Laryngeal cancer: epidemiological data from Νorthern Greece and review of the literature.

BACKGROUND: Cancer of the larynx accounts for 1% to 2.5% of all human neoplasms and is the most common malignancy of the Head and Neck region. The purpose of this study is to analyze epidemiological data of patients with laryngeal cancer and to point out the geographical variations. METHODS: This is the first systematic recording of the laryngeal cancer epidemiological data in Northern Greece. During the period 1992-2010 1,638 patients were diagnosed with and treated for malignant head and neck tumors. One thousand one hundred and four cases (67.4%) were malignant laryngeal tumors, 98.4% of which (1,088 cases) were squamous cell carcinomas (SCC). Only 16 patients (1.5%) presented with other types of malignancies. RESULTS: The average age of the SCC patients was 62.1 years. Only 35 patients were women (3.2%). More than 60% of the patients were farmers or labor workers, 86.9%, were smokers, 43.2% were consuming alcohol on a daily basis and 36.1% had a positive family history of malignancy. Concerning tumor location, 60.2% were glottic cancers. T staging revealed that 1.2% of the cases were carcinomas in situ, 28% T1 tumors, 19% T2, 32 % T3, and 20% T4. Tumor grading showed that 43% of the cases were G1, 42.1% were G2, and 11.8% were G3. CONCLUSIONS: The pathogenesis of laryngeal carcinoma is the result of the combined action of endogenous and environmental factors. The recording and analysis of the epidemiology of the disease is important for its better study and understanding.

Monitoring of dissolved oxygen and cellular bioenergetics within a pancreatic substitute.

This work investigated the use of nuclear magnetic resonance (NMR) spectroscopy in combination with a mathematical model of an encapsulated cell system as a method for rapidly assessing the status of a pancreatic substitute. To validate this method, an in vitro experiment was performed in which the encapsulated cells were perfused in an NMR-compatible system and the dissolved oxygen (DO) concentration of the perfusing medium was lowered from 0.20 to 0.05 mM, then returned to 0.20 mM in a stepwise fashion. The cellular metabolic activity and bioenergetics were evaluated by measuring the oxygen consumption rate (via DO sensors) and nucleotide triphosphate levels (via (31)P NMR). By incorporating a perfluorocarbon emulsion into the alginate beads, the cellular oxygenation state was monitored by measuring the average intrabead DO (AIDO) concentration by (19)F NMR. The in vitro measurements were then compared with model predictions based on the measured external DO concentration and time. Model-predicted cell growth and AIDO closely matched the experimentally acquired data. As the DO concentrations both external to and within the pancreatic substitute are needed to apply this methodology in vivo, the feasibility of measuring the DO concentration from two distinct bead populations implanted in the peritoneal cavity of mice was established. It is concluded that PFC incorporation and (19)F NMR measurements, in combination with a mechanistic model of the encapsulated system, allow the tracking of the state of a pancreatic substitute in vitro and potentially in vivo.

Modeling of encapsulated cell systems.

Tissue engineered substitutes consisting of cells in biocompatible materials undergo remodeling with time as a result of cell growth and death processes. With inert matrices that do not directly influence cell growth, remodeling is driven mainly by the concentration of dissolved oxygen (DO). Insulin-secreting cell lines encapsulated in alginate-based beads and used as a pancreatic substitute represent such a case. Beads undergo remodeling with time so that an initially homogeneous distribution of cells is eventually replaced by a dense peripheral ring of primarily viable cells, whereas inner cells are mostly necrotic. This paper develops and analyzes a mathematical model of an encapsulated cell system of spherical geometry that tracks the viable and dead cell densities and the concentration of DO within the construct as functions of radial position and time. Model simulations are compared with experimental histology data on cell distribution. Correlations are then developed between the average intrabead DO concentration (AIDO) and the total viable cell number, as well as between AIDO and the radial cell and DO distributions in beads. As AIDO can be measured experimentally by incorporating a perfluorocarbon emulsion in the beads and acquiring (19)F nuclear magnetic resonance (NMR) spectroscopic data, these correlations can be used to track the remodeling that occurs in the construct in vitro and potentially in vivo. The usefulness of mathematical models in describing the dynamic changes that occur in tissue constructs with time, and the value of these models at obtaining additional information on the system when used interactively with experimental measurements, are discussed.

