Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.

CD44 is a multifunctional protein involved in cell adhesion and signaling. The role of CD44 in prostate cancer (PCa) development and progression is controversial with studies showing both tumor-promoting and tumor-inhibiting effects. Most of these studies have used bulk-cultured PCa cells or PCa tissues to carry out correlative or overexpression experiments. The key experiment using prospectively purified cells has not been carried out. Here we use FACS to obtain homogeneous CD44(+) and CD44(-) tumor cell populations from multiple PCa cell cultures as well as four xenograft tumors to compare their in vitro and in vivo tumor-associated properties. Our results reveal that the CD44(+) PCa cells are more proliferative, clonogenic, tumorigenic, and metastatic than the isogenic CD44(-) PCa cells. Subsequent molecular studies demonstrate that the CD44(+) PCa cells possess certain intrinsic properties of progenitor cells. First, BrdU pulse-chase experiments reveal that CD44(+) cells colocalize with a population of intermediate label-retaining cells. Second, CD44(+) PCa cells express higher mRNA levels of several 'stemness' genes including Oct-3/4, Bmi, beta-catenin, and SMO. Third, CD44(+) PCa cells can generate CD44(-) cells in vitro and in vivo. Fourth, CD44(+) PCa cells, which are AR(-), can differentiate into AR(+) tumor cells. Finally, a very small percentage of CD44(+) PCa cells appear to undergo asymmetric cell division in clonal analyses. Altogether, our results suggest that the CD44(+) PCa cell population is enriched in tumorigenic and metastatic progenitor cells.

[Evaluation of the vagal activity by the Deep-Breathing test]. / Evaluation de l'activité vagale par le test de la respiration profonde (Deep-Breathing).

OBJECTIVE: The Deep-Breathing (DB) test is of major importance in the evaluation of the vagal response (VR). We applied this test to assess the VR in a group of subjects with functional (neurological, cardiovascular or digestive) symptoms unexplained by standard cardiac examination and to compare it with the VR measured in a group of healthy controls. PATIENTS AND METHODS: The following groups were considered: a C-Group of healthy controls (n=50), and three groups each consisting of 50 symptomatic patients (S1, S2, S3). Subjects in the S1-Group had a postural orthostatic tachycardia syndrome (POTS), while members of the S2-Group had arterial hypertension, and members of S3-Group had neither POTS nor arterial hypertension. The VR was expressed as a percentage variation of RR intervals 100x[(RR(max)-RR(min))/RR(min)], and was correlated with age and sex in the C-Group before any comparison. RESULTS: In controls the VR was 31.0%+/-8.2. It was negatively correlated with age (r=-0.42, p=0.003) and there was no significant difference between males (31.2%+/-5.7) and females (30.9%+/-9.0) (p=0.12). Compared to the C-Group, the VR was 51.6%+/-20.4 in the S1-Group (p<0.001), 26.9%+/-11.3 in the S2-Group (p<0.001), and 47.2%+/-22.7 in the S3-Group (p<0.001). CONCLUSION: The VR was independent of sex but was negatively correlated with age. In comparison with healthy controls, it was significantly increased in the patients with POTS and significantly decreased in hypertensives.

Increased cell growth and tumorigenicity in human prostate LNCaP cells by overexpression to cyclin D1.

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the effects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in S-phase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo.

[Autonomic profile of patients with migraine]. / Profil autonomique des patients migraineux.

