Haematopathology of 'Sjögren-mice': histopathological changes in spleens after semiallogeneic cell transfer.

Haematopoietic transplantation chimeras may be readily produced in adult mice, using F1-hybrids of selected inbred strains as recipients and mice from one of the parental strains as donors. We transplanted spleen cells from BALB/c donors into nonirradiateded F1-hybrids of BALB/c and CBA/H-T6. Both female and male recipients developed a primary Sjögren's syndrome-like exocrinopathy without signs of kidney disease. At long-term follow-up, 7(1/2) months after cell transfer, lymph nodes were enlarged, and spleens were diminished and irregular in shape. In general, changes in haematopoietic organs were more prominent in males. The results verify that although hybrid mice of either sex develop glandular manifestations comparable with primary Sjögren's syndrome, when the immune system is stimulated by semiallogeneic immunocytes, the evoked reactions in haematopoietic tissues show gender difference.

Photoreceptor allografts in a feline model of retinal degeneration.

BACKGROUND: Photoreceptor transplants provide a potential means to restore function in a degenerate retina and/or rescue degenerating host photoreceptors by trophic influences. We have examined photoreceptor allografts in the Abyssinian cat model of hereditary photoreceptor degeneration to determine the viability and influence of such transplants on the host retina. METHODS: Small pieces of 3- to 5-day-old normal kitten retina containing undifferentiated photoreceptors were injected into the subretinal space of adult Abyssinian cats at an early stage of retinal degeneration using standard vitreo-retinal surgical techniques. The retinas were examined by ophthalmoscopy and fundus photography, then by light and electron microscopy at different times after surgery. RESULTS: Such allografts survive for at least 6 months after surgery. The photoreceptors develop outer segments, invariably in rosettes. The transplants gradually integrate with the host retina but detach the host photoreceptor layer from the retinal pigment epithelium (RPE), which tends to reduce the number of host photoreceptors over the transplant. There is no slowing of the photoreceptor degeneration in neighboring non-detached retina. Inflammation or rejection was not detected. CONCLUSION: Undifferentiated, neonatal photoreceptor allografts survive and develop outer segments in the subretinal space of the Abyssinian mutant feline retina. The allografts gradually integrate with the host neural retina without inducing rejection. In the vicinity of the transplant there is increased loss of host photoreceptors, considered to be due to their detachment from the RPE layer. There is no evidence of any rescue of host photoreceptors elsewhere in this mutant retina.

Increased accuracy in MR diagnosis of uveal melanoma by carbohydrate loading. A simple method that reflects rapid metabolic change.

PURPOSE: To evaluate patients with clinically diagnosed uveal melanoma. MATERIAL AND METHODS: Forty-eight consecutive patients were examined with spin-echo (SE) and fast spin-echo (FSE) MR sequences that utilized glucose-fructose enhancement together with a subtraction technique on a 1.5 T unit. RESULTS: Twenty-seven patients were enucleated and the eyes histologically examined for tumours (spindle cell, mixed cell, and epitheloid cell). The remaining patients were referred for other treatment. There were no significant differences in T2 although T2 was longer in the amelanotic lesions. Carbohydrate loading in combination with a subtraction technique gave: an increased signal intensity; a prolongation of T2; and an increased tumour size. The FSE sequences were as good as the SE sequences in the visualization of uveal malignant melanoma. CONCLUSION: MR imaging performed with carbohydrate loading registers metabolic changes induced in the tumour. This gives the method great validity in the diagnosis of uveal melanoma. The method is especially useful in amelanotic tumours that have longer relaxation times than melanotic tumours. The SE technique can be replaced with the FSE technique.

Proton irradiation of malignant uveal melanoma. A five year follow-up of patients treated in Uppsala, Sweden.

