Effect of Ca2+, Ba2+, and Sr2+ on alginate microbeads.

When encapsulated with alginate derivatives that resist the foreign-body response, human embryonic stem cell–derived beta cells restore long-term normoglycemia in immunocompetent mice without the need for immunosuppression.

Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice

Cell microencapsulation technology: towards clinical application.

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-gal(pH6)), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-gal(pH6)-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-gal(pH6)-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-gal(pH6)-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-gal(pH6)-positive cells and reconsideration of potential cellular target for anti-aging treatment.

/pdf/aging-of-mice-is-associated-with-p16-ink4a-and-b-46egrj5utj.pdf

Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells

The insulin-like growth factor 1 (IGF1) signaling pathway is implicated in the development of cancer. High levels of circulating IGF1 and certain genetic polymorphisms of IGF1 and IGFBP3 are associated with an increased risk of several common cancers. The IGF1 receptor (IGF1R) has been shown to be expressed in a wide range of tumors, and IGF1R signaling is crucial for tumor transformation and the survival of malignant cells. Several monoclonal antibodies and small-molecule inhibitors have been tested in preclinical studies and early-phase clinical studies. IGF1R signaling interferes with numerous growth factors and receptors such as VEGF and EGFR. In the experimental system, IGF1R signaling has been found to correlate with resistance to therapies based on the inhibition of EGFR and HER2. This Review highlights the most relevant studies in this exciting area of research, focusing in particular on the role of IGF1R in resistance to other receptor-targeted therapies for cancer.

Mechanisms of disease: signaling of the insulin-like growth factor 1 receptor pathway--therapeutic perspectives in cancer.

We describe the results of the first study to show that adult rat and human islets can be protected against xenogenic rejection in immunocompetent diabetic mice by encapsulating them in a novel alginate-based microcapsule system with no additional permselective membrane. Nonencapsulated islets lost function within 4-8 days after being transplanted into diabetic Balb/c mice, whereas transplanted encapsulated adult rat or human islets resulted in normoglycemia for >7 months. When rat islet grafts were removed 10 and 36 weeks after transplantation, the mice became immediately hyperglycemic, thus demonstrating the efficacy of the encapsulated islets. The explanted capsules showed only a mild cellular reaction on their surface and a viability of >85%, and responded to a glucose stimulus with a 10-fold increase in insulin secretion. Furthermore, transplanted mice showed a slight decrease in the glucose clearance rate in response to intraperitoneal glucose tolerance tests 3-16 weeks after transplantation; after 16 weeks, the rate remained stable. Similar results were obtained for encapsulated human islets. Thus we provide the first evidence of successful transplantation of microencapsulated human islets. In conclusion, we have developed a novel microcapsule system that enables survival and function of adult rat and human islets in immunocompetent mice without immunosuppression for >7 months.

https://diabetes.diabetesjournals.org/content/diabetes/54/3/687.full.pdf

Long-Term Graft Function of Adult Rat and Human Islets Encapsulated in Novel Alginate-Based Microcapsules After Transplantation in Immunocompetent Diabetic Mice

Background: Reliable PCR amplification of DNA fragments is the prerequisite for most genetic assays. We investigated the impact of G-quadruplex– or i-motif–like sequences on the reliability of PCR-based genetic analyses.

Methods: We found the sequence context of a common intronic polymorphism in the MEN1 gene (multiple endocrine neoplasia I) to be the cause of systematic genotyping errors by inducing preferential amplification of one allelic variant [allele dropout (ADO)]. Bioinformatic analyses and pyrosequencing-based allele quantification enabled the identification of the underlying DNA structures.

Results: We showed that G-quadruplex– or i-motif–like sequences can reproducibly cause ADO. In these cases, amplification efficiency strongly depends on the PCR enzyme and buffer conditions, the magnesium concentration in particular. In a randomly chosen subset of candidate single-nucleotide polymorphisms (SNPs) defined by properties deduced from 2 originally identified ADO cases, we confirmed preferential PCR amplification in up to 50% of the SNPs. We subsequently identified G-quadruplex and i-motifs harboring a SNP that alters the typical motif as the cause of this phenomenon, and a genomewide search based on the respective motifs predicted 0.5% of all SNPs listed by dbSNP and Online Mendelian Inheritance in Man to be potentially affected.

Conclusions: Undetected, the described phenomenon produces systematic errors in genetic analyses that may lead to misdiagnoses in clinical settings. PCR products should be checked for G-quadruplex and i-motifs to avoid the formation of ADO-causing secondary structures. Truly affected assays can then be identified by a simple experimental procedure, which simultaneously provides the solution to the problem. .

Identification and Prevention of Genotyping Errors Caused by G-Quadruplex– and i-Motif–Like Sequences

In order to determine the role of the IGF-I receptor (IGF-IR) in human pheochromocytomas we have compared the expression of the IGF-IR in normal tissues and in pheochromocytomas with regard to the IGF-IR mRNA levels and ligand binding. By semiquantitative reverse transcription polymerase chain reaction (RT-PCR), the mRNA of the IGF-IR could be detected in all samples of normal adrenomedullary cells (n=13) and pheochromocytomas (n=16). However, pheochromocytomas exhibited 2·8-fold higher mean IGF-IR mRNA levels than normal adrenomedullary cells (2·8±0·5×105 molecules/µg RNA vs 7·8±1·2×105 molecules/µg RNA; P,0·001). This overexpression of the IGF-IR in pheochromocytomas could be confirmed at the protein level by binding studies. Radioligand assays and Scatchard analysis revealed a single class of high affinity IGF-IR binding sites with a similar dissociation constant (Kd: 0·32±0·1 nmol/l vs 0·22±0·08 nmol/l) for both normal adrenomedullary cells and pheochromocytomas. However, specific 125I-labeled IGF-I binding and the calculated receptor concentration were significantly elevated in pheochromocytomas as compared with normal adrenomedullary cells (58·3±5 vs 24·3±12 nmol/kg protein; P,0·05). In summary, our results demonstrate significant overexpression of the IGF-IR in human pheochromocytomas. This suggests a possible role of the IGF system in the pathogenesis of adrenal neoplasia and thus IGF-IR may be a target for future therapeutic approaches.

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Timo Minnemann

Papers

Long-Term Graft Function of Adult Rat and Human Islets Encapsulated in Novel Alginate-Based Microcapsules After Transplantation in Immunocompetent Diabetic Mice

Identification and Prevention of Genotyping Errors Caused by G-Quadruplex– and i-Motif–Like Sequences

Overexpression of the insulin-like growth factor I receptor in human pheochromocytomas