In vitro labeling of pancreatic islets with iron nanoparticles enables their direct posttransplant visualization by magnetic resonance; however, there is still a discrepancy in the fate of iron ...nanoparticles. This study was performed to detail the labeling process, consequently to improve the labeling efficacy and to confirm safety for islet cells. The islets were visible on T2*-weighted magnetic resonance images as hypointense spots immediately after 1-hr cultivation. Although at this time already the sufficient superparamagnetic effect was achieved, most of the particles were deposed in islet macrophages and only later were they found in endosomes of endocrine islet cells. The iron content depended on length of culture period. The labeled islets showed an intact ultrastructure, responded normally to glucose stimulation in vitro, and were able to treat experimental diabetes. For purpose of subsequent magnetic resonance imaging, a 24-hr culture with ferucarbotran leads to sufficient labeling with no apparent adverse effect on beta cell morphology or function.
Lyme borreliosis (LB) is a serious infectious disease of humans and some domestic animals in temperate regions of the Northern Hemisphere. It is caused by certain spirochetes in the Borrelia ...burgdorferi sensu lato (s.l.) species complex. The complex consists of 11 species (genospecies). Borrelia burgdorferi sensu stricto (s.s.), Borrelia garinii and Borrelia afzelii are the major agents of human disease. Borrelia burgdorferi s.l. species are transmitted mainly by ticks belonging to the Ixodes ricinus species complex plus a few additional species not currently assigned to the complex. B. burgdorferi infections may produce an acute or chronic disease with a wide array of clinical symptoms such as erythema migrans (EM), carditis, arthritis, neuroborreliosis, and acrodermatitis chronica atrophicans (ACA). Differences in LB spirochetes 'genospecies' and strains/isolates determine the occurrence and severity of this multi-system disease. Accurate and reliable identification of the LB spirochetes in ticks as well as knowledge of their prevalence are essential for prevention against the disease and development of an effective vaccine. An overview of the knowledge of molecular factors with emphasis on potential protein-carbohydrate interactions in the tick-borrelia system is the main focus of this review.
Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic ...β-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (Δ
Ψ
m) during GSIS are lacking. Using high resolution respirometry and parallel Δ
Ψ
m monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and Δ
Ψ
m during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the “toxic” effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, Δ
Ψ
m jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10–20
mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.
Background: Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis.
Objectives: The aim of our study was to monitor the in vitro production of cytokines by peripheral blood ...mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens.
Subjects: Ten T1D patients (tested at the time of diagnosis and 6 and 12 months later), 10 first‐degree relatives of the T1D patients, and 10 controls underwent the study.
Methods: PBMCs were stimulated with glutamic acid decarboxylase 65 (GAD65) amino acids (a.a.) 247–279, 509–528, and 524–543; proinsulin a.a. 9–23; and tyrosine phosphatase (islet antigen‐2)/R2 a.a. 853–872. Interleukin (IL)‐2, IL‐4, IL‐5, IL‐6, IL‐10, IL‐13, interferon (IFN)‐γ, tumor necrosis factor β, transforming growth factor β1, and granulocyte colony‐stimulating factor (GCSF) were analyzed by protein microarray.
Results: Differences in cytokine(s) poststimulatory and mainly in basal production were observed in all groups. The most prominent findings were in controls, the higher basal levels of IL‐2, IL‐4, IL‐5, IL‐13, and GCSF were observed when compared with relatives (p < 0.05, for all). After stimulation in controls, there was a significant decrease in IL‐2, IL‐13, GCSF, and IFN‐γ (p < 0.05, for all). The group of relatives was the most variable in poststimulatory production. A strong correlation between cytokines production was found but groups differed in this aspect.
Conclusion: By multiplex analysis, it may be possible, for example, to define the risk immunological response pattern among relatives or to monitor the immune response in patients on immune modulation therapy.
A site-specific glycosylation of salivary glands (SGs) isolated from unfed and partially fed Ixodes ricinus females was identified with the use of lectin affinity labeling on sections and western ...blots of SDS-PAGE gels. The results revealed that secretory granules of a, b, and c cells of the type II acinus and e and f cells of the type III acinus are glycosylated. In partially engorged tick SGs, 2 subtypes of c cells were distinguished. The granules of c1 cells contained mannose, N-acetyl-d-glucosamine, and sialic acid residues. The granules of b, c2, and e cells exhibited complex glycoconjugates rich in mannose, N-acetyl-D-glucosamine, galactose, N-acetyl-d-galactosamine, and a moderate amount of sialic acid. The granules of f cells contained N-acetyl-d-glucosamine and mannose moieties. Type III acini surfaces were covered with mannose-specific ConA binding sites. Except the granules of salivary cells, sialic acid–specific lectins MAA II and SNA strongly bound cuticular structures of alveolar ducts, and weakly with the cuticular spiral thread of excretory salivary ducts. The total sialic acid level in SG homogenates isolated from partially fed females was determined by the thiobarbituric acid method. Sialic acid, which has been found during the development of a few insect species, has not been reported in ticks as yet.
During treatment of diabetes mellitus by immunointervention or transplantation, it is necessary to monitor the markers of immune destruction or rejection of surviving insulin producing cells. An aim ...of this thesis is to improve the possibilities of following autoimmunity and to detect the survival of transplanted pancreatic islet in vivo. Partial aims included vitality testing of isolated islets for transplantation by measurement of respiration activity, observing the process of in vitro labeling of isolated islets with superparamagnetic iron oxide (SPIO) contrast agent for subsequent magnetic resonance imaging (MRI) of islets and observing SPIO particles transport after transplantation. We also studied a new dual paramagnetic contrast agent combined with fluorescein intended for identification of the MRI contrast agent in samples for histology. Further, we assessed autoimmune reaction by evaluation of cytokine response to specific stimulation with auto-antigens. We tried to affect beta-cells destruction by polyclonal anti- thymocyte antibodies in a mouse experimental model. A new method of the islet respiration measurement correlated with other methods of islet quality testing and it was suggested as a diagnostic test before clinical transplantation. Results obtained studying the intercellular...
During treatment of diabetes mellitus by immunointervention or transplantation, it is necessary to monitor the markers of immune destruction or rejection of surviving insulin producing cells. An aim ...of this thesis is to improve the possibilities of following autoimmunity and to detect the survival of transplanted pancreatic islet in vivo. Partial aims included vitality testing of isolated islets for transplantation by measurement of respiration activity, observing the process of in vitro labeling of isolated islets with superparamagnetic iron oxide (SPIO) contrast agent for subsequent magnetic resonance imaging (MRI) of islets and observing SPIO particles transport after transplantation. We also studied a new dual paramagnetic contrast agent combined with fluorescein intended for identification of the MRI contrast agent in samples for histology. Further, we assessed autoimmune reaction by evaluation of cytokine response to specific stimulation with auto-antigens. We tried to affect beta-cells destruction by polyclonal anti- thymocyte antibodies in a mouse experimental model. A new method of the islet respiration measurement correlated with other methods of islet quality testing and it was suggested as a diagnostic test before clinical transplantation. Results obtained studying the intercellular...
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