[Integration of palliative care into intensive care : Systematic review]. / Integration der Palliativmedizin in die Intensivmedizin : Systematische Übersichtsarbeit.

BACKGROUND: One of the prime aims of intensive care is to cure patients or at least to extend life duration, sometimes to the extent of losing quality of life. Palliative care aims to improve the quality of life of patients with life-limiting conditions; however, some patients need both intensive and palliative care. About 5-10% of all deaths in Germany and 20% of all deaths in the USA occur in an intensive care unit (ICU) and many of those as well as other patients may benefit from palliative care consultation. Palliative care consultations are increasingly available for intensive care patients but are still infrequently used. OBJECTIVES: We aimed to determine the current situation of palliative patients in ICU settings: what is the impact of palliative care interventions on the quality of care of ICU patients? To what extent is palliative care support at ICUs available and to what extent is it used? Which factors trigger palliative care consultations? METHOD: We set out with a search of PubMed, Scopus and other databases in English and on a) the impact of palliative care interventions on the quality of care of ICU patients, b) the utilization of palliative care support in ICUs and c) the factors which trigger palliative care consultations. We included both quantitative and qualitative studies to reflect the views of all parties involved. To emphasize the situation in German-speaking countries we also searched Google Scholar with search terms in German and added those results to the review. Additionally, hand-searched studies in English and in German were included. RESULTS: We screened 695 abstracts and identified 18 relevant articles of which 15 were from the USA and Great Britain, 1 each was from Austria, Germany and Switzerland. Palliative care is a meaningful addition to ICU standard treatment: it can improve quality of care and helps reduce length of stay in an ICU. It is unclear if the reduced length of stay leads to economic benefits; however, the utilization of palliative care is inconsistent and infrequent as is its acceptance among ICU physicians. Trigger factors can be used to improve the integration of palliative care support in ICUs and point out patients' unmet palliative needs. DISCUSSION: Trigger factors can reduce barriers which hold back the integration of palliative care in ICUs. Early integration of palliative care can improve quality of care by offering psychological support to patients and their families and by providing collegial consultation. An ongoing prospective study is investigating the acceptance of trigger factors in the daily routine among ICU physicians in Germany.

IGRP and insulin vaccination induce CD8+ T cell-mediated autoimmune diabetes in the RIP-CD80GP mouse.

Autoimmune diabetes is characterized by autoantigen-specific T cell-mediated destruction of pancreatic islet beta cells, and CD8(+) T cells are key players during this process. We assessed whether the bitransgenic RIP-CD80 x RIP-LCMV-GP (RIP-CD80GP) mice may be a versatile antigen-specific model of inducible CD8(+) T cell-mediated autoimmune diabetes. Antigen-encoding DNA, peptide-loaded dendritic cells and antigen plus incomplete Freund's adjuvant were used for vaccination. Of 14 pancreatic proteins tested by DNA vaccination, murine pre-proinsulin 2 (100% of mice; median time after vaccination, 60 days) and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) (77%, 58 days) could induce diabetes. Vaccination with DNA encoding for zinc transporter 8, Ia-2, Ia-2ß, glutamic acid decarboxylase 67 (Gad67), chromogranin A, insulinoma amyloid polypeptide and homeobox protein Nkx-2.2 induced diabetes development in 25-33% of mice. Vaccination with DNA encoding for Gad65, secretogranin 5, pancreas/duodenum homeobox protein 1 (Pdx1), carboxyl ester lipase, glucagon and control hepatitis B surface antigen (HBsAg) induced diabetes in <20% of mice. Diabetes induction efficiency could be increased by DNA vaccination with a vector encoding a ubiquitin-antigen fusion construct. Diabetic mice had florid T cell islet infiltration. CD8(+) T cell targets of IGRP were identified with a peptide library-based enzyme-linked immunospot assay, and diabetes could also be induced by vaccination with major histocompatibility complex (MHC) class I-restricted IGRP peptides loaded on mature dendritic cells. Vaccination with antigen plus incomplete Freund's adjuvant, which can prevent diabetes in other models, led to rapid diabetes development in the RIP-CD80GP mouse. We conclude that RIP-CD80GP mice are a versatile model of antigen specific autoimmune diabetes and may complement existing mouse models of autoimmune diabetes for evaluating CD8(+) T cell-targeted prevention strategies.

