model. FINDINGS The tumour necrosis factor (TNF) -308G-->A polymorphism was associated with increased risk of non-Hodgkin lymphoma (p for trend=0.005), particularly for diffuse large B-cell lymphoma, the main histological subtype (odds ratio 1.29 [95% CI 1.10-1.51] for GA and 1.65 [1.16-2.34] for AA, p for trend <0.0001), but not for follicular lymphoma. The interleukin 10 (IL10) -3575T-->A polymorphism was also associated with increased risk of non-Hodgkin lymphoma (p for trend=0.02), again particularly for diffuse large B-cell lymphoma (p for trend=0.006). For individuals homozygous for the TNF -308A allele and carrying at least one IL10 -3575A allele, risk of diffuse large B-cell lymphoma doubled (2.13 [1.37-3.32], p=0.00083). INTERPRETATION Common polymorphisms in TNF and IL10, key cytokines for the inflammatory response and Th1/Th2 balance, could be susceptibility loci for non-Hodgkin lymphoma. Moreover, our results underscore the importance of consortia for investigating the genetic basis of chronic diseases like cancer.

Tumor vascularity and tryptase‐positive mast cells correlate with a poor prognosis in melanoma

See commentary on page 361.

Effects of Drugs of Abuse on Putative Rostromedial Tegmental Neurons, Inhibitory Afferents to Midbrain Dopamine Cells

Recent findings have underlined the rostromedial tegmental nucleus (RMTg), a structure located caudally to the ventral tegmental area, as an important site involved in the mechanisms of aversion. RMTg contains γ-aminobutyric acid neurons responding to noxious stimuli, densely innervated by the lateral habenula and providing a major inhibitory projection to reward-encoding midbrain dopamine (DA) neurons. One of the key features of drug addiction is the perseverance of drug seeking in spite of negative and unpleasant consequences, likely mediated by response suppression within neural pathways mediating aversion. To investigate whether the RMTg has a function in the mechanisms of addicting drugs, we studied acute effects of morphine, cocaine, the cannabinoid agonist WIN55212-2 (WIN), and nicotine on putative RMTg neurons. We utilized single unit extracellular recordings in anesthetized rats and whole-cell patch-clamp recordings in brain slices to identify and characterize putative RMTg neurons and their responses to drugs of abuse. Morphine and WIN inhibited both firing rate in vivo and excitatory postsynaptic currents (EPSCs) evoked by stimulation of rostral afferents in vitro, whereas cocaine inhibited discharge activity without affecting EPSC amplitude. Conversely, nicotine robustly excited putative RMTg neurons and enhanced EPSCs, an effect mediated by α7-containing nicotinic acetylcholine receptors. Our results suggest that activity of RMTg neurons is profoundly influenced by drugs of abuse and, as important inhibitory afferents to midbrain DA neurons, they might take place in the complex interplay between the neural circuits mediating aversion and reward.

Non-Hodgkin lymphoma and obesity: A pooled analysis from the InterLymph Consortium

Nutritional status is known to alter immune function, a suspected risk factor for non‐Hodgkin lymphoma (NHL). To investigate whether long‐term over, or under, nutrition is associated with NHL, self‐reported anthropometric data on weight and height from over 10,000 cases of NHL and 16,000 controls were pooled across 18 case‐control studies identified through the International Lymphoma Epidemiology Consortium. Study‐specific odds ratios (OR) were estimated using logistic regression and combined usinga random‐effects model. Severe obesity, defined as BMI of 40 kg m−2 or more, was not associated with NHL overall (pooled OR = 1.00, 95% confidence interval (CI) 0.70–1.41) or the majority of NHL subtypes. An excess was however observed for diffuse large B‐cell lymphoma (pooled OR = 1.80, 95% CI 1.24–2.62), although not all study‐specific ORs were raised. Among the overweight (BMI 25–29.9 kg m−2) and obese (BMI 30–39.9 kg m−2), associations were elevated in some studies and decreased in others, while no association was observed among the underweight (BMI < 18.5 kg m−2). There was little suggestion of increasing ORs for NHL or its subtypes with every 5 kg m−2 rise in BMI above 18.5 kg m−2. BMI components height and weight were also examined, and the tallest men, but not women, were at marginally increased risk (pooled OR = 1.19, 95% CI 1.06–1.34). In summary, whilst we conclude that there is no evidence to support the hypothesis that obesity is a determinant of all types of NHL combined, the association between severe obesity and diffuse large B‐cell lymphoma may warrant further investigation.