Insights into the role of anaplerosis in insulin secretion: A 13C NMR study.

AIMS/HYPOTHESIS: Defining mechanisms and enzymatic paths critical to fuel-regulated insulin secretion are key goals of diabetes research. In this study, 13C-nuclear magnetic resonance spectroscopy and isotopomer analysis were used to investigate the link between insulin secretion and metabolic pathways associated with the tricarboxylic acid (TCA) cycle. MATERIALS AND METHODS: To this end, four insulinoma cell lines (betaTC3, betaTC-tet, INS-1 [832/13], R7T1) and porcine islets were examined under a variety of culture conditions (i.e. presence vs absence of amino acids and sera, and low vs high glucose). RESULTS: Glucose consumption, insulin release, and glutamate isotopomeric patterns were influenced by media complexity (e.g. PBS, plain culture media, fully supplemented culture media). The 13C-labelled metabolites increased with media complexity and increasing glucose concentration, with the notable exception of aspartate, which was always higher under low-glucose conditions. The 13C-glutamate isotopomeric fractions were fitted to metabolic models to estimate the relative metabolic fluxes to the TCA cycle through key enzymatic processes. These indices of metabolism were compared with insulin secretion to determine correlative links. A model containing a single pool of pyruvate, an entrance to the TCA cycle via the pyruvate dehydrogenase complex, and two anaplerotic entrances, one through pyruvate carboxylase and another through an undefined (by the modelling program) source, provided the best fit to the data under all conditions tested, for all cell lines. CONCLUSIONS/INTERPRETATION: On the basis of our findings, a strong correlation may exist between stimulated insulin secretion and non-pyruvate carboxylase anaplerosis for the four cell lines examined in this study.

Use of glucose-responsive material to regulate insulin release from constitutively secreting cells.

Genetically-engineered cells offer a solution to the cell availability problem in tissue engineering a pancreatic substitute for the treatment of insulin-dependent diabetes. These cells can be non-beta cells, such as hepatocytes or myoblasts, retrieved as a biopsy from the same patient and genetically engineered to secrete recombinant insulin constitutively or under transcriptional regulation. However, the continuous or slowly responsive insulin secretion dynamics from these cells cannot provide physiologic glucose regulation in patients. Our objective consists of using such cells as an insulin source and of regulating insulin release by incorporating a glucose-responsive material, which acts as a control barrier for insulin in a cell-material hybrid device. Experiments were performed with insulinoma betaTC3 cells, HepG2 hepatomas, and C2C12 myoblasts, the latter two genetically-modified to constitutively secrete insulin. The control barrier consisted of concanavalin A (con A)-based glucose-responsive material, which forms a gel at low and a sol at high glucose concentrations. Results demonstrated that the device released insulin at a higher rate in response to glucose challenges. In contrast, a device containing an inert hydrogel instead of glucose-responsive material released insulin at an essentially constant rate, irrespective of the surrounding glucose concentration. Necessary material improvements include increased sensitivity to glucose, so that the material responds to physiologically relevant glucose concentrations, and increased stability. The prospects of developing a properly functional, implantable substitute based on engineered non-beta cells and glucose-responsive material, and the material and device improvements that need to be made prior to in vivo experiments, are discussed.

Alginate assessment by NMR microscopy.

Alginate hydrogels have long been used to encapsulate cells for the purpose of cell transplantation. However, they also have been criticized because they fail to consistently maintain their integrity for extended periods of time. Two issues of critical importance that have yet to be thoroughly addressed concerning the long-term integrity of alginate/poly-L-lysine/alginate microcapsules are: (i) are there temporal changes in the alginate/poly-L-lysine interaction and (ii) are there temporal changes in the alginate gel structure. NMR microscopy is a non-invasive analytical technique that can address these issues. in this report, we present data to demonstrate the utility of (1)H NMR microscopy to (i) visualize the poly-L-lysine layer in an effort to address the first question, and (ii) to observe temporal changes in the alginate matrix that may represent changes in the gel structure.