Diagnostic accuracy of the BACs-on-Beads™ assay versus karyotyping for prenatal detection of chromosomal abnormalities: a retrospective consecutive case series.

OBJECTIVE: To evaluate the diagnostic performance of the BACs-on-Beads(™) (BoBs(™)) assay for prenatal detection of chromosomal abnormalities. DESIGN: Retrospective study. SETTING: Tertiary prenatal diagnosis centre. POPULATION: Women referred for prenatal diagnosis. METHODS: We retrieved 2153 archived DNA samples collected between January 2010 and August 2011 for the BoBs(™) assay. These samples had previously been tested by quantitative fluorescence polymerase chain reaction (QF-PCR) and karyotyping. In the BoBs(™) assay a sample was defined as normal disomic when the ratio of the fluorescence intensities in a chromosome locus lay within the threshold (mean ratio ± 2SD), and as deleted or duplicated when the ratio was below the lower threshold (0.6-0.8) or above the upper threshold (1.3-1.4), respectively. The BoBs(™) results were further validated by microarray and compared in a blinded manner with the original QF-PCR and karyotyping results. MAIN OUTCOME MEASURES: Concordance of any numerical, structural, and submicroscopic chromosomal abnormalities between the methods. RESULTS: BACs-on-Beads(™) was similar to karyotyping and QF-PCR in detecting trisomy 13, trisomy 18, trisomy 21, and sex chromosomal aneuploidies, and superior to QF-PCR in detecting major structural abnormalities (53.3 versus 13.3%) and mosaicism (28.6 versus 0%) involving chromosomal abnormalities other than the common aneuploidies. BoBs(™) detected six microdeletion syndromes missed by karyotyping and QF-PCR; however, BoBs(™) missed two cases of triploidy identified by QF-PCR. Therefore, the sensitivity of BoBs(™) is 96.7% (95% CI 92.6-98.7%), and its specificity is 100% (95% CI 99.8-100%). CONCLUSIONS: BACs-on-Beads(™) can replace QF-PCR for triaging in prenatal diagnosis, and gives a better diagnostic yield than current rapid aneuploidy tests.

Predicting 5-fluorouracil toxicity in colorectal cancer patients from peripheral blood cell telomere length: a multivariate analysis.

BACKGROUND: Identifying various pretreatment factors that predict chemotherapy-induced toxicity in colorectal cancer (CRC) patients undergoing treatment for their disease is crucial to optimising patient care. METHODS: Seventy-three patients received adjuvant 5-fluorouracil (5FU)/leucovorin using either the Mayo Clinic (n=42) or a weekly schedule (n=31) and evaluated for clinical toxicity. Pretreatment blood analysis included measures of plasma uracil and dihydrouracil, peripheral blood mononuclear cell (PBMNC) telomere length (TL), standard biochemistry and cell differential analysis. On the first day of treatment 5FU-pharmacokinetic variables of area under the curve, half life and clearance were also measured. These variables together with age and gender were used in univariate and multivariate analysis as predictors of clinical toxicity. RESULTS: For the Mayo schedule the primary toxicities were neutropenia (69%), mucositis (58%) and leukopenia (46%), with 70% of patients presenting with haematological toxicity ≥grade 1 (neutropenia and/or leukopenia). Multivariate analysis showed that haematological toxicity was predicted by short TL, high platelet lymphocyte ratio (PLR) and low neutrophil count (R(2)=0.38, P<0.0006), whereas mucositis was predicted by age, TL and PLR (R(2)=0.34, P<0.001). For the weekly schedule diarrhoea predominated (16%), with female gender as the only predictive factor. Although measures of uracil metabolism correlated well with 5FU metabolism (r=0.45-0.49), they did not indicate abnormal pyrimidine metabolism in this cohort and not surprisingly failed to predict for 5FU toxicity. CONCLUSION: Short TL of PBMNC and an increased PLR were strong predictors of mucositis and haematological toxicity in CRC patients undergoing 5FU treatment in the adjuvant setting.