Lymphoma risk and occupational exposure to pesticides: results of the EPILYMPH study

Objectives We investigated the role of occupational exposure to specific groups of agrochemicals in the aetiology of lymphoma overall, B cell lymphoma and its most prevalent subtypes. Methods In 1998–2003, 2348 incident lymphoma cases and 2462 controls were recruited to the EPILYMPH case-control study in six European countries. A detailed occupational history was collected in cases and controls. Job modules were applied for farm work including specific questions on type of crop, farm size, pests being treated, type and schedule of pesticide use. In each study centre, industrial hygienists and occupational experts assessed exposure to specific groups of pesticides and individual compounds with the aid of agronomists. We calculated the OR and its 95% CI associated with lymphoma and the most prevalent lymphoma subtypes with unconditional logistic regression, adjusting for age, gender, education and centre. Results Risk of lymphoma overall, and B cell lymphoma was not elevated, and risk of chronic lymphocytic leukaemia (CLL) was elevated amongst those ever exposed to inorganic (OR=1.6, 95% CI 1.0 to 2.5) and organic pesticides (OR=1.5, 95% CI 1.0 to 2.1). CLL risk was highest amongst those ever exposed to organophosphates (OR=2.7, 95% CI 1.2 to 6.0). Restricting the analysis to subjects most likely exposed, no association was observed between pesticide use and risk of B cell lymphoma. Conclusions Our results provide limited support to the hypothesis of an increase in risk of specific lymphoma subtypes associated with exposure to pesticides.

Nonrandom chromosomal imbalances in primary mediastinal B-cell lymphoma detected by arbitrarily primed PCR fingerprinting.

We used arbitrarily primed polymerase chain reaction (AP‐PCR) fingerprinting to identify chromosomal imbalances in six primary mediastinal B‐cell lymphomas (PMBLs). Seventy‐four chromosomal imbalances were detected, consisting of 49 sequence gains and 25 losses. Amplifications on chromosome X were seen in five cases, four of which involved the same chromosomal locus. Nonrandom gains at the same locus were also identified on chromosomes 2 and 7 in four cases and on chromosomes 5, 9, and 12 in three cases. Five PMBLs were also analyzed by comparative genomic hybridization (CGH), which found chromosome arm 9p amplification as the only nonrandom imbalance. Our data demonstrate that chromosomal amplifications outnumber losses in PMBL. These mainly involve chromosomes 9 and X and may reflect more complex phenomena, such as translocations or other chromosomal rearrangements, as AP‐PCR found coexistent gains and losses on these chromosomes. Comparison between AP‐PCR and CGH suggests that anomalies affecting the same chromosomal regions may occur at much higher frequencies than expected by CGH, suggesting that genomic amplifications are usually confined to DNA segments smaller than the megabase long segments required for detection in CGH. Modest increases in genetic material may be as effective as higher‐level amplifications when affecting sites where a proto‐oncogene resides. Genes Chromosomes Cancer 26:203–209, 1999.

PPARα regulates cholinergic-driven activity of midbrain dopamine neurons via a novel mechanism involving α7 nicotinic acetylcholine receptors.

Ventral tegmental area dopamine neurons control reward-driven learning, and their dysregulation can lead to psychiatric disorders. Tonic and phasic activity of these dopaminergic neurons depends on cholinergic tone and activation of nicotinic acetylcholine receptors (nAChRs), particularly those containing the β2 subunit (β2*-nAChRs). Nuclear peroxisome proliferator-activated receptors type-α (PPARα) tonically regulate β2*-nAChRs and thereby control dopamine neuron firing activity. However, it is unknown how and when PPARα endogenous ligands are synthesized by dopamine cells. Using ex vivo and in vivo electrophysiological techniques combined with biochemical and behavioral analysis, we show that activation of α7-nAChRs increases in the rat VTA both the tyrosine phosphorylation of the β2 subunit of nAChRs and the levels of two PPARα endogenous ligands in a Ca2+-dependent manner. Accordingly, in vivo production of endogenous PPARα ligands, triggered by α7-nAChR activation, blocks in rats nicotine-induced increased firing activity of dopamine neurons and displays antidepressant-like properties. These data demonstrate that endogenous PPARα ligands are effectors of α7-nAChRs and that their neuromodulatory properties depend on phosphorylation of β2*-nAChRs on VTA dopamine cells. This reveals an autoinhibitory mechanism aimed at reducing dopamine cell overexcitation engaged during hypercholinergic drive. Our results unveil important physiological functions of nAChR/PPARα signaling in dopamine neurons and how behavioral output can change after modifications of this signaling pathway. Overall, the present study suggests PPARα as new therapeutic targets for disorders associated with unbalanced dopamine–acetylcholine systems.

