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.     

Dissolved oxygen concentration regulates human hepatic organoid formation from pluripotent stem cells in a fully controlled bioreactor

Developing technologies for scalable production of human organoids has gained increased attention for “organoid medicine” and drug discovery. We developed a scalable and integrated differentiation process for generation of hepatic organoid from human pluripotent stem cells (hPSCs) in a fully controlled stirred tank bioreactor with 150 ml working volume by application of physiological oxygen concentrations in different liver tissue zones. We found that the 20–40% dissolved oxygen concentration [DO] (corresponded to 30–60 mmHg pO₂ within the liver tissue) significantly influences the process outcome via regulating the differentiation fate of hPSC aggregates by enhancing mesoderm induction. Regulation of the [DO] at 30% DO resulted in efficient generation of human fetal-like hepatic organoids that had a uniform size distribution and were comprised of red blood cells and functional hepatocytes, which exhibited improved liver-specific marker gene expressions, key liver metabolic functions, and, more important, higher inducible cytochrome P450 activity compared to the other trials. These hepatic organoids were successfully engrafted in an acute liver injury mouse model and produced albumin after implantation. These results demonstrated the significant impact of the dissolved oxygen concentration on hPSC hepatic differentiation fate and differentiation efficacy that should be considered ascritical translational aspect of established scalable liver organoid generation protocols for potential clinical and drug discovery applications.     

Loss and rescue of osteocalcin and osteopontin modulate osteogenic and angiogenic features of mesenchymal stem/stromal cells

Noncollagenous proteins in the bone extracellular matrix, such as osteocalcin (OC) and osteopontin (OPN), inherent to evolution of bone as a skeletal tissue, are known to regulate bone formation and mineralization. However, the fundamental basis of this regulatory role remains unknown. Here, for the first time, we use mouse mesenchymal stem/stromal cells (MSC) lacking both OC and OPN to investigate the mechanistic roles of OC and OPN on the proliferation capacity and differentiation ability of MSC. We found that the loss of OC and OPN reduces stem cells self-renewal potential and multipotency, affects their differentiation into an osteogenic lineage, and impairs their angiogenic potential while maintaining chondrogenic and adipogenic lineages. Moreover, loss of OC and OPN compromises the extracellular matrix integrity and maturation, observed by an unexpected enhancement of glycosaminoglycans content that are associated with a more primitive skeletal connective tissue, and by a delay on the maturation of mineral species produced. Interestingly, exogenously supplemented OC and OPN were able to rescue MSC proliferative and osteogenic potential along with matrix integrity and mineral quality. Taken together, these results highlight the key contributions of OC and OPN in enhancing osteogenesis and angiogenesis over primitive connective tissue, and support a potential therapeutic approach based on their exogenous supplementation.     

Process,correlation and parameter fitting in species distribution models: a response to Kriticos et al

In a recent article (Dormann et al., 2012, Journal of Biogeography, 39, 2119–2131), we compared different approaches to species distribution modelling and depicted modelling approaches along an axis from purely ‘correlative’ to ‘forward process‐based’ models. In their correspondence, Kriticos et al. (2013, Journal of Biogeography, doi: 10.1111/j.1365‐2699.2012.02791.x ) challenge this view, claiming that our continuum representation neglects differences among models and does not consider the ability of fitted process‐based models to combine the advantages of both process‐based and correlative modelling approaches. Here we clarify that the continuum view resulted from recognition of the manifold differences between models. We also reinforce the point that the current trend towards combining different modelling approaches may lead not only to the desired combination of the advantages but also to the accumulation of the disadvantages of those approaches. This point has not been made sufficiently clear previously.     

Mapping Dominican transnationalism: narrow and broad transnational practices

This article maps the structure for understanding the Dominican transnational field. By transnational field we refer to a web of linkages that affects the lives of Dominicans in their places of residence in every social field. We find that social boundaries of the nation do not coincide with political ones and the degree of participation in transnational exchanges varies. We suggest that the structure of the transnational social field is better understood by establishing and defining broad and narrow transnational social practices.