Twenty patients with malignant uveal melanoma were treated at the The Svedbergh cyclotron in Uppsala from 1989 to 1991. Each tumour received a total dose of 54.6 Gy in four equal fractions on four following days. After treatment the melanoma in all eyes showed decrease in size combined with irradiation retinopathy. In eight patients the treatment was successful after five years. Nine eyes had to be enucleated, two due to recurrence and seven due to neovascular glaucoma. Three patients died, two from metastases and one from heart disease. In all patients the visual acuity was dependent on the distance between the irradiation field and the macula or optic nerve. Each patient suffered from transient post irradiation skin erythema and permanent loss of eyelashes and eyebrows when these were included in the irradiation field. The development of secondary glaucoma was positively correlated with tumour volume, but not to the age or sex of the patients. Histological examination of all the enucleated eyes revealed residual viable tumour without obvious radiation damage: mitotic figures were not identified. MRI examination, performed before and after treatment, demonstrated a marked shift in water binding properties after irradiation. The final visual acuity was dependent on the location of the tumour.

Long-term follow-up of proton irradiated malignant melanoma by glucose-fructose enhanced magnetic resonance imaging.

MR imaging is frequently used to diagnose uveal melanomas due to the characteristic short T2 relaxation time. T2 may be significantly prolonged within 2 h after ingestion of glucose and fructose due to changed water distribution in the melanoma. This method is used to follow melanomas for up to 6 years after proton beam irradiation. In the tumours, T2 was shortened in parallel in all the lesions during the first 9 months. After this, T2 increased only in tumours which showed recurrence. T2 determination and histopathological examination revealed no signs of recurrence in eyes which were enucleated due to neovascular glaucoma. It is concluded that MR imaging performed with carbohydrate loading, registers metabolic changes induced in the tumour, giving this method great validity in the follow-up of choroidal malignant melanoma after irradiation. Eighteen patients treated with proton beam for uveal melanoma at the cyclotron in Uppsala, Sweden, were followed.

Insulin receptors in human ocular tissues. Immunohistochemical demonstration in normal and diabetic eyes.

The alpha- and beta-subunits of the insulin receptor have been localised in human eyes by immunohistochemistry. In the normal eye staining for both receptor subunits was distinct at the same sites of the anterior part of the eye, i.e. cornea, smooth muscle and epithelium of the ciliary body and the lens capsule. In the retina, the receptor was clearly demonstrated in the nerve fibre layer, the ganglion cells and Müller cells, the outer nuclear layer, inner segments of rods and cones, the outer limiting membrane and in the pigment epithelium. In eyes with diabetic retinopathy, the receptor did not stain in the inner segments of the rods and cones and the staining of the other layers was weak. Endothelial cells stained positively in normal and diabetic eyes, but pericytes of normal and new vessels did not stain. The receptor staining did not change in cornea, iris, ciliary body and lens. All together, the study shows that alpha- and beta-subunits of the insulin receptor are present in the retina, and that the staining reaction for the receptor is reduced in diabetes. To what extent these findings are of importance for the development of diabetic retinopathy, remains to be clarified.

Recurrence of malignant melanoma after irradiation diagnosed by glucose-fructose enhanced magnetic resonance imaging.

Two patients who had been given ruthenium plaque treatment for uveal melanoma were MR examined due to suspected recurrence. Spin-echo sequences were applied using a 1.5 T equipment. The examinations were performed in both patients two times with a 2-h interval. Immediately after the first examination the patients were perorally given 20 g glucose and 10 g fructose. An increase of signal intensity (prolongation of the relaxation times) and size of the uveal lesions could be visualized by a subtraction technique in both patients after the carbohydrate loading. In agreement with our previous studies of malignant melanoma the changed metabolism in the uveal lesions indicated recurrence of the tumour. One eye was available for histological examination. The morphological difference between areas of recurring and degenerating tumour was clearly seen. Similar changes were not observed by ultrasound.

Cranio-orbital teratoma.

A new-born baby presented with a large, rapidly growing tumour. The tumour involved the right orbit, the anterior half of the right intracranial space and extended through the skull of the temporal region. The proptotic eye ruptured due to exposure. The tumour was extirpated when the patient was 12 days old. A large recurrence was extirpated two months later. The tumour was firmly bound to the medial aspect of the superior orbital fissure which probably was the location of origin of the tumour. Based on the size of the intracranial-and the orbital part of the tumour, it was classified as a secondary orbital teratoma. The tumour was macroscopically composed of cystic and solid areas. Microscopically it was seen to be a benign teratoma with tissue from all three germinal layers.