OBJECT: Dysfunction of autonomic nervous system (ANS) is implicated in the genesis and persistence of migraine. The objective of this study was to compare autonomic nervous system (ANS) profile of migraineurs during headache-free periods to a group of normal subjects based on cardio-vascular reactivity. METHODS: Patients with migraine according to the criteria of IHS 2004 were selected for the study. After a 30 min resting blood pressure (BP), the following standard tests were performed: deep-breathing (DB), hand grip (HG) of 15 s and 3 min, valsalva maneuver, echo stress, (ES) and tilt test (TT). Results were compared to 44 normal subjects, age similar, 37 female, (84.1%) using the Student test, with P < 0.005 as significant. RESULTS: Thirty-two patients (27 female (84.38%), 16-51 years, mean 40.41 +/- 7.8) were studied. Twenty-two patients (69%) had systolic blood pressure below 94 mmHg and 25 patients (78%) had diastolic blood pressure below 60 mmHg. Compared to normal, migraineurs exhibited a significantly higher vagal response (P < 0.001) and a significantly lower alpha sympathetic response, central by using ES as well as peripheral by using HG of 3 min (P < 0.001). CONCLUSIONS: Autonomic cardiovascular reactivity of patients with migraine showed a vagal hyperactivity and a deficiency of the alpha sympathetic system. This leads to further studies with new therapeutical approaches.

Ventricular coupling via the pericardium: normal versus tamponade.

OBJECTIVE: The aim was to examine how regional variations in pericardial pressure affect the mechanical coupling between the ventricles. METHODS: Canine hearts from 14 dogs (14.5-18 kg) were removed and placed in cold cardioplegia solution. Balloons were inserted into the left and right ventricles and the atria. Pericardial pressure over the left ventricle (Pclv) and the right ventricle (Pcrv) was measured with thin balloon catheters. Ventricular and pericardial pressures were measured, and ventricular and pericardial coupling was calculated, under control conditions and with increases in pericardial tension and fluid. RESULTS: At baseline, regional differences in pericardial pressure occurred [Pclv > Pcrv, 4.0(SD 0.9) v 2.9(0.6) mm Hg, p < 0.05]. Ventricular coupling via the pericardium was defined as delta Pclv/delta Pcrv for right ventricular volume increases and delta Pcrv/delta Pclv for left ventricular volume increases. This ratio increased more after increasing right ventricular volume than after increasing left ventricular volume [delta Pclv/delta Pcrv > delta Pcrv/delta Pclv, 1.14(0.33) v 0.51(0.15), p < 0.05]. Increasing the pericardial tension by clamping the pericardium increased pericardial pressures, yet did not alter the regional variations in pressure [Pclv > Pcrv, 8.4(2.2) v 6.4(2.5) mm Hg, p < 0.05] or pericardial coupling [delta Pclv/delta Pcrv > delta Pclv/delta Pcrv, 1.18(0.46) v 0.54(0.16), p < 0.05]. In contrast, creating a mild tamponade increased pericardial pressures, eliminated regional differences in pressure, and altered the coupling between ventricles [delta Pclv/delta Pcrv approximately delta Pclv/delta Pcrv, 0.95(0.11) v 1.05(0.08), p = NS]. These regional differences in pericardial pressure might have a geometrical basis. In four in vivo canine experiments using cine magnetic resonance, the short axis radius of curvature for the right ventricle was greater than for the left ventricle [38.3(4.4) mm v 29.2(3.8) mm, p < 0.05]. CONCLUSIONS: The pericardium partially protects right ventricular filling: regional differences in pericardial pressure normally occurred with lower pericardial pressure over the right ventricle, and left to right ventricular coupling was less. This protection of right ventricular filling was lost with even a small pericardial effusion.

Subcutaneous immunization of rabbits with nitrocellulose paper strips impregnated with microgram quantities of protein.

The desire to produce specific antibodies to substances available only in minute quantities and increased concern for laboratory animal welfare have each contributed to heightened interest in alternative immunization methods. In this report, we describe the production of polyclonal antibodies against microgram quantities of a weakly immunogenic tumor-associated protein of canine origin. Our technique employs the subcutaneous implantation of nitrocellulose electroblot strips without the use of adjuvant. The method is simple, appears reliable, can be used to improve antigen purity, and is applicable for either polyclonal or monoclonal antibody production in different host species. In addition, because traditional adjuvants are not required with this system, severe inflammatory responses and associated animal discomfort are reduced.

Correlation of thallium uptake with left ventricular wall thickness by cine magnetic resonance imaging in patients with acute and healed myocardial infarcts.

