Characterizing Adversity of Lysosomal Accumulation in Nonclinical Toxicity Studies: Results from the 5th ESTP International Expert Workshop.

Lysosomes have a central role in cellular catabolism, trafficking, and processing of foreign particles. Accumulation of endogenous and exogenous materials in lysosomes represents a common finding in nonclinical toxicity studies. Histologically, these accumulations often lack distinctive features indicative of lysosomal or cellular dysfunction, making it difficult to consistently interpret and assign adverse dose levels. To help address this issue, the European Society of Toxicologic Pathology organized a workshop where representative types of lysosomal accumulation induced by pharmaceuticals and environmental chemicals were presented and discussed. The expert working group agreed that the diversity of lysosomal accumulations requires a case-by-case weight-of-evidence approach and outlined several factors to consider in the adversity assessment, including location and type of cell affected, lysosomal contents, severity of the accumulation, and related pathological effects as evidence of cellular or organ dysfunction. Lysosomal accumulations associated with cytotoxicity, inflammation, or fibrosis were generally considered to be adverse, while those found in isolation (without morphologic or functional consequences) were not. Workshop examples highlighted the importance of thoroughly characterizing the biological context of lysosomal effects, including mechanistic data and functional in vitro readouts if available. The information provided here should facilitate greater consistency and transparency in the interpretation of lysosomal effects.

[Non-cardiac and non-pulmonary causes of dyspnea]. / Nichtkardiale und nichtpulmonale Ursachen der Dyspnoe.

In addition to cardiac and pulmonary diseases there is a broad variety of different underlying causes of dyspnea. The spectrum includes the different forms of anemia, all causes of upper airway obstructions, neuromuscular diseases and psychopathological disorders. This article gives a brief review of the entire spectrum by providing information about differential diagnostics as well as the main therapeutic principles. A field of growing interest is dyspnea in the context of palliative care.

A genome-wide scan for human obesity genes reveals a major susceptibility locus on chromosome 10.

Obesity, a common multifactorial disorder, is a major risk factor for type 2 diabetes, hypertension and coronary heart disease (CHD). According to the definition of the World Health Organization (WHO), approximately 6-10% of the population in Westernized countries are considered obese. Epidemiological studies have shown that 30-70% of the variation in body weight may be attributable to genetic factors. To date, two genome-wide scans using different obesity-related quantitative traits have provided candidate regions for obesity. We have undertaken a genome-wide scan in affected sibpairs to identify chromosomal regions linked to obesity in a collection of French families. Model-free multipoint linkage analyses revealed evidence for linkage to a region on chromosome 10p (MLS=4.85). Two further loci on chromosomes 5cen-q and 2p showed suggestive evidence for linkage of serum leptin levels in a genome-wide context. The peak on chromosome 2 coincided with the region containing the gene (POMC) encoding pro-opiomelanocortin, a locus previously linked to leptin levels and fat mass in a Mexican-American population and shown to be mutated in obese humans. Our results suggest that there is a major gene on chromosome 10p implicated in the development of human obesity, and the existence of two further loci influencing leptin levels.

Toxicological evaluation of brominated vegetable oil in Sprague Dawley rats.

Brominated vegetable oil (BVO) has been approved by the US Food and Drug Administration on an interim basis as a food additive. Past studies have raised concerns about potential toxicities from consuming BVO. To investigate further these toxicities, we conducted a 90-day dietary exposure study in Sprague Dawley rats and analyzed tissue distribution of the main metabolites. Six-week-old male and female rats were fed diets containing 0 (control), 0.002%, 0.02%, 0.1%, or 0.5% BVO by weight. Statistically significant increases were observed in the serum bromide in the high-dose group of both sexes and in the incidence of thyroid follicular cell hypertrophy in the two highest dose groups of males and the high-dose group of females. An increase in serum TSH was observed in the high-dose group for both sexes, as well as a decrease in serum T4 in the high-dose males. A clear dose-response was observed in di- and tetra-bromostearic acid levels in the heart, liver, and inguinal fat. These data expand upon previous observations in rats and pigs that oral exposure to BVO is associated with increased tissue levels of inorganic and organic bromine, and that the thyroid is a potential target organ of toxicity.

An important role for Myb-MuvB and its target gene KIF23 in a mouse model of lung adenocarcinoma.

The conserved Myb-MuvB (MMB) multiprotein complex has an important role in transcriptional activation of mitotic genes. MMB target genes are overexpressed in several different cancer types and their elevated expression is associated with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. However, although MMB has been extensively characterized biochemically, the requirement for MMB in tumorigenesis in vivo has not been investigated. Here we demonstrate that MMB is required for tumor formation in a mouse model of lung cancer driven by oncogenic K-RAS. We also identify a requirement for the mitotic kinesin KIF23, a key target gene of MMB, in tumorigenesis. RNA interference-mediated depletion of KIF23 inhibited lung tumor formation in vivo and induced apoptosis in lung cancer cell lines. Our results suggest that inhibition of KIF23 could be a strategy for treatment of lung cancer.