Effects of alginate encapsulation on mitochondrial activity.

The long-term objective of our research is to study the biochemical consequences of primary genetic defects of the Pyruvate Dehydrogenase Complex, a key mitochondrial enzyme complex, by NMR spectroscopy. An established method to obtain energetic and metabolic information from intact cells involves the use of 31P and 13C NMR spectroscopic techniques. NMR spectra from live and fully functional cells can be obtained from cells encapsulated within alginate beads and maintained in a perfusion bioreactor throughout the NMR experiment. However, before spectroscopic studies can commence, the effects of alginate encapsulation on the general metabolism and mitochondrial activity of fibroblasts need to be determined. in this study we report glucose consumption and flow cytometry measurements (with the fluorescent markers MitoTracker GreenFM and Nonyl-acridine Orange to determine the mitochondrial status and mass) of healthy human fibroblasts encapsulated in a mannuronic acid-rich alginate matrix. The results show that alginate encapsulation of fibroblasts does not affect the glucose consumption, the mitochondrial integrity, or the mitochondrial mass during 21 days of in vitro culture.

Noninvasive measurement of viable cell number in tissue-engineered constructs in vitro, using 1H nuclear magnetic resonance spectroscopy.

Noninvasive monitoring of tissue-engineered constructs is of critical importance for accurate characterization of constructs and their remodeling in vitro and in vivo. This study investigated the utility of (1)H NMR spectroscopy to noninvasively quantify viable cell number in tissue-engineered substitutes in vitro. Agarose disk-shaped constructs containing betaTC3 cells were employed as the model tissue-engineered system. Two construct prototypes containing different initial cell numbers were monitored by localized, water-suppressed 1H NMR spectroscopy over the course of 13 days. (1)H NMR measurements of the total choline resonance at 3.2 ppm were compared with results from the traditional cell viability assay MTT and with insulin secretion rates. Results show a strong linear correlation between total choline and MTT (R (2) = 0.86), and between total choline and insulin secretion rate (R (2) = 0.90). Overall, this study found noninvasive measurement of total choline to be an accurate and nondestructive assay for monitoring viable betaTC3 cell numbers in tissue-engineered constructs. The applicability of this method to in vivo monitoring is also discussed.

In vivo noninvasive monitoring of a tissue engineered construct using 1H NMR spectroscopy.

Direct, noninvasive monitoring of tissue engineered substitutes containing live, functional cells would provide valuable information on dynamic changes that occur postimplantation. Such changes include remodeling both within the construct and at the interface of the implant with the surrounding host tissue, and may result in changes in the number of viable cells in the construct. This study investigated the use of 1H NMR spectroscopy in noninvasively monitoring the viable cell number within a tissue engineered construct in vivo. The construct consisted of mouse betaTC3 insulinomas in a disk-shaped agarose gel, surrounded by a cell-free agarose gel layer. Localized 1H NMR spectra were acquired from within implanted constructs, and the total choline resonance was measured. Critical issues that had to be addressed in accurately quantifying total choline from the implanted cells included avoiding signal from host tissue and correcting for interfering signal from diffusing solutes. In vivo NMR measurements were correlated with MTT assays and NMR measurements performed in vitro on explanted constructs. Total choline measurements accurately and noninvasively quantified viable betaTC3 cell numbers in vivo, in the range of 1 x 10(6) to more than 14 x 10(6) cells, and monitored changes in viable cell number that occurred in the same construct over time. This is the first study using NMR techniques to monitor viable cell numbers in an implanted tissue substitute. It established architectural characteristics that a construct should have to be amenable to NMR monitoring, and it set the foundation for future in vivo investigations with other tissue engineered implants.

In vitro effects of transcatheter injection on structure, cell viability, and cell metabolism in fibroblast-impregnated alginate microspheres.

