Vascular density in melanoma xenografts correlates with vascular permeability factor expression but not with metastatic potential

Previous studies have shown that human airway epithelial cells (AEC) can stimulate allogeneic peripheral blood T-lymphocyte (PBT) proliferation. We now sought to determine which AEC surface molecule/T-cell coreceptors are involved in this process. AEC-induced PBT proliferation was inhibited by 25 microM genestein or herbamycin A (0.9 and 1.8 microM), both tyrosine kinase inhibitors. Anti-phosphotyrosine immunoblots performed on PBT lysates after coculture with AEC demonstrated phosphorylation of 56kD and 60kD bands. To determine whether CD3 associated p59fyn, or CD4 and CD8 associated p56lck phosphotyrosine kinases (PTK) were involved, we assayed kinase activity in lymphocyte lysates immunoprecipitated with anti-p56lck and p59fyn mAbs. PBT cells or murine T-cell line transfectants expressing human CD4 (3G4) or human CD8alpha (3G8) were cocultured with AEC or A549, an alveolar-like cell line lacking class II Ag expression. After A549 or AEC coculture, p56lck activity in PB T-cells peaked at 2 min whereas p59fyn kinase activity continued to rise at 8 min. AEC (expressing class II Ags) stimulate PTK activity in both 3G8 and 3G4 cells. A549 stimulated p56lck in 3G8, but not in 3G4 cells. This activation of p56lck was not blocked by preincubation of A549 with anti-class I or anti-CD1d mAbs. An antibody generated in our laboratory, which recognizes an epithelial specific surface molecule (mAb L12) and which blocks AEC driven PBT proliferation, was shown to block PTK activity of peripheral blood T-cell lysates, though not of 3G8 lysates. These studies suggest that AEC are capable of stimulating CD4 and CD8 associated lck and CD3 associated fyn kinases through class II dependent and independent pathways.     

Angiogenesis correlates with vascular endothelial growth factor expression but not with Ki-ras oncogene activation in non-small cell lung carcinoma

A total of 195 non-small cell lung carcinoma (NSCLC) specimens were studied for the presence of mutations in their ras family genes, for tumor vascularity, and for their immunostaining pattern with an antibody to vascular endothelial growth factor (VEGF). ras mutation was found in 37 of 104 (34.6%) adenocarcinoma specimens, in 0 of 64 squamous cell carcinomas, and in 2 of 27 (7.4%) large cell undifferentiated carcinomas. All mutations were found on the Ki-ras gene, with 37 (95%) of them on codon 12 and the remaining 2 on codon 13. Thirty (77%) of the mutations were G to T transversions. There was a correlation between increasing tumor vascularity and VEGF immunostaining score, but there was no correlation between either of them with the activation of the ras oncogene. A study of VEGF mRNA expression in 14 NSCLC cell lines also demonstrated a lack of correlation between the constitutive expression levels of VEGF and the presence or absence of ras mutation in these cell lines. The results suggest that VEGF is a major angiogenesis factor in NSCLC but that other factors beside ras mutations may influence tumor vascularity in these tumors. The two parameters may potentially serve as independent prognostic factors in NSCLC.     

Elevated soluble fas production in SLE correlates with HLA status not with disease activity

CONTEXT: Medical educators have had a growing sense that proficiency in physical diagnostic skills is waning, but few data have examined the question critically. OBJECTIVE, DESIGN, AND SETTING: To compare the cardiac auscultatory proficiency of medical students and physicians in training. A multicenter cross-sectional assessment of students and house staff. A total of 8 internal medicine and 23 family practice programs of the mid-Atlantic area. PARTICIPANTS: A total of 453 physicians in training and 88 medical students. INTERVENTIONS: All participants listened to 12 cardiac events directly recorded from patients, which they identified by completing a multiple-choice questionnaire. MAIN OUTCOME MEASURES: scores were expressed as the percentage of participants, for year and type of training, who correctly identified each event. Cumulative scores were expressed as the total number of events correctly recognized. An adjusted score was calculated whenever participants selected not only the correct finding but also findings that are acoustically similar and yet absent. RESULTS: Trainees' cumulative scores ranged between 0 and 7 for both internal medicine and family practice residents (median, 2.5 and 2.0, respectively). Internal medicine residents had the highest cumulative adjusted scores for the 6 extra sounds and for all 12 cardiac events tested (P=.01 and .02, respectively). On average, internal medicine and family practice residents recognized 20% of all cardiac events; the number of correct identifications improved little with year of training and was not significantly higher than the number identified by medical students. CONCLUSIONS: Both internal medicine and family practice trainees had a disturbingly low identification rate for 12 important and commonly encountered cardiac events. This study suggests a need to improve the teaching and assessment of cardiac auscultation during generalists' training, particularly with the advent of managed care and its search for more cost-effective uses of technology.     