Effects of fructose on D-[6-3H]-glucose uptake and sorbitol metabolism of bovine retina in vitro.

In order to study the influence of fructose on sorbitol formation, bovine retinal tissue was incubated with different concentrations of glucose and fructose, and supplemented with tracer amounts of D-[6-3H]-glucose. Combining high-performance liquid chromatography (HPLC) with radioactivity determinations allowed detection of sorbitol and fructose derived from glucose in the incubation medium. In addition, the total amount of sorbitol was measured with a sensitive bioluminescence method. In this way, it was possible to distinguish between sorbitol formation from glucose and fructose. High concentrations of glucose in the medium increased the formation of sorbitol and fructose from glucose. Addition of fructose to the incubation medium diminished the sorbitol and fructose formation from glucose although the total amount of sorbitol increased significantly. Incubating retinal tissue with an aldose reductase inhibitor decreased sorbitol formation from glucose but did not influence the formation of sorbitol from fructose. Thus, the present findings clearly demonstrate the important influence exerted by fructose on sorbitol formation. The possible significance of the present finding is discussed with respect to diabetes retinopathy and retinal osmoregulation.

Increased lenticular aldose reductase activity and high incidence of congenital cataract in the offspring of diabetic rats.

In previous experimental studies we have found a 47% incidence of congenital cataracts among the fetuses of manifestly diabetic (MD) rats, and 4% of the offspring of normal (N) rats. The aim of the present study was to clarify the role of the sorbitol shunt in the pathogenesis of this congenital defect. Light microscopical evaluation of the fetal lenses revealed excessive formation of vacuoles in offspring of MD rats compared to N offspring. On gestational day 16 we found that aldose reductase (AR) activity was doubled in the lenses of fetuses of MD rats as compared to that of N fetuses. This difference as well as the net activity of AR declined in both groups during subsequent development. The sorbitol concentration was elevated more than ten-fold in the MD fetal lenses compared to N fetal lenses at all time points. The sorbitol concentration in both the MD and the N group decreased from day 16 to day 20 and increased again slightly on day 22. We conclude that the diabetic uterine milieu induces elevated AR activity and sorbitol formation in the fetal lens. This over-activity of the sorbitol shunt may produce metabolic and osmotic imbalances in the fetal lens, resulting in excessive vacuole formation and subsequent development of congenital cataract.

Facial nerve enhancement in Bell's palsy demonstrated by different gadolinium-enhanced magnetic resonance imaging techniques.

Twenty-one patients with an acute complete peripheral facial palsy, Bell's palsy, were examined by medium- and high-Tesla magnetic resonance imaging. Three contrast techniques were used: intravenous gadolinium; oral carbohydrate and intravenous gadolinium; and gadolinium, carbohydrate, and readministration of gadolinium. Three to 22 days after the onset of palsy, 12 of the 21 patients demonstrated ipsilateral facial nerve enhancement, most consistently in the meatal region, which is indicative of an inflammatory reaction. Two to 4.5 months after the onset, the enhancement had disappeared in 10 of the 12 patients. For the individual patient, contrast-enhanced magnetic resonance imaging gave little or no help in predicting the outcome of palsy. It is speculated that the intake of carbohydrate and readministration of gadolinium may improve the sensitivity of medium-high-Tesla magnetic resonance imaging in some cases.

Bioluminescence analysis of NAD(P)H and NAD(P)+ preventing mutual interference by selective nucleotide destruction and enhanced specific light emission.

