Effect of insulin pump treatment for one year on renal function and retinal morphology in patients with IDDM.

The effect of strict metabolic control for 1 yr on renal function and retinal morphology was estimated in 24 insulin-dependent diabetic individuals (age 29 +/- 8 yr, diabetes duration 10 +/- 6 yr) with Albustix negative urine and minimal or no background retinopathy before the study. They were randomized to conventional insulin treatment (CIT) or continuous subcutaneous insulin infusion (CSII) with a portable pump. During CSII treatment the metabolic status was significantly improved and glomerular filtration rates (GFR) were found to decline (from 130 +/- 18 to 116 +/- 15 ml/min/1.73 m2, P less than 0.01). The mean value of urinary albumin excretion (UAE) was not statistically significantly reduced (from 15.3 +/- 24.1 to 6.8 +/- 1.5 mg/24 h, P greater than 0.1). In the CIT group both GFR and UAE values were unchanged. In all subjects before treatment we found a positive correlation between the metabolic status, estimated by the HbA1c values, and GFR as well as UAE values, respectively (r = 0.64, P less than 0.01 and r = 0.42, P less than 0.05). We conclude that elevated GFR values can be reduced toward normal level by insulin pump treatment for 1 yr. Retinal morphology was found to deteriorate in four of 12 CIT subjects and in three of 12 CSII subjects. However, the progression was mild and in most individuals only due to a slight increase in the number of microaneurysms. Pump treatment did not induce proliferation and "cotton-wool" exudates were not detectable in any pump subject after 1 yr of treatment. We conclude that CSII treatment for 1 yr was unable to stop the progression of retinopathy in individuals with minimal or no retinopathy on entering the study. On the other hand, pump treatment did not induce cotton-wool exudates or proliferation in these subjects as previously reported in subjects with more severe retinopathy before CSII treatment.

Significant effect of high-resolution HLA-DRB1 matching in high-risk corneal transplantation.

The effect of HLA matching in corneal transplantation is still--after numerous of studies--disputable. We investigated the effect of DRB1 matching in high-risk cases with vascularization and/or retransplantation. Only class II antigens were matched because we were unable to obtain donor lymphocytes for HLA typing. Typing was performed on DNA isolated from the ocular tissues up to 24 hr after death. When this study was initiated, DNA-based methods had been developed only for class II typing. The first part of the study concerns 74 cases with at least 3 years of observation fully matched for 17 DRB1 specificities detected using restriction fragment-length polymorphism. This showed an improved long-term graft survival of 72% compared with 45% in a historical control group of 23 comparable cases. In the second part of the study, stored DNA samples from the restriction fragment-length polymorphism-matched donor-recipient pairs were subjected to retyping with a new method based on sequence-specific polymerase chain reaction. It was possible to split DRB1*01, *04, and *11 in 3, 14, and 5 alleles, respectively. The matching was then re-assigned taking all splits into account. This showed that 36 cases had at least one incompatibility, whereas 38 cases were fully compatible. The long-term graft survival rate was 79% in the matched group compared with only 59% in the mismatched group, which is significantly different at P=0.032. This retrospective, but blinded, randomized study is strong evidence for the effect of matching and may give scope for international collaboration to obtain completely matched corneas for this group of patients.

Human lens thioltransferase: cloning, purification, and function.

PURPOSE: To clone the human lens thioltransferase (TTase) gene and to purify, characterize and study the possible function of the recombinant human lens thioltransferase (RHLT). METHODS: The human lens TTase gene was cloned by using RT-PCR and verified by sequence and RNase protection assay. TTase overexpressed in Escherichia coli was isolated and purified to homogeneity by column chromatography and identified by Western blot analysis. The activity was assayed with a synthetic substrate hydroxyethyl disulfide. Its function in dethiolating and reactivating other key metabolic enzymes was studied by using pure glutathione S:-transferase (GST) and glutathione peroxidase (GPx) from commercial source and also with the cell extract of rabbit lens epithelial cells preexposed to H2O2. RESULTS: The cloned human lens TTase gene showed identical sequence to the TTase gene from other human tissues. The RNase protection assay displayed a single transcript from the total RNA of human lens epithelial cells. The purified RHLT had a molecular weight of 11.8 kDa and reacted positively with anti-pig liver TTase. It displayed similar structural, functional, and kinetic characteristics to those of TTases from other sources. It was shown that RHLT effectively regenerated the activities of GST and GPx, after each was inactivated by S-thiolation with cystine in vitro. Furthermore, RHLT was able to restore the activity of the oxidatively inactivated glyceraldehyde-3-phosphate dehydrogenase (G-3PD) in H2O2-exposed rabbit lens epithelial cells. CONCLUSIONS: The human lens TTase gene has been cloned for the first time. Its gene product showed the characteristics which support our speculation that TTase may play a major role in maintaining the homeostasis of lens protein thiols thus protecting against oxidative stress.