Early expression of Ig mu chain from a transgene significantly reduces the duration of the pro-B stage but does not affect the small pre-B stage

During B cell development, V-J rearrangements at the Ig heavy mu chain (IgH mu chain) locus occur in early cycling precursors (pro-B stage). Subsequently, rearrangements at the Ig light (IgL) chain locus occur in late resting precursors (small pre-B stage). To study the effects of mu chain expression on the rate of B cell development, purified hematopoietic stem cells (HSC) bearing a mu chain transgene or wild-type HSC were transferred into immunodeficient RAG-2-/- mice and B cell development was followed over time. In addition, cycling B cell precursors were pulse-labeled by the injection of BrdU into transgenic and wild-type mice, and the production of BrdU-labeled kappa + and lambda + B cells was followed over time. These experiments suggested that early expression of the mu chain from the transgene significantly shortened the duration of the pro-B stage and immediately drove the precursors to differentiate into small pre-B cells. By contrast, the presence of the transgene did not affect the small pre-B stage, where IgL rearrangements occur. Thus, kappa and lambda rearrangements occurred only after the arrest of cell cycling as previously shown in wild-type mice, even when the mu chain is artificially expressed earlier in B cell development.     

Primary DNA damage but not mutagenicity correlates with ciprofloxacin concentrations in German hospital wastewaters

BACKGROUND: Auscultation of patients with mitral annular calcification on echocardiography revealed a particular constellation of findings. OBJECTIVE: To test the hypothesis that a particular auscultatory constellation provides a high degree of certainty in diagnosing the combination of mitral annular calcification and aortic sclerosis so often found in the elderly. METHODS: Two groups of patients were studied to evaluate the particular auscultatory constellation under consideration which consisted of: (1) a harsh ejection systolic murmur heard from the 2nd right interspace to the cardiac apex and usually loudest between the 3rd left interspace and the apex; (2) the murmur radiates from the apex towards the left axilla and radiates poorly or not at all from the 2nd right interspace to the neck, and (3) the 2nd heart sound at the cardiac base is normal in intensity, and no ejection clicks are present. Group 1 consisted of patients with mitral annular calcification on echocardiographic examination, and group 2 consisted of patients in whom the particular constellation of auscultatory findings was present and who were then referred for echocardiographic assessment. RESULTS: The particular auscultatory constellation under investigation allowed the diagnosis of the presence of the combination of mitral annular calcification and aortic sclerosis with substantial accuracy. CONCLUSION: The findings in this exploratory study suggest that the pathologic combination of mitral annular calcification and aortic sclerosis can be diagnosed with a reasonably high degree of certainty in elderly patients, if the particular auscultatory configuration is identified.     

Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities

Glutathione has been implicated to function in cytoprotection against cadmium toxicity. The mechanism by which glutathione plays this role has not been well understood. Because glutathione is an important antioxidant and several studies have shown that cadmium induces oxidative stress, this study was undertaken to determine whether development of cadmium resistance is linked to enhanced antioxidant activities. A cadmium-resistant subpopulation of human lung carcinoma A549 cells, which was developed by repeatedly exposing the cells to step-wise increased cadmium concentrations, was compared to a cadmium-sensitive one. The acquired cadmium resistance resulted from neither decreased cadmium uptake nor enhanced cellular metallothionein synthesis. Glutathione content, however, was markedly elevated in the cadmium-resistant cells. In contrast, the activities of the glutathione redox cycle related enzymes, glutathione peroxidase and reductase, were unchanged. Two other antioxidant enzymes, superoxide dismutase and catalase, were also not altered. The results suggest that the development of cadmium resistance in A549 cells unlikely results from enhanced antioxidant enzyme activities, although it is associated with elevated cellular glutathione levels. In addition, measurement of the mRNA and DNA levels for gamma-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione biosynthesis, revealed that enhanced expression of the enzyme but not gene amplification is likely responsible for the elevation of cellular glutathione levels.     

