Generation of transgenic mice with mild and severe retinal neovascularisation.

AIM: To generate a mouse model for slow progressive retinal neovascularisation through vascular endothelial growth factor (VEGF) upregulation. METHODS: Transgenic mice were generated via microinjection of a DNA construct containing the human VEGF165 (hVEGF) gene driven by a truncated mouse rhodopsin promoter. Mouse eyes were characterised clinically and histologically and ocular hVEGF levels assayed by ELISA. RESULTS: One transgenic line expressing low hVEGF levels showed mild clinical changes such as focal fluorescein leakage, microaneurysms, venous tortuosity, capillary non-perfusion and minor neovascularisation, which remained stable up to 3 months postnatal. Histologically, there were some disturbance and thinning of inner and outer nuclear layers, with occasional focal areas of neovascularisation. By contrast, three other lines expressing high hVEGF levels presented with concomitantly severe phenotypes. In addition to the above, clinical features included extensive neovascularisation, haemorrhage, and retinal detachment; histologically, focal to extensive areas of neovascularisation associated with retinal folds, cell loss in the inner and outer nuclear layers, and partial retinal detachment were common. CONCLUSIONS: The authors generated four hVEGF overexpressing transgenic mouse lines with phenotypes ranging from mild to severe neovascularisation. These models are a valuable research tool to study excess VEGF related molecular and cellular changes and provide additional opportunities to test anti-angiogenic therapies.

Identification and quantitation of steroids in sulfate fractions from plasma of pregnant chimpanzee, orangutan, and rhesus monkey.

Steroids in the mono- and disulfate fractions from plasma of pregnant chimpanzees (Pan troglodytes), orangutans (Pongo pygmaeus), and a rhesus monkey (Macaca mulatta) were identified by gas chromatography/mass spectrometry and quantitated by gas-liquid chromatography on open tubular glass capillary columns. Whereas the average total concentrations were 4-5 times lower, 2.3-5.5 mumol X 1(-1) vs. 10.7-19.8 mumol X 1(-1), the pattern of steroid sulfates in the chimpanzees and orangutans were very similar to that previously found in pregnant women. Twenty one steroids were identified. The 3 beta-hydroxy-5-ene steroids were the same as in humans. Saturated pregnane derivatives were predominant and increased with time during pregnancy. Four isomers each of 3-hydroxypregnan-20-one and pregnane-3,20 alpha-diol were found, having 3 alpha, 5 alpha, 3 beta, 5 beta, 3 alpha, 5 beta, and 3 beta, 5 alpha stereochemistry, respectively. The relative proportion of disulfates was slightly lower in the great apes (15-28% of the total steroid sulfates) than in humans (23-33%). The monosulfate of 5 beta-pregnane-3 alpha, 20 alpha-diol constituted 12-14% of the total in chimpanzees and 3-4% in orangutans and humans. The monosulfate of 5 alpha-pregnane-3 beta, 20 alpha-diol constituted 5-7% in chimpanzees and 11-16% in orangutans and humans, whereas the disulfate was relatively less abundant in the great apes, 4-8%, than in humans, 10-18%. Although difficult to quantitate accurately, the chromatograms indicated that the proportion of 3 beta, 5 beta-isomers was higher in great apes than in women. The presence of 5 alpha-pregnane-3 beta, 16 alpha, 20 alpha-triol and 5 alpha-pregnane-3 alpha, 20 alpha, 21-triol indicated that hydroxylations of steroid sulfates in the great apes were similar to those in pregnant women. The steroid sulfate pattern in the rhesus monkey was completely different, 3 beta-hydroxy-5-ene steroids constituting over 95% of the total. Dehydroepiandrosterone sulfate was by far the predominant steroid, followed by the disulfates of 5-androstene-3 beta, 17 beta-diol and 5-pregnene-3 beta, 20 alpha-diol and the monosulfate of 5-androstene-3 beta, 16 alpha, 17 beta-triol. The results are discussed in relation to previous knowledge of progesterone metabolism in different animal species. So far, great apes are the only species showing the same pattern of steroid sulfates in plasma as humans.

Identification and measurement of urinary estrone, estradiol-17 beta, estriol, pregnanediol and androsterone during the menstrual cycle of the orangutan.

Urinary estrone, estradiol-17beta, estriol, pregnanediol and androsterone were identified and measured during 3 menstrual cycles in 2 female orangutans. In 2 of the cycles, the animals excreted 1-8 mug/day estrone, 0.5-6 mug/day estradiol-17beta, 1-8 mug/day estriol, 20-206 mug/day pregnanediol and 120-522 mug/day androsterone during the first half of the menstrual cycle. In the second half of the cycle, corresponding values were 3-21 mug/day estrone, 2-10 mug/day estradiol-17beta, 1-9 mug/day estriol, 54-800 mug/day pregnanediol and 90-1158 mug/day androsterone. In 1 cycle, the estrogen values for the second half were considerably higher, possibly due to the animal becoming pregnant just before this study commenced. The values for estrone and estradiol-17beta are similar to those found in the human and chimpanzee menstrual cycle. The values for estriol were lower than in the human but higher than in the chimpanzee. Levels for urinary pregnanediol and androsterone were significantly lower than in the human. Variations during the menstrual cycle for estrone were characterized by a midcycle peak followed by a second peak in the luteal phase. No definite pattern was apparent for estradiol-17beta or estriol. Both urinary pregnanediol and androsterone levels were low during the first half of the cycle, started to rise just after midcycle, and showed a peak during the second half of the menstrual cycle.

Adrenarche: a survey of rodents, domestic animals, and primates.

The concentrations of the adrenal steroids dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and delta 4-androstenedione (delta 4-A) have been measured by RIA before and after sexual maturation in plasma of rodents, domestic animals, and primates to determine whether these species exhibit and adrenarchal process comparable to man. The average concentrations of DHA and DHAS were less than 60 ng/dl and 5 microgram/dl, respectively, in plasma of sexually mature rodents and domestic animals, and a significant increase in the plasma DHA level after sexual maturation was seen only in the rabbit and dog. The concentrations of DHA, DHAS, and delta 4-A in 21 rhesus monekeys from 0-3 yr of age were 2021 +/- 235 ng/dl (mean +/- SE), 357 +/- 60 microgram/dl, and 107 +/- 9 ng/dl, respectively, and did not increase during sexual maturation. By contrast, DHA, DHAS, and delta 4-A levels in plasma of chimpanzees were 5.9-fold, 3.3-fold, and 4.8-fold greater, respectively, in 7- to 22-compared to 0- to 3-yr-old animals. Temporally, the increase in DHA levels in the chimpanzee is apparent at 5 yr and this precedes the increase in gonadal steroids, as is characteristic of human adrenarche. It is apparent that adrenal androgen levels and their developmental patterns differ markedly among species, and that among the species examined, only the chimpanzee exhibits an adrenarche comparable to that of man.

Effect of contact lens removal or displacement on intraocular pressure.

We conducted a controlled, prospective study involving 102 healthy volunteers to determine the effects of contact lens removal and contact lens displacement on subsequent intraocular pressure measurements. Intraocular pressure was measured at baseline, immