Megalencephalic leukoencephalopathy with subcortical cysts protein-1: A new calcium-sensitive protein functionally activated by endoplasmic reticulum calcium release and calmodulin binding in astrocytes.

BACKGROUND: MLC1 is a membrane protein highly expressed in brain perivascular astrocytes and whose mutations account for the rare leukodystrophy (LD) megalencephalic leukoencephalopathy with subcortical cysts disease (MLC). MLC is characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling which cause cognitive and motor dysfunctions and epilepsy. In cultured astrocytes, lack of functional MLC1 disturbs cell volume regulation by affecting anion channel (VRAC) currents and the consequent regulatory volume decrease (RVD) occurring in response to osmotic changes. Moreover, MLC1 represses intracellular signaling molecules (EGFR, ERK1/2, NF-kB) inducing astrocyte activation and swelling following brain insults. Nevertheless, to date, MLC1 proper function and MLC molecular pathogenesis are still elusive. We recently reported that in astrocytes MLC1 phosphorylation by the Ca2+/Calmodulin-dependent kinase II (CaMKII) in response to intracellular Ca2+ release potentiates MLC1 activation of VRAC. These results highlighted the importance of Ca2+ signaling in the regulation of MLC1 functions, prompting us to further investigate the relationships between intracellular Ca2+ and MLC1 properties. METHODS: We used U251 astrocytoma cells stably expressing wild-type (WT) or mutated MLC1, primary mouse astrocytes and mouse brain tissue, and applied biochemistry, molecular biology, video imaging and electrophysiology techniques. RESULTS: We revealed that WT but not mutant MLC1 oligomerization and trafficking to the astrocyte plasma membrane is favored by Ca2+ release from endoplasmic reticulum (ER) but not by capacitive Ca2+ entry in response to ER depletion. We also clarified the molecular events underlining MLC1 response to cytoplasmic Ca2+ increase, demonstrating that, following Ca2+ release, MLC1 binds the Ca2+ effector protein calmodulin (CaM) at the carboxyl terminal where a CaM binding sequence was identified. Using a CaM inhibitor and generating U251 cells expressing MLC1 with CaM binding site mutations, we found that CaM regulates MLC1 assembly, trafficking and function, being RVD and MLC-linked signaling molecules abnormally regulated in these latter cells. CONCLUSION: Overall, we qualified MLC1 as a Ca2+ sensitive protein involved in the control of volume changes in response to ER Ca2+ release and astrocyte activation. These findings provide new insights for the comprehension of the molecular mechanisms responsible for the myelin degeneration occurring in MLC and other LD where astrocytes have a primary role in the pathological process.

Local treatment with deep percutaneous electrochemotherapy of different tumor lesions: pain relief and objective response results from an observational study.

OBJECTIVE: The aim of this investigation focuses on the evaluation of the efficacy of deep-seated Electrochemotherapy (ECT) in terms of pain relief and local objective response, in pre-treated patients with neither further available pharmacological treatments nor eligible for surgery. PATIENTS AND METHODS: Deep percutaneous ECT has been performed in 20 patients subjected to systemic anaesthesia. Bleomycin was administrated intravenously before the application of the electrical pulses on the target area, employing multiple single needles depending on the size and location of the target tumor. RESULTS: Pain assessment based on Visual Analogue Scale showed significant pain relief one month after treatment in all patients, reducing from 7.5 to 3 as a median value (p-value at Wilcoxon test <0.001). Local symptom-free survival median value was 5.5 months. At the first follow-up (1-2 months), a local disease control rate (LDCR) was observed in 19/20 (95%) patients: complete responses in 2 (10%), partial responses in 8 (40%) and stable disease in 9 (45%). Local progression-free survival median value was 5.7 months. Overall, no major adverse effects were observed. CONCLUSIONS: Our study indicates that deep percutaneous ECT can produce a significant pain reduction and a high LDCR in different tumor lesions, for anatomical site or histotype. In particular, ECT has demonstrated to be effective in various histotypes and deep-seated tumor lesions never treated before by this approach giving a new chance to physicians for reducing oncological pain in patients not eligible to other therapeutic routes. The innovative peculiarity of our study was the successful application of deep percutaneous ECT on adrenal metastasis, malignant pleural mesothelioma, uterine leiomyosarcoma and the uncommon case of a male müllerian tumor.

Prebiotic supplementation over a cold season and during antibiotic treatment specifically modulates the gut microbiota composition of 3-6 year-old children.