No increase in arteriolosclerotic retinopathy or activity in tests for circulating immune complexes 5 years after vasectomy.

On the basis of studies in monkeys, the hypothesis has been proposed that vasectomy induces the formation of circulating immune complexes (CICs), which--via activation of the complement system--may result in immune injury of the endothelium and thereby exacerbate atherosclerosis; the same mechanism has been suggested to cause retinal arteriolar changes in vasectomized men. We compared 46 men, 5 years after vasectomy, with 46 age-matched control subjects and found no difference in the distribution of arteriolosclerotic retinopathy gradings evaluated by ophthalmoscopy and fundus photography. Blood samples from the two groups were collected and handled identically, and no significant difference in activity was found in four different tests for CICs and two tests for split products of complement factor C3. Thus, the results do not support the hypothesis that changes mediated by CICs occur in vessels after human vasectomy.

Acute tissue deformation of the human cornea after radial keratotomy.

PURPOSE: The regional deformation pattern of the cornea after radial keratotomy, which is essential for understanding the mode of action of the procedure, has not previously been studied in detail. METHODS: Up to 90 tiny mercury droplets were placed from center to limbus on the epithelial and endothelial corneal surfaces of eight eviscerated human donor eyes with four radial keratotomies (depth 100% of central corneal thickness, 3.5-mm clear zone). From digital images obtained under pressure loads ranging from 2 to 100 mm Hg, the distances between the fixed droplets were measured with an accuracy of 1 micron. After transforming the data to polar coordinates, regional meridional and circumferential strain patterns were calculated. Regional meridional and circumferential radii of curvatures were calculated from corneal profile images obtained at different pressure loads before and after keratotomy. RESULTS: Increasing the intraocular pressure from 2 to 100 mm Hg induced: an epithelial side wound gape of 44 mm; epithelial side circumferential tissue compression between incisions; considerable epithelial side meridional tissue elongation at and between incisions; little endothelial side circumferential strain across incisions; and little endothelial side meridional strain at and between incisions. The radial keratotomy induced 2.30 diopters (D) of central corneal flattening at an intraocular pressure of 2 mm Hg. The degree of central flattening correlated linearly with the amount of wound gape. In the physiological pressure range of the central cornea flattened 0.05 D for each millimeter-of-mercury increment in intraocular pressure. Pronounced meridional steepening was induced corresponding to the middle and peripheral parts of the keratotomy incisions. CONCLUSIONS: Our study suggests that the peripheral "tissue addition" seen after radial keratotomy is a net result of wound gape and circumferential tissue compression. Local bending of intact stromal tissue below the incisions plays an important role for the generation of the wound gape at the corneal surface. These data may help verify finite-element computer models of the human cornea.

Mechanical stability of the cornea after radial keratotomy and photorefractive keratectomy.

BACKGROUND: Corneal refractive surgical procedures alter the shape and structure of the tissue, possibly compromising its mechanical stability. METHODS: One or both eyes of 87 subjects were studied: 26 of these 87 had been treated for myopia by excimer laser ablation and 36 by radial keratotomy; 25 without previous corneal surgery functioned as controls. Corneal topography was evaluated by TMS-1 videophotokeratography before and after 180 degrees tilting of the patient, and the pressure-induced ring-wise changes in spherical equivalent power (axial power) were calculated. Changes in corneal shape also were evaluated by computing the instantaneous radius of curvature. The intraocular pressure was measured before and after tilting by a hand-held applanation tonometer. RESULTS: The mean intraocular pressure increased from 13.9 +/- 2.3 mm Hg before tilting to 30.0 +/- 3.8 mm Hg during tilting. In the control eyes, the mean power of the central cornea during tilting decreased 0.187 +/- 0.045 diopters (D) (p < .05); in the excimer-laser-ablated eyes, 0.038 +/- 0.056 D (not statistically significant); and in those treated with radial keratotomy, 0.523 +/- 0.054 D (p < .01). After radial keratotomy, the cornea steepened outside the clear zone in response to pressure loading, whereas it did not change significantly in the laser-ablated or control eyes. CONCLUSIONS: Pressure-induced deformation of normal and excimer laser-ablated human cornea is small, whereas radially incised corneas have significantly decreased mechanical stability.

