Noncanonical Role of Telomerase in Regulation of Microvascular Redox Environment With Implications for Coronary Artery Disease.

Telomerase reverse transcriptase (TERT) (catalytic subunit of telomerase) is linked to the development of coronary artery disease (CAD); however, whether the role of nuclear vs. mitchondrial actions of TERT is involved is not determined. Dominant-negative TERT splice variants contribute to decreased mitochondrial integrity and promote elevated reactive oxygen species production. We hypothesize that a decrease in mitochondrial TERT would increase mtDNA damage, promoting a pro-oxidative redox environment. The goal of this study is to define whether mitochondrial TERT is sufficient to maintain nitric oxide as the underlying mechanism of flow-mediated dilation by preserving mtDNA integrity.Immunoblots and quantitative polymerase chain reaction were used to show elevated levels of splice variants α- and ß-deletion TERT tissue from subjects with and without CAD. Genetic, pharmacological, and molecular tools were used to manipulate TERT localization. Isolated vessel preparations and fluorescence-based quantification of mtH2O2 and NO showed that reduction of TERT in the nucleus increased flow induced NO and decreased mtH2O2 levels, while prevention of mitochondrial import of TERT augmented pathological effects. Further elevated mtDNA damage was observed in tissue from subjects with CAD and initiation of mtDNA repair mechanisms was sufficient to restore NO-mediated dilation in vessels from patients with CAD. The work presented is the first evidence that catalytically active mitochondrial TERT, independent of its nuclear functions, plays a critical physiological role in preserving NO-mediated vasodilation and the balance of mitochondrial to nuclear TERT is fundamentally altered in states of human disease that are driven by increased expression of dominant negative splice variants.

[The role of uveitis in demyelinating diseases of the central nervous system]. / Stellenwert der Uveitis im Rahmen demyelinisierender Erkrankungen des Zentralnervensystems.

An involvement of the visual system can be found in many neurologic diseases. Especially demyelinating processes of the central nervous system (CNS) and multiple sclerosis (MS) in particular present with a variety of ophthalmological abnormalities. While optic neuritis (ON) is known to be a positive predictor for the development of MS and can be considered a symptom of the disease, the high frequency of uveitis observed in MS patients seems to occur rather in the context of a general predisposition for autoimmune disorders. However, MS-associated uveitis can precede the onset of neurological symptoms by many years and shows response to treatment with steroids and interferons, suggesting the presence of similar underlying pathogenic mechanisms. Therefore, further studies are warranted in order to reveal whether administration of early immunomodulatory therapy can delay or even prevent the clinical manifestation of MS in a distinct subgroup of patients presenting with uveitis.

Xenogenic humoral responses to islets transplanted in biohybrid diffusion chambers.

It has been hypothesized that chronic antigen leakage from the hybrid artificial pancreas could stimulate a host humoral response. Such antibodies could be induced by antigens shed from the islet cell surface, or by proteins secreted by live cells or liberated after cell death. To determine if this humoral response occurs, porcine (n = 15) or canine (n = 7) islets were seeded (2-5 x 10(4) equivalent islet number, density 30 islets/mm3) into diffusion chambers fabricated from permselective acrylic membranes (nominal M(r) exclusion of 80,000). The chambers were implanted intraperitoneally into streptozotocin-induced diabetic rats. Sera were collected at various intervals (0-12 weeks) and tested against isolated canine and porcine islets, for tissue specificity and interspecies cross-reactivity by fluorescence immunocytochemistry. No immunofluorescence (or only weak background staining) was obtained when islets were exposed to horse sera, or to sera obtained before to xenodevice implantation. Within 2-6 weeks, however, the postimplantation sera showed strong immunoreactivity. The antibodies were found to be reactive to multiple tissues, and to possess little or no interspecies cross-reactivity. The appearance of these xenoantibodies coincided with the appearance of circulating soluble immune complexes. However, none of the respiratory, cutaneous, or gastrointestinal manifestations that are characteristic of an anaphylactic reaction, or of the diseases of immediate-type hypersensitivity, were observed, even after intraperitoneal injection of additional naked islet tissue. Renal glomeruli did not stain for IgG or C3 in islet recipients. These results suggest that islet cell antigens crossed the membrane and stimulated antibody formation in the host, although they did not appear to cause renal or immune complex disease during the course of this study.