Hippocampal, but not amygdala, activity at encoding correlates with long-term, free recall of nonemotional information

Participation of two medial temporal lobe structures, the hippocampal region and the amygdala, in long-term declarative memory encoding was examined by using positron emission tomography of regional cerebral glucose. Positron emission tomography scanning was performed in eight healthy subjects listening passively to a repeated sequence of unrelated words. Memory for the words was assessed 24 hr later with an incidental free recall test. The percentage of words freely recalled then was correlated with glucose activity during encoding. The results revealed a striking correlation (r = 0.91, P < 0.001) between activity of the left hippocampal region (centered on the dorsal parahippocampal gyrus) and word recall. No correlation was found between activity of either the left or right amygdala and recall. The findings provide evidence for hippocampal involvement in long-term declarative memory encoding and for the view that the amygdala is not involved with declarative memory formation for nonemotional material.     

Testin secreted by Sertoli cells is associated with the cell surface, and its expression correlates with the disruption of Sertoli-germ cell junctions but not the inter-Sertoli tight junction

Testin is a testosterone-responsive Sertoli cell secretory product. In the present study, we demonstrated that the amount of testin secreted by Sertoli cells in vitro was comparable with several other Sertoli cell secretory products. However, virtually no testin was found in the luminal fluid and cytosols of the testis and epididymis when the intercellular junctions were not previously disrupted, suggesting that secreted testin may be reabsorbed by testicular cells in vivo. Studies using Sertoli cells with and without a cell surface cross-linker and radioiodination in conjunction with immunoprecipitation illustrated the presence of two polypeptides of 28 and 45 kDa, which constitute a binding protein complex that anchors testin onto the cell surface. The 28- and 45-kDa peptide appear to be residing on and inside the cell surface, respectively. Immunogold EM studies illustrated testin was abundantly localized on the Sertoli cell side of the ectoplasmic specialization (a modified adherens junction) surrounding developing spermatids. In contrast, very few testin gold particles were found at the site of inter-Sertoli tight junctions. When the inter-Sertoli tight junctions were formed or disrupted, no significant change in testin expression was noted. This is in sharp contrast to the disruption of Sertoli-germ cell junctions, which is accompanied by a surge in testin expression. These results demonstrate the usefulness of testin in examining Sertoli-germ cell interactions.     

Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.     

Bioactive tumour necrosis factor alpha but not granulocyte-macrophage colony-stimulating factor correlates inversely with Langerhans''s cell numbers in skin tumours

Human cytomegalovirus (CMV) infection of smooth muscle cells generates reactive oxygen species (ROS) and thereby activates nuclear factor kappaB (NFkappaB), which causes expression of viral and cellular genes involved in immune and inflammatory responses. These changes could account for the mounting evidence suggesting that CMV may contribute causally to restenosis and atherosclerosis. We found that CMV induces ROS, at least partly, through a cyclooxygenase-2 (COX-2)-dependent pathway. Moreover, the viral immediate-early (IE) gene products, IE72 and IE84, have the capacity to transactivate the COX-2 promoter. Aspirin and indomethacin, both cyclooxygenase inhibitors as well as direct ROS scavengers, reduce CMV-induced ROS, probably through both of these activities. Sodium salicylate also has antiviral effects as the result of its potent antioxidant properties. Furthermore, by reducing ROS, aspirin and sodium salicylate inhibit CMV-induced NFkappaB activation, the ability of IE72 to transactivate its promoter, CMV IE gene expression after infection of SMCs, and CMV replication in SMCs. This is the first time aspirin has been shown to have antiviral effects. Thus, it is possible that aspirin has previously unrecognized therapeutic effects in various clinical situations, such as in viral infections (when used as an antipyretic agent) and in atherosclerosis (when used as an antiplatelet agent).     

Expression of a CD3 epsilon transgene in CD3 epsilon(null) mice does not restore CD3 gamma and delta expression but efficiently rescues T cell development from a subpopulation of prothymocytes

The TCR-associated CD3 complex consists of four subunits, i.e. CD3 gamma, delta, epsilon and zeta, which are expressed very early in T cell development prior to the expression of the TCR and the pre-TCR alpha chain. It is unclear whether the expression of each CD3 protein is independent of, or is influenced by, other CD3 subunits. To study whether CD3 epsilon regulates expression of CD3 gamma and delta genes, we generated a strain of CD3 epsilon-deficient mice termed CD3 epsilon(delta P/delta P) (epsilon(delta P)), in which the promoter of CD3E was disrupted, and subsequently reconstituted these mice with a CD3 epsilon transgene. In the epsilon(delta P) mice, T cell development is arrested at the double-negative stage and targeting the CD3 epsilon gene caused severe inhibition of CD3 gamma and delta gene expression. Introduction of the CD3 epsilon transgene did not restore CD3 gamma and delta expression. However, a very small fraction of prothymocytes that expressed CD3 gamma and delta was rescued upon reconstitution of the CD3 epsilon transgene. Remarkably, this rescue led to a very efficient differentiation and maturation of thymocytes, resulting in a significant T cell population in the periphery. These results demonstrate that CD3 epsilon does not regulate expression of CD3 gamma and delta genes, and underscore the capacity of each prothymocyte to give rise to a large number of mature peripheral T cells.     