Clinical copper metabolism parameters in patients with retinitis pigmentosa and other tapeto-retinal degenerations.

Reports have appeared of abnormal copper metabolism in retinitis pigmentosa, and of a family with vitelliform retinal degeneration in which other members suffered from hepatolenticular degeneration. In the present study 15 patients with retinitis pigmentosa, 4 with various other retinal degenerations, and 1 with a family disposition to retinitis pigmentosa were examined. The copper concentration in serum and the coeruloplasmin concentration in plasma were found to be within normal limits. In 9 of the patients with retinitis pigmentosa the urinary excretion of copper per 24 hours was determined and was found to be normal. The results of the present study lend no support to the hypothesis of abnormal copper metabolism in retinitis pigmentosa.

Evaluation of potential organ culture media for eye banking using human donor corneas.

AIM: To evaluate the ability of different commercially available cell culture solutions to preserve human donor corneas during 3 weeks of "closed system" organ culture at physiological temperature. This screening was performed in an attempt to establish a rational basis for the development of a serum-free organ culture medium for eye banking. METHODS: 72 normal human donor corneas were organ cultured for 21 days at 31 degrees C in eight different test media (nine corneas in each group). The basic culture solutions included: minimal essential medium (MEM), MEM with stabilised L-glutamine, M199, DIF-1000, SFM, F99, and F99 with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids (termed F99-Sr). All media were supplemented with 2% fetal calf serum (FCS), except for MEM, which was also studied at 8% FCS. The evaluation parameters included: (1) the endothelial cell loss as evaluated using trypan blue staining; (2) the ability of keratocytes and endothelial cells to incorporate tritiated uridine into RNA as evaluated using autoradiography and digital image analysis; (3) the leakage of immunogenic keratan sulphate as assessed using ELISA; and (4) changes in storage medium pH, glucose, and lactate content. RESULTS: SFM induced the lowest endothelial cell loss of 14% (SD 2%) and the highest RNA synthesis rates of all test solutions supplemented with 2% FCS. Corneas stored in SFM also showed the least leakage of keratan sulphate and the highest glucose consumption and lactate production. In five media (MEM with 2% FCS, MEM with stabilised L-glutamine, M199, F99, and F99-Sr), comparable and intermediate potentials for organ culture were observed with endothelial cell loss of 16-19%. By contrast, 29% (4%) of the endothelium was lost after storage in DIF-1000. Interestingly, the use of 8% FCS (in MEM) had a marked protective effect on the endothelium, which showed the highest RNA synthetic activity combined with a cell loss of only 11% (4%), compared with 19% (6%) at 2% FCS (p<0.05). CONCLUSION: Among the present test solutions, SFM appears to be the most prominent candidate for a new corneal organ culture medium and should be further tested and possibly refined to effectively substitute serum addition.

A three-dimensional study of the human corneal keratocyte density.

The population of stromal keratocytes represents a rarely examined topic. In the present paper we investigated the three-dimensional distribution of keratocytes in human donor corneas. The keratocyte density was calculated from biochemical measurements of the DNA content in samples of the corneal (and scleral) stroma obtained from well-defined regions. The DNA content of the central stroma (0-2 mm from apex) had a mean (+/- SD) of 1.25 +/- 0.30 micrograms DNA/mg dry tissue weight, corresponding to a cellularity of 4.6 +/- 1.1 x 10(4) cells/mm3 (n = 13). Towards the corneal periphery, the cellularity gradually increased to a 60-70% higher cell density at limbus. In the central stroma, an anterior-posterior cell gradient was found with a 30% lower cellularity in the subendothelial region compared to the subepithelial stroma (n = 8). In the four main quadrants of the central stroma, a uniform cell density was found while the peripheral areas showed a 10% higher cellularity in the superior quadrant. A close intraindividual correlation was observed between data obtained from 40 paired corneas ('cornea 1' versus 'cornea 2'); the stromal cellularity within 0-3.75 mm from apex (r = 0.81), the stromal cellularity within 3.75-5.5 mm (r = 0.92), the stromal cellularity within 5.5-8.0 mm (r = 0.85), and the endothelial cell density (r = 0.84). However, within a given cornea no correlation was found between the density of endothelial cells and keratocytes. These data define normal values for the regional density of keratocytes in the human cornea.