The effect of gestation and fetal mismatching on the development of autoimmune diabetes in non-obese diabetic mice.

The impact of gestation and fetal-maternal interactions on pre-existent autoimmune beta cell destruction is widely unknown. The aim of this study was to investigate the influence of gestation per se and fetal mismatching on the onset of autoimmune diabetes in female non-obese diabetic (NOD) mice. We examined cumulative diabetes frequencies of NOD dams mated to syngeneic NOD, haploidentical CByB6F1/J and fully mismatched C57BL/6J male mice. Pregnancy from NOD males neither increased nor accelerated the diabetes onset of NOD dams (71% by age 28 weeks) compared to unmated female NOD mice (81% by age 28 weeks; P = 0·38). In contrast, delayed diabetes onset was observed when NOD dams were mated at 10 weeks of age with major histocompatibility complex (MHC) haploidentical CByB6F1/J male mice (38% at age 28 weeks; P = 0·01). Mating with fully MHC mismatched C57BL/6J male mice (72% diabetes by age 28 weeks; P = 0·22) or mating with the haploidentical males at the later time-point of age 13 weeks (64% versus 91% in unmated litter-matched controls; P = 0·13) did not delay diabetes significantly in NOD females. Because infusion of haploidentical male mouse splenocytes was found previously to prevent diabetes in NOD mice we looked for, but found no evidence of, persistent chimeric lymphocytes from haploidentical paternal origin within the dams' splenocytes. Gestation per se appears to have no aggravating or ameliorating effects on pre-existent autoimmune beta cell destruction, but pregnancy from MHC partially mismatched males delays diabetes onset in female NOD mice.

Insulin autoantibodies with high affinity to the bovine milk protein alpha casein.

Insulin autoantibodies (IAA) can appear in children within months of introducing solid foods to the diet and before clinical type 1 diabetes. The aim of this study was to determine whether infant dietary antigens could be immunizing agents of IAA. To this end, IAA binding to [(125) I]insulin was competed with food preparations and extracts of foods encountered in the infant diet (milk formulas, bovine milk, wheat flour, fowl meal). Bovine milk powder extracts inhibited IAA-positive samples from six of 53 children (age 0·3-14·0 years) participating in German prospective cohorts. Inhibition in these sera ranged from 23 to 100%. Competition was abolished when hydrolyzed milk powder was used. Competition with protein components of bovine milk found that two of the six milk-reactive sera were inhibited strongly by alpha- and beta-casein; none were inhibited by the milk proteins bovine serum albumin or lactoglobulins. The two casein-reactive sera had high affinity to alpha-casein (1·7×10(9) ; 3·1×10(9) l/mol), and lesser affinity to beta-casein (4·0×10(8) ; 7·0×10(7) l/mol) and insulin (2·6×10(8) ; 1·6×10(8) l/mol). No children with milk-reactive IAA developed autoantibodies to other islet autoantigens or diabetes (median follow-up 9·8 years). These results suggest that autoimmunity to insulin can occur infrequently via cross-reactivity to food proteins, but this form of IAA immunization does not appear to be associated with progression to diabetes.

Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-alpha subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation.

Stimulation of CD4(+) helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-alpha/Ig-beta heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-alpha-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-alpha cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide- MHC class II complexes through antigen targeting by BCR subunits.

Extraoptic phase shifting of circadian locomotor rhythm in salamanders.

Timing of locomotor rhythm in the slimy ralamander, Plethodon glutinosus, can be shifted in phase by the environmental light cycle, whether the animals have eyes or not. Rhythmicity persists at least for the first day when animals are transferred to constant conditions, with a period of about 24 hours, and is therefore circadian in nature. An extraoptic photoreceptor site in the brain is suggested.

Spatial orientation by Salamanders using plane-polarized light.

