Resistance to the tyrosine kinase inhibitor axitinib is associated with increased glucose metabolism in pancreatic adenocarcinoma.

Alterations in energy (glucose) metabolism are key events in the development and progression of cancer. In pancreatic adenocarcinoma (PDAC) cells, we investigated changes in glucose metabolism induced by resistance to the receptor tyrosine kinase inhibitor (RTKI) axitinib. Here, we show that human cell lines and mouse PDAC cell lines obtained from the spontaneous pancreatic cancer mouse model (Kras(G12D)Pdx1-cre) were sensitive to axitinib. The anti-proliferative effect was due to a G2/M block resulting in loss of 70-75% cell viability in the most sensitive PDAC cell line. However, a surviving sub-population showed a 2- to 3-fold increase in [C-14]deoxyglucose ([C-14]DG) uptake. This was sustained in axitinib-resistant cell lines, which were derived from parental PDAC. In addition to the axitinib-induced increase in [C-14]DG uptake, we observed a translocation of glucose transporter-1 (Glut-1) transporters from cytosolic pools to the cell surface membrane and a 2-fold increase in glycolysis rates measured by the extracellular acidification rate (ECAR). We demonstrated an axitinib-induced increase in phosphorylated Protein Kinase B (pAkt) and by blocking pAkt with a phosphatidylinositol-3 kinase (PI3K) inhibitor we reversed the Glut-1 translocation and restored sensitivity to axitinib treatment. Combination treatment with both axitinib and Akt inhibitor in parental pancreatic cell line resulted in a decrease in cell viability beyond that conferred by single therapy alone. Our study shows that PDAC resistance to axitinib results in increased glucose metabolism mediated by activated Akt. Combining axitinib and an Akt inhibitor may improve treatment in PDAC.

Characterization of different courses of atopic dermatitis in adolescent and adult patients.

BACKGROUND: Atopic dermatitis (AD) starts most often during the first years of life and goes into remission in a high proportion of cases during childhood. However, in severe cases, AD persists until adulthood or starts and relapses later in life. So far, studies investigating the natural course of AD during adolescence and adulthood are rare. The aim of our study was to classify different courses of AD and to correlate these with specific risk factors for severe variants of AD. METHODS: A detailed clinical examination and retrospective evaluation of the history of the disease were performed in a collective of 725 adolescent and adult patients with AD. Laboratory data including total and specific IgE were evaluated. RESULTS: Six hundred and seven patients of 725 patients could be classified into course types. Of these 607 patients 85.7% could be classified into five main different course types of all 31 course types recorded. The highest differences in the number of sensitizations, total immunoglobulin E serum levels and predilection of the skin lesions were observed between patients with an early type of onset of AD and a chronic persisting course until adulthood and patients with a late type of onset of AD, that is, after the 20th year of life. CONCLUSION: Our data show that the natural course of AD can be divided into subgroups that display different clinical features. The data support the assumption of a broad heterogeneity of AD in adolescence and adulthood and emphasize the future need for careful stratification of patients with AD.

Relationships between four measures of genetic distance and breeding behavior in spring wheat.

We estimated the genetic distances among 10 spring wheat genotypes based on pedigree data, morphological traits and AFLP markers, used individually and combined with morphological traits, to find the best predictors of general- and specific-combining abilities among parental genotypes. Ten wheat parents were crossed in a diallel form, disregarding reciprocal hybrids, totaling 45 combinations. The F1 hybrids, F2 populations and parents were evaluated in the field in 2007. The experimental plots consisted of 20 plants for F1 hybrids and 40 plants for parental and F2 populations. All methods (pedigree data, AFLP markers and morphological traits, used individually and combined) were found to be useful for the assessment of genetic diversity. The significant coefficient correlations ranged from low (0.45) to moderate (0.67) between the distance measures and hybrid performance. There was significant agreement between the distance measures based on AFLP markers vs morphological traits + AFLP markers (r = 0.47) and between pedigree data vs morphological traits + AFLP markers (r = 0.43). The pedigree distance was positively associated with traits 100-kernel weight and grain yield per plant in F1 (correlations of 0.67 and 0.62, respectively) and F2 (correlations of 0.62 and 0.59, respectively) generations. These correlation values indicate that the genetic distance, based on pedigree data, could replace diallel crosses for the selection of parents with higher combining ability and with moderate reliability.