Supplementing kindergarten children during a cold season with a prebiotic inulin-type fructans product with shorter and longer fructan chains has been shown to reduce febrile episodes requiring medical attention and to lower the incidence of sinusitis. These beneficial effects may be connected to the specific modulation of children's gut microbiota. By applying quantitative and qualitative microbiota analysis this study aimed at characterising the gut microbiota composition and at exploring effects of prebiotic intervention on the gut microbiota during a 24-weeks intervention and during antibiotic treatment in healthy children. The study was a randomised, placebo-controlled trial with 258 healthy children aged 3 to 6 years consuming 6 g/day prebiotic inulin-type fructans or maltodextrin. During the course of the study, faecal samples were collected and subject to targeted qPCR analysis and phylogenetic profiling by multiplexed high throughput sequencing of the prokaryotic 16S rRNA gene PCR amplicons. The microbiota composition of the cohort could be clustered into three distinct constellations (enterotypes). Prebiotic intake resulted in a selective modulation of the gut microbiota composition. Relative abundance of Bifidobacterium was significantly higher in the prebiotic group (n=104) compared to control group (n=105) and this effect was found for all three enterotypes. Antibiotic administration decreased the relative abundance of Bifidobacterium in both groups. Nonetheless, children of the prebiotic group receiving antibiotic treatment displayed significantly higher levels of Bifidobacterium than children receiving the placebo control. Prebiotic supplementation induced specific changes in the gut microbiota composition of children aged 3 to 6 years. Moreover, it attenuated antibiotic-induced disturbances in the gut microbiota composition as shown by higher relative abundance of bifidobacteria at the end of the antibiotic treatment in the prebiotic group. With the previously reported benefits on immune function, the study contributes to the evidence on the immune-modulating effects of prebiotics through gut microbiota modifications. The study was registered as NCT03241355 ( https://clinicaltrials.gov/show/NCT03241355 ).

Production of rotavirus-like particles in Spodoptera frugiperda larvae.

A strategy for the production of virus-like particles (VLPs) from simian rotavirus in larvae of the lepidopteran Spodoptera frugiperda is described. VP2 and VP6 coding sequences were co-expressed in larvae co-infected with recombinant baculovirus and these structural proteins self-assembled into VLPs that were secreted and accumulated in the haemolymph. Under electron microscopy, VLPs produced in larvae were indistinguishable from those produced in Sf9 insect cell cultures. The results showed that it is possible to obtain rotavirus VLPs in larvae reducing significantly the costs of production, making this approach an alternative for the manufacture of live rotavirus vaccines.

Effect of a single oral dose of oxymetholone on the metabolism of human erythrocytes.

Androgenic steroids have been shown to enhance erythrocyte 2,3-DPG production in vivo and in vitro, and to stimulate the pentose shunt oxidative reactions in vitro. Furthermore, a 3 beta- and a 17 beta-hydroxysteroid dehydrogenase have been identified in red cells. The present study was carried out to explore a cumulative effect of androgens on glycolysis and androgen reduction in human erythrocytes in vivo following a single 50 mg oral dose of 17 beta-hydroxy-2 (hydroxymethylene)-17 methyl-5 alpha-androstan-3-one (oxymetholone). The rate of erythrocyte glycolysis was measured by quantitative determination of: fructose-1,6-diphosphate (FDP); dehydroxyacetone phosphate (DAP); 2,3-diphosphoglycerate (2,3-DPG); adenosine triphosphate (ATP); and lactate. Serum and erythrocyte steroids were separated by thin layer chromatography. The reduction of 5 alpha-androstan-17 beta-ol-3-one by red cell hemolysate was measured in the presence of NADPH as an index of 3(17)beta-hydroxysteroid dehydrogenase activity. Our results show that oxymetholone administration is followed by the appearance of an unidentified steroid fraction in chromatograms of serum and erythrocytes, simultaneously with the enhancement of glycolysis and of hydroxysteroid dehydrogenase activity in erythrocytes. A direct effect of androgen on erythrocyte metabolism, which is independent of the hormone erythropoietic effect, is discussed.

Effect of androgens on maturation and metabolism of erythroid tissue.