Antiviral therapy reduces viral burden but does not prevent thymic involution in young cats infected with feline immunodeficiency virus.

The thymus is a major target organ in human immunodeficiency virus type 1 (HIV-1)-infected children and feline immunodeficiency virus (FIV)-infected young cats (G. A. Dean and N. C. Pedersen, J. Virol. 72:9436-9440, 1998; J. L. Heeney, Immunol. Today 16:515-520, 1995; S. M. Schnittman et al., Proc. Natl. Acad. Sci. USA 87:7727-7731, 1990; T. A. Seemayer et al., Hum. Pathol. 15:469-474, 1984; H.-J. Shuurn et al., Am. J. Pathol. 134:1329-1338, 1989; J. C. Woo et al., J. Virol. 71:8632-8641, 1997; J. C. Woo et al., AIDS Res. Hum. Retrovir. 15:1377-1388, 1999). It is likely that the accelerated disease process in children and cats is due to infection of the thymus during the time when generation of naive T lymphocytes is needed for development of the mature immune system. Zidovudine (ZDV) monotherapy, which is used to prevent and treat perinatal HIV-1 infection (R. Sperling, Infect. Dis. Obstet. Gynecol. 6:197-203, 1998), previously had been shown to reduce viral burden in FIV-infected young cats (K. A. Hayes et al., J. Acquir. Immune Defic. Syndr. 6:127-134, 1993). The purpose of this study was to evaluate the effect of drug-induced reduction of viral burden in the thymus on virus-mediated thymic involution and peripheral blood CD4 decline using FIV-infected cats as a model for pediatric HIV-1 infection. Eight-week-old cats were randomly assigned to uninfected, saline-treated; uninfected, ZDV-treated; FIV-infected, saline-treated; and FIV-infected, ZDV-treated groups. Parameters measured included blood lymphocyte numbers, viral load in blood and thymic tissue, and thymic histopathology. While the viral burden was significantly reduced by ZDV monotherapy in peripheral blood lymphocytes, plasma, and thymus, thymic lesions were similar for the treated and untreated FIV-infected cats. Further, markedly lowering the viral burden did not increase blood CD4 lymphocyte numbers or prevent their decline. The data suggest that an inflammatory process continued in spite of reduced virus replication. These observations imply that reducing virus load and limiting thymic inflammation are separate factors that must be addressed when considering therapeutic strategies aimed at preserving thymic function.

Early suppression of viremia by ZDV does not alter the spread of feline immunodeficiency virus infection in cats.

Prophylactic zidovudine (ZDV) therapy in feline immunodeficiency virus (FIV) inoculated cats was evaluated for 12 months postinfection (pi) and 11 months post drug treatment. Plasma FIV antigenemia was prevented in six of six ZDV-treated and none of six untreated cats during the initial phase of infection. The present study is a continuation of that earlier work. CD4 lymphocyte numbers from ZDV-treated cats were higher than in the untreated cats. CD8 lymphocytes numbers were maintained within control limits in the ZDV-treated cats, while they declined in the untreated cats. Anti-FIV antibody titers were comparable between the ZDV-treated and the untreated cats. Histologically, lymphoid tissues for the untreated and ZDV-treated cats were unremarkable and similar to those of the uninfected control cats. Low-level FIV antigen was detected by enzyme-linked immunosorbent assay in thymus or lymph node homogenates from 3 of 11 cats tested. Polymerase chain reaction analysis showed FIV DNA in blood, lymph node, bone marrow, spleen, thymus, and brain from FIV-inoculated cats irrespective of ZDV treatment. Therefore, while prophylactic ZDV treatment prevented detectable plasma antigenemia and FIV-induced CD8 lymphocyte decline, it did not slow infection of tissues and blood cells of FIV-inoculated cats.

Effect of zidovudine on the primary cytolytic T-lymphocyte response and T-cell effector function.