Up for Mischief? IL-17/Th17 in the tumour microenvironment.

The role of interleukin (IL)-17 and the IL-17-producing T helper (Th)17 cells in cancer has recently become the focus of extensive investigation. An expanding body of literature implicates Th17 cells and their hallmark cytokine in both pro- and anti-tumourigenic processes. In this review we describe their biological activities and outline the reciprocal interactions between Th17 cells and other cells of the immune system. We also discuss the evidence regarding their dual role in the tumour microenvironment. An understanding of the processes that regulate the pro- or anti-tumour activities of Th17 cell and IL-17 will allow the development of more effective means for cancer immunotherapy.

Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression.

OBJECTIVE: Cell immortalization is considered to be a prerequisite status for carcinogenesis. Normal human ovarian surface epithelial (OSE) cells, which are thought to be the origin of most of human ovarian carcinomas, have a very limited lifespan in culture. Establishment of immortalized OSE cell lines has, in the past, required inactivation of pRb and p53 functions. However, this often leads to increased chromosome instability during prolonged culture. MATERIALS AND METHODS: In this study, we have used a retroviral infection method to overexpress human telomerase reverse transcriptase (hTERT) gene, in primary normal OSE cells, under optimized culture conditions. RESULTS: In vitro and in vivo analysis of hTERT-immortalized cell lines confirmed their normal epithelial characteristics. Gene expression profiles and functional analysis of p16(INK4A), p15(INK4B), pRb and p53 confirmed the presence of their intact functions. Our study suggests that inactivation of pRb and p53 is not necessary for OSE immortalization. Furthermore, down-regulation of p15(INK4B) in the immortalized cells may indicate a functional role for this protein in them. CONCLUSION: These immortal OSE cell lines are likely to be an important tool for studying human OSE biology and carcinogenesis.

Putative association of a TLR9 promoter polymorphism with atopic eczema.

BACKGROUND: Toll-like receptors (TLR) play a pivotal role in the induction of first-line defense mechanisms of the innate immune system and trigger adaptive immune responses to microbial pathogens. Genetic variations in innate immunity genes have been reported to be associated with a range of inflammatory disorders. Deficiencies on the level of immunity receptors such as pathogen-recognition receptors are suspected to affect the maturation of our immune system and to avail thereby the high prevalence of atopic diseases and susceptibility of atopic patients to microbial infections. AIMS OF THE STUDY: We evaluated TLR9 as susceptibility gene for atopic eczema (AE). METHODS: Analyses of four tag single-nucleotide polymorphisms in two panels of families containing a total of 483 parent-affected offspring trios as well as a cohort of 274 unrelated adult AE cases and 252 hypernormal population-based controls have been performed. RESULTS: In both family cohorts, polymorphism C-1237T, which is located within the promoter region of the TLR9 gene, was significantly associated with AE, in particular the intrinsic subtype of AE. No associations were seen in the case-control cohort. Luciferase reporter gene assays revealed significantly higher promoter activity of the TT allelic variant at this single nucleotide polymorphism site. CONCLUSION: These observations suggest that the TLR9 promoter polymorphism C-1237T might affect AE susceptibility in particular in patients with the intrinsic variant of AE.

[Associated diseases and differential diagnostic considerations in childhood atopic eczema]. / Differenzialdiagnose des atopischen Ekzems in der Kindheit. Erkrankungen und Syndrome in Assoziation.

Atopic eczema (AE) is a chronic inflammatory skin disease which affects 10 to 20% of children and 1 to 3% of adults. AE is usually diagnosed based on standard criteria such as those of Hanifin and Rajka, whereby the age-related variation must be considered. There are numerous other diseases which go along with AE or show a very similar clinical picture and represent important differential diagnostic considerations including parasitic diseases, immunodeficiency, nutritional diseases, certain neoplastic disorders and various corneal abnormalities. Additionally, it is important to consider diseases which can occur in association with AE, such as keratosis pilaris, alopecia areata or sweat disturbances.

Molecular profiling of cervical cancer progression.

