Insights into the physiological function of cellular prion protein

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc) and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction.

Slaughterhouse blood as a perfusate for studying myocardial function under ischemic conditions

Metabolic studies using the in vitro non-recirculating blood-perfused isolated heart model require large volumes of blood. The present study was designed to determine whether heterologous pig blood collected from a slaughterhouse can be used as perfusate for isolated pig hearts perfused under aerobic and constant reduced flow conditions. Eight isolated working pig hearts perfused for 90 min at a constant flow of 1.5 ml g-1 min-1 with non-recirculated blood diluted with Krebs-Henseleit bicarbonate buffer at a hematocrit of 23% were compared to eight hearts subjected to the same protocol but perfused only with Krebs-Henseleit bicarbonate buffer solution. Hearts were paced at 100 bpm and subjected to aerobic perfusion at 38ºC. Hearts were weighed before perfusion and at the end of the experiment and the results are reported as percent weight gain (mean ± SD). Comparisons between groups were performed by the Student t-test (P<0.05). After 90 min of perfusion with modified Krebs-Henseleit, perfused hearts presented a larger weight gain than blood-perfused hearts (39.34 ± 9.27 vs 23.13 ± 5.42%, P = 0.003). Left ventricular end-diastolic pressure was higher in the modified Krebs-Henseleit-perfused group than in the blood group (2.8 ± 0.4 vs 2.3 ± 0.3 mmHg, respectively, P = 0.01). We conclude that heterologous blood perfusion, by preserving a more physiological myocardial water content, is a better perfusion fluid than modified Krebs-Henseleit solution for quantitative studies of myocardial metabolism and heart function under ischemic conditions.

Pulmonary function, cholinergic bronchomotor tone, and cardiac autonomic abnormalities in type 2 diabetic patients

This prospective study analyzed the involvement of the autonomic nervous system in pulmonary and cardiac function by evaluating cardiovascular reflex and its correlation with pulmonary function abnormalities of type 2 diabetic patients. Diabetic patients (N = 17) and healthy subjects (N = 17) were evaluated by 1) pulmonary function tests including spirometry, He-dilution method, N2 washout test, and specific airway conductance (SGaw) determined by plethysmography before and after aerosol administration of atropine sulfate, and 2) autonomic cardiovascular activity by the passive tilting test and the magnitude of respiratory sinus arrhythmia (RSA). Basal heart rate was higher in the diabetic group (87.8 ± 11.2 bpm; mean ± SD) than in the control group (72.9 ± 7.8 bpm, P<0.05). The increase of heart rate at 5 s of tilting was 11.8 ± 6.5 bpm in diabetic patients and 17.6 ± 6.2 bpm in the control group (P<0.05). Systemic arterial pressure and RSA analysis did not reveal significant differences between groups. Diabetes intragroup analysis revealed two behaviors: 10 patients with close to normal findings and 7 with significant abnormalities in terms of RSA, with the latter subgroup presenting one or more abnormalities in other tests and clear evidence of cardiovascular autonomic dysfunction. End-expiratory flows were significantly lower in diabetic patients than in the control group (P<0.05). Pulmonary function tests before and after atropine administration demonstrated comparable responses by both groups. Type 2 diabetic patients have cardiac autonomic dysfunction that is not associated with bronchomotor tone alterations, probably reflecting a less severe impairment than that of type 1 diabetes mellitus. Yet, a reduction of end-expiratory flow was detected.

Modulation of B lymphocyte function by an aqueous fraction of the ethanol extract of Cissampelos sympodialis Eichl (Menispermaceae)