Chemotaxis of human keratocytes is increased by platelet-derived growth factor-BB, epidermal growth factor, transforming growth factor-alpha, acidic fibroblast growth factor, insulin-like growth factor-I, and transforming growth factor-beta.

PURPOSE: Following corneal wounding, early migration of keratocytes into the wound area is of pivotal importance in the healing process, but the nature of this migration is not well understood. The influence of peptide growth factors on the chemotactic and chemokinetic migration of human corneal keratocytes was investigated, using the following growth factors: platelet derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and transforming growth factor-beta-1 (TGF-beta 1). METHODS: The chemotactic stimulation was investigated in the Boyden blind-well chemotaxis chamber, and the chemokinetic effect of the growth factors determined by a modified checker-board analysis. RESULTS: PDGF-BB, EGF and TGF-beta 1 stimulated chemotaxis towards a peak value, with a subsequent decline at higher concentrations. PDGF-BB and EGF peaked at 1 ng/ml with a 2.0 and a 2.5-fold increase respectively in the number of keratocytes migrating, whereas TGF-beta 1 reached a maximum response at 0.1 ng/ml, with a 1.7-fold increase. Chemotaxis reached an early plateau and remained constant at concentrations between 1 ng/ml and 100 ng/ml when stimulating with TGF-alpha (2.7-fold), bFGF (2.0-fold), aFGF (2.7-fold), and IGF-I (4.5-fold). Checkerboard analysis revealed that all growth factors were chemotactic agents for human keratocytes, except bFGF, which principally stimulated chemokinesis. CONCLUSION: These in vitro results demonstrate that PDGF-BB, EGF, TGF-alpha, aFGF, IGF-I, and TGF-beta 1 increase keratocyte chemotaxis, and they may play an important role in the early recruitment of keratocytes to the corneal wound site in vivo.

Keratocyte migration and peptide growth factors: the effect of PDGF, bFGF, EGF, IGF-I, aFGF and TGF-beta on human keratocyte migration in a collagen gel.

PURPOSE: Peptide growth factors are known accelerators of corneal wound healing, probably mediated through increased proliferation of the cells; however, information about their effect on keratocyte motility is lacking. The influence of peptide growth factors on keratocyte migratory activity was investigated, using the following growth factors: platelet derived growth factor (PDGF-BB), epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and transforming growth factor-beta-1 (TGF-beta 1). METHODS: Keratocytes were seeded on gels of type 1 collagen, growth factor added, and the cells left to migrate for 72 hours. Subsequently, the number of keratocytes at the different levels in the collagen gel was evaluated by optically sectioning the gel at 20 microns, intervals, with an inverted phase contrast microscope. RESULTS: PDGF, EGF and bFGF at 10 ng/ml, all increased the number of keratocytes at the different levels of the gel as compared to a non-stimulated control (p < 0.05 or p < 0.01, students t-test). TGF-beta proved to be a strong inhibitor of keratocyte migration, decreasing the number of keratocytes observed at every level in the gel (p < 0.05 and p < 0.01, students t-test), whereas no effect of IGF-I and aFGF was found. During the 72 hours of migration, no contraction of the collagen gels was observed. Autoradiography of histological sections of the gels showed that during the 72-hour period only TGF-beta and 10% fetal bovine serum induced an increase in keratocyte proliferation. CONCLUSION: PDGF, EGF and bFGF increase keratocyte migration, independent of proliferation in a collagen gel invasion assay and might promote corneal wound healing, not only by increasing cell proliferation, but also through increased motility.

The keratocyte density of human donor corneas.

The cellularity of the human corneal stroma has not been described in the literature. In the present study we calculated the density of keratocytes in human donor corneas using a new method for biochemical measurement of the stromal DNA content (sDNA). The DNA measurements were compared to morphological counts of the number of keratocyte nuclei per area (KNPA) obtained from histological sections. A significant correlation was found between the data achieved by the two methods (r = +0.52, p < 0.001, n = 46). No significant change in either sDNA or KNPA was found during 28 days of organ culture, and no influence of donor age, sex, or post mortem time was found on either sDNA or KNPA. Both sDNA and KNPA approximated a normal distribution with a mean sDNA of 1.10 +/- 0.25 micrograms DNA/mg dry tissue weight and an average KNPA of 200 +/- 53 nuclei/mm2 (n = 35). Between paired corneas the sDNA were closely correlated (r = +0.83, p < 0.005, n = 11 pairs) with an intra-individual variation of only 0.5%. Using the sDNA data, the keratocyte density in the central region of human donor corneas was calculated to be 129,000 +/- 29.000 per mg dry tissue weight (n = 35). Thus, when corneal grafting is performed (using a 7 mm trephine) an average of 818,000 +/- 186,000 donor keratocytes are transplanted. Assuming a uniform cellularity throughout the stroma, the average number of keratocytes was calculated to be 2,430.000 +/- 551,000 per human donor cornea.(ABSTRACT TRUNCATED AT 250 WORDS)