Specific changes taking place in the erythroid tissue following depletion or replacement of androgens were studied in rats. The reduction of testosterone levels in blood of orchiectomized animals did occur in conjunction with a decline of erythrocyte glucose-6-phosphate (G6P) and lactate levels. No evidence of anemia was observed. The subcutaneous administration of testosterone propionate (16.0 mg/kg) to orchiectomized rats restored, within 12 hours, blood testosterone levels as well as erythrocyte G6P levels and lactate production. The in vitro incorporation of glucose-1-14C into rat erythrocytes incubated with testosterone was comparable to that of control cells. A radioautographic study of rat erythroid marrow pulsed with glucose-1-14C showed a lower labeling when testosterone propionate was administered. The authors conclude that testosterone does directly affect glucose metabolism of erythroid cells, via the pentose shunt pathway. The possible role of the androgen-dependent enhancement of erythroid glycolysis is discussed in relation to the function of testosterone receptocytes present in marrow cells and a 17beta-hydroxysteroid dehydrogenase present in erythrocytes.

[Bilateral intermediate uveitis and acute interstitial nephritis (TINU syndrome). Ultrasonographic documentation of a case]. / Uvéite intermédiaire bilatérale et néphrite interstitielle aiguë (TINU syndrome). Documentation échographique d'un cas.

Clinical features and natural course of acute interstitial nephritis and uveitis (TINU Syndrome) in a 9 years old boy are reported. The patient's ocular symptoms began seven month after the renal disease was diagnosed. Intermediate uveitis was present bilaterally with vitreous infiltration. Optic disc drusen were documented by echography. An immunological disorder causing the syndrome is most likely, as some laboratory data demonstrate. No systemic or infectious diseases were found in our case but an etiology to drugs considered likely.

[Clinical notes on 3 years of treating open bite]. / Considerazioni cliniche su tre anni di trattamento del morso aperto.

Personal experience in the treatment of open bite is described, with particular reference to cases complicated by interposition of the tongue. A classification felt to be of importance in diagnosis and prognosis is proposed.

Trends in health legislation and human rights.

For the past three or four decades many countries have ranked health as one of their social priorities and have embarked on comprehensive programmes to ensure universal access to health care. In the same period, mostly under the influence of the international community, the protection and promotion of human rights have become an integral part of modern legal theories and, in many countries or regions, these rights have been framed as constitutional norms or as rules paramount to local legislation. Trends observed in recent legislation may lead to infringements of human rights. At the core of these trends are measures, derived from express legislative sources or implemented by having recourse to the use of delegated powers, concerning the allocation of resources and the duty to render assistance. These measures often impact on the rights of an individual and are thus questionable under human rights theories. This article assesses the consequences of these legislative trends and questions the implicit proposal that the balance between individual rights and collective interests should be weighed in favour of the latter.

Co-inoculation of baculovirus and FMDV vaccine in mice, elicits very early protection against foot and mouth disease virus without interfering with long lasting immunity.

Baculoviruses (Bvs) potentiate the immune response against soluble antigens. We investigated whether Bv could be used as immunoactivator in foot-and-mouth disease (FMD) vaccines using the BALB/c mouse model. Mice were vaccinated with a single dose of inactivated FMDV (iFMDV), iFMDV+Bv, Bv, or culture medium. Humoral and cellular immune responses were higher in animals immunized with iFMDV+Bv than in mice vaccinated with iFMDV alone. Animals receiving iFMDV+Bv had significantly lower viremia at 2, 4 and 7dpv, than those immunized with iFMDV alone. In order to prolong the immune response, iFMDV oil vaccine was co-inoculated with Bv. Animals receiving iFMDV oil vaccine+Bv were protected two days earlier than those receiving the iFMDV oil vaccine alone. Both formulations protected until 14dpv, the last day of the experiment. This is the first report in which Bv is used as an adjuvant in a FMDV vaccine.

VP1 protein of Foot-and-mouth disease virus (FMDV) impairs baculovirus surface display.

The Foot-and-mouth disease virus (FMDV) causes important economical losses in livestock farming. In order to develop a novel subunit vaccine against FMDV, we constructed recombinant baculoviruses that display the protein VP1 of FMDV on their surface, with either polar (fused to gp64) or nonpolar (fused to anchor membrane from VSV-G protein) distribution. Insect cells infected with the different recombinant baculoviruses expressed VP1 fusion protein to high levels. However, the recombinant VP1 protein was not carried by budded virions. Subcellular localization of VP1 revealed that the trafficking of the fusion protein to the cell plasma membrane was impaired. Our results suggest that VP1 contains cryptic domains that interfere with protein secretion and subsequent incorporation into budded baculoviruses.

[Dmt1]N/OFQ(1-13)-NH2: a potent nociceptin/orphanin FQ and opioid receptor universal agonist.

