Intraclonal heterogeneity is a critical early event in the development of myeloma and precedes the development of clinical symptoms.

The mechanisms involved in the progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to malignant multiple myeloma (MM) and plasma cell leukemia (PCL) are poorly understood but believed to involve the sequential acquisition of genetic hits. We performed exome and whole-genome sequencing on a series of MGUS (n=4), high-risk (HR)SMM (n=4), MM (n=26) and PCL (n=2) samples, including four cases who transformed from HR-SMM to MM, to determine the genetic factors that drive progression of disease. The pattern and number of non-synonymous mutations show that the MGUS disease stage is less genetically complex than MM, and HR-SMM is similar to presenting MM. Intraclonal heterogeneity is present at all stages and using cases of HR-SMM, which transformed to MM, we show that intraclonal heterogeneity is a typical feature of the disease. At the HR-SMM stage of disease, the majority of the genetic changes necessary to give rise to MM are already present. These data suggest that clonal progression is the key feature of transformation of HR-SMM to MM and as such the invasive clinically predominant clone typical of MM is already present at the SMM stage and would be amenable to therapeutic intervention at that stage.

Deletions of CDKN2C in Multiple Myeloma: Biological and Clinical Implications

Purpose: Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material.Experimental Design: We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis.Results: By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor.Conclusions: Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.

Toxicity of mildly modified low-density lipoproteins to cultured retinal capillary endothelial cells and pericytes

To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37 degrees C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P <0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal <glycated <or = minimally oxidized <glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.

An Immunohistochemical Study of the Distribution of the Measles Virus Receptors, CD46 and SLAM, In Normal Human Tissues and Subacute Sclerosing Panencephalitis

We have compared the expression of the known measles virus (MV) receptors, membrane cofactor protein (CD46) and the signaling lymphocyte-activation molecule (SLAM), using immunohistochemistry, in a range of normal peripheral tissues (known to be infected by MV) as well as in normal and subacute sclerosing panencephalitis (SSPE) brain. To increase our understanding of how these receptors could be utilized by wild-type or vaccine strains in vivo, the results have been considered with regard to the known route of infection and systemic spread of MV. Strong staining for CD46 was observed in endothelial cells lining blood vessels and in epithelial cells and tissue macrophages in a wide range of peripheral tissues, as well as in Langerhans' and squamous cells in the skin. In lymphoid tissues and blood, subsets of cells were positive for SLAM, in comparison to CD46, which stained all nucleated cell types. Strong CD46 staining was observed on cerebral endothelium throughout the brain and also on ependymal cells lining the ventricles and choroid plexus. Comparatively weaker CD46 staining was observed on subsets of neurons and oligodendrocytes. In SSPE brain sections, the areas distant from lesion sites and negative for MV by immunocytochemistry showed the same distribution for CD46 as in normal brain. However, cells in lesions, positive for MV, were negative for CD46. Normal brain showed no staining for SLAM, and in SSPE brain only subsets of leukocytes in inflammatory infiltrates were positive. None of the cell types most commonly infected by MV show detectable expression of SLAM, whereas CD46 is much more widely expressed and could fulfill a receptor function for some wild-type strains. In the case of wild-type stains, which are unable to use CD46, a further as yet unknown receptor(s) would be necessary to fully explain the pathology of MV infection.

In vitro phototoxicity of 5-aminolevulinic acid and its methyl ester and the influence of barrier properties on their release from a bioadhesive patch.

opical administration of excess exogenous 5-aminolevulinic acid (ALA) leads to selective accumulation of the potent photosensitiser protoporphyrin IX (PpIX) in neoplastic cells, which can then be destroyed by irradiation with visible light. Due to its hydrophilicity, ALA penetrates deep lesions, such as nodular basal cell carcinomas (BCCs) poorly. As a result, more lipophilic esters of ALA have been employed to improve tissue penetration. In this study, the in vitro release of ALA and M-ALA from proprietary creams and novel patch-based systems across normal stratum corneum and a model membrane designed to mimic the abnormal stratum corneum overlying neoplastic skin lesions were investigated. Receiver compartment drug concentrations were compared with the concentrations of each drug producing high levels of PpIX production and subsequent light-induced kill in a model neoplastic cell line (LOX). LOX cells were found to be quite resistant to ALA- and M-ALA-induced phototoxicity. However, drug concentrations achieved in receiver compartments were comparable to those required to induce high levels of cell death upon irradiation in cell lines reported in the literature. Patches released significantly less drug across normal stratum corneum and significantly more across the model membrane. This is of major significance since the selectivity of PDT for neoplastic lesions will be further enhanced by the delivery system. ALA/M-ALA will only be delivered in significant amounts to the abnormal tissue. PpIX will only then accumulate in the neoplastic cells and the normal surrounding tissue will be unharmed upon irradiation.

Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

Characterization of outward currents in interstitial cells from the guinea pig bladder.