Tiger salamanders (Ambystoma tigrinum) can perceive the plane of polarization in linearly polarized light and can learn to use that e-vector direction for spatial orientation in indoor orientation tests.

Mycoplasma pneumoniae induces airway epithelial cell expression of MUC5AC in asthma.

As excess mucin expression can contribute to the exacerbation of asthma, the present authors hypothesised that Mycoplasma pneumoniae significantly induces MUC5AC (the major airway mucin) expression in airway epithelial cells isolated directly from asthmatic subjects. A total of 11 subjects with asthma and six normal controls underwent bronchoscopy with airway brushing. Epithelial cells were cultured at an air-liquid interface and incubated with and without M. pneumoniae for 48 h, and in the presence and absence of nuclear factor (NF)-kappaB and a toll-like receptor (TLR)2 inhibitor. Quantitative PCR was performed for MUC5AC and TLR2 mRNA. MUC5AC protein and total protein were determined by ELISA. M. pneumoniae exposure significantly increased MUC5AC mRNA and protein expression after 48 h in epithelial cells isolated from asthmatic, but not from normal control subjects, at all concentrations as compared to unexposed cells. TLR2 mRNA expression was significantly increased in asthmatic epithelial cells at 4 h compared with unexposed cells. NF-kappaB and TLR2 inhibition reduced MUC5AC expression to the level of the unexposed control in both groups. Mycoplasma pneumoniae exposure significantly increased MUC5AC mRNA and protein expression preferentially in airway epithelial cells isolated from asthmatic subjects. The toll-like receptor 2 pathway may be involved in this process.

MARCKS regulates neuritogenesis and interacts with a CDC42 signaling network.

Through the process of neuronal differentiation, newly born neurons change from simple, spherical cells to complex, sprawling cells with many highly branched processes. One of the first stages in this process is neurite initiation, wherein cytoskeletal modifications facilitate membrane protrusion and extension from the cell body. Hundreds of actin modulators and microtubule-binding proteins are known to be involved in this process, but relatively little is known about how upstream regulators bring these complex networks together at discrete locations to produce neurites. Here, we show that Myristoylated alanine-rich C kinase substrate (MARCKS) participates in this process. Marcks-/- cortical neurons extend fewer neurites and have less complex neurite arborization patterns. We use an in vitro proteomics screen to identify MARCKS interactors in developing neurites and characterize an interaction between MARCKS and a CDC42-centered network. While the presence of MARCKS does not affect whole brain levels of activated or total CDC42, we propose that MARCKS is uniquely positioned to regulate CDC42 localization and interactions within specialized cellular compartments, such as nascent neurites.

The lipoprotein abnormality in Tangier disease: quantitation of A apoproteins.

In this study we have determined by radioimmunoassay and double immunoelectrophoresis the total quantities and distributions of A apoproteins in three adult patients affected with Tangier disease (hereditary alpha-lipoprotein deficiency). Compared with normal plasma, the total quantities of apoproteins A-I and A-II in Tangier plasma were determined to be less than 1% and 5-7%, respectively. In Tangier patients, approximately 90% of the apoprotein A-I sedimented when ultracentrifugations of plasma were carried out at density 1.21 g/ml KBr. By contrast, more than 95% of the apoprotein A-II floated under those conditions. In normal plasma, approximately 90% of both apoproteins A-I and A-II is found in the 1.063-1.21-g/ml KBr density fraction. These findings suggest that complete dissociation of A apoproteins occurs in Tangier plasma. This dissociation of apoproteins was confirmed by double immunoelectrophoresis with monospecific antisera. Immunochemical and electrophoretic experiments did not provide evidence for a structural abnormality of apoprotein A-I in these patients, The results taken together strongly suggest that normal high-density lipoproteins are absent from Tangier plasma.

Oxygen metabolites stimulate release of high-molecular-weight glycoconjugates by cell and organ cultures of rodent respiratory epithelium via an arachidonic acid-dependent mechanism.