Cissampelos sympodialis Eichl species are used in folk medicine for the treatment of asthma, arthritis and rheumatism. In the present study, we investigated the immunomodulatory effect of an aqueous fraction of a 70% (v/v) ethanol extract of C. sympodialis leaves on B lymphocyte function. The hydroalcoholic extract inhibited the in vitro proliferative response of resting B cells induced by LPS (IC50 = 17.2 µg/ml), anti-delta-dextran (IC50 = 13.9 µg/ml) and anti-IgM (IC50 = 24.3 µg/ml) but did not affect the anti-MHC class II antibody-stimulated proliferative response of B cell blasts obtained by stimulation with IL-4 and anti-IgM. Incubation with the hydroalcoholic extract used at 50 µg/ml induced a 700% increase in intracellular cAMP levels. IgM secretion by resting B cells (obtained from normal mice) and polyclonally activated B cells (obtained from Trypanosoma cruzi-infected animals) was inhibited by the hydroalcoholic extract. The latter were more sensitive to the hydroalcoholic extract since 6.5 µg/ml induced a 20% inhibition in the response of cells from normal mice while it inhibited the response of B cells from infected animals by 75%. The present data indicate that the alcoholic extract of C. sympodialis inhibited B cell function through an increase in intracellular cAMP levels. The finding that the hydroalcoholic extract inhibited immunoglobulin secretion suggests a therapeutic use for the extract from C. sympodialis in conditions associated with unregulated B cell function and enhanced immunoglobulin secretion. Finally, the inhibitory effect of the hydroalcoholic extract on B cells may indicate an anti-inflammatory effect of this extract.

Alternative parameters for echocardiographic assessment of fetal diastolic function

Alternative methods to assess ventricular diastolic function in the fetus are proposed. Fetal myocardial hypertrophy in maternal diabetes was used as a model of decreased left ventricular compliance (LVC), and fetal respiratory movements as a model of increased LVC. Comparison of three groups of fetuses showed that, in 10 fetuses of diabetic mothers (FDM) with septal hypertrophy (SH), the mean excursion index of the septum primum (EISP) (ratio between the linear excursion of the flap valve and the left atrial diameter) was 0.36 ± 0.09, in 8 FDM without SH it was 0.51 ± 0.09 (P = 0.001), and in the 8 normal control fetuses (NCF) it was 0.49 ± 0.12 (P = 0.003). In another study, 28 fetuses in apnea had a mean EISP of 0.39 ± 0.05 which increased to 0.57 ± 0.07 during respiration (P < 0.001). These two studies showed that the mobility of the septum primum was reduced when LVC was decreased and was increased when LVC was enhanced. Mean pulmonary vein pulsatility was higher in 14 FDM (1.83 ± 1.21) than in 26 NCF (1.02 ± 0.31; P = 0.02). In the same fetuses, mean left atrial shortening was decreased (0.40 ± 0.11) in relation to NCF (0.51 ± 0.09; P = 0.011). These results suggest that FDM may have a higher preload than normal controls, probably as a result of increased myocardial mass and LV hypertrophy. Prenatal assessment of LV diastolic function by fetal echocardiography should include analysis of septum primum mobility, pulmonary vein pulsatility, and left atrial shortening.

Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients

In most mammals, male development is triggered by the transient expression of the SRY gene, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Mutation studies have identified several genes essential for early gonadal development. We report here a molecular study of the SRY, DAX1, SF1 and WNT4 genes, mainly involved in sexual determination, in Brazilian 46,XX and 46,XY sex-reversed patients. The group of 46,XX sex-reversed patients consisted of thirteen 46,XX true hermaphrodites and four 46,XX males, and was examined for the presence of the SRY gene and for the loss of function (inactivating mutations and deletions) of DAX1 and WNT4 genes. In the second group consisting of thirty-three 46,XY sex-reversed patients we investigated the presence of inactivating mutations in the SRY and SF1 genes as well as the overexpression (duplication) of the DAX1 and WNT4 genes. The SRY gene was present in two 46,XX male patients and in none of the true hermaphrodites. Only one mutation, located outside homeobox domain of the 5' region of the HMG box of SRY (S18N), was identified in a patient with 46,XY sex reversal. A novel 8-bp microdeletion of the SF1 gene was identified in a 46,XY sex-reversed patient without adrenal insufficiency. The dosage of DAX1 and WNT4 was normal in the sex-reversed patients studied. We conclude that these genes are rarely involved in the etiology of male gonadal development in sex-reversed patients, a fact suggesting the presence of other genes in the sex determination cascade.