[Biomechanical study of corneal stability after photorefractive keratectomy]. / Biomechanische Untersuchung der Hornhautstabilität nach photorefraktiver Keratektomie.

BACKGROUND: Photorefraktive keratectomy (PRK) is the most frequent refractive surgical procedure worldwide. The central corneal thickness is reduced due to removal of the anterior stroma, including Bowman's layer, with a laser beam. This procedure results in considerable alterations of the corneal structure. What does this mean for the mechanical properties of the cornea? METHODS: Intraocular pressure was increased via a 180 degrees tilt. Before and during this procedure, corneal topography was measured by photokeratoscopy. We examined 26 patients after PRK and 25 controls who had not undergone any surgical procedure. RESULTS: The corneal center flattened by 0.038 +/- 0.05 dpt (P > 0.05) in the PRK patients and by 0.187 +/- 0.045 dpt (P < 0.05) in the control group. PRK patients within 1 year after operation showed a minimal central corneal steepening, whereas PRK patients after more than 1 year showed a reaction similar to that in normal corneas (P < 0.05). CONCLUSION: Corneal stability is altered after PRK. After 1 year corneal stability seems to normalize due to stromal remodelling.

[Control of refraction in cataract surgery]. / Refraktionskontrol ved operation for grå staer.

The optical problems associated with cataract surgery are reviewed and methods to control the refractive state of the eye after the operation are presented. The clinical benefit of a calculation of intraocular lens (IOL) power from measurements of corneal curvature and eye length is demonstrated in a series of operated patients from two Danish eye departments. It is concluded, that the postoperative refraction can be controlled with a reasonable accuracy. Large and unexpected deviations in the postoperative refraction are avoided in this way, and in a number of cases it is possible to reduce the need for spectacles after the operation.

[Excimer laser therapy of recurrent corneal erosions]. / Excimer-laserbehandling af recidiverende korneale erosioner.

Recurrent corneal erosions have been recognized as a clinical problem for many years. Many patients with erosions do not respond satisfactorily to the standard treatment. In this paper we present a study of treatment of 24 patients with recurrent corneal erosions with the excimer laser, where 75% of the patients reported the treatment as a success.

[Surgery for nearsightedness]. / Operation for naersynethed.

Myopia can today be reduced or eliminated by refractive surgery. Excimer laser surgery of the cornea by surface sculpturing (photorefractive keratectomy) or intrastromal tissue removal (LASIK) are the most widely used techniques, although implantation of intra corneal ring segments for low myopia also appears promising. Treatment of high myopia (> 10 diopters) is still difficult although epikeratoplasty or phakic IOL implantation are present possibilities. The perfect surgery for myopia remains to be developed, but the existing techniques will without doubt be further optimised. In 10 years time, supra normal visual acuity may even be obtained when surgical, optical, and biological variables can be described and controlled in each individual undergoing refractive surgery.

[Diagnosis and treatment of conjuncitivitis]. / Diagnostik og behandling af conjunctivitis.

Conjunctivitis is a frequent reason for consulting a doctor. Most often treatment consist of antibiotics, even though sensitive bacteria are only rarely demonstrated. In our part of the world conjunctivitis is self-limiting, and it may be worth considering the effect of treatment, if any. With this in mind, diagnosis and differential diagnoses are reviewed, and with regard to treatment distinctions are made between neonatal conjunctivitis (gonococcus, chlamydia, virus), conjunctivitis in children (symptoms of upper respiratory tract infections), and conjunctivitis in adults and the elderly (dry eyes, epiphora in ectropion).

Corneatransplantation and matching for HLA-A and B.

Analysis of 140 corneatransplantations performed without selection showed a significant correlation between the HLA-A and B match and the clinical outcome. Furthermore, a highly significant correlation was found between rejection episodes and a positive direct cell-mediated lympholysis.