Azidothymidine (AZT) and other nucleoside analogues, used to treat AIDS, can cause severe clinical side effects and are suspected of suppressing immune cell proliferation and effector immune cell function. The purpose of the present study was to quantitatively measure the effects of AZT on cytotoxic T-lymphocyte (CTL) priming and to determine if the major histocompatibility complex-restricted CTL killing was affected by AZT exposure. For this purpose, we employed a murine alloantigen model and limiting-dilution analysis (LDA) to estimate cytotoxic effector cell frequencies of alloreactive splenocytes treated with drug during antigen sensitization. This noninfectious model was chosen to avoid analysis of a virus-compromised immune system. Exposure of splenocytes to therapeutic concentrations of AZT (2 to 10 microM) caused a two- to threefold dose-dependent reduction in CLT precursor frequency. This reduction was caused by decreased proliferation of alloantigen-specific CTLs rather than loss of function, because full cytolytic function could be restored by adjusting the AZT-treated effector/target cell ratios to that of untreated cells. In addition, when AZT was added to the assay system at various times during antigen sensitization there was a time-related loss of the suppressive effect on the generation of cytolytic effector function, suggesting that functional CTLs are not affected by even high doses of AZT. Taken together, the data indicate that the reduction of CTL function associated with AZT treatment is due to a quantitative decrease of effector cell precursor frequency rather than to direct drug cytotoxicity or interference with mediation of cytolysis. Furthermore, antigen-naive immune cells were most sensitive to this effect during the first few days following antigen encounter.

Genetic and environmental nature of the insulin resistance syndrome in Indo-Mauritian subjects with premature coronary heart disease: contribution of beta3-adrenoreceptor gene polymorphism and beta blockers on triglyceride and HDL concentrations.

AIMS/HYPOTHESIS: Insulin resistance syndrome is an important risk factor for developing premature coronary heart disease. It is a complex syndrome which could arise from the interaction of several genes modulated by environmental factors. The Trp64Arg polymorphism in the beta3-adrenoreceptor gene has been found to be associated with insulin resistance, obesity or with earlier onset of Type II (non-insulin-dependent) diabetes mellitus in different populations. We aimed to study whether features of the insulin resistance syndrome are associated with this polymorphism in Indo-Mauritian patients with premature coronary heart disease. METHODS: We carried out a case control study using PCR-RFLP techniques, of consecutive Indo-Mauritian patients (n = 338) with premature coronary heart disease (onset below age of 60 years) and unrelated control subjects (n = 148) of the same ethnicity. RESULTS: In Indo-Mauritian patients with premature coronary heart disease who were not treated by beta blockers, triglyceride concentrations were lower (median: 1.44 vs 1.93 mmol/l, p < 0.008) and HDL cholesterol concentrations higher (mean: 0.98 vs 0.85 mmol/l, p < 0.015) in those carrying the wild type of the beta3-adrenoreceptor gene than in those carrying the Trp64Arg variant. The latter genotype specific effect on HDL was inverse in patients treated with beta blockers. CONCLUSION/INTERPRETATION: The beta3-adrenoreceptor gene variant seems to modulate the effects of beta blockers on triglyceride and HDL cholesterol concentrations in this group of Indo-Mauritian population. Dyslipidaemia, a key component of the metabolic syndrome has a heterogeneous nature in Indo-Mauritian subjects. This potent risk factor for early-onset coronary heart disease is influenced by the interaction between genetic and environmental effects.

Genetic studies of the leptin receptor gene in morbidly obese French Caucasian families.

Family studies have shown that in some populations up to 75% of the variation of body mass index can be explained by genetic factors. However, in humans, no major obesity gene has been identified to date. In contrast, there are a number of genetically well defined animal models for obesity. In two of those models (ob/ob and db/db), defects in the same pathway are responsible for obesity. Recently, some evidence has been found for the OB gene also being involved in human obesity. In this study we investigated the potential role of the OB receptor (OBR) in the etiology of massive obesity in humans using familial linkage analyses and case-control association studies. The typing of two microsatellite markers (D1S198 and D1S209), flanking the OBR gene, in 256 sib pairs showed no evidence for linkage with obesity. In order to be able to detect small gene effects, association studies with a 3'-UTR insertion/deletion polymorphism were carried out. The results of these analyses remained non-significant (chi 2 = 3.442, P = 0.18). However, subjects heterozygous for the insertion/deletion polymorphism showed a slight trend towards lower insulin values 30 min after an oral glucose load compared to homozygous individuals (P = 0.02). In summary, our results do not support a major role of the human OBR gene in the development of morbid obesity in our population.

A polymorphism in the 5' untranslated region of the human ob gene is associated with low leptin levels.

OBJECTIVE: To search the human ob gene for mutations and evaluate their role in massive obesity. DESIGN: Direct mutation screening of the gene and case-control association study. Multivariate analyses for evaluation of differences in clinical parameters. SUBJECTS: Primary mutation screening: 24 morbidly obese subjects (body mass index (BMI) > 40 kg/m2). Association study: 395 unrelated morbidly obese subjects (BMI > 40 kg/m2), 121 lean, non-diabetic control individuals, 72 women of a random sample with an average BMI 32.5 kg/m2. RESULTS: We report the finding of a DNA variant in exon 1 of the human ob gene (A --> G substitution, base + 19). This variant showed a prevalence of 62% in our study population. Association analyses under different genetic models (dominant, co-dominant, recessive) showed no significant evidence for an association of this variant with BMI. However, obese individuals homozygous for the G-allele showed significantly lower leptin concentrations compared to obese patients either heterozygous or homozygous for the A-allele after correction for BMI. CONCLUSION: Recent linkage studies have shown evidence for linkage of the hsob locus with obesity. Our study provides further evidence that a defect in the ob gene in linkage disequilibrium with the G-allele of exon 1 might be involved in obesity by affecting leptin concentrations.