Several common pulmonary disorders characterized by mucus hypersecretion and airway obstruction may relate to increased levels of inhaled or endogenously generated oxidants (O2 metabolites) in the respiratory tract. We found that O2 metabolites stimulated release of high-molecular-weight glycoconjugates (HMG) by respiratory epithelial cells in vitro through a mechanism involving cyclooxygenase metabolism of arachidonic acid. Noncytolytic concentrations of chemically generated O2 metabolites (purine + xanthine oxidase) stimulated HMG release by cell and explant cultures of rodent airway epithelium, an effect which is inhibitable by coaddition of specific O2 metabolite scavengers or inhibitors of arachidonic acid metabolism. Addition of O2 metabolites to epithelial cells provoked production of PGF2a, an effect also inhibitable by coaddition of O2 metabolite scavengers or inhibitors of arachidonic acid metabolism. Finally, addition of exogenous PGF2a to cell cultures stimulated HMG release. We conclude that O2 metabolites increase release of respiratory HMG through a mechanism involving cyclooxygenase metabolism of arachidonic acid with production mainly of PGF2a. This mechanism may be fundamental to the pathogenesis of a variety of lung diseases associated with hypersecretion of mucus and/or other epithelial fluids, as well as a basic cellular response to increased oxidants.

HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway.

HMG-CoA reductase inhibitors (statins) have been developed as lipid-lowering drugs and are well established to reduce morbidity and mortality from coronary artery disease. Here we demonstrate that statins potently augment endothelial progenitor cell differentiation in mononuclear cells and CD34-positive hematopoietic stem cells isolated from peripheral blood. Moreover, treatment of mice with statins increased c-kit(+)/Sca-1(+)--positive hematopoietic stem cells in the bone marrow and further elevated the number of differentiated endothelial progenitor cells (EPCs). Statins induce EPC differentiation via the PI 3-kinase/Akt (PI3K/Akt) pathway as demonstrated by the inhibitory effect of pharmacological PI3K blockers or overexpression of a dominant negative Akt construct. Similarly, the potent angiogenic growth factor VEGF requires Akt to augment EPC numbers, suggesting an essential role for Akt in regulating hematopoietic progenitor cell differentiation. Given that statins are at least as potent as VEGF in increasing EPC differentiation, augmentation of circulating EPC might importantly contribute to the well-established beneficial effects of statins in patients with coronary artery disease.

Inhibition of mucin secretion with MARCKS-related peptide improves airway obstruction in a mouse model of asthma.

Allergic asthma is associated with airway epithelial cell mucous metaplasia and mucin hypersecretion, but the consequences of mucin hypersecretion on airway function are unclear. Recently, a peptide derived from the myristoylated alanine-rich C kinase substrate protein NH(2)-terminal sequence (MANS) was shown to inhibit methacholine (MCh)-induced mucin secretion from airway mucous cells by >90%. We studied the effect of intranasal pretreatment with this peptide on specific airway conductance (sGaw) during challenge with MCh in mice with allergen-induced mucous cell metaplasia. sGaw was noninvasively measured in spontaneously breathing restrained mice, using a double-chamber plethysmograph. Pretreatment with MANS peptide, but not a control peptide [random NH(2)-terminal sequence (RNS)], resulted in partial inhibition of the fall in sGaw induced by 60 mM MCh (mean +/- SE; baseline 1.15 +/- 0.06; MANS/MCh 0.82 +/- 0.05; RNS/MCh 0.55 +/- 0.05 cmH(2)O/s). The protective effect of MANS was also seen in mice challenged with allergen for 3 consecutive days to increase airway hyperresponsiveness, although the degree of protection was less (baseline 1.1 +/- 0.08; MANS/MCh, 0.65 +/- 0.06; RNS/MCh 0.47 +/- 0.03 cmH(2)O/s). Because routine sGaw measurement in mice includes nasal airways, the effectiveness of MANS was also confirmed in mice breathing through their mouths after nasal occlusion (baseline 0.92 +/- 0.05; MANS/MCh 0.83 +/- 0.06; RNS/MCh 0.61 +/- 0.03 cmH(2)O/s). In all instances, sGaw in the MANS-pretreated group was approximately 35% higher than in RNS-treated controls, and mucous obstruction accounted for approximately 50% of the MCh-induced fall in sGaw. In summary, mucin secretion has a significant role in airway obstruction in a mouse model of allergic asthma, and strategies to inhibit mucin secretion merit further investigation.

