Improving the success rate of human corneal endothelial cell cultures from single donor corneas with stabilization medium.

Main objective of this study was to improve the success rate of human corneal endothelial cell (hCEC) cultures from single donor corneas. We could show that the use of stabilization medium prior to cell isolation may have a positive effect on the success rate of hCEC cultures from single research-grade donor corneas by allowing growth of otherwise possibly not successful cultures and by improving their proliferative rate. hCEC were obtained from corneo-scleral rims of 7 discarded human research-grade cornea pairs. The Descemet membrane-endothelium (DM-EC) sheets of each pair were assigned to 2 experimental conditions: (1) immediate cell isolation after peeling, and (2) storage of the DM-EC sheet in a growth factor-depleted culture medium (i.e. stabilization medium) for up to 6 days prior to cell isolation. hCEC isolated by enzymatic digestion were then induced to proliferate on pre-coated culture plates. The success rate of primary cultures established from single donor corneas were higher for DM-EC sheets kept in stabilization medium before cell isolation. All cultures (7/7) initiated from stabilized DM-EC sheets were able to proliferate up to the third passage, while only 4 out of 7 cultures initiated from freshly peeled DM-EC sheets reached the third passage. In addition, for the 4 successful paired cultures we observed a faster growth rate if the DM-EC sheet was pre-stabilized prior to cell isolation (13.8 ± 1.8 vs 18.5 ± 1.5 days, P < 0.05). Expression of the phenotypical markers Na+/K+-ATPase and ZO-1 could be shown for the stabilized cultures that successfully proliferated up to the third passage.

[Standardized Descemet membrane endothelial keratoplasty (DMEK): technique and latest results]. / Standardisierte Descemet-Membran-Endothelkeratoplastik (DMEK) : Technik und aktuelle Ergebnisse.

BACKGROUND: The procedure of posterior lamellar keratoplasty has now become established as first choice therapy for the treatment of corneal endothelial diseases, with Descemet membrane endothelial keratoplasty (DMEK) as the most recent refinement of endothelial keratoplasty techniques. In this study the outcome of 400 consecutive cases after standardized no-touch DMEK was evaluated. METHODS: This was a non-randomized retrospective clinical study of 400 consecutive eyes in 321 patients who underwent standard DMEK. Outcome measures included best corrected visual acuity (BCVA) preoperatively and postoperatively at 1, 3 and 6 months, endothelial cell density (ECD), pachymetry and complications within the first 6 months. In addition two subgroups were analyzed (subgroup I cases 1-200 and subgroup II cases 201-400). RESULTS: At 6 months postoperatively 80 % of all eyes had a BCVA of ≥ 20/25 (≥ 0.8), 44 % of ≥ 20/20 (≥ 1.0) and 14 % of ≥ 20/18 (≥ 1.2). There were no significant differences between the two subgroups. The mean ECD preoperatively was 2,542 (± 217) cells/mm(2) and 6 months postoperatively 1,622 (± 500) cells/mm(2). The mean ECD decrease was 36 % 6 months after DMEK. No difference between the subgroups was noted. In 5 % a re-operation was needed of which 75 % were performed in subgroup I. CONCLUSION: With the standardized no-touch DMEK technique a rapid and nearly complete visual rehabilitation as well as ECD values similar to earlier endothelial keratoplasty techniques can be achieved. Therefore DMEK may become the preferred treatment for corneal endothelial disorders.

[Visual acuity following Descemet-membrane endothelial keratoplasty (DMEK): first 100 cases operated on for Fuchs endothelial dystrophy]. / Visus nach Descemet-Membran Endothelkeratoplastik (DMEK): Ergebnisse der ersten 100 Eingriffe bei Fuchs'scher Endotheldystrophie.