PURPOSE: To determine if microsphere-encapsulated cell preparations can be delivered through a microcatheter without compromising microsphere structure, cell viability, or metabolism. MATERIALS AND METHODS: Fibroblast-impregnated microspheres were fabricated by using 1.0% alginate and rabbit synovial fibroblasts. Fibroblast-impregnated alginate microspheres injected through microcatheters were analyzed in parallel with identical noninjected microspheres. The effects of transcatheter injection on structure and cell viability (percentage of viable cells per microsphere) were correlated with microsphere size. Structural effects were analyzed by using light microscopy, and 7-day percentage (ratio of live cells to dead cells) cell viability was assessed with confocal microscopy and fluorescent staining. In a second series of experiments, the metabolism of small microspheres was studied during a course of 7 days by using a spectrophotometric bioanalyzer. RESULTS: Transcatheter injection caused fracturing and/or fragmentation of large (800-1,000 microm) and medium (500-750 microm) microspheres, while small (250-400 microm) microspheres were structurally unaffected by transcatheter injection. Fracturing and fragmentation were associated with cell release from the alginate matrix. Although transcatheter injection reduced cell viability by 17%-23% in all size categories, it did not cause a detectable alteration in the rate of glucose metabolism. CONCLUSION: Transcatheter injection was physiologically well tolerated by fibroblasts encapsulated in alginate microspheres; however, when microsphere diameter exceeded the catheter diameter, fracturing and fragmentation of microspheres compromised the sequestration function of the microsphere vector.

The effects of alginate composition on encapsulated betaTC3 cells.

The effects of alginate composition on the growth of murine insulinoma betaTC3 cells encapsulated in alginate/poly-L-lysine/alginate (APA) beads, and on the overall metabolic and secretory characteristics of the encapsulated cell system, were investigated for four different types of alginate. Two of the alginates used had a high guluronic acid content (73% in guluronic acid residues) with varying molecular weight, while the other two had a high mannuronic acid content (68% in mannuronic acid residues) with varying molecular weight. Each composition was tested using two different polymer concentrations. Our data show that betaTC3 cells encapsulated in alginates with a high guluronic acid content experienced a transient hindrance in their metabolic and secretory activity because of growth inhibition. Conversely, betaTC3 cells encapsulated in alginates with a high mannuronic acid content experienced a rapid increase in metabolic and secretory activity as a result of rapid cell growth. Our data also demonstrate that an increase in either molecular weight or concentration of high mannuronic acid alginates did not alter the behavior of the encapsulated betaTC3 cells. Conversely, an increase in molecular weight and concentration of high guluronic acid alginates prolonged the hindrance of glucose metabolism, insulin secretion and cell growth. These observations can be best interpreted by changes in the microstructure of the alginate matrix, i.e., interaction between the contiguous guluronic acid residues and the Ca2+ ions, as a result of the different compositions.

Non-Invasive monitoring of a bioartificial pancreas in vitro and in vivo.

Monitoring biochemical processes relevant to the function, survival, and longevity of tissue-engineered pancreatic constructs is important for the development of an optimum construct design as well as patient care management after implantation. In this report we demonstrate the ability of nuclear magnetic resonance (NMR) techniques to monitor aspects of intracellular metabolism, overall morphology, and distribution of a microencapsulation based bioartificial pancreas in vitro and in vivo.

MRS methodology.

There are several options in the MRS methods that require consideration before proceeding with the acquisition of the data. The decision to select a nucleus should be based solely on the metabolic information sought. For instance, if an investigator is interested in the effects of medication on the bioenergetic status of the brain, then 31P MRS is indicated. Similarly, selecting the TE for 1H MRS should be based on the metabolite sought. Resonances assigned to myo-In and Glx can only be detected at short TE. Selecting between single-voxel and spectroscopic imaging depends on the requirements of the study. For example, if spatial information is needed, then MRS data ought to be acquired via spectroscopic imaging. However, if the spatial resolution is not critical and interest is to compare a pathologic area with a contralateral VOI, then the acquisition of two single-voxel spectra may be preferred. MRS is widely used. All major vendors of MRI instruments provide some spectroscopic capabilities as a commercially available package with their equipment. Its application to epilepsy has been significant, to which the 40 plus research papers from more than a dozen centers around the world attest.

Effects of short-term hypoxia on a transformed cell-based bioartificial pancreatic construct.