Most cancer patients die of metastatic or recurrent disease, hence the importance to identify target genes upregulated in these lesions. Although a variety of gene signatures associated with metastasis or poor prognosis have been identified in various cancer types, it remains a critical problem to identify key genes as candidate therapeutic targets in metastatic or recurrent cancer. The aim of our study was to identify genes consistently upregulated in both lymph node micrometastases and recurrent tumours compared to matched primary tumours in human cervical cancer. Taqman Low-Density Arrays were used to analyse matched tumour samples, obtained after laser-capture microdissection of tumour cell islands for the expression of 96 genes known to be involved in tumour progression. Immunohistochemistry was performed for a panel of up- and downregulated genes. In lymph node micrometastases, most genes were downregulated or showed expressions equal to the levels found in primary tumours. In more than 50% of lymph node micrometastases studied, eight genes (AKT, BCL2, CSFR1, EGFR1, FGF1, MMP3, MMP9 and TGF-beta) were upregulated at least two-fold. Some of these genes (AKT and MMP3) are key regulators of epithelial-mesenchymal transition in cancer. In recurrent tumours, almost all genes were upregulated when compared to the expression profiles of the matched primary tumours, possibly reflecting their aggressive biological behaviour. The two genes showing a consistent downregulated expression in almost all lymph node metastases and recurrent tumours were BAX and APC. As treatment strategies are very limited for metastatic and recurrent cervical cancer, the upregulated genes identified in this study are potential targets for new molecular treatment strategies in metastatic or recurrent cervical cancer.

Successful treatment of acrodermatitis continua suppurativa with topical tacrolimus 0.1% ointment.

Acrodermatitis continua suppurativa of Hallopeau (ACS) is a rare pustular variant of psoriasis in which numerous treatment modalities have been used without any consistent long-term effect. We report for the first time two patients with ACS which was successfully treated with topical tacrolimus 0.1% ointment. Our observations raise hopes that this new treatment strategy for ACS may constitute a novel effective therapeutic option for this recalcitrant condition.

[Somatization and alexithymia in male infertility. A replication study]. / Alexithymie und Somatisierung bei infertilen Männern. Eine Replikationsstudie.

BACKGROUND AND OBJECTIVE: In a previous study, we found a higher degree of alexithymia and somatization in infertile men (Hum Reprod 2001 Vol.16(3),587-592). This study was designed to confirm the findings of the former study PATIENTS/METHODS: 88 infertile men (48 idiopathic infertility, 40 somatic infertility) were consecutively enrolled in the study. The control group consisted of 44 healthy men. Alexithymia was measured by the 20-item-Toronto-Alexithymia-Scale;somatization was measured by the Symptom Checklist-90-R. RESULTS: There were no differences between the subgroups of infertile men. The study group showed a significantly higher sum score in the TAS-20 (p<0.01) and a higher score on the scale somatization in the Symptom Checklist-90-R (p<0.05) compared to the control group. CONCLUSIONS: Our study replicated the findings of the previous investigation. Alexithymia and somatization are discussed as consequences of coping with male infertility.

Procalcitonin: a useful discriminator between febrile conditions of different origin in hemato-oncological patients?

Plasma concentrations of procalcitonin (PCT) have been shown to be elevated in bacterial and fungal infections. In contrast to C-reactive protein (CRP), PCT is not elevated in inflammations of noninfectious origin. Febrile inflammatory conditions are frequent in patients with hemato-oncological diseases. A reliable marker to discriminate infectious inflammations from drug-related and tumor-associated fever is still lacking. To evaluate the impact of PCT in this setting, PCT and CRP were prospectively measured in 95 febrile hemato-oncological patients. Infections could be identified in 40 of 95 patients: 38 of 95 had fever of unknown origin (FUO), 9 patients were suspected to suffer from drug-related fever, and 8 patients from tumor-associated fever. In the noninfection group (drug-related and tumor-associated fever), PCT levels were significantly lower than in patients with infections (P<0.001) or FUO (P<0.001). Differences were still highly significant comparing patients with suspected drug-related or tumor-associated fever alone with the infection or the FUO cohort. All eight patients with tumor-associated fever as well as eight of the nine patients with drug-related fever had PCT levels within the normal range (<0.5 micro g/l). CRP values only partially allowed discrimination between the various subgroups. Differences were significant between patients with drug-related fever and the infection (P=0.001) or FUO group (P=0.004). However, as CRP levels were far above the normal range also in the patients with drug-related fever, the significance of individual values was rather limited. In conclusion, PCT may provide useful additional information to assess the clinical significance of febrile conditions. PCT may facilitate the decision on when to initiate antimicrobial or cytotoxic therapy.

mRNA expression of matrix metalloproteases and their inhibitors differs in subtypes of renal cell carcinomas.