Widespread expression of an autoantigen-GAD65 transgene does not tolerize non-obese diabetic mice and can exacerbate disease

Glutamic acid decarboxylase (GAD)65 is a pancreatic beta cell autoantigen implicated as a target of T cells that initiate and sustain insulin-dependent diabetes mellitus (IDDM) in humans and in non-obese diabetic (NOD) mice. In an attempt to establish immunological tolerance toward GAD65 in NOD mice, and thereby to test the importance of GAD in IDDM, we generated three lines transgenic for murine GAD65 driven by a major histocompatibility complex class I promoter. However, despite widespread transgene expression in both newborn and adult mice, T cell tolerance was not induced. Mononuclear cell infiltration of the islets (insulitis) and diabetes were at least as bad in transgenic mice as in nontransgenic NOD mice, and in mice with the highest level of GAD65 expression, disease was exacerbated. In contrast, the same transgene introduced into mouse strain, FvB, induced neither insulitis nor diabetes, and T cells were tolerant to GAD. Thus, the failure of NOD mice to develop tolerance toward GAD65 reflects at minimum a basic defect in central tolerance, not seen in animals not predisposed to IDDM. Hence, it may not be possible experimentally to induce full tolerance toward GAD65 in prediabetic individuals. Additionally, the fact that autoimmune infiltration in GAD65 transgenic NOD mice remained largely restricted to the pancreas, indicates that the organ-specificity of autoimmune disease is dictated by tissue-specific factors in addition to those directing autoantigen expression.     

Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine

Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10-50 microM of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21(ras) (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels of S-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21(ras) and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21(ras)-MAP kinase pathway may represent one of the mechanisms that mediates homocysteine's effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process.     

Clonidine blocks acquisition but not expression of conditioned opiate withdrawal in rats

Previous studies in rodents have reported that clonidine, an alpha 2-adrenergic receptor agonist, attenuated conditioned aversions to naloxone-precipitated opiate withdrawal when administered prior to each withdrawal conditioning episode. The current study was designed to determine whether clonidine could modify the expression of previously established conditioned place aversions and conditioned suppression of operant responding. Dose- and time-dependent effects of clonidine on activity and suppression of operant responding for food identified appropriate treatment parameters for subsequent studies in which rats rendered dependent on opiates through implantation of morphine pellets were tested for: (1) conditioned place aversion; and (2) conditioned suppression of operant responding for food (fixed ratio-15 schedule), in a paradigm wherein rats received four pairings of naloxone with a distinct tone and odor stimulus. Clonidine dose-dependently blocked the acquisition of both conditioned behaviors when administered prior to naloxone on each conditioning trial, but was ineffective in blocking the expression of these conditioned withdrawal signs when administered prior to the test session.     

Low nitrogen diets preserve nutritional status but not residual renal function in rats with severe renal failure

The effects of low nitrogen diets (oral and parenteral) on nutritional status and residual renal function were evaluated in rats with experimentally induced severe renal failure (partial nephrectomy) and compared with the effects of a normal protein diet. Non-uremic rats were used as controls. Muscle protein synthesis was significantly lower in uremic rats fed a normal protein diet than in those fed low protein diets. Values for serum insulin-like growth factor 1 (IGF-1) were significantly lower than for controls in all uremic groups as were the wet and dry weights of skeletal muscle. The glomerular filtration rate was studied after 3 wk of nutritional intervention. Serum creatinine concentration was lower and creatinine clearance greater in uremic rats given the low nitrogen dietary treatments (both oral and parenteral) compared with those fed the normal protein diet. However, the glomerular filtration rate determined with 51Cr-EDTA clearance did not differ between uremic rats with normal and low protein intake. Thus, a low nitrogen diet had positive effects on uremic toxicity and did not compromise nutritional status. The results do not, however, establish a favorable influence on residual renal function and emphasize the need for reliable methods for studying dietary effects on renal function in uremia.