Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells.

The low-density lipoprotein receptor-related protein (LRP1B), encoding an endocytic LDL-family receptor, is among the 10 most significantly deleted genes across 3312 human cancer specimens. However, currently the apparently crucial role of this lipoprotein receptor in carcinogenesis is not clear. Here we show that LRP1B inactivation (by chromosomal, epigenetic and microRNA (miR)-mediated mechanisms) results in changes to the tumor environment that confer cancer cells an increased growth and invasive capacity. LRP1B displays frequent DNA copy number loss and CpG island methylation, resulting in mRNA underexpression. By using CpG island reporters methylated in vitro, we found that DNA methylation disrupts a functional binding site for the histone-acetyltransferase p300 located at intron 1. We identified and validated an miR targeting LRP1B (miR-548a-5p), which is overexpressed in cancer cell lines as a result of 8q22 DNA gains. Restoration of LRP1B impaired in vitro and in vivo tumor growth, inhibited cell invasion and led to a reduction of matrix metalloproteinase 2 in the extracellular medium. We emphasized the role of an endocytic receptor acting as a tumor suppressor by modulating the extracellular environment composition in a way that constrains the invasive behavior of the cancer cells.

An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues.

Interference by autofluorescence is one of the major shortcomes of immunofluorescence analysis by confocal laser scanning microscopy (CLSM). CLSM requires minimal tissue autofluorescence and reduced unspecific fluorescence background, requisites that become more critical when direct immunofluorescence studies are concerned. To control autofluorescence, different reagents and treatments can be used. Until now, the efficacy of the processes described depended on the tissue type and on the processing technique, no general recipe for the control of autofluorescence being available. Using paraffin sections of archival formalin-fixed murine liver, kidney and pancreas, we have found that previously described techniques were not able to reduce autofluorescence to levels that allowed direct immunofluorescence labelling. In this work, we aimed at improving currently described methodologies so that they would allow reduction of the autofluorescent background without affecting tissue integrity or direct immunofluorescence labelling. We have found that the combination of short-duration, high-intensity UV irradiation and Sudan Black B was the best approach to reduce autofluorescence in highly vascularised, high lipofuscins' content tissues, such as murine liver and kidney, and poorly vascularised, low lipofuscins' content tissues such as the pancreas. In addition, we herein show that this methodology is highly effective in reducing autofluorescent background to levels that allow detection of specific signals by direct immunofluorescence.

Absence of the aortic valve associated with hypoplastic left-sided heart syndrome.

In this report we describe the twelfth case in the literature of absence of the aortic valve cusps, associated with hypoplastic left-sided heart syndrome in a neonate. Clinical and hemodynamic conditions in our patient resemble the classical features of this syndrome except for a greater development of the ascending aorta and the left ventricular cavity, due to aortic insufficiency. A patch was unsuccessfully inserted at the aortic annulus to exclude the left ventricle from the circulation. In addition the Norwood operation was performed.

NOD mice are resistant to depletion of thymic cells caused by acute stress or infection.

NOD mice spontaneously develop autoimmune diabetes; disease onset in females of our colony of NOD mice usually takes place around the 4th month of age. Diabetes of NOD mice can be modulated by different stress protocols, even though these animals were shown to be resistant to the effects of glucocorticoids on their lymphocytes. We have recently found that the early host inflammatory response to mycobacteria can be strongly modified by stress, the autoimmunity-prone NOD and NZB/W mice being particularly affected. These mice show reduced numbers of granulocytes in the inflammatory cavity after exposure to stress. Mycobacterium avium is an opportunistic agent that is responsible for disseminated infections seen in AIDS patients. Here, we investigated whether the early immune response to M. avium was altered by stress in NOD mice and we compared the stress response of these mice with a non-autoimmune strain, BALB/c mice. The effects of stress on infected BALB/c mice, which like AIDS patients are susceptible to M. avium infection, offers experimental evidence that M. avium infection, if coupled with stress of the host, may accelerate loss of T helper cells. In contrast, in NOD mice, stress or infection significantly increased the number of cells of the thymuses of the animals. Data obtained with NOD mice support the previously reported resistance of NOD mice lymphocytes to glucocorticoids and suggest that there are two distinct signalling pathways involved in the response of NOD lymphocytes to these stress hormones: one leading to apoptosis and the other mediating glucocorticoid inhibition of activation-induced cell death.