PURPOSE: Outward currents were characterized from cells resembling interstitial cells of Cajal (ICCs) isolated from the detrusor of the guinea pig bladder. MATERIALS AND METHODS: ICC-like cells were studied using the whole cell patch clamp technique and K+ filled pipettes. Outward currents were evoked by stepping positively from a holding potential of -80 mV. RESULTS: ICC-like cells were distinguished from smooth muscle cells by the presence of lateral branches and an inability to contract spontaneously or when depolarized. Depolarization elicited large outward currents. Penitrem A, a blocker of large conductance, Ca activated K+ channels, significantly decreased the outward current. Its Ca dependence was demonstrated by significant inhibition with nifedipine and Ca-free solution. When large conductance, Ca activated K+ and Ca currents were blocked with penitrem A and nifedipine, a voltage dependent current was unmasked, which activated positive to -50 mV and displayed voltage dependent inactivation with half-maximal inactivation occurring at -71 mV. It was blocked in concentration dependent fashion by tetraethylammonium but unaffected by 4-aminopyridine, charybdotoxin or apamin, suggesting that small and intermediate conductance, calcium activated potassium channels, and Kv1.2 and Kv1.3 channels are unlikely to be involved. At maximal concentrations of tetraethylammonium a portion of the voltage dependent K+ current remained that was not affected by any of the blockers tested. CONCLUSIONS: ICC-like cells from the detrusor possess calcium activated and voltage dependent K+ currents.

Large carbon isotope fractionation associated with oxidation of methyl halides by methylotrophic bacteria

The largest biological fractionations of stable carbon isotopes observed in nature occur during production of methane by methanogenic archaea. These fractionations result in substantial (as much as 70) shifts in 13C relative to the initial substrate. We now report that a stable carbon isotopic fractionation of comparable magnitude (up to 70) occurs during oxidation of methyl halides by methylotrophic bacteria. We have demonstrated biological fractionation with whole cells of three methylotrophs (strain IMB-1, strain CC495, and strain MB2) and, to a lesser extent, with the purified cobalamin-dependent methyltransferase enzyme obtained from strain CC495. Thus, the genetic similarities recently reported between methylotrophs, and methanogens with respect to their pathways for C1-unit metabolism are also reflected in the carbon isotopic fractionations achieved by these organisms. We found that only part of the observed fractionation of carbon isotopes could be accounted for by the activity of the corrinoid methyltransferase enzyme, suggesting fractionation by enzymes further along the degradation pathway. These observations are of potential biogeochemical significance in the application of stable carbon isotope ratios to constrain the tropospheric budgets for the ozone-depleting halocarbons, methyl bromide and methyl chloride.

Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells. (C) 2008 by Radiation Research Society.

CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells.

Recent studies have challenged the view that Langerhans cells (LCs) constitute the exclusive antigen-presenting cells of the skin and suggest that the dermal dendritic cell (DDC) network is exceedingly complex. Using knockin mice to track and ablate DCs expressing langerin (CD207), we discovered that the dermis contains five distinct DC subsets and identified their migratory counterparts in draining lymph nodes. Based on this refined classification, we demonstrated that the quantitatively minor CD207+ CD103+ DDC subset is endowed with the unique capability of cross-presenting antigens expressed by keratinocytes irrespective of the presence of LCs. We further showed that Y-Ae, an antibody that is widely used to monitor the formation of complexes involving I-Ab molecules and a peptide derived from the I-E alpha chain, recognizes mature skin DCs that express I-Ab molecules in the absence of I-E alpha. Knowledge of this extra reactivity is important because it could be, and already has been, mistakenly interpreted to support the view that antigen transfer can occur between LCs and DDCs. Collectively, these data revisit the transfer of antigen that occurs between keratinocytes and the five distinguishable skin DC subsets and stress the high degree of functional specialization that exists among them.

Induction and rejoining of DNA double-strand breaks in bladder tumor cells

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose-response curves (0-60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0-10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24), The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this. (C) 2000 by Radiation Research Society.

Acute and chronic effects of dietary fatty acids on cholecystokinin expression, storage and secretion in enteroendocrine STC-1 cells

Cholecystokinin (CCK) is a peptide hormone secreted from the I-cells of the intestine and it has important physiological actions related to appetite regulation and satiety. In this study we used STC-1 cells to investigate the effects of common dietary-derived fatty acids (FAs) on I-cell secretory function and metabolism. We extend earlier studies by measuring the acute and chronic effects of 11 FAs on CCK secretion, cellular CCK content, CCK mRNA levels, cellular DNA synthesis, cellular viability and cytotoxicity. FAs were selected in order to assess the importance of chain length, degree of saturation, and double bond position and conformation. The results demonstrate that secretory responses elicited by dietary FAs are highly selective. For example, altering the conformation of a double bond from cis to trans (i.e. oleic acid versus elaidic acid) completely abolishes CCK secretion. Lauric acid appears to adversely affect I-cell metabolism and arachidonic acid suppresses DNA synthesis. Our studies reveal for the first time that conjugated linoleic acid isoforms are particularly potent CCK secretagogues, which also boost intracellular stores of CCK. These actions of conjugated linoleic acid may explain satiating actions observed in dietary intervention studies.