Altered expression of matrix metalloproteases (MMPs) and their inhibitors, the tissue inhibitors of matrix metalloproteases (TIMPs), has been demonstrated in various tumour tissues. mRNA expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, MMP-11, MMP-12, MMP-14 and TIMP-1, TIMP-2, TIMP-3 and TIMP-4 were evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 30 renal cell carcinomas (RCC), as well as in the surrounding tissues. Expression of the MMPs was significantly stronger in the carcinomas than in non-malignant tissues. High levels were demonstrated particularly in clear cell RCCs (CC-RCC). Except for MMP-1, MMP expression in the papillary RCCs (P-RCC) was, for most MMPs, significantly lower. Expression of the TIMPs in malignant cells of both subtypes was weak, with the exception of TIMP-4 which was strongly expressed in the P-RCCs and downregulated in the CC-RCCs. The latter was correlated with chromosomal loss of 3p, harbouring the TIMP-4 gene locus. In conclusion, deregulated expression of the MMPs and TIMPs in RCCs differs according to histology, grade, size and cytogenetic characteristics, suggesting that MMP and TIMP expression patterns play an important role for the typical histomorphological features of RCC subtypes and their respective biological behaviour.

Gene regulation of Wiskott-Aldrich syndrome protein and the human homolog of the Drosophila Su(var)3-9: WASP and SUV39H1, two adjacent genes at Xp11.23.

The region Xp11.23 is a gene-rich, light giemsa-staining segment on the short arm of the X chromosome. In this study, we have characterized the transcriptional regulatory elements in this interval for two adjacent genes: SUV39H1, a regulator of chromatin organization, and the Wiskott-Aldrich syndrome protein (WASP). The WASP gene exhibits two alternate promoters, both of which demonstrate transcription factor binding elements specific to blood cell lineages. Reporter gene expression analyses indicate that both WASP promoters show high levels of expression in different hematopoietic cell lines. The human homolog of the Drosophila Su(var)3-9 gene was identified by sequence analysis of the region downstream from WASP. SUV39H1 is ubiquitously expressed, and the promoter sequence consists mostly of general transcription factors. The presence of putative binding sites for GAGA and Adf1 transcription factors may indicate a cross regulatory mechanism with other chromatin regulators.

The identification and characterization of two promoters and the complete genomic sequence for the Wiskott-Aldrich syndrome gene.

The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by immunodeficiency, eczema and thrombocytopenia. The gene responsible for WAS was identified through positional cloning, and the function of the encoded protein (WASP) is still the subject of much speculation. WASP is currently thought to be involved in the regulation of actin polymerization in hematopoietic cells. To study the elements that regulate the WASP gene, we have identified the sites for transcription initiation. We found that two promoters were responsible for controlling WASP expression. Multiple transcription initiation sites were found immediately adjacent to the translation start site, however an alternate exon with a second promoter region was identified 6 kb upstream. Examination of the 5' sequence adjacent to the initiation sites in both promoters failed to reveal a TATA or CCAAT box, but numerous putative transcription factor binding sites including Sp1, Ets, c-Myb and PU.1 were apparent. Reporter constructs generated from each promoter showed functional activity in the Jurkat T-cell and HEL erythro-megakaryocytic cell lines. Although the alternate exon sequence was extremely GC rich and contained several potential binding elements, the primary promoter was stronger than the upstream promoter in the cell lines assayed. The transcription factor binding site profiles within each promoter suggested that they may play different roles in regulating WASP expression depending on the stage of differentiation and development, and the cell lineage. In this study we have also reported the complete nucleotide sequence of the coding and intervening sequences for the WASP gene. A comprehensive knowledge of the genomic structure and the further characterization of WASP gene expression will facilitate the continued investigation of mutations in WAS patients, and the eventual prospect of gene therapy.

ABI sequencing analysis. Manipulation of sequence data from the ABI DNA sequencer.