Genomewide search for type 2 diabetes-susceptibility genes in French whites: evidence for a novel susceptibility locus for early-onset diabetes on chromosome 3q27-qter and independent replication of a type 2-diabetes locus on chromosome 1q21-q24.

Despite recent advances in the molecular genetics of type 2 diabetes, the majority of susceptibility genes in humans remain to be identified. We therefore conducted a 10-cM genomewide search (401 microsatellite markers) for type 2 diabetes-related traits in 637 members of 143 French pedigrees ascertained through multiple diabetic siblings, to map such genes in the white population. Nonparametric two-point and multipoint linkage analyzes-using the MAPMAKER-SIBS (MLS) and MAXIMUM-BINOMIAL-LIKELIHOOD (MLB) programs for autosomal markers and the ASPEX program for chromosome X markers-were performed with six diabetic phenotypes: diabetes and diabetes or glucose intolerance (GI), as well as with each of the two phenotypes associated with normal body weight (body-mass index<27 kg/m(2)) or early age at diagnosis (<45 years). In a second step, high-resolution genetic mapping ( approximately 2 cM) was performed in regions on chromosomes 1 and 3 loci showing the strongest linkage to diabetic traits. We found evidence for linkage with diabetes or GI diagnosed at age <45 years in 92 affected sib pairs from 55 families at the D3S1580 locus on chromosome 3q27-qter using MAPMAKER-SIBS (MLS = 4.67, P=.000004), supported by the MLB statistic (MLB-LOD=3.43, P=.00003). We also found suggestive linkage between the lean diabetic status and markers APOA2-D1S484 (MLS = 3. 04, P=.00018; MLB-LOD=2.99, P=.00010) on chromosome 1q21-q24. Several other chromosomal regions showed indication of linkage with diabetic traits, including markers on chromosome 2p21-p16, 10q26, 20p, and 20q. These results (a) showed evidence for a novel susceptibility locus for type 2 diabetes in French whites on chromosome 3q27-qter and (b) confirmed the previously reported diabetes-susceptibility locus on chromosome 1q21-q24. Saturation on both chromosomes narrowed the regions of interest down to an interval of <7 cM.

A genome-wide scan for coronary heart disease suggests in Indo-Mauritians a susceptibility locus on chromosome 16p13 and replicates linkage with the metabolic syndrome on 3q27.

Prevalence of coronary heart disease (CHD), of type 2 diabetes (T2DM) and of the metabolic syndrome are in Mauritius amongst the highest in the world. As T2DM and CHD are closely associated and have both a polygenic basis, we conducted a 10 cM genome scan with 403 microsatellite markers in 99 independent families of North-Eastern Indian origin including 535 individuals. Families were ascertained through a proband with CHD before 52 years of age and additional sibs with myocardial infarction (MI) or T2DM. Model-free two-point and multipoint linkage analysis were performed using the Mapmarker-Sibs (MLS) and maximum-likelihood-binomial (MLB) programs for autosomal markers and the Aspex program for chromosome X markers. In a second step, additional markers were studied to increase the genetic map density in three regions on chromosomes 3, 8 and 16 where initial indication for linkage was found. Our data show suggestive linkage with CHD on chromosome 16p13-pter with the MLS statistics at 8.69 cM (LOD = 3.06, P = 0.00017) which partially overlaps with a high pressure (HBP) peak. At the same locus, a nominal indication for linkage with T2DM was found in 35 large T2DM Pondicherian families also having Indian origin. With respect to region 8q23, we found suggestive linkage with T2DM (LOD = 2.55, P = 0.00058) as well as with HBP. On 3q27, we replicated previous indication for linkage found in Caucasians (for the metabolic syndrome and for diabetes) according to the categorized trait for CHD and MI with the MLB statistics (LOD = 2.13, P = 0.0009). The genome scan also revealed nominal evidence of linkage with CHD on 10q23 (LOD = 2.06, P = 0.00188). Interestingly, we detected in the same region overlapping linkages with three QTLs: age of onset of CHD (LOD = 2.03), HDL cholesterol (LOD = 1.48) and LDL/HDL ratio (LOD = 1.34). Ordered-subset analysis based on family body mass index ranking replicated finding on 2q37 for T2DM (at Calpain 10 locus). These results show the first evidence for susceptibility loci that predispose to CHD, T2DM and HBP in the context of the metabolic syndrome.