Hypoxia is an adverse condition that can jeopardize the function of a bioartificial pancreatic construct. In this study we have investigated the effects of short-term hypoxic exposure (up to 24 h) on the bioenergetic status, metabolism, and insulin secretion of perfused pancreatic constructs composed of alginate/poly-L-lysine/alginate (APA) encapsulated mouse insulinoma betaTC3 cells. The bioenergetic status of the encapsulated cells was monitored noninvasively with the aid of 31P NMR spectroscopy, while glucose, lactate, and insulin concentrations were measured with off-line assays from media samples removed from the perfusion loop. Our results demonstrate that in freshly prepared constructs insulin secretion was not affected by the hypoxic conditions, although intracellular ATP concentration decreased and glucose consumption increased. Alternatively, in constructs that were maintained in our perfusion system for at least 10 days, identical hypoxic conditions resulted in a decreased insulin secretion concomitant to a decreased intracellular ATP concentration and increased glucose consumption. These results suggest that the effects of hypoxia on a transformed cell-based pancreatic construct are not constant throughout the duration of an in vitro culture. The observed differences are attributed to the significant cell growth and rearrangement that occurs with time during an in vitro culture of the constructs.

In vitro monitoring of total choline levels in a bioartificial pancreas: (1)H NMR spectroscopic studies of the effects of oxygen level.

This investigation implements specifically designed solvent-suppressed adiabatic pulses whose properties make possible the long-term monitoring of (1)H NMR detectable metabolites from alginate/poly-l-lysine/alginate (APA)-encapsulated betaTC3 cells. Our encapsulated preparations were maintained in a perfusion bioreactor for periods exceeding 30 days. During this prolonged cultivation period, the cells were exposed to repetitive hypoxic episodes of 4 and 24 h. The ratio of the total choline signal (3.20 ppm) to the reference signal (observed at 0.94 ppm assigned to isoleucine, leucine, and valine) decreased by 8-10% for the 4-h and by 20-32% for the 24-h episodes and returned to its prehypoxic level upon reoxygenation. The decrease in the mean value of total choline to reference signal ratio for three 4-h and two 24-h episodes in two different cultures was highly significant (P<0.01). The rate of recovery by this ratio was slower than the rates of recovery by oxygen consumption, lactate production, or glucose consumption. A step-up in oxygen level led to a new, higher value for the total choline to reference ratio. From spectra of extracts at 400 MHz, it was determined that 63.6% of the total choline signal is due to intracellular phosphorylcholine. Therefore, it is inferred that the observed changes in total choline signal are linked to an oxygen level dependence of the intracellular phosphorylcholine. Several possible mechanisms in which oxygen may influence phosphorylcholine metabolism are suggested. In addition, the implications of these findings to the development of a noninvasive monitoring method for tissue-engineered constructs composed of encapsulated cells are discussed.

Effects of pH on murine insulinoma betaTC3 cells.

Confluent monolayer cultures of betaTC3 cells were exposed for 4 h to acidic, neutral, or alkaline pH media. Studies determined the impact of pH on viability, insulin secretion rate, glucose consumption rate, lactate production rate, and ATP content. Cell viability was not affected by exposure to media of different pH (>95% for all groups). Insulin release from cells exposed to acidic media (pH of 6.4) was approximately 75% higher than that from cells exposed to either neutral (pH of 7.1) or alkaline (pH of 7.8) conditions. Conversely, ATP content was significantly reduced in cultures exposed to acidic conditions, although there was no statistical difference between neutral and alkaline conditions. Glucose consumption and lactate production rates increased linearly with increasing pH.

Development of a bioartificial pancreas: I. long-term propagation and basal and induced secretion from entrapped betaTC3 cell cultures.

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion.

Development of a bioartificial pancreas: II. Effects of oxygen on long-term entrapped betaTC3 cell cultures.