Comparison of angular frequency contrast sensitivity in young and older adults

The aim of the present study was to measure contrast sensitivity curves for angular frequencies in the range between 2 and 96 cycles/360º in older human adult volunteers and to compare these measurements with the more usual contrast sensitivity functions for sine-wave gratings. All subjects were free of identifiable ocular disease and had normal acuity. We measured the contrast thresholds for young adults (N = 6; age range, 20-26 years) and older adults (N = 6; age range, 60-67 years) using the psychophysical forced-choice method. In this paradigm the volunteers had to choose the stimulus containing a test frequency at low contrast (e.g., either a sine-wave grating or an angular frequency stimulus), or another neutral stimulus at mean luminance (without any contrast). Older adults presented a loss in contrast sensitivity at high and medium angular frequencies compared to the young adults (i.e., from 8 to 96 cycles/360º). Contrary to expectation, contrast sensitivity at low angular frequencies, i.e., 2 and 4 cycles/360º, was better for the older group than for the younger group. On the other hand, contrast sensitivity for sine-wave gratings at 3 and 4 cpd was higher for young adults as expected. These results suggest age-related changes in the contrast sensitivity function for angular frequencies.

Normal skin of HIV-infected individuals contains increased numbers of dermal CD8 T cells and normal numbers of Langerhans cells

Dysregulation of the skin immune system (SIS) could explain the high prevalence of skin disorders in HIV+ individuals. The present study was carried out to determine whether alterations in the cell population of SIS and epidermal immunoactivation occur in the normal skin of HIV+ individuals. Forty-five biopsies were taken from the normal upper arm skin of 45 HIV+ patients and of 15 healthy controls. HIV+ individuals were divided into three categories according to their CD4 cell blood count (<200, 200-499 and ³500/µl). Hematoxylin-eosin was used to stain tissue sections for morphological analysis and immunohistochemistry was used for the evaluation of the frequency of macrophages, Langerhans cells, and CD lymphocyte subsets. In addition, semiquantitative analysis of LFA-1, ICAM-1 and HLA-DR was determined in epidermal cells. Macrophages, Langerhans cells, and CD lymphocyte subsets did not differ significantly between any of the patient categories and the control group. When all HIV+ individuals were compared as a group to the control group, a significant increase in dermal CD8+ T lymphocytes (P < 0.01) and lower CD4-CD8 ratios (P < 0.01) were observed in the HIV+ individuals. Epidermal ICAM-1 and HLA-DR expression was negative in both HIV+ and normal skin biopsies. No evidence of a depletion of the SIS population or of epidermal immunoactivation in normal skin from HIV+ individuals was demonstrable, suggesting that alterations in the central immune system are not necessarily reflected in the SIS of HIV-infected patients.

Preservation of graft function in low-risk living kidney transplant recipients treated with a combination of sirolimus and cyclosporine

The use of sirolimus (SRL) in combination with full doses of cyclosporin A (CsA) results in reduced one-year kidney allograft function, which is associated with shorter long-term allograft survival. We determined the effect of reduced CsA exposure on graft function in patients receiving SRL and prednisone. Ninety recipients of living kidney transplants receiving SRL (2 mg/day, po) were compared to 35 recipients receiving azathioprine (AZA, 2 mg kg-1 day-1, po). All patients also received CsA (8-10 mg kg-1 day-1, po) and prednisone (0.5 mg kg-1 day-1). Efficacy end-point was a composite of biopsy-confirmed acute rejection, graft loss, or death at one year. Graft function was measured by creatinine, creatinine clearance, and graft function deterioration between 3 and 12 months (delta1/Cr). CsA concentrations in patients receiving SRL were 26% lower. No differences in one-year composite efficacy end-point were observed comparing SRL and AZA groups (18 vs 20%) or in the incidence of biopsy-proven acute rejection (14.4 and 14.3%). There were no differences in mean ± SD creatinine (1.65 ± 0.46 vs 1.60 ± 0.43 mg/dl, P = 0.48) or calculated creatinine clearances (61 ± 15 vs 62 ± 13 ml/min, P = 0.58) at one year. Mean ± SD delta1/Cr (-11 ± 17 vs -14 ± 15%, P = 0.7) or the percentage of patients with >20% (26 vs 31%, P = 0.6) or >30% delta1/Cr (19 vs 17%, P = 1) did not differ between the two groups. The use of 2-mg fixed oral doses of SRL and reduced CsA exposure was effective in preventing acute rejection and preserving allograft function.

Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-gamma) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-gamma receptor (IFN-gammaR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gammaR1 was normal in all 4 patients. The genetic analysis of IFN-gammaR1 and IFN-gammaR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-gamma activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gammaR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-gamma interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

Mesopic radial frequency contrast sensitivity function for young and older adults

The objective of the present study was to determine contrast sensitivity curves of concentric circular patterns with radial frequencies of 0.25, 0.5, 1.0, 2.0, and 4.0 cycles per degree in young and older adult volunteers. These parameters were also compared with sensitivity contrasts for sine-wave gratings. All participants had normal acuity vision and were free of identifiable ocular illness. Contrast sensitivity was measured in 6 young adults aged 19 to 23 years and 6 older adults aged 60 to 69 years using the psychophysical forced-choice method. In this paradigm the volunteers had to decide which of two stimuli contained the above radial frequencies at low contrast levels. The other neutral stimulus was gray with homogeneous luminance. We detected a decline in contrast sensitivity for older adults at all radial frequencies compared to young adults. Also, contrast sensitivity for sine-wave gratings at all measured frequencies was better, as predicted, for all young adults. Maximum sensitivities in the radial frequency contrast sensitivity function and contrast sensitivity function occurred at 0.25 and 0.5 cycles per degree, respectively, for both young and older adults. These results suggest age-related changes in the contrast sensitivity function for concentric symmetrical stimuli.

Imaging Neuroinflammation in Progressive Multiple Sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system CNS), where inflammation and neurodegeneration lead to irreversible neuronal damage. In MS, a dysfunctional immune system causes auto‐reactive lymphocytes to migrate into CNS where they initiate an inflammatory cascade leading to focal demyelination, axonal degeneration and neuronal loss. One of the hallmarks of neuronal injury and neuroinflammation is the activation of microglia. Activated microglia are found not only in the focal inflammatory lesions, but also diffusely in the normal‐appearing white matter (NAWM), especially in progressive MS. The purine base, adenosine is a ubiquitous neuromodulator in the CNS and also participates in the regulation of inflammation. The effect of adenosine mediated via adenosine A2A receptors has been linked to microglial activation, whereas modulating A2A receptors may exert neuroprotective effects. In the majority of patients, MS presents with a relapsing disease course, later advancing to a progressive phase characterised by a worsening, irreversible disability. Disease modifying treatments can reduce the severity and progression in relapsing MS, but no efficient treatment exists for progressive MS. The aim of this research was to investigate the prevalence of adenosine A2A receptors and activated microglia in progressive MS by using in vivo positron emission tomography (PET) imaging and [11C]TMSX and [11C](R)‐PK11195 radioligands. Magnetic resonance imaging (MRI) with diffusion tensor imaging (DTI) was performed to evaluate structural brain damage. Non‐invasive input function methods were also developed for the analyses of [11C]TMSX PET data. Finally, histopathological correlates of [11C](R)‐PK11195 radioligand binding related to chronic MS lesions were investigated in post‐mortem samples of progressive MS brain using autoradiography and immunohistochemistry. [11C]TMSX binding to A2A receptors was increased in NAWM of secondary progressive MS (SPMS) patients when compared to healthy controls, and this correlated to more severe atrophy in MRI and white matter disintegration (reduced fractional anisotropy, FA) in DTI. The non‐invasive input function methods appeared as feasible options for brain [11C]TMSX images obviating arterial blood sampling. [11C](R)‐PK11195 uptake was increased in the NAWM of SPMS patients when compared to patients with relapsing MS and healthy controls. Higher [11C](R)‐PK11195 binding in NAWM and total perilesional area of T1 hypointense lesions was associated with more severe clinical disability, increased brain atrophy, higher lesion load and reduced FA in NAWM in the MS patients. In autoradiography, increased perilesional [11C](R)‐PK11195 uptake was associated with increased microglial activation identified using immunohistochemistry. In conclusion, brain [11C]TMSX PET imaging holds promise in the evaluation of diffuse neuroinflammation in progressive MS. Being a marker of microglial activation, [11C](R)‐ PK11195 PET imaging could possibly be used as a surrogate biomarker in the evaluation of the neuroinflammatory burden and clinical disease severity in progressive MS.