A role for CD45RBlow CD38+ T cells and costimulatory pathways of T-cell activation in protection of non-obese diabetic (NOD) mice from diabetes.

Non-obese diabetic (NOD) mice spontaneously develop autoimmune insulin-dependent diabetes mellitus (IDDM). Infection of the animals with mycobacteria, or immunization with mycobacteria-containing adjuvant, results in permanent protection of NOD mice from diabetes and we have recently reported that the phenomenon is associated with increased numbers of interferon-gamma-producing T cells, possessing increased cytotoxic activity, and also with augmented numbers of activated immunoglobulin M-positive (IgM+) B cells. Here, we have investigated whether protection of NOD mice from IDDM was associated with changes on costimulatory pathways of T and B cells, namely CD28/CTLA-4-B7 and CD40-CD40 ligand (CD40L) and we also further characterized protective T helper (Th) cells with regards to the expression of the differentiation markers CD45RB and CD38. We report that Th cells involved in diabetes vaccination of NOD mice by mycobacterial infection seem to belong to CD45RBlo CD38+ phenotype. The protective effect of Mycobacterium avium infection is also associated with increased CD40L and CTLA-4- expressing Th cells and with the generation of a CD40- IgG+ B cells. Our data are consistent with induction by mycobacterial infection of regulatory CD45RBlo CD38+ Th cells with the ability to trigger deletion or anergy of peripheral self-reactive lymphocytes, with shutting down of IgG+ B-cell response. They also implicate a role for IgG+ B cells in the autoimmune aggression of the endocrine pancreas of NOD mice.

Mechanisms of Mycobacterium avium-induced resistance against insulin-dependent diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice: role of Fas and Th1 cells.

NOD mice spontaneously develop autoimmune diabetes. One of the manipulations that prevent diabetes in NOD mice is infection with mycobacteria or immunization of mice with mycobacteria-containing adjuvant. Infection of NOD mice with Mycobacterium avium, done before the mice show overt diabetes, results in permanent protection of the animals from diabetes and this protective effect is associated with increased numbers of CD4+ T cells and B220+ B cells. Here, we investigate whether the M. avium-induced protection of NOD mice from diabetes was associated with changes in the expression of Fas (CD95) and FasL by immune cells, as well as alterations in cytotoxic activity, interferon-gamma (IFN-gamma) and IL-4 production and activation of T cells of infected animals. Our data indicate that protection of NOD mice from diabetes is a Th1-type response that is mediated by up-regulation of the Fas-FasL pathway and involves an increase in the cytotoxicity of T cells. These changes are consistent with induction by the infection of regulatory T cells with the ability of triggering deletion or anergy of peripheral self-reactive lymphocytes that cause the autoimmune disease of NOD mice.

Involvement of c-myc in the resistance of non-obese diabetic mice to glucocorticoid-induced apoptosis.

Non-obese diabetic (NOD) mice spontaneously develop insulin-dependent diabetes mellitus (IDDM) as a consequence of autoimmune aggression of beta cells of the endocrine pancreas by T cells. T lymphocytes of NOD mice are resistant to apoptosis induced by glucocorticoids, or by starving or DNA-damaging treatments, a feature that was interpreted as being linked to escape of autoreactive T cells from thymic negative selection. c-myc is one of the gene targets of glucocorticoids (GC), its expression being down-regulated by the activated GC-GC receptor complex. We investigated here whether expression of Myc protein, in response to dexamethasone stimulation, was the same in NOD mice and in non-autoimmune strains, namely NON, BALB/c and C57Bl.6. We found a consistent increase in the levels of Myc protein after GC-treatment of lymphocytes of NOD mice, a finding that was in contrast to the down-regulation of c-myc that we observed in lymphocytes from mice not prone to diabetes. We also report that, rather than a absolute resistance to GC-induced cell death, NOD mice display a delayed apoptotic response to GC. We propose that the resistance of NOD mice lymphocytes to GC-induced apoptosis is because of inhibition of the repressive action of GC-GR complexes at the level of c-myc transcription. This deficient action of GC-GR results in increased production of nuclear Myc protein, peculiar to NOD mice cells, following their treatment with GC.