Evaluation of glycated glucagon-like peptide-1(7-36)amide in intestinal tissue of normal and diabetic animal models

Glucagon-like peptide-1(7-36)amide (tGLP-1) is an important insulin-releasing hormone of the enteroinsular axis which is secreted by endocrine L-cells of the small intestine following nutrient ingestion. The present study has evaluated tGLP-1 in the intestines of normal and diabetic animal models and estimated the proportion present in glycated form. Total immunoreactive tGLP-1 levels in the intestines of hyperglycaemic hydrocortisone-treated rats, streptozotocin-treated mice and ob/ob mice were similar to age-matched controls. Affinity chromatographic separation of glycated and non-glycated proteins in intestinal extracts followed by radioimmunoassay using a fully crossreacting anti-serum demonstrated the presence of glycated tGLP-1 within the intestinal extracts of all control animals (approximately 19%., of total tGLP-1 content). Chemically induced and spontaneous animal models of diabetes were found to possess significantly greater levels of glycated tGLP-1 than controls, corresponding to between 24-71% of the total content. These observations suggest that glycated tGLP-1 may be of physiological significance given that such N-terminal modification confers resistance to DPP IV inactivation and degradation, extending the very short half-life (

Fasciola hepatica: Disruption of the vitelline cells in vitro by the sulphoxide metabolite of triclabendazole

The effects of the active sulphoxide metabolite of the fasciolicide triclabendazole (Fasinex, Ciba-Geigy) on the vitelline cells of Fusciola hepatica were determined in vitro by transmission electron microscopy using both intact flukes and tissue-slice material. At a triclabendazole concentration of 15 mu g/ml the vitelline cells of intact flukes showed ultrastructural changes only after prolonged incubation periods (12-24 h). The changes observed were a swelling of the granular endoplasmic reticulum (GER) cisternae with decreased ribosomal covering in the intermediate-type cells and condensation of chromatin and disappearance of the nucleolus in the nucleus of the stem cell. Similar changes were evident more quickly (by 6 h) in whole flukes treated at the higher concentration of 50 mu g/ml. The shell globule clusters were loosely packed in the intermediate type-2 cells, and the number of intermediate type-1 cells declined with more prolonged incubation. Disruption of the nurse-cell cytoplasm was also observed from 12 h onwards. After only 6 h incubation of tissue-slice material at 50 mu g/ml, intermediate type-1 cells were absent, shell globule clusters in mature cells were loosely packed and the nurses cell cytoplasm was badly disrupted. By 12 h the vitelline cells were vacuolated and grossly abnormal. The results are discussed in relation to postulated actions of triclabendazole against the microtubule component of the cytoskeleton and against protein synthesis in the fluke.

Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats

Advanced glycation end products (AGEs), formed from the nonenzymatic glycation of proteins and lipids with reducing sugars, have been implicated in many diabetic complications; however, their role in diabetic retinopathy remains largely unknown. Recent studies suggest that the cellular actions of AGEs may be mediated by AGE-specific receptors (AGE-R). We have examined the immunolocalization of AGEs and AGE-R components R1 and R2 in the retinal vasculature at 2, 4, and 8 months after STZ-induced diabetes as well as in nondiabetic rats infused with AGE bovine serum albumin for 2 weeks. Using polyclonal or monoclonal anti-AGE antibodies and polyclonal antibodies to recombinant AGE-R1 and AGE-R2, immunoreactivity (IR) was examined in the complete retinal vascular tree after isolation by trypsin digestion. After 2, 4, and 8 months of diabetes, there was a gradual increase in AGE IR in basement membrane. At 8 months, pericytes, smooth muscle cells, and endothelial cells of the retinal vessels showed dense intracellular AGE IR. AGE epitopes stained most intensely within pericytes and smooth muscle cells but less in basement membrane of AGE-infused rats compared with the diabetic group. Retinas from normal or bovine-serum-albumin-infused rats were largely negative for AGE IR. AGE-R1 and -R2 co-localized strongly with AGEs of vascular endothelial cells, pericytes, and smooth muscle cells of either normal, diabetic, or AGE-infused rat retinas, and this distribution did not vary with each condition. The data indicate that AGEs accumulate as a function of diabetes duration first within the basement membrane and then intracellularly, co-localizing with cellular AGE-Rs. Significant AGE deposits appear within the pericytes after long-term diabetes or acute challenge with AGE infusion conditions associated with pericyte damage. Co-localization of AGEs and AGE-Rs in retinal cells points to possible interactions of pathogenic significance.