Myocardial infarction (MI) is characterized by cellular necrosis which undergoes fibrotic transformation over time. Cine magnetic resonance imaging (MRI) offers high-resolution 3-dimensional images of the left ventricular myocardium, allowing sampling of the myocardial wall thickness over the entire left ventricle. Tomographic (single-photon emission computed tomography [SPECT]) thallium images also provide 3-dimensional information on the location and level of thallium uptake, which has been shown to correlate with myocardial viability. The purposes of this study were: (1) to examine the relation between both end-diastolic and end-systolic wall thickness and normalized thallium-201 uptake over the left ventricle in a group of patients with MI, (2) to examine the relation between regional wall thickening and normalized thallium uptake, and (3) to examine the relation between thallium uptake and wall thickness both early and late after infarction. Twenty-four patients with MI underwent stress, redistribution, and reinjection thallium SPECT imaging and cine MRI within several days. Seventeen patients underwent imaging late after infarction and 7 underwent imaging early after infarction. Normalized thallium activity was correlated with MRI wall thicknesses at both end-diastole and end-systole for 18 segments for each ventricle. In addition, end-diastolic and end-systolic wall thicknesses were grouped by their corresponding thallium activity levels into percentiles. End-systolic wall thickness correlated significantly with normalized thallium uptake in 14 of 18 segments, end-diastolic wall thickness in only 4 of 18 segments, and wall thickening in only 3 of 18 segments. Mean values for end-diastolic and end-systolic wall thicknesses corresponding to severely reduced (<50%) normalized thallium activity were 9.9 +/- 1.1 and 8.5 +/- 0.6, respectively. Using receiver-operating curve analysis, end-systolic wall performed as a better diagnostic parameter than end-diastolic wall for identifying severely reduced thallium activity levels. For all levels of thallium activity, end-diastolic wall thicknesses were all thinner late versus early after MI, whereas end-systolic wall thickness was thinner only in the segments corresponding to severely reduced thallium activity. Based on these results, end-systolic wall thickness is the best noninvasive anatomic parameter of myocardial scar.

Optimal fluorescence excitation wavelengths for detection of squamous intra-epithelial neoplasia: results from an animal model.

Using the hamster cheek pouch carcinogenesis model, we explore which fluorescence excitation wavelengths are useful for the detection of neoplasia. 42 hamsters were treated with DMBA to induce carcinogenesis, and 20 control animals were treated only with mineral oil. Fluorescence excitation emission matrices were measured from the cheek pouches of the hamsters weekly. Results showed increased fluorescence near 350-370 nm and 410 nm excitation and decreased fluorescence near 450-470 nm excitation with neoplasia. The optimal diagnostic excitation wavelengths identified using this model - 350-370 nm excitation and 400-450 nm excitation - are similar to those identified for detection of human oral cavity neoplasia.

Cardiac architecture: Gothic versus Romanesque. A cardiologist's view.

The healthy left ventricle, with remarkable mechanical efficiency, has a gothic architecture, which results from the disposition of the myocardial fibers supported and maintained by a normal collagen matrix scaffold. This conclusion, arising from the analysis of roman and gothic buildings and from comparative biology of the left ventricles of different species, has been substantiated by the study of three-dimensional images obtained by MRI and analyzed with mathematic methods for measurements of the curvature and thickness of the ventricular walls. The assessment of left ventricular functional reserve based on the architecture has been very important in making therapeutic and surgical decisions in our patients and has important implications for the design of surgical strategies designed to try to improve ventricular function by restoring an architecture that allows more efficient ventricular mechanics. The structural approach and its combination with important advances in the knowledge of membrane channels, signaling pathways, cytokines, growth factors, neuroregulation, and targeted pharmacology, and with the advances in methods for reducing hemodynamic load and its cellular and structural consequences, is certain to bring about a dramatic change in the very serious and highly prevalent congestive failure associated with the Romanesque transformation of the diseased left ventricle.

Hepatic pathology of the colon carcinogen, azoxymethane, in Hanford-Moore miniature pigs.