BACKGROUND AND PURPOSE: Intrathecally (i.t.) administered nociceptin/orphanin FQ (N/OFQ) evokes antinociceptive effects in rodents. Recent studies in monkeys demonstrated that i.t. co-application of N/OFQ and morphine elicits synergistic antinociceptive actions suggesting mixed N/OFQ peptide (NOP) and µ opioid receptor agonists as innovative spinal analgesics. Thus, novel N/OFQ related peptides were synthesized in order to identify and pharmacologically characterize a mixed NOP/ µ opioid receptor agonist. EXPERIMENTAL APPROACH: The following in vitro assays were used: calcium mobilization in cells expressing the human NOP or classical opioid receptors and chimeric G proteins, receptor and [(35)S]-GTPγS binding, [(35)S]-GTPγS binding in rat spinal cord membranes, guinea pig ileum bioassay. In vivo experiments were performed in monkeys using the tail withdrawal assay. KEY RESULTS: From calcium mobilization studies [Dmt(1)]N/OFQ(1-13)-NH(2) was selected as the most potent and least selective compound. The mixed NOP/opioid full agonist activity and high affinity of [Dmt(1)]N/OFQ(1-13)-NH(2) was confirmed at human recombinant receptors in receptor binding, calcium mobilization and/or [(35)S]-GTPγS binding studies, at rat spinal cord receptors in [(35)S]-GTPγS binding experiments, and at guinea pig receptors inhibiting neurogenic contractions in the ileum. In vivo in the tail withdrawal assay in monkeys i.t. [Dmt(1) ]N/OFQ(1-13)-NH(2) was able to elicit robust and long-lasting antinociceptive effects. CONCLUSIONS AND IMPLICATIONS: Collectively, these results demonstrate that [Dmt(1)]N/OFQ(1-13)-NH(2) behaves as NOP/opioid receptor universal agonist and substantiate the suggestion that such mixed ligands are worthy of development as innovative spinal analgesics.

Mastitis in the transition period: identification of potential blood markers / Mastite no período de transição: identificação de potenciais marcadores no sangue

This study identified potential blood markers associated with mastitis in dairy cows under different dry therapies during the transition period, using a logistic regression model. Thirty-four Holstein dairy cows were divided into three groups: untreated controls (13 cows, 42 quarters); animals that received an antimicrobial at drying-off (10 cows, 40 quarters); and animals that were administered an internal teat sealant at drying-off (11 cow, 44 quarters). Blood and quarter milk samples were collected 60 days before the expected day of calving, on the day of calving, and three, seven, 15, 21, and 30 days after calving. Milk samples were submitted for bacteriological analysis and somatic cell count. Blood samples were collected for analyses of the following: the erythrogram and leucogram; plasm fibrinogen concentration; hepatic and renal functions; metabolic profile; serum calcium and phosphorous levels; total serum protein and albumin concentrations. The concentration of total serum proteins was associated with a high somatic cell count. Similarly, the concentrations of total serum proteins and triglycerides were associated to milk bacteriological positive samples during the transition period. Thus, the occurrence of mastitis in dairy cows during the transition period was greater in animals that showed higher concentrations of serum total proteins and triglycerides, in contrast to the use of dry cow therapy.(AU)

O presente estudo identificou potenciais marcadores sanguíneos associados tanto à mastite, durante o período de transição em vacas leiteiras submetidas à antimicrobianoterapia, quanto à utilização do selante interno de teto na secagem por modelo de regressão logística. Trinta e quatro vacas da raça Holandesa foram divididas em três grupos experimentais, a saber: 13 animais (42 quartos mamários) que não receberam tratamento na secagem; 10 animais (40 quartos mamários) tratados por via intramamária com uma bisnaga do antimicrobiano para vaca seca à base de cefalônio anidro após a última ordenha em cada quarto mamário; e 11 animais (44 quartos mamários) que receberam, por via intramamária, uma bisnaga de selante à base de subnitrato de bismuto após a última ordenha em cada quarto mamário. As amostras de leite e sangue foram coletadas 60 dias antes da data prevista do parto, na secagem, e no dia do parto e após três, sete, 15 e 21 dias após o parto. As amostras de leite foram utilizadas para o exame microbiológico e a determinação da contagem de células somáticas. As amostras de sangue foram utilizadas para determinação do eritrograma, leucograma, fibrinogênio plasmático, funções hepáticas e renais, perfil metabólico, proteína total e albuminas séricas e concentração sérica de cálcio e fósforo. A concentração total de proteínas séricas foi associada à alta contagem de células somáticas. Similarmente, a concentração total de proteínas séricas e triglicérides foi associada ao isolamento de patógenos causadores de mastite nas amostras de leite durante o período de transição. Desse modo, conclui-se que vacas leiteiras com concentrações séricas maiores de proteína total e triglicérides têm maior chance de apresentar mastite durante o período de transição; em contraste, o uso da antimicrobianoterapia de vaca seca reduz esse risco.(AU)

