Development and Validation of a Discriminative Dissolution Test for Betamethasone Sodium Phosphate and Betamethasone Dipropionate Intramuscular Injectable Suspension

The intramuscular administration of the injectable suspension betamethasone sodium phosphate (BSP) and betamethasone dipropionate (BD) has immediate therapeutic activity due to solubilized BSP and prolonged activity resulting from the slow release of BD micro-crystals. The purpose of this study was to develop and validate a dissolution method for BD in intramuscular injectable suspensions with detection by high-performance liquid chromatography (HPLC) method. Five commercial products presented a distribution of particle sizes, ranging between 7.43 and 40.25 μm as measured by laser diffraction. It was also found that particle sizes differed between batches of the same product. The different products were tested using the paddle apparatus, with stirring speeds of 25 and 50 rpm in 300 mL of phosphate buffer; simulated body fluid, muscle fluid, and synovial fluid were used as biorelevant dissolution media at 37 ± 0.5°C. It was verified that not only does average particle size affect the dissolution rate, but also the mode and the polydispersity index of the particles. Discriminatory power was obtained using the in vitro dissolution method with 0.1 M sodium phosphate buffer pH 7.4 containing 0.1% sodium lauryl sulfate and a stirring speed of 50 rpm. The HPLC-method is linear, precise, selective, and accurate for the quantification of BSP and BD in dissolution profile testing. This dissolution method can be utilized as a method to control the quality of these injectable suspensions.Key words: dipropionate betamethasone, dissolution test, intramuscular injectable suspensions, simulated muscular fluid, sodium phosphate betamethasone     

The core of affects: namer and named in Bahia (Brazil)

This article is a study of personal naming using drawings made by secondary school students from two small towns in coastal Bahia (Brazil) of the persons who gave them their names. The article explores the relation between namer and named as one of continued identity created by imaginative processes of surrogation akin to pretend‐play. In early ontogeny, the person is constituted through a series of mutualities of being that come to constitute a core of affects. The article aims to contribute towards a better understanding of the processes of constitution of personhood in the context of human relatedness.     

Characterization,subsite mapping and N-terminal sequence of miliin,a serine-protease isolated from the latex of Euphorbia milii

Miliin is a serine protease purified from the latex of Euphorbia milii. This work reports the effect of pH and temperature on the catalytic activity of miliin, using fluorescence resonance energy transfer (FRET) substrates. Miliin displayed the highest activity at pH 9 and 35 °C. Subsite mapping shows that subsites S₂ to S₂′ prefer uncharged residues. The S₂ subsite prefers hydrophobic aliphatic amino acids (Val, Pro and Ile) and defines the cleavage site. This work is the first one that reports subsite mapping of Euphorbiacea proteases. The N-terminal sequence showed higher similarity (40%) with the serine protease LIM9 isolated from Lilium. The presence of Tyr, Pro and Lys at positions 2, 5 and 10 respectively, were observed for most of the serine proteases used for comparison. The N-terminal sequence has striking differences with those reported previously for milin and eumiliin, other serine proteases isolated from the latex of E. milii.     

Comparison of vitrification and slow cooling for umbilical tissues

The tissue cryopreservation maintains the cellular metabolism in a quiescence state and makes the conservation possible for an indefinite period of time. The choice of an appropriate cryopreservation protocol is essential for maintenance of cryopreserved tissue banks. This study evaluated 10 samples of umbilical cord, from which small fragments of tissue (Wharton’s jelly and cord lining membrane) were subjected to two protocols of cryopreservation: slow cooling and vitrification. The samples were frozen for a period of time ranging from 5 to 78 days. The efficiency of cryopreservation was evaluated by testing cell viability, histological analysis, cell culture, cytogenetic analysis and comparison with the results of the fresh samples. The results showed that the slow cooling protocol was more efficient than the vitrification for cryopreservation of umbilical cord tissue, because it has caused fewer changes in the structure of tissue (edema and degeneration of the epithelium) and, despite the significant decrease cell viability compared to fresh samples, the ability of cell proliferation in vitro was preserved in most samples. In conclusion, this study showed that it is possible to cryopreserve small fragments of tissue from the umbilical cord and, to obtain viable cells capable of proliferation in vitro after thawing, contributing to the creation of a frozen tissue bank.     

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.     