Analytic evaluation of three-phase short circuit demagnetization and hysteresis loss risk in rotor-surface-magnet Permanent-Magnet Synchronous Machines

Permanent magnet synchronous machines (PMSM) have become widely used in applications because of high efficiency compared to synchronous machines with exciting winding or to induction motors. This feature of PMSM is achieved through the using the permanent magnets (PM) as the main excitation source. The magnetic properties of the PM have significant influence on all the PMSM characteristics. Recent observations of the PM material properties when used in rotating machines revealed that in all PMSMs the magnets do not necessarily operate in the second quadrant of the demagnetization curve which makes the magnets prone to hysteresis losses. Moreover, still no good analytical approach has not been derived for the magnetic flux density distribution along the PM during the different short circuits faults. The main task of this thesis is to derive simple analytical tool which can predict magnetic flux density distribution along the rotor-surface mounted PM in two cases: during normal operating mode and in the worst moment of time from the PM’s point of view of the three phase symmetrical short circuit. The surface mounted PMSMs were selected because of their prevalence and relatively simple construction. The proposed model is based on the combination of two theories: the theory of the magnetic circuit and space vector theory. The comparison of the results in case of the normal operating mode obtained from finite element software with the results calculated with the proposed model shows good accuracy of model in the parts of the PM which are most of all prone to hysteresis losses. The comparison of the results for three phase symmetrical short circuit revealed significant inaccuracy of the proposed model compared with results from finite element software. The analysis of the inaccuracy reasons was provided. The impact on the model of the Carter factor theory and assumption that air have permeability of the PM were analyzed. The propositions for the further model development are presented.

Determination of renal function in long-term heart transplant patients by measurement of urinary retinol-binding protein levels

Significant improvements have been noted in heart transplantation with the advent of cyclosporine. However, cyclosporine use is associated with significant side effects, such as chronic renal failure. We were interested in evaluating the incidence of long-term renal dysfunction in heart transplant recipients. Fifty-three heart transplant recipients were enrolled in the study. Forty-three patients completed the entire evaluation and follow-up. Glomerular (serum creatinine, creatinine clearance measured, and creatinine clearance calculated) and tubular functions (urinary retinol-binding protein, uRBP) were re-analyzed after 18 months. At the enrollment time, the prevalence of renal failure ranged from 37.7 to 54% according to criteria used to define it (serum creatinine > or = 1.5 mg/dL and creatinine clearance <60 mL/min). Mean serum creatinine was 1.61 ± 1.31 mg/dL (range 0.7 to 9.8 mg/dL) and calculated and measured creatinine clearances were 67.7 ± 25.9 and 61.18 ± 25.04 mL min-1 (1.73 m²)-1, respectively. Sixteen of the 43 patients who completed the follow-up (37.2%) had tubular dysfunction detected by increased levels of uRBP (median 1.06, 0.412-6.396 mg/dL). Eleven of the 16 patients (68.7%) with elevated uRBP had poorer renal function after 18 months of follow-up, compared with only eight of the 27 patients (29.6%) with normal uRBP (RR = 3.47, P = 0.0095). Interestingly, cyclosporine trough levels were not different between patients with or without tubular and glomerular dysfunction. Renal function impairment is common after heart transplantation. Tubular dysfunction, assessed by uRBP, correlates with a worsening of glomerular filtration and can be a useful tool for early detection of renal dysfunction.

Impact of marked weight loss induced by bariatric surgery on bone mineral density and remodeling

Data about the impact of bariatric surgery (BS) and subsequent weight loss on bone are limited. The objective of the present study was to determine bone mineral density (BMD), bone remodeling metabolites and hormones that influence bone trophism in premenopausal women submitted to BS 9.8 months, on average, before the study (OGg, N = 16). The data were compared to those obtained for women of normal weight (CG, N = 11) and for obese women (OG, N = 12). Eight patients in each group were monitored for one year, with the determination of BMD, of serum calcium, phosphorus, magnesium, parathyroid hormone, 25-hydroxyvitamin D, insulin-like growth factor-I (IGF-I) and osteocalcin, and of urinary calcium and deoxypyridinoline. The biochemical determinations were repeated every three months in the longitudinal study and BMD was measured at the end of the study. Parathyroid hormone levels were similar in the three groups. IGF-I levels (CG = 332 ± 62 vs OG = 230 ± 37 vs OGg = 128 ± 19 ng/mL) were significantly lower in the operated patients compared to the non-operated obese women. Only OGg patients presented a significant fall in BMD of 6.2% at L1-L4, of 10.2% in the femoral neck, and of 5.1% in the forearm. These results suggest that the weight loss induced by BS is associated with a significant loss of bone mass even at sites that are not influenced by weight overload, with hormonal factors such as IGF-I being associated with this process.