The ABI Sequencing Analysis application is designed specifically for the analysis of data produced by the ABI DNA Sequencer. The ABI sequencer is a laser-based instrument that utilizes fluorescent labels to analyze the products of a sequencing reaction as they migrate through a gel. After the data are collected from a sequencing run, the Analysis program identifies and tracks the sample lanes of the gel and subsequently normalizes and integrates the raw data into a chromatogram of the final sequence. For the user, there are basically two types of files that can be manipulated to potentially improve the analysis results. The Gel File consists of a computer generated image of the sequencing gel with the fluorescent DNA banding patterns. This image allows the user to view and edit the tracking lines generated and used by Analysis to collect data points for each sample. Individual Sample Files are stored for each of the samples analyzed and include the chromatogram, raw data, and annotations and information regarding the sample and sequence run. Generally, the products of a sequencing reaction are easily resolved and the Analysis software interprets the correct nucleotide sequence. Ambiguous base calls tend to occur near the end of the sequence and may be either edited or deleted by the user before exporting the data for further comparisons or alignments. Occasionally the tracking lines within the gel image may need to be adjusted or moved. The sample data are then reextracted from the Gel File and analyzed again. This review explains the general operation of Analysis in terms of viewing and editing a chromatogram, retracking the lanes of a Gel File, and analyzing the final sample data. The three versions 1.2.1, 2.1.2, and 3.3 are discussed.

Wiskott-Aldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation.

The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations in a gene (WASP) encoding a cytoplasmic protein implicated in regulating the actin cytoskeleton. To elucidate WASP function, we disrupted the WASP gene in mice by gene-targeted mutation. WASP-deficient mice showed apparently normal lymphocyte development, normal serum immunoglobulin levels, and the capacity to respond to both T-dependent and T-independent type II antigens. However, these mice did have decreased peripheral blood lymphocyte and platelet numbers and developed chronic colitis. Moreover, purified WASP-deficient T cells showed markedly impaired proliferation and antigen receptor cap formation in response to anti-CD3epsilon stimulation. Yet, purified WASP-deficient B cells showed normal responses to anti-Ig stimulation. We discuss the implications of our findings regarding WASP function in receptor signaling and cytoskeletal reorganization in T and B cells and compare the effects of WASP deficiency in mice and humans.

Gastrointestinal medications and breastfeeding.

Medications used to treat gastrointestinal symptoms are increasingly being used as more have been gained nonprescription status. Most of the gastrointestinal medications, such as laxatives, antacids, and antidiarrheal agents, are used short term. Women who breastfeed should be aware of the risks of taking any medications, whether prescription or nonprescription. There is little information describing transfer into breast milk for many of these products. Cimetidine, atropine, cascara, cisapride, loperamide, magnesium sulfate, and senna are the only products identified by the AAP as compatible with breast feeding. Metoclopramide is listed by the AAP as a drug whose effect on nursing infants is unknown but may be of potential concern, although studies published to date have not reported any adverse effects. The safest laxatives and antidiarrheals are those that are not absorbed and should be considered first-line therapy for conditions of constipation or loose stools. Famotidine and nizatidine are excreted into breast milk to a lesser extent than cimetidine or ranitidine and may be the preferred histamine antagonists. Despite the limited data on the use of cisapride in nursing women, it is considered safe by the AAP and may be preferred over metoclopramide for first-line prescription treatment of heartburn. Although most of these agents appear safe in the nursing infant, caretakers should be aware of the potential adverse reactions that may occur in infants whose mothers require these products.

Variable expression of WASP in B cell lines of Wiskott-Aldrich syndrome patients.

The Wiskott-Aldrich syndrome (WAS) arises from defects of the X-chromosome gene WASP. Severe platelet defects, thrombocytopenia with small platelets, are a hallmark of the disease, but clinical immunodeficiency based in lymphocyte dysfunction varies from negligible to life threatening among WAS patients. To address the connection between WASP mutations and clinical outcomes, we generated and characterized a panel of patient B cell lines. Three cell lines from patients with exon 2 missense mutations and mild immune dysfunction were found to express substantial levels of WASP mRNA and protein. On the other hand, 8 of 10 cell lines from patients with moderate or severe immune dysfunction lack detectable WASP protein. The findings suggest that the clinical variability of the WAS can partially be explained by the level of WASP protein in the patient's cells.