Improved bioluminescence analysis of pyridine nucleotides has been designed based on the fact that the luminescence intensity expresses the velocity of the light formation. The bacterial luciferase system is, in principle, composed of two reactions with two different velocities, one for energy supply by the oxidation of NAD(P)H and the other for the subsequent light generation. The rate setting can be arranged such that an emission maximum is produced 30 to 40 s after mixing the sample with the light-yielding solution, hence providing for a convenient analytical performance. The maximal intensity which is easily recorded, e.g., by a tracking volt-meter, is proportional to the concentration of the reduced nucleotide. Discriminative analysis of the various pyridine nucleotides is facilitated by selective destruction of the oxidized forms with alkali and the reduced forms with acid. Erroneous conversion of NAD(P)H to NAD(P)+ may be induced by haemoglobin in a tissue sample but this is prevented by the presence of 2 mM ascorbic acid at the instant of the acidification. Simultaneous coupling of the ongoing reduction of a pyridine nucleotide to the oxidation in the bacterial luciferase system generates a light-yielding cycle which offers important advantages. With NAD(P)+ as the analytic target compound, direct measurement replaces a preceding separate conversion to NAD(P)H. The four nucleotide forms become determinable in a sample by combining selective destruction of either the reduced or oxidized species with a nucleotide-specific reduction in the cycle. Discriminative analyses are furthermore facilitated by the enhanced emission which is due to the energy derived from the continuous specific reduction, whereas initial light signals from side reactions fade out. It is often possible to suppress disturbing analytical errors by the design of the light-yielding cycle. If the rate of the dehydrogenase reactions is kept low compared with the overall rate of the luciferase system, moderately impaired function of some of its components may only give rise to a slight and tolerable decrease in emission intensity. Kinetic evaluations and model experiments are presented and supplemented with applications to tissue samples.

Concomitant changes in MR image and morphology induced by glucose and fructose in B16 mouse melanomas.

The short proton relaxation times in the MR images of malignant melanomas make them different from most other tumors. We have previously shown that the T1 and T2 signals may be significantly influenced when the water distribution of the tumor is changed in vivo and in vitro. In the present work T1 and T2 were estimated and compared with the electron microscopy picture in subcutaneously implanted B16 melanomas in mice. Two hours after the mice were given an i.p. injection of 0.9% NaCl containing 10% glucose and 10% fructose (9 mice) both the T2 components were markedly and the T1 slightly prolonged. At the same time the electron microscopy picture displayed swelling of the melanocytes together with a marked decrease in number and size of their mitochondriae. There were no changes in the MR image or the melanocyte structure in control mice injected with 0.9% NaCl (9 mice) or 0.9% NaCl containing 10% fructose. It is concluded that the changed MR image may be coupled to the metabolism in melanoma.

Influence of glucose, fructose and aldose reductase inhibition on retinal sorbitol metabolism.

The sorbitol shunt has been studied in bovine retinal tissue at incubation times from 1 to 24 h. It was shown that an elevated glucose concentration (22 mM) of the medium was accompanied by a slight increase in sorbitol content already after 3 h. At longer incubation times, but lower glucose concentration (11.1 mM) there was a similar increase. Addition of an aldose reductase (AR) inhibitor prevented the sorbitol increase. Addition of fructose to the medium significantly increased the sorbitol accumulation above the effect seen with glucose alone and this effect was not influenced by the AR inhibitor. Thus the sorbitol concentration in the retina may be increased after a short incubation time and further enhanced by the presence of fructose.

Changes in MR of malignant melanomas induced by glucose and fructose. A clinical and experimental investigation.

MR imaging has been performed on malignant melanomas in vitro and in vivo. Changes of the water content in an enucleated malignant melanoma in vitro were followed by significant changes of the T1 and T2 values. In mice with implanted subcutaneous melanoma similar changes could be obtained after injection of glucose and fructose intraperitoneally. Malignant melanoma of the eye could be influenced in the same way in 10 consecutive patients after oral intake of glucose and fructose. The present study shows that the MR images may be significantly changed after a few hours by altered metabolism induced by glucose and fructose. It is anticipated that this is due to changes within the tumor leading to different water distribution. The finding may be of importance as a further help for diagnosing malignant melanoma of the eye.

The narrow proton beam therapy unit at the the Svedberg Laboratory in Uppsala.