[Complete atrioventricular septal defect. Anatomo-functional correlation between patients with and without Down's syndrome]. / Defeito total do septo atrioventricular. Correlação anatomofuncional entre pacientes com e sem síndrome de Down.

PURPOSE: To compare clinical course, causes and symptoms beginning enset time in children with complete atrioventricular canal with and without Down's syndrome. METHODS: Records of 80 patients < 2 years of age, were reviewed. There were 55 (69%) with Down's syndrome-group I (GI) and 25 (31%) without-group II (GII). Age at synpton enset intensity, functional class, clinical repercussion and anatomic variations in patients undergoing corrective surgery were evaluated. RESULTS: Mean age at symptoms onset was similar for the two groups (50 +/- 75 days). Class II (NYHA) was more frequent in GI (31 patients-56.5%) and class III-IV (NYHA) in GII (19 patients-76%) p < 0.005. Clinical repercussion evaluation showed that congestive heart failure was present in 34 (62%) patients of GI and 21 (84%) of GII; and, pulmonary hypertension was in 21 (38%) patients of GI and 4 (16%) patients of GII p < 0.04. Mean pulmonary arterial pressure of 50 mmHg or more was present in 68% of children with Down's syndrome and in 35% of GII. Clinical course until surgical correction was down hill in 33 (60%) from GI and 21 (84%) from GII p < 0.03. Seventy seven patients underwent surgical correction. CAVC type A of the Rastelli classification was predominant in both groups, GI 37 (67%)-GII 25 (100%). There or more severe valvar morphologic lesions in group II (38%) than in group I (8%). CONCLUSION: There seems to be a pulmonary vascular hyperreactivity predominance in Down's children and cardiac insufficiency signs in the normal genetic group.

Changes in B and T lymphocytes associated with mycobacteria-induced protection of NOD mice from diabetes.

Most female NOD mice spontaneously develop insulin-dependent diabetes mellitus (IDDM) after the 4th month of age. We have recently reported that infection of 2-month-old NOD mice with Mycobacterium avium prevents IDDM expression in these mice. We have searched here for changes in splenic lymphocytes that are associated with the effect of M. avium vaccination. Three experimental groups of female NOD mice were studied: (i) animals infected with 10B viable M. avium bacteria (mice that become protected from IDDM); (ii) mice inoculated with 10B heat-killed (HK) M. avium bacilli, and (iii) untreated age-matched NOD mice. Similar treatments were given to mice of the NON strain which are related to NOD mice but do not develop IDDM. Flow cytometry was used to compare M. avium-infected, HK M. avium inoculated and untreated NOD and NON mice with regard to subpopulations of splenic lymphocytes bearing the surface antigens CD3, CD4, CD8, IgM and B220. We found that M. avium infection of NOD mice caused a sustained enhancement in T cells that was due to an early and transient increase in CD8+ T cells (detected at day 7 of infection). This was followed by marked augmentation in the number of CD4+ T cells at days 14 and 30. There was also elevation in B220+ B cells at days 14 and 30, and of IgM+ B cells at day 30 of infection. Inoculation of NOD mice with HK mycobacteria, which did not prevent IDDM, failed to produce significant changes in the number of T and B cells. No significant enhancement in T and B cells was observed in NON mice that were injected with either viable or HK M. avium bacilli. In NOD mice that reached 16 months of age because of being protected from IDDM (due to the M. avium infection) there was an increase in B220+ B cells. We conclude that: (i) M. avium-induced protection of NOD mice from diabetes depends on the viability of the bacteria; (ii) the protective effect of the infection is associated with an early and marked increase in helper T cells and with a smaller elevation in B cells; (iii) elevation in B cells, but not in T cells, is associated with long term mycobacteria-induced protection of NOD mice from IDDM.