Azoxymethane (AOM) is commonly used in colon carcinogenesis studies in rodents. In an attempt to develop a large animal model of human colon cancer, AOM was given to Hanford-Moore miniature pigs. Six pigs were injected intraperitoneally with a single dose of AOM of either 5, 10, 20, 40 or 55 mg per kg body weight. Within 48 h, severe signs of toxicity and death occurred in animals receiving greater than 20 mg per kg AOM. After 30 days, all surviving animals were killed and necropsied. Acute hepatic necrosis with haemorrhage was the major toxic effect of AOM in all animals receiving doses exceeding 20 mg per kg. In a second, longer-term experiment, eight pigs were injected with either 20 mg per kg AOM weekly or 10 mg per kg AOM every other week or a combination of both treatments. Chronic toxic effects were limited to the liver. No colon tumours were observed. It is concluded that this particular species demonstrates marked hepatic sensitivity to the toxic effects of AOM.

PCR-based microsatellite analysis for differentiation and genetic monitoring of nine inbred SENCAR mouse strains.

Sixteen DNA microsatellites or simple sequence length polymorphisms (SSLPs), generated by polymerase chain reaction (PCR) were selected for use in the genetic quality control of the nine inbred SENCAR strains currently available. The SENCAR strains constitute a powerful tool for mechanistic studies of multi-stage skin carcinogenesis, as well as for studies to understand the underlying genetic basis of resistance to tumour promotion and progression. SSLP analysis is a fast and economical way for detecting genetic contamination (unexpected outcrosses) among these closely-related albino strains, where standard immunological and biochemical markers have been shown to be insufficient.

Data-based organizational change: the use of administrative data to improve child welfare programs and policy.

Administrative databases hold the potential to have a significant impact on the development of effective child welfare programs and policies. This article discusses the strengths and weaknesses of administrative databases, issues with their implementation and data analysis, and effective presentation of their data at different levels in child welfare organizations.

[Cardiovascular autonomic reflexes on the postural orthostatic tachycardia syndrome]. / Réflexes autonomiques cardiovasculaires dans le syndrome de tachycardie orthostatique idiopathique.

Postural orthostatic tachycardia syndrome (POTS) is an inadequately understood pathology because its diagnosis is not based on the conventional methods of investigation. The orthostatic test allows to make the diagnosis easily. The objective of this study is to determine cardiovascular autonomic reflexes of 70 patients having POTS. The tests of exploration of the autonomic nervous system practised are: deep breathing, hand grip, mental stress and orthostatic test. The analysis of orthostatic test showed that the increase of the cardiac frequency, relative to the state of "beta" peripheral sympathetic hyperactivity occurred before the 2nd minute in 80% of patients. The POTS was considered "florid" in 43% of patients and had complicated of a rough and severe fall of systolic blood pressure inferior to 70 mmHg in four patients, after the fifth minute of the test. The analysis of the different tests had shown vagal hyperactivity in 63% of patients on deep breathing, in 93% of patients on hand grip and in 100% on orthostatic test. The "alpha" central sympathetic activity was increased in 76% of the cases and "beta" central sympathetic activity was high in 83% of cases. The "alpha" peripheral hyperactivity was observed in 63% of patients on hand grip, and in 44% on orthostatic test. The analysis of cardiovascular autonomic reflexes in patients affected by POTS allowing the determination of their autonomic profile, will contribute probably to a better understanding of this pathology and to a better orientation of its care.

[Sympathetic response in primary hypertension]. / Les réponses sympathiques dans l'hypertension artérielle essentielle.