The synchrocyclotron at the The Svedberg Laboratory (TSL) in Uppsala is now reconstructed and can presently operate with fixed frequency and proton energies up to 100 MeV. A first treatment room with a narrow proton beam unit for therapy of eye tumours is now in operation. Therapy of eye melanomas started in April, 1989 and during 1989 and 1990, 19 patients were treated with 72 MeV protons. The narrow beam unit provides a fixed horizontal beam and the patient is treated in a seated position. The present paper describes mainly the technical aspects of the unit which so far has been used only for eye melanomas. In the future, modifications of the unit will allow therapy of intracranial targets when higher proton energies are available. In its final form, the proton therapy facility at TSL will harbour a second treatment unit. Here a rotating gantry for 200 MeV protons will provide a broad beam, which will enable treatment of tumours located anywhere in the body.

Effects of long-term chloroquine exposure on the phospholipid metabolism in retina and pigment epithelium of the mouse.

The influence of 6 month's treatment of normal mice with chloroquine on neuroretina and retinal pigment epithelium has been investigated biochemically and morphologically. All classes of neuroretinal phospholipids, except lysophosphatidylcholine, showed increased 14C-glucose incorporation after chloroquine treatment. No metabolic changes were observed in the pigment epithelium after the chloroquine treatment. Morphological signs of phospholipidosis were only evident in the ganglion cells of the neuroretina. It is concluded that long-term treatment with chloroquine does not affect pigment epithelium phospholipid metabolism but leads to morphological and biochemical signs of phospholipidosis in the neuroretina.

DNA replication in transplanted and endogenous pancreatic islets of obese-hyperglycemic mice at different stages of the syndrome.

It was the aim of the present study to examine, at different stages of the obese-hyperglycemic syndrome, rates of islet-cell DNA replication in endogenous pancreatic and grafted islets of such mice. For this purpose obese-hyperglycemic mice were given an islet transplant prepared from lean mice under the kidney capsule. Two weeks later the animals were given an IP injection of 3H-thymidine one hour before being killed. Autoradiography of pancreas and kidney sections indicated the highest labeling index (LI) for the endogenous islet cells in the youngest obese mice (6 weeks old), which was more than fivefold higher than that of lean, normoglycemic controls. The LI, however, decreased extensively with age. The transplanted islets had LI, which were higher and constant during the most intense period of the syndrome, but again there was a decrease in the oldest mice. By isolating and transplanting islets of the oldest obese mice (greater than 12 months) into younger obese mice, it was possible to revive the high cell replicatory activity of the ob/ob islets. Starvation for three days was found to markedly decrease the rate of islet cell DNA replication. Adrenalectomy of obese-hyperglycemic mice resulted in a decrease of the serum glucose and insulin concentrations; concomitantly, there was a decrease of LI of both transplanted and endogenous islets. Thus, it seems as if the attenuation of the hyperglycemia is most probably responsible for the decline of the islet cell replication with increasing age in obese-hyperglycemic mice.

Design of coupled reactions for simplification of bioluminescence analysis.

Luminescence analysis may be applied to many substances by arranging a prior reaction producing a species entering the light-emitting reaction. Under favourable conditions the two consecutive reactions are carried out simultaneously as a one-step procedure. In a bioluminescence assay, luciferase stability is frequently a problem, making it desirable to develop analytical schemes where the analytical response becomes largely independent of any impaired luciferase activity. The value of maximal emission or an approached steady-state level is a convenient and usually well-defined analytical parameter. When recording this level it is important to design the participating reactions in a way that compensates for changes in luciferase activity.

Influence of sorbitol accumulation on growth and development of embryos cultured in elevated levels of glucose and fructose.

Day-9 rat embryos, cultured for 48 hours in 67 mmol/l D-glucose, showed impaired growth, increased malformation rate and elevated concentration of sorbitol compared to embryos cultured in medium without additional glucose supplement. Supplementing the high-glucose cultures with an aldose reductase inhibitor markedly decreased the sorbitol levels without affecting the malformations or the retarded growth of the embryos. Since embryos cultured in 6.6 mmol/l D-fructose showed normal growth and morphology despite increased accumulation of sorbitol, this study suggests a dissociation between raised sorbitol levels and glucose-induced maldevelopment in rat embryos.