Stress modulates acute inflammation triggered by mycobacteria in autoimmunity-prone and normal mice.

We have investigated the effect of a single stress treatment of mice on the early cellular response (granulocyte influx) of acute inflammation that was produced by intraperitoneal inoculation of the animals with mycobacteria (10(6) bacilli of M. avium). Autoimmunity-prone (NZB/W) and normal (BALB/c, MRL, NZW) mice were submitted to the same stress (20 min of swimming) this given either before or simultaneously with the induction of the intraperitoneal mycobacteria-induced inflammation. Local inflammation was evaluated by the quantification of leukocytes harvested from the peritoneal cavity of the mice; this was done 30 and 60 minutes after the end of the stress treatment. We found that the stressor alone was able to increase the number of cells that were harvested from the peritoneal cavity; this may be due to stress-induced detachment of resident macrophages from the peritoneal walls. The autoimmunity-prone mice (NZB/W) showed a marked decrease in the number of inflammatory cells (mostly of granulocytes) when stress treatment was performed immediately before the triggering of inflammation; these stress-induced alterations were attenuated in normal mice (BALB/c and MRL strains), as well as in the non-autoimmune parent strain (NSW) of NZB/W mice. Modulation of the acute inflammatory response by stress was smaller if the stress was induced concomitantly with the triggering of inflammation; here, NZB/W were again the mice most affected by stress. Our data indicate that [1] stress is able to modify the acute inflammatory response of mice; [2] autoimmunity-prone mice are more sensitive to stress-induced modulation of inflammation than normal animals; and [3] the timing of stress (with regard to the initiation of inflammatory phenomena) is an important factor in the intensity of changes produced by stress in the early cellular response of acute inflammation.

Left ventricular performance in children: transthoracic versus transoesophageal measurement of M mode derived indices.

OBJECTIVE: To compare M mode derived indices of left ventricular performance obtained with transthoracic and transoesophageal echocardiography in children with congenital heart disease. DESIGN: Transthoracic and transoesophageal M mode echocardiograms were obtained under general anaesthesia before cardiac catheterisation. Recordings were digitised by dedicated software. Indices of cavity dimension and left ventricular wall dynamics were compared. PATIENTS: 16 unselected patients with congenital heart disease. RESULTS: Group data for simple measurements of ventricular dimension and wall thickness were similar with the two techniques and had acceptable coefficients of repeatability, but there were considerable individual differences. Correlation was poor with unacceptable repeatability for derived indices of cavity and ventricular wall dynamics. CONCLUSION: Both transthoracic and transoesophageal echocardiography can be used to obtain M mode derived indices of left ventricular performance but the resultant measurements are not directly comparable, presumably because of regional non-uniformity of function.

[Do mitral valve components have the same predictive value in percutaneous balloon mitral valvuloplasty? Doppler echocardiography study]. / São os componentes do aparelho valvar mitral de mesmo valor preditivo na valvoplastia mitral por cateter balão? Estudo ecodopplercardiográfico.

PURPOSE: To evaluate the predictive value of mitral valve components in percutaneous balloon mitral valvuloplasty (PBMV). METHODS: 53 patients undergoing PBMV were submitted to an echocardiographic analysis of mitral valve in order to note mobility, thickness, calcification of leaflets and subvalvar apparatus (SV). Mitral valve area (VA) before and after PBMV was obtained using continuous wave Doppler. Patients were divided in group 1 (VA enhance inferior to 50%) and 2 (VA enhance equal or superior to 50%) and subgroups A (VA post PBMV inferior to 1.5 sqcm) and B (VA post PBMV equal or superior to 1.5 sqcm). Correlations between the score of each component of mitral valve and the results were established. RESULTS: Concerning to the total score, there was no significant difference between the groups and subgroups. Differences were significant when SV was analysed separately (p less than or equal to 0.001). VA average in patients with SV compromising grade 3 (1.28 +/- 0.26 sqcm) was inferior to those with grade 1 or 2 (p less than or equal to 0.001). CONCLUSION: SV has a higher predictive value in the success of PBMV.