Biohybrid artificial pancreas. Long-term function of discordant islet xenografts in streptozotocin diabetic rats.

Long-term function of canine, bovine, and porcine islet xenografts implanted in streptozotocin-induced diabetic rats has been achieved by islet encapsulation within permselective acrylic membrane chambers. Intraperitoneal implants of 1 x 10(4) (n = 11) or 2 x 10(4) (n = 2) encapsulated canine islets reversed the diabetic state of the recipients within 24 hr, with plasma glucose levels dropping from a preimplantation level of 480 +/- 26 (mean +/- SEM) to 97 +/- 4 mg/dl during the first month. Chambers from 2 of the animals were removed, bisected, and reimplanted at 1 week and 2 months; both animals reverted to hyperglycemia (glucose, > 200 mg/dl) in < 2 weeks. The remaining implants maintained function for a mean time of 138 +/- 16 days, whereas the 2 animals that received the higher islet dose maintained function for > 260 days. Membranes containing 2 x 10(4) bovine (n = 6) or porcine (n = 10) islets also normalized glucose concentrations, with plasma glucose levels dropping from 468 +/- 61 to 91 +/- 10 (bovine) and 97 +/- 11 (porcine) mg/dl during the first month (vs. 94 +/- 3 mg/dl for nondiabetic control rats). Three of the latter implants were removed at 1 month. All 3 animals promptly reverted to diabetes. The 3-, 6-, 9-, and 12-month graft survival rates for the remaining animals were 100%, 100%, 60%, and 40%, and 100%, 75%, 50%, and 25%, respectively. The transplant recipients showed an approximately 38-54% gain in body weight during the first 100 days after implantation, compared with < 1% (P < 0.001) and 86% (P < 0.001) for the untreated diabetic (n = 5) and normal control (n = 6) groups. Immunohistochemical staining of long-term grafts (1-20 months) revealed varying degrees of alpha-, beta-, and delta-cell granulation; the external membrane surfaces were generally free of fibrotic overgrowth and exhibited only occasional host cell adherence. Despite a problem of membrane breakage in long-term implants, these results suggest that prolonged survival of discordant transplants of porcine, bovine, and canine islets in diabetic rats can be achieved without immunosuppression.

Relationship between insulin secretion and oxygen tension in hybrid diffusion chambers.

Oxygen tension is of potential importance in hybrid diffusion chambers, where both islet density and the site chosen for implantation can significantly affect the pO2 within the chambers. To investigate this, isolated islets were incubated at oxygen tensions of 38 mmHg ("low") and 154 mmHg ("ambient"). The mean (+/- SD) ratio between insulin secretion at low and at ambient oxygen tensions was 0.92 +/- 0.27 (n = 10) for porcine islets and 0.80 +/- 0.08 (n = 5) for canine islets. The pO2 was then determined in vivo in cylindrical diffusion chambers (inner diameter 4.5 mm) fabricated from acrylic copolymer, seeded with isolated porcine islets at densities of 0, 15, 30, and 45 islets/mm3, and implanted intraperitoneally in rats. The pO2 levels inside the chambers after 2 weeks were 57 +/- 6 (n = 12), 39 +/- 5 (n = 6), 38 +/- 6 (n = 6), and 43 +/- 3 (n = 6) mmHg, respectively. After 6 weeks, the results were 51 +/- 3 (n = 6), 26 +/- 8 (n = 3), 29 +/- 12 (n = 3), and 40 +/- 5 (n = 3) mmHg, respectively. In comparison, similar chambers containing 10 islets/mm3 cultured for 1 week at ambient oxygen had a pO2 of 120 +/- 9 (n = 4) mmHg immediately underneath the membrane and 67 +/- 7 (n = 4) mmHg at the axial center.(ABSTRACT TRUNCATED AT 250 WORDS)

Medium-scale production and purification of monoclonal antibodies in protein-free medium.

Hybridoma cell lines can be adapted to grow in a totally protein-free tissue culture medium and cultured in spinner flasks to generate moderate-to-high quantities of monoclonal antibodies. Such antibodies are easily purified by ammonium sulfate precipitation. This system was shown to be useful for growth of 23 different hybridoma cell lines from different sources to yield an average of 40 mg of highly purified antibody per liter of tissue culture medium.