Evaluation of the efficacy and safety of a novel formulation of metaflumizone in cats naturally infested with fleas in Europe.

The efficacy and safety of a novel spot-on formulation of metaflumizone (ProMeris for Cats, Fort Dodge Animal Health, Overland Park, KS) was assessed in cats naturally infested with fleas in a multiregional, clinical field study. Sixteen veterinary clinics in Germany and eight clinics in France enrolled patients to the study. A total of 173 cats with flea infestation qualified as primary patients and were randomly allocated to one of the two treatments in a ratio of approximately 2:1 for metaflumizone (minimum dosage of 40mg/kg) or fipronil (at the recommended label rate). Clinical examinations and baseline parasite counts were performed on Day 0 prior to treatment. Flea counts and safety evaluations were repeated at approximately 2-week intervals for 8 weeks. Both treatments resulted in consistent reductions (>84%) in flea numbers throughout the study, but metaflumizone resulted in numerically higher reductions on most count days. Within groups the flea reduction was highly significant (p<0.0001) compared to baseline at all observation periods. The efficacy of metaflumizone against fleas compared to baseline was 91.0%, 89.4%, 90.8% and 90.7% at Day 14, 28, 42 and 56, respectively. The corresponding efficacies for fipronil were 91.7%, 86.9%, 84.6% and 87.7%. Metaflumizone was highly effective in controlling existing infestations of fleas on cats and was effective against reinfestation for at least 56 days. Metaflumizone showed a good tolerance profile in cats.

Evaluation of the efficacy and safety of a novel formulation of metaflumizone plus amitraz in dogs naturally infested with fleas and ticks in Europe.

The efficacy and safety of a novel spot-on formulation of metaflumizone plus amitraz (ProMeris/ProMeris Duo for Dogs, Fort Dodge Animal Health, Overland Park, KS) was assessed in dogs naturally infested with ticks and/or fleas in a multiregional, clinical field study. Nineteen veterinary clinics in Germany and 11 clinics in France enrolled patients to the study. One hundred eighty one dogs with tick infestation and 170 dogs with flea infestation (plus three dogs harboring both ticks and fleas) qualified as primary patients and were randomly allocated to one of two treatments in a ratio of approximately 2:1 for metaflumizone plus amitraz (minimum dosage of 20 plus 20mg/kg) or fipronil (at the recommended label rate). Clinical examinations and baseline parasite counts were performed on Day 0 prior to treatment. Tick and/or flea counts and safety evaluations were repeated at intervals of about 2 weeks for 8 weeks. Both products resulted in consistent reductions in tick numbers (>81%) throughout the study, with metaflumizone plus amitraz giving consistently higher reductions in tick numbers. The efficacy against tick count compared with Day 0 was 97.6%, 93.5%, 89% and 94% at Day 14, 28, 42 and 56, respectively, for metaflumizone plus amitraz. The corresponding efficacies for fipronil were 86.3%, 81.1%, 84.8% and 86.1%. Within groups, the tick reduction was highly significant (P<0.0001) compared to baseline at all observation periods. Both treatments resulted in consistent (>89%) and highly significant (P<0.0001) reductions in flea numbers relative to the baseline counts throughout the study, although fipronil resulted in numerically higher reductions on each count day. The efficacy against fleas compared to baseline was 91.8%, 88.7%, 91.5% and 92.0% at Day 14, 28, 42 and 56, respectively, for metaflumizone plus amitraz. The corresponding efficacies for fipronil were 98.2%, 96.3%, 95.9% and 96.7%. Metaflumizone plus amitraz was highly effective in controlling existing infestations of fleas and ticks on dogs and was effective against reinfestation for at least 56 days. Metaflumizone plus amitraz showed a good tolerance profile in dogs.

Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease.

Recent studies provide increasing evidence that postnatal neovascularization involves bone marrow-derived circulating endothelial progenitor cells (EPCs). The regulation of EPCs in patients with coronary artery disease (CAD) is unclear at present. Therefore, we determined the number and functional activity of EPCs in 45 patients with CAD and 15 healthy volunteers. The numbers of isolated EPCs and circulating CD34/kinase insert domain receptor (KDR)-positive precursor cells were significantly reduced in patients with CAD by approximately 40% and 48%, respectively. To determine the influence of atherosclerotic risk factors, a risk factor score including age, sex, hypertension, diabetes, smoking, positive family history of CAD, and LDL cholesterol levels was used. The number of risk factors was significantly correlated with a reduction of EPC levels (R=-0.394, P=0.002) and CD34-/KDR-positive cells (R=-0.537, P<0.001). Analysis of the individual risk factors demonstrated that smokers had significantly reduced levels of EPCs (P<0.001) and CD34-/KDR-positive cells (P=0.003). Moreover, a positive family history of CAD was associated with reduced CD34-/KDR-positive cells (P=0.011). Most importantly, EPCs isolated from patients with CAD also revealed an impaired migratory response, which was inversely correlated with the number of risk factors (R=-0.484, P=0.002). By multivariate analysis, hypertension was identified as a major independent predictor for impaired EPC migration (P=0.043). The present study demonstrates that patients with CAD revealed reduced levels and functional impairment of EPCs, which correlated with risk factors for CAD. Given the important role of EPCs for neovascularization of ischemic tissue, the decrease of EPC numbers and activity may contribute to impaired vascularization in patients with CAD. The full text of this article is available at http://www.circresaha.org.

Isolation and spontaneous transformation of cloned lines of hamster tracheal epithelial cells.

In respiratory carcinogenesis studies using rodents, the tracheal epithelium is the target tissue for the induction of tumors after exposure of animals to chemical carcinogens. In the studies described below, tracheal epithelial cells were isolated to evaluate their biological and biochemical features. Epithelial cells derived from the tracheal mucosa of Syrian golden hamsters were established in culture. Three morphological types of polygonal cells were observed as mixed populations in four clonally derived lines. One type of cell is mucin secreting since membrane-bound vesicles that stain positively using the alcian blue:periodic acid-Schiff reaction are present in the cytoplasm and increased amounts of mucin constituents are demonstrable in the culture medium. Cells of a second type possess both intracytoplasmic and surface cilia, but they lack mucin vesicles. The third type exhibits no differentiating features. Four density-dependent inhibited cloned cell lines were established. After repeated passage, these cells: (a) grew in soft agar; (b) released proteases that were activators of plasminogen; (c) demonstrated measurable basal and inducible aryl hydrocarbon hydroxylase activity; and (d) produced anaplastic carcinomas in syngeneic hamsters. Factors affecting the transformation and differentiation of respiratory epithelial cells have not been elucidated. The availability of these cell lines will permit studies that focus on these questions.

Detection of gene amplification by genomic hybridization to cDNA microarrays.

Gene amplification is one of the major mechanisms of oncogene activation in tumorigenesis. To facilitate the identification of genes mapping to amplified regions, we have used a technique based on the hybridization of total genomic DNA to cDNA microarrays. To aid detection of the weak signals generated in this complex hybridization, we have used a tyramide-based technique that allows amplification of a fluorescent signal up to 1000-fold. Dilution experiment suggests that amplifications of 5-fold and higher can be detected by this approach. The technique was validated using cancer cell lines with several known gene amplifications, such as those affecting MYC, MYCN, ERBB2, and CDK4. In addition to the detection of the known amplifications, we identified a novel amplified gene, ZNF133, in the neuroblastoma cell line NGP. Hybridization of NGP cDNA on an identical array also revealed over expression of ZNF133. Parallel analysis of genomic DNA for copy number and cDNA for expression now provides rapid approach to the identification of amplified genes and chromosomal regions in tumor cells.