Increased Frequency of CD4 and CD8 Regulatory T Cells in Individuals under 15 Years with Multibacillary Leprosy

Background

Leprosy is a chronic disease, caused by Mycobacterium leprae, which poses a serious public health problem worldwide. Its high incidence in people under 15 years old in Ceará state, Brazil, reflects the difficulty of its control. The spectrum of clinical manifestations is associated with the immune response developed, with the Th1 and Th2 responses being related to the paucibacillary and multibacillary forms, respectively. Regulatory T cells (Treg), which can suppress Th1 and Th2 response, have received special attention in the literature and have been associated with development of chronic infections. However, their role in leprosy in individuals under 15 years old has not yet been elucidated. We evaluated the frequency of CD4⁺/CD8⁺CD25^highFOXP3⁺ and CD4⁺/CD8⁺CD25^highFOXP3^high cells in leprosy patients and household contacts, in both cases under 15 years old.

Methodology/Principal Findings

PBMC from 12 patients and 17 contacts were cultured for 72 hours with anti-CD3 and anti-CD28 (activators) or with activators associated with total sonicated fraction of M. leprae. After culture, the frequency of CD4⁺/CD8⁺ Treg was identified by flow cytometry. Cells stimulated by activators and antigen from multibacillary patients showed Treg frequencies almost two times that of the contacts: CD4⁺FOXP3⁺ (21.93±8.43 vs. 13.79±8.19%, p = 0.0500), CD4⁺FOXP3^high (10.33±5.69 vs. 5.57±4.03%, p = 0.0362), CD8⁺FOXP3⁺ (13.88±9.19 vs. 6.18±5.56%, p = 0.0230) and CD8⁺FOXP3^high (5.36±4.17 vs. 2.23±2.68%, p = 0.0461). Furthermore, the mean fluorescence intensity of FOXP3 in Treg was higher in multibacillary patients than in the contacts. Interestingly, there was a positive correlation of the bacillary index and number of lesions with the frequency of all Treg evaluated in patients.

Conclusions/Significance

We have demonstrated for the first time that multibacillary leprosy patients under 15 years old have greater CD4⁺ and CD8⁺ Treg frequencies and these correlate with clinical and laboratorial aspects of disease. These findings suggest the involvement of these cells in the perpetuation of M. leprae infection.     

Glycosaminoglycan remodeling during chondrogenic differentiation of human bone marrow−/synovial-derived mesenchymal stem/stromal cells under normoxia and hypoxia

Glycosaminoglycans (GAGs) are major components of cartilage extracellular matrix (ECM), which play an important role in tissue homeostasis not only by providing mechanical load resistance, but also as signaling mediators of key cellular processes such as adhesion, migration, proliferation and differentiation. Specific GAG types as well as their disaccharide sulfation patterns can be predictive of the tissue maturation level but also of disease states such as osteoarthritis. In this work, we used a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to perform a comparative study in terms of temporal changes in GAG and disaccharide composition between tissues generated from human bone marrow- and synovial-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) after chondrogenic differentiation under normoxic (21% O₂) and hypoxic (5% O₂) micromass cultures. The chondrogenic differentiation of hBMSC/hSMSC cultured under different oxygen tensions was assessed through aggregate size measurement, chondrogenic gene expression analysis and histological/immunofluorescence staining in comparison to human chondrocytes. For all the studied conditions, the compositional analysis demonstrated a notable increase in the average relative percentage of chondroitin sulfate (CS), the main GAG in cartilage composition, throughout MSC chondrogenic differentiation. Additionally, hypoxic culture conditions resulted in significantly different average GAG and CS disaccharide percentage compositions compared to the normoxic ones. However, such effect was considerably more evident for hBMSC-derived chondrogenic aggregates. In summary, the GAG profiles described here may provide new insights for the prediction of cartilage tissue differentiation/disease states and to characterize the quality of MSC-generated chondrocytes obtained under different oxygen tension culture conditions.

     

Expression of the Arcelin 1 gene from Phaseolus vulgaris L. in cowpea seeds (Vigna unguiculata L.) confers bruchid resistance

Persistent decrease in the productivity of cowpea (Vigna unguiculata L.) has been partly due to attack by bruchids including Zabrotes subfasciatus and Callosobruchus maculatus. Resistance to these insects in Phaseolus vulgaris L. has been shown to be associated with arcelins, a family of seed proteins encoded by a multigenic family of lectins on the APA locus. In this work, we report the construction of an expression vector containing Arc1 gene isolated from P. vulgaris and introduced into cowpea as a strategy to confer resistance to insect attack. Following transformation and selection, feeding experiments in which C. maculatus and Z. subfasciatus were fed with transgenic (L3 and L5) and non-transgenic (control) grains showed that introduced gene protected the transgenic line. Significant differences (p < .05 and p < .01) were found in the number of eggs laid, the number of emerging insects and the loss of grain mass in L3, compared with control, for both insects. Similar observations were made in L5 with the exception of the number of laid eggs. The strategy here described may form the basis for the development of a cowpea variety tolerant to bruchids in a crop cultivated by farmers throughout Latin America and Africa.