PURPOSE: The aim of this study was to evaluate the visual outcome and endothelial cell density in the first series of 100 eyes after Descemet membrane endothelial keratoplasty (DMEK) for the treatment of Fuchs endothelial dystrophy. DESIGN: This was a non-randomised prospective clinical trial. METHODS: The first 100 consecutive eyes (87 patients) that underwent DMEK (i. e., transplantation of an isolated donor Descemet membrane carrying its endothelium) for Fuchs endothelial dystrophy were evaluated. In all eyes, the best corrected visual acuity (BCVA) as well as the endothelial cell density (ECD) were measured before and at one, three and six months after surgery. RESULTS: In eyes with a functional DMEK graft and good visual potential (n = 78), the BCVA was > or = 20 / 40 (> or = 0.5) in 79 % after 1 month, 94 % after 3 months, and 96 % after six months; > or = 20 / 25 (> or = 0.8) in respectively 54 %, 64 %, and 74 %, and > or = 20 / 20 (> or = 1.0) in respectively 26 %, 36 %, and 45 %. In this group, ECD averaged 2630 (+/- 190) cells/mm (2) before, and 1800 (+/- 540) cells/mm (2) at six months after surgery (n = 76). In 15 eyes, a secondary "Descemet stripping endothelial keratoplasty" (DSEK) was performed. When the outcomes of DMEK and secondary DSEK procedures were combined, 91 % reached a BCVA of > or = 20 / 40 (> or = 0.5), 65 % > or = 20 / 25 (> or = 0.8) and 38 % > or = 20 / 25 (> or = 1.0) at six months after surgery (n = 93). For the whole group, ECD averaged 2620 (+/- 190) cells/mm (2) before, and 1730 (+/- 590) cells/mm (2) at six months after surgery (n = 89). CONCLUSION: DMEK may provide relatively quick and nearly complete visual rehabilitation in a majority of patients operated on for Fuchs endothelial dystrophy. Endothelial cell survival may be similar to earlier types of endothelial keratoplasty. Early graft detachment (19 cases) was the main complication in this first series of DMEK surgeries.

Spectral diffusion and electron-phonon coupling of the B800 BChl a molecules in LH2 complexes from three different species of purple bacteria.

We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the alphabeta-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.

Symmetry matters for the electronic structure of core complexes from Rhodopseudomonas palustris and Rhodobacter sphaeroides PufX-.

Low-temperature (1.4 K), single-molecule fluorescence-excitation spectra have been recorded for individual reaction center-light-harvesting 1 complexes from Rhodopseudomonas palustris and the PufX(-) strain of Rhodobacter sphaeroides. More than 80% of the complexes from Rb. sphaeroides show only broad absorption bands, whereas nearly all of the complexes from Rps. palustris also have a narrow line at the low-energy end of their spectrum. We describe how the presence of this narrow feature indicates the presence of a gap in the electronic structure of the light-harvesting 1 complex from Rps. palustris, which provides strong support for the physical gap that was previously modeled in its x-ray crystal structure.

Observation of the energy-level structure of the low-light adapted B800 LH4 complex by single-molecule spectroscopy.

Low-light adapted B800 light-harvesting complex 4 (LH4) from Rhodopseudomonas palustris is a complex in which the arrangement of the bacteriochloropyll a pigments is very different from the well-known B800-850 LH2 complex. For bulk samples, the main spectroscopic feature in the near-infrared is the occurrence of a single absorption band at 802 nm. Single-molecule spectroscopy can resolve the narrow bands that are associated with the exciton states of the individual complexes. The low temperature (1.2 K) fluorescence excitation spectra of individual LH4 complexes are very heterogeneous and display unique features. It is shown that an exciton model can adequately reproduce the polarization behavior of the complex, the experimental distributions of the number of observed peaks per complex, and the widths of the absorption bands. The results indicate that the excited states are mainly localized on one or a few subunits of the complex and provide further evidence supporting the recently proposed structure model.

Effect of recombinant human insulin-like growth factor I and early total parenteral nutrition on immune depression following severe head injury.

OBJECTIVE: To determine the effects of insulin-like growth factor I (IGF-I) and aggressive nutrition on CD4/CD8 ratios following head injury. DESIGN: Randomized controlled trial. SETTING: An urban level 1 trauma center. PARTICIPANTS: Head-injured patients with a Glasgow Coma Scale score of 4 to 10 within 6 hours of hospital admission requiring no major extracranial surgery with the exception of isolated lower-extremity fracture fixation. Fourteen patients were recruited and 11 completed the study. INTERVENTIONS: Patients were randomized to a continuous infusion of saline or 0.01 mg/kg per hour of recombinant human (rh) IGF-I. Both groups received parenteral nutrition and rapidly advanced to a total protein intake of 2 g/kg per day and a maximum nonprotein calorie intake of 40 kcal/kg per day. The nonprotein prescription was 1.25 times the metabolic energy expenditure determined by metabolic cart not to exceed a nonprotein calorie intake of 40/kcal. MAIN OUTCOME MEASURES: The CD4/CD8 ratios and serum IGF-I levels on days 1, 7, and 14. RESULTS: Administration of early aggressive nutrition eliminated the depressed CD4/CD8 ratio usually seen after head injury; administration of IGF-I increased the CD4/CD8 ratio while IGF-I levels were elevated. CONCLUSIONS: Infusion of rhIGF-I and aggressive early intravenous nutrition affects the immunologic response of patients with severe head injury.