Tissue-engineered pancreatic constructs based on immunoisolated, insulin-secreting cells are promising in providing an effective, relatively inexpensive, long-term treatment for type I (insulin-dependent) diabetes. An in vitro characterization of construct function under conditions mimicking the in vivo environment is essential prior to any extensive animal experimentation. Encapsulated cells may experience hypoxic conditions postimplantation as a result of one or more of the following: the design of the construct; the environment at the implantation site; or the development of fibrosis around the construct. In this work, we studied the effects of 3- and 4-day-long hypoxic episodes on the metabolic and secretory activities and on the levels of intracellular metabolites detectable by phosphorus-31 nuclear magnetic resonance ((31)P NMR) of alginate/poly-L-lysine/alginate entrapped betaTC3 mouse insulinomas continuously perfused with culture medium. Results show that, upon decreasing the oxygen concentration in the surrounding medium, the encapsulated cell system reached a new, lower metabolic and secretory state. Hypoxia drove the cells to a more anaerobic glycolytic metabolism, increased the rates of glucose consumption (GCR) and lactate production (LPR), and reduced the rates of oxygen consumption (OCR) and insulin secretion (ISR). Furthermore, hypoxia reduced the levels of intracellular nucleotide triphosphates (NTP) and phosphorylcholine (PC) and caused a rapid transient increase in inorganic phosphate (P(i)). Upon restoration of the oxygen concentration in the perfusion medium, all parameters returned to their prehypoxic levels within 2 to 3 days following either gradual unidirectional changes (ISR, NTP, PC) or more complicated dynamic patterns (OCR, GCR, LPR). A further increase in oxygen concentration in the perfusion medium drove OCR, ISR, NTP, PC, and P(i) to new, higher levels. It is concluded that (31)P NMR spectroscopy can be used for the prolonged noninvasive monitoring of the bioenergetic changes of encapsulated betaTC3 cells occurring with changes in oxygen tension. The data also indicate that the oxygen-dependent states might be related to the total number of viable, metabolically active cells supported by the particular oxygen level to which the system is exposed. These findings have significant implications in developing and non-invasively monitoring a tissue-engineered bioartificial pancreas based on transformed beta cells, as well as in understanding the biochemical events pertaining to insulin secretion from betaTC3 insulinomas.

Effects of alginate composition on the metabolic, secretory, and growth characteristics of entrapped beta TC3 mouse insulinoma cells.

The effects of alginate composition on cell growth as well as the metabolic and secretory profile of transformed beta-cells entrapped in alginate/poly-L-lysine/alginate (APA) solid beads were investigated following entrapment of beta TC3 mouse insulinoma cells in alginate composed of either high mannuronic acid or high guluronic acid residues. Entrapped cultures were maintained in spinner flasks for 40-60 days. The pattern of cell growth and the overall rates of glucose consumption and insulin secretion were investigated. Cultures of beta TC3 cells entrapped in alginate composed predominantly of mannuronic acid units (77%) displayed a linear increase in the rates of glucose consumption and insulin secretion concomitant with an increase in cell population in the periphery of the beads. Conversely, cultures of beta TC3 cells entrapped in alginate composed predominantly of high guluronic acid units (69%) displayed a decrease in the rates of glucose consumption and insulin secretion during the first three weeks of culture, followed by a rapid recovery that surpassed the initial rates by day 40. This biphasic pattern was concomitant to a decrease in viable cells during the first three weeks as ascertained by histology, followed by an increase in cell proliferation. Cell growth in high guluronic acid alginate took place at random locations throughout the solid bead and not in the periphery, as was the case in high mannuronic acid alginate preparations. Possible reasons for these differences and the significance of these findings in the context of a bioartificial pancreas composed of APA entrapped transformed cells are discussed.

Age-related brain changes in rhesus monkeys: a magnetic resonance spectroscopic study.

Brain metabolites were measured by proton magnetic resonance spectroscopy in five young (4-10 years of age) and six old (24-30 years of age) adult rhesus monkeys. The two age groups had similar levels of N-acetylaspartate and of choline relative to creatine, but the ratio of myo-inositol/creatine was higher in each old monkey than in any of the young animals. There was no significant relationship between the metabolite ratios and cognitive performance. The findings indicate that a consistent pattern of non-invasively detectable biochemical changes occurs in the brain with ageing. Whether these changes have functional significance in age-related pathologies, or are simply markers of brain ageing will be the subject of future studies.