Multidimensional Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers, 1

Summary: The chromatographic analysis of hydrophilic copolymers is complicated due to the fact that in most cases aqueous eluents must be used. In aqueous eluents different polar and ionic effects may disturb the selective interactions between the macromolecules and the stationary phase making it impossible to separate such copolymers with regard to chemical composition. Therefore, 2D chromatography combining a separation according to composition with a separation according to molar mass has been applied mostly to polymers that are soluble in organic solvents. The present contribution describes experimental approaches to analyze such hydrophilic copolymers by 2D‐chromatography. For a model polymer system resulting from the copolymerization of methacrylic acid and a poly(ethylene glycol) macromonomer, it is shown that different analytical techniques including SEC, LC‐CC, MALDI‐TOF MS and 2D chromatography can be used to analyze the different parameters of molecular heterogeneity of such copolymers.

2D separation of poly(MPEG‐MM 2), 1^st dimension: LC‐CC, 2^nd dimension: SEC.     

Cytotoxic T cell responses to haptenated cells III. Isolation and specificity analysis of continuously growing clones

Various procedures were used to derive continuously growing cytotoxic T lymphocyte (CTL) clones from a primary culture containing responder cells from immunized mice and 3-(p-sulfophenyldiazo)-4-hydroxylphenyl acetic acid (SP)-or fluorescein isothiocyanate (FL)-coupled stimulator cells. It seems likely that CTL have to undergo some change, possibly genetic, to be able to grow continuously in T cell growth factor conditioned medium in the absence of any stimulator or filler cells. The most convenient and reliable procedure to generate CTL clones with different specificities was to establish from several aliquots of a primary culture cell populations continuously growing in medium conditioned with T cell growth factor(s). Clones with different specificities segregated in the different populations. SP-and FL-specific CTL clones restricted to H-2K^k, and H-2D^d and two FL-specific CTL clones with no apparent H-2 restriction are described.     

Three-dimensional in vitro model of adipogenesis: comparison of culture conditions

In vivo and in vitro studies have demonstrated both promise and current limitations in tissue engineering of fat. Herein, we report the establishment of a well-defined three-dimensional (3-D) in vitro model useful for systematic investigations of 3-D adipogenesis. Polyglycolic acid fiber meshes were dynamically seeded with 3T3-L1 preadipocytes; subsequently, cell-polymer constructs were hormonally induced and cultivation under three different conditions was evaluated. Regarding tissue coherence and intracellular lipid content, culture of cell-polymer constructs either dynamically in well plates or in stirred bioreactors yielded similar results, which were distinctly improved compared with static conditions in well plates. At the protein and mRNA levels, significantly increased expression of genes characteristic for a mature adipose phenotype was demonstrated for constructs dynamically cultured in well plates, as compared with static conditions. Furthermore, investigation of lipolysis under stimulating and inhibiting conditions demonstrated functionality of the dynamically differentiated constructs. Using dynamic culture conditions, the presented in vitro model system is suggested as a valuable tool serving both fat tissue engineering and basic research by facilitating investigations of tissue-inherent features not possible under conventional 2-D culture conditions.     

Effects of combined renovascular hypertension and diabetes mellitus on myocardial cells,non-vascular interstitium and capillaries: a stereological study on rat hearts

Summary The effects of combined renovascular hypertension and diabetes mellitus on the rat heart were investigated in order to detect possible synergistic effects of the two conditions. Hypertensive diabetic and hypertensive non-diabetic animals were compared to diabetic and non-diabetic controls. Hypertension was established for 12 weeks by a surgical stenosis of the left renal artery; diabetes mellitus was maintained for 8 weeks by a single intraperitoneal injection of 60 mg/kg streptozotocin. Light microscopic stereology did not reveal significant divergences between diabetic hypertensives and non-diabetic hypertensives. Hypertension induced a focal perivascular and interstitial fibrosis with increased volume densities of non-vascular interstitium and fibrosis (P<0.001). Capillary density (Q_A) was decreased in transverse sections (P<0.01) and increased in longitudinal sections (P<0.01). This indicates a three-dimensional remodelling of the capillary bed with an increased number of obliquely running capillaries. At least the length density (L_V) of capillaries (mm/mm³) tends to be normalized in long-term renovascular hypertension. At the ultrastructural level, a synergism of hypertension and diabetes mellitus was observed: the volume ratio of mitochondria to myofibrils was significantly decreased in hypertensive diabetics, but not in non-diabetic hypertensives or in diabetics. This may enhance the risk of cardiac deterioration. We conclude that the primary target of the synergistic damage in hypertensive diabetic heart muscle disease is the myocardial cell and not the cardiac interstitium.Preliminary results of this study have been published in: Mall G (1991) Morphometric study on the rat heart in combined renovascular hypertension and diabetes mellitus. In: Nagano N, Dhalla NS (eds) The diabetic heart. Raven Press, New York, pp 115–124Dedicated to Prof. Dr. med. G. Seifert on the occasion of his 70th birthday     

Sequence analysis and comonomer interactions in poly[(4-chlorophenyl methacrylate)-co-(methyl methacrylate)]s

Copolymers of 4-chlorophenyl methacrylate and methyl methacrylate were prepared and investigated by ¹H and ¹³C NMR spectroscopy. The copolymer composition, determined by chlorine analysis, ¹H and ¹³C NMR, was found to be close to the initial composition of the monomer mixture. The sequence analysis was carried out by analyzing the methoxy signals of the ¹H NMR spectra. Six out of ten tactic and compositional triads could be resolved. It was found that the copolymers are predominantly syndiotactic and the compositional and tactic triad populations are given. The aromatic carbon atoms are sensitive towards compositional and tactic sequence effects, which results in a switch of the order of the tactic signals at different aromatic carbon atoms.     

A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes

CD8(hi+) cytotoxic T lymphocytes (CTL) are major players in immune defense. In addition, they contribute to the maintenance of immune homeostasis. We now describe a hitherto unavailable, but simple assay to determine ex vivo lytic granule-based cytotoxic functions of human CD8(hi+) CTL subgroups in a clinical setting, under target cell free conditions. Ficoll-isolated peripheral blood lymphocytes from 17 healthy volunteers were stimulated either by phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin or by antibody mediated crosslinking of the CD3 molecule on the T cell surface. Using perforin as a marker for lytic granules, the reduction of CTL granules over time intervals up to 120 min was quantified by FACScan flow cytometry. The kinetics of perforin reduction were compared to the kinetics of NA-CBZ-L-lysine-thiobenzyl ester hydrochloride (BLT)-esterase release and of CD63 upregulation. The reduction in the perforin(+) portion of CD8(hi+) CTLs was correlated inversely with BLT-esterase release and CD63 upregulation. At 30 and 120 min after PMA/ionomycin stimulation, 55 +/- 14% and 42 +/- 14%, respectively, of CD8(hi+) CTLs still stained perforin(+) (time point 0 min = 100%). Perforin-granule release induced by CD3-crosslinking occurred as fast within 30 min (55 +/- 17%), but over the 120 min time interval it was not as complete when compared to PMA/ionomycin-stimulated perforin-reduction. Thus, the combination of an established degranulation assay with the power of immuno flow cytometry allows one to investigate the cytotoxic capability of CTL-subtypes and the kinetics of perforin-granule release. In addition, the assay may prove useful in the elucidation of intracellular signaling cascades governing the perforin-granule release process.