Regional distribution of 5α-reductase type 2 in the adult rat brain: An immunohistochemical analysis

The enzyme 5α-reductase (5αR) catalyzes the conversion of testosterone and other Δ(4)-3-ketosteroids into their 5α-reduced metabolites. Of the five members of the 5αR family, the type 2 enzyme (5αR2) plays a key role in androgen metabolism, and is abundantly distributed in the urogenital system. Although 5αR2 has been reported to be highly expressed in the brain during early developmental stages, little is currently known on its anatomical and cellular distribution in the adult brain. Thus, the present study was designed to determine the detailed localization of 5αR2 in the adult rat brain, using a highly specific polyclonal antibody against this isoform. Parasagittal and coronal sections revealed 5αR2 immunoreactivity throughout most brain regions, with strong immunolabeling in the layers III and VI of the prefrontal and somatosensory cortex, olfactory bulb, thalamic nuclei, CA3 field of hippocampus, basolateral amygdala and Purkinje cell layer of cerebellum. Lower 5αR2 levels were detected in the hypothalamus and midbrain. Moreover, double labeling fluorescence with confocal laser scanning microscopy (CLSM) revealed that 5αR2 is localized in neurons, but not in glial cells. Specifically, the enzyme was documented in the pyramidal neurons of the cortex by CLSM analysis of simultaneous Golgi-Cox and immunofluorescent staining. Finally, low levels of 5αR2 expression were identified in GABAergic cells across the cortex, hippocampus and striatum. These findings show that, in the adult brain, 5αR2 is distributed in critical regions for behavioral regulation, suggesting that the functional role of this isoform is present throughout the entire lifespan of the individual.

Interleukin‐1B (IL1B) and interleukin‐6 (IL6) gene polymorphisms are associated with risk of chronic lymphocytic leukaemia

Common polymorphisms in genes encoding for cytokines implicated in the inflammatory response and Th1/Th2 balance might play a role in the development and prognosis of chronic lymphocytic leukaemia (CLL). To test the hypothesis, we investigated 13 single nucleotide polymorphisms (SNPs) in nine of such genes in a population‐based case‐control study, conducted in the Italian region of Sardinia in 1999–2003. Forty incident CLL cases and 113 population controls were available for study. The following SNPs were selected: IL1A‐889C > T, IL1RN 9589A > T, IL1B‐31C > T, IL1B‐511C > T, IL2‐384T > G, IL6‐174G > C, IL6‐597G > A, IL10‐1082A > G, IL10‐3575T > A, TNF‐308G > A, LTA‐ 91A > C, LTA 252A > G and CARD15 nt1007. After adjusting by age and gender, individuals homozygous for the IL1B‐511T allele run a lower risk of CLL (OR = 0.1, 95% CI 0.0, 0.8, p = 0.032), while risk showed a 4.5‐fold increase associated with the genotype homozygous for the IL6‐174C allele (OR = 4.5; 95% CI 1.1, 19.3, p = 0.041). Individuals homozygous for the IL6‐174C allele and carrying the homozygous IL1B‐511C allele showed an 11‐fold increase in CLL risk (OR = 11.4, 95% CI 1.9, 69.4, p = 0.008). None of the other interleukin SNPs evaluated showed any association with CLL risk. Large multicentre pooled studies are warranted, achieving the statistical power required to confirm whether IL6 and IL1B gene polymorphisms might play a role in CLL development and prognosis, as well as the null associations herein reported. Copyright

Inhibition of cytokines expression in human microglia infected by virulent and non-virulent mycobacteria

The pathogenesis of tuberculosis (TBC) meningitis is still unknown. As shown by previous studies, human microglia can be the target of mycobacteria, but no data are available about their cellular response to infection. Consequently, we studied the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-10 in human microglia pure cultures infected with the two variants of Mycobacterium avium (domed-opaque (SmD) and transparent (SmT)) and with Mycobacterium tuberculosis. Results showed that microglia was productively infected by mycobacteria which could grow inside the cells. Mycobacteria internalization was more rapid for M. avium, but M. tuberculosis infection turned out to be more efficient due to the incorporation of densely packed bacteria. TNF-alpha expression was not affected by M. avium, whereas an increase followed by a decrease was observed in M. tuberculosis. Both IL-1 and IL-10 cytokine expression was rapidly inhibited by infection with the more virulent bacteria, whereas the non-pathogenic one had almost no effect. Also, the expression of the co-stimulatory molecule CD137, a member of tumor necrosis factor receptor family, was affected by infection with virulent mycobacteria. Our results show that microglia response to mycobacterial infection is modulated in correlation with virulence, mainly toward inhibition of inflammatory response. This observation might be one of the mechanisms by which non-pathogenic mycobacteria are quickly eliminated, explaining one of the bases of virulence.