UNLABELLED: High blood pressure (BP) is a major cause of cardiovascular disease and primary hypertension is a frequent pathological condition. Sympathetic hyperactivity may be involved in primary hypertension. The purpose of this study was mainly to evaluate sympathetic activity when performing cardiovascular autonomic profile examination in patients with primary hypertension in comparison with normotensive subjects. PATIENTS AND METHODS: This prospective study included one group of hypertensive patients (n=120, mean age 54 years) compared with a control group (n=120, mean age 52 years) of normotensive subjects. Autonomic tests included deep-breathing (DB), hand-grip (HG) and echostress test (ES). Comparison tests between the two groups, similar in age, were expressed as mean+/-SE and made using the t Student test, p<0.05 was considered significant. RESULTS: Alpha-adrenergic sympathetic response using ES method produced a BP response of 20,0%+/-9,8 in hypertensive patients group and 15,2%+/-8,6 in the control group (p<0.001). Alpha-adrenergic sympathetic response using three minutes HG test was of 16,7%+/-7,5 in hypertensive patients group and 13,3%+/-6,5 in the control group (p<0.001). Vagal stimulation in hypertensive group after DB showed that electrocardiographic: ECG (EKG) waves R (RR) interval variation was of 30,2%+/-8,1 meanwhile in the control group this RR variation was of 46,1%+/-21,1 p<0.001, and the one of HG of 15 seconds was 17,6%+/-10,2 versus 32,5%+/-12,7 p<0.001. CONCLUSION: Hypertensive patients had a significantly higher sympathetic response to central and peripheral stimulations and a significantly lower parasympathetic response when compared to normotensive controls.

Effect of protein and zinc deficiencies on vaccine efficacy in guinea pigs following pulmonary infection with Listeria.

Specific pathogen-free guinea pigs were maintained for 3 weeks on purified diets containing 30% protein (ovalbumin) and 50 ppm added zinc (Control-C), 10% protein and 50 ppm added zinc (low protein-LP), or 30% protein and no added zinc (low zinc-LZ). Half of the animals in each diet group were vaccinated intraperitoneally with 2.5 x 10(3) viable Listeria monocytogenes organisms after 8 days of diet treatment. Ten days later, all animals received an aerosol challenge of 250 L. monocytogenes organisms and were killed 4 days later. Both zinc and protein deficiency resulted in animals that were growth retarded as compared to controls. Specific nutrient effects were observed as significant reductions in total serum proteins (LP group) and plasma zinc concentrations (LZ group). In vaccinated guinea pigs, both protein and zinc deprivation resulted in significant impairment of delayed-type hypersensitivity (DTH) responses following the intradermal injection of listeria antigen. Diet did not exert a measurable impact on the response of nonvaccinated guinea pigs to pulmonary listeriosis. Prior vaccination allowed both malnourished groups to control the challenge infection successfully as measured by significant reductions in viable bacilli recovered from the lung, spleen and hilar lymph nodes. The diet and vaccine effect varied depending on the tissue examined. Thus, although both protein and zinc deficiencies resulted in loss of peripheral antigen-specific T lymphocyte function (DTH), vaccine efficacy was not impaired.

Chronic zinc deficiency and listeriosis in rats: acquired cellular resistance and response to vaccination.

The functional significance of zinc deficiency on primary and secondary host responses to infection with a facultative intracellular pathogen was studied in specific pathogen free rats. Groups of female rats fed either a low zinc or normal diet for 8 or 10 weeks were infected with Listeria monocytogenes five days prior to sacrifice. Zinc-deficient rats demonstrated thymic atrophy, reduced delayed hypersensitivity responses to listeria antigen, and impaired lymphocyte response of spleen cells to phytohemagglutinin, but not to Concanavalin A. Separate groups of zinc-deficient or control rats were vaccinated with viable L. monocytogenes 10 days prior to respiratory challenge. Vaccination resulted in successful control of bacteria in both dietary groups.

Murine T-lymphomas corresponding to the immature CD4-8+ thymocyte subset.

N-methyl-N-nitrosourea induces murine CD4-8+ T-lymphomas that express high levels of J11d and low levels of CD5 antigens, a phenotype characteristic of immature CD4-8+ thymocytes. This assignment is supported by the fact that CD4-8+ lymphoma cell lines acquire CD4 expression after intrathymic (i.t.) transfer, a finding consistent with the established precursor potential of the normal immature CD4-8+ subset. CD4+8+ lymphomas recovered after i.t. transfer maintain a CD4+8+ phenotype in long-term culture. Northern blot analyses reveal that CD4 expression is regulated at the transcriptional level in immature CD4-8+ and CD4+8+ cell lines. CD4-8+ lymphomas express low levels of functional CD3/TCR complexes that mediate intracellular Ca2+ mobilization in response to CD3 or alpha/beta-TCR monoclonal antibody. These data suggest that the immature CD4-8+ subset contains cells capable of undergoing TCR-mediated signaling and selection events. In contrast to normal immature CD4-8+ cells, which comprise a heterogeneous and transient subset, the CD4-8+ lymphoma lines provide stable, monoclonal models of the immature CD4-8+ stage of thymocyte development.

