Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice.

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.

Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.

Proteome-wide modulation of S-nitrosylation in Trypanosoma cruzi trypomastigotes upon interaction with the host extracellular matrix.

Trypanosoma cruzi trypomastigotes adhere to extracellular matrix (ECM) to invade mammalian host cells regulating intracellular signaling pathways. Herein, resin-assisted enrichment of thiols combined with mass spectrometry were employed to map site-specific S-nitrosylated (SNO) proteins from T. cruzi trypomastigotes incubated (MTy) or not (Ty) with ECM. We confirmed the reduction of S-nitrosylation upon incubation with ECM, associated with a rewiring of the subcellular distribution and intracellular signaling pathways. Forty, 248 and 85 SNO-peptides were identified only in MTy, Ty or in both conditions, respectively. SNO proteins were enriched in ribosome, transport, carbohydrate and lipid metabolisms. Nitrosylation of histones H2B and H3 on Cys64 and Cys126, respectively, is described. Protein-protein interaction networks revealed ribosomal proteins, proteins involved in carbon and fatty acid metabolism to be among the enriched protein complexes. Kinases, phosphatases and enzymes involved in the metabolism of carbohydrates, lipids and amino acids were identified as nitrosylated and phosphorylated, suggesting a post-translational modifications crosstalk. In silico mapping of nitric oxide synthase (NOS) genes, previously uncharacterized, matched to four putative T. cruzi proteins expressing C-terminal NOS domain. Our results provide the first site-specific characterization of S-nitrosylated proteins in T. cruzi and their modulation upon ECM incubation before infection of the mammalian hosts. SIGNIFICANCE: Protein S-nitrosylation represents a major molecular mechanism for signal transduction by nitric oxide. We present for the first time a proteomic profile of S-nitrosylated proteins from infective forms of T. cruzi, showing a decrease in SNO proteins after incubation of the parasite with the extracellular matrix, a necessary step for the parasite invasion of the host mammalian cells. We also show for the first time nitrosylation of H2B (Cys64) and H3 (Cys126) histones, sites not conserved in higher eukaryotic cells, and suggest that some specific histone isoforms are sensitive to NO signaling. S-nitrosylation in H2B and H3 histones are more abundant in MTy. Moreover, proteins involved in translation, glycolytic pathway and fatty acid metabolism are enriched in the present dataset. Comparison of the SNO proteome and the phosphoproteome, obtained previously under the same experimental conditions, show that most of the proteins sharing both modifications are involved in metabolic pathways, transport and ribosome function. The data suggest that both PTMs are involved in reprogramming the metabolism of T. cruzi in response to environmental changes. Although NO synthesis was detected in T. cruzi, the identification of NOS remains elusive. Analysis in silico showed two genes similar in domains to NADPH-dependent cytochrome-P450 reductase and two putative oxidoreductases, but no oxygenase domain of NOS was mapped in the T. cruzi genome. It is tempting to speculate that NO synthase-like from T. cruzi and its early NO-mediated pathways triggered in response to host interaction constitute potential diagnostic and therapeutic targets.

Direct identification of trypanosomatids by matrix-assisted laser desorption ionization-time of flight mass spectrometry (DIT MALDI-TOF MS).

Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI-TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI-TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI-TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software-assisted identification at the strain level. Overall, this study shows the importance of MALDI-TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry-based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.