Mastitis in the transition period: identification of potential blood markers / Mastite no período de transição: identificação de potenciais marcadores no sangue

This study identified potential blood markers associated with mastitis in dairy cows under different dry therapies during the transition period, using a logistic regression model. Thirty-four Holstein dairy cows were divided into three groups: untreated controls (13 cows, 42 quarters); animals that received an antimicrobial at drying-off (10 cows, 40 quarters); and animals that were administered an internal teat sealant at drying-off (11 cow, 44 quarters). Blood and quarter milk samples were collected 60 days before the expected day of calving, on the day of calving, and three, seven, 15, 21, and 30 days after calving. Milk samples were submitted for bacteriological analysis and somatic cell count. Blood samples were collected for analyses of the following: the erythrogram and leucogram; plasm fibrinogen concentration; hepatic and renal functions; metabolic profile; serum calcium and phosphorous levels; total serum protein and albumin concentrations. The concentration of total serum proteins was associated with a high somatic cell count. Similarly, the concentrations of total serum proteins and triglycerides were associated to milk bacteriological positive samples during the transition period. Thus, the occurrence of mastitis in dairy cows during the transition period was greater in animals that showed higher concentrations of serum total proteins and triglycerides, in contrast to the use of dry cow therapy.(AU)

O presente estudo identificou potenciais marcadores sanguíneos associados tanto à mastite, durante o período de transição em vacas leiteiras submetidas à antimicrobianoterapia, quanto à utilização do selante interno de teto na secagem por modelo de regressão logística. Trinta e quatro vacas da raça Holandesa foram divididas em três grupos experimentais, a saber: 13 animais (42 quartos mamários) que não receberam tratamento na secagem; 10 animais (40 quartos mamários) tratados por via intramamária com uma bisnaga do antimicrobiano para vaca seca à base de cefalônio anidro após a última ordenha em cada quarto mamário; e 11 animais (44 quartos mamários) que receberam, por via intramamária, uma bisnaga de selante à base de subnitrato de bismuto após a última ordenha em cada quarto mamário. As amostras de leite e sangue foram coletadas 60 dias antes da data prevista do parto, na secagem, e no dia do parto e após três, sete, 15 e 21 dias após o parto. As amostras de leite foram utilizadas para o exame microbiológico e a determinação da contagem de células somáticas. As amostras de sangue foram utilizadas para determinação do eritrograma, leucograma, fibrinogênio plasmático, funções hepáticas e renais, perfil metabólico, proteína total e albuminas séricas e concentração sérica de cálcio e fósforo. A concentração total de proteínas séricas foi associada à alta contagem de células somáticas. Similarmente, a concentração total de proteínas séricas e triglicérides foi associada ao isolamento de patógenos causadores de mastite nas amostras de leite durante o período de transição. Desse modo, conclui-se que vacas leiteiras com concentrações séricas maiores de proteína total e triglicérides têm maior chance de apresentar mastite durante o período de transição; em contraste, o uso da antimicrobianoterapia de vaca seca reduz esse risco.(AU)

A chimeric baculovirus displaying bovine herpesvirus-1 (BHV-1) glycoprotein D on its surface and their immunological properties.

The ability of a recombinant baculovirus containing the ectodomain of the mature sequence of glycoprotein D (gD) fused to the amino-terminus of baculoviral glycoprotein gp64 to display gD on its surface and to serve as an improved immunogen against bovine herpesvirus-1 was tested. The gD-gp64 fusion protein was correctly expressed on the virus particles as revealed by immunomicroscopy assays. Mice immunized with 5 x 10(8) plaque forming units developed antibodies that specifically reacted in an enzyme-linked immunosorbent assay with recombinant gD and whole bovine herpesvirus-1. These antibodies were able to neutralize bovine herpesvirus-1 in vitro, whereas those elicited by a version of gD expressed in Escherichia coli did not. Our data demonstrated that the display on the virion surface of recombinant baculovirus can provide a tool for the development of recombinant vaccines against bovine herpesvirus-1.