Practical and effective eradication of pinworms (Syphacia muris) in rats by use of fenbendazole.

Oxyurid parasites are common contaminants of laboratory rodents, and despite many described treatments, no method has assumed preeminence. Limitations in drug efficacy and the general inability to control for exposure to infective eggs are the primary contributors to treatment failure. In addition, some effective drugs must be eliminated from consideration because of narrow safety margins, other toxic aspects, or concerns related to particular uses of the experimental animals. As an alternative to currently described treatments or surgical derivation, we conducted an efficacy study against Syphacia muris in rats with a new fenbendazole-based protocol. Fenbendazole is a highly efficacious broad-spectrum anthelmintic with adulticidal, larvicidal, and ovicidal actions. Its pharmacokinetic behavior, ovicidal activity, and exceptionally wide safety margin in rats and mice make it an attractive choice for pinworm treatment. We used a 150-ppm medicated feed formulation to reach a targeted dose of 8.0 to 12.0 mg/kg/day in three separate studies designed to assess drug intake and efficacy under different housing conditions and in breeding and nonbreeding populations of ACI rats. In all cases, drug was given on alternating weeks, and nonbreeding populations were medicated for a cumulative period of 14 days. The same schedule was used for breeding populations, but the treatment was repeated after a 2-week rest period to ensure sufficient exposure for newly weaned animals. The results of our study indicate that our described treatment, in combination with environmental control measures against pinworm eggs, is capable of eliminating S. muris.

Rhesus thymic/liver xenografts in severe combined immunodeficient mice: immunologic reconstitution and intrathymic infection with simian immunodeficiency virus.

By serving as host recipients of xenografts from both humans and animals, severe combined immunodeficient (SCID) mice have become valuable to many laboratories interested in examining the pathophysiology of different diseases. To gain insight into the usefulness of the SCID mutation in retrovirus research, rhesus monkey fetal hematolymphoid tissues (liver and thymus) were used to construct a SCID-rhesus chimeric mouse (SCID-rh) and were engrafted in the renal capsule. The size and maturation of the thymic engrafts were monitored grossly, histologically, and immunologically. SCID mice were tolerant to rhesus tissues, and thymic engrafts contained thymocytes at different stages of maturation and differentiation that had morphologic features similar to age-matched rhesus thymus. Mature single positive CD2+, CD4+, and CD8+ T lymphocytes that were phenotypically similar to rhesus T lymphocytes were present at low levels (2% to 5%) in the peripheral blood and at moderately higher levels (7% to 15%) in the spleens of SCID-rh mice obtained between 12 and 15 weeks after thymus/liver engraftment. Within 3 weeks after engraftment, > 85% of the thymocytes in the thymic engrafts were immature double positive CD4+CD8+ T cells. The highest number of positive cells were seen in thymic engrafts obtained at 12 to 18 weeks. During these weeks, > 90% of the cells were double positive (CD2+CD4+, CD2+CD8+, and CD4+CD8+). After infection of the engrafted thymus tissue with simian immonodeficiency virus (SIVmac239), PCR analysis revealed successful viral infection of engrafts at 2 and 4 weeks after infection. No significant histopathologic and flow cytometric changes were observed in the thymic engrafts at 2 and 4 weeks after infection. An unrelated lesion of thymic lymphomas involving the SCID host thymus was seen in 12% of the mice. The data presented herein suggest that the SCID-rh is a valuable model for specific studies related to thymus-retrovirus interaction and that it could be used for further studies. The results are discussed in relation to current knowledge of thymus involvement during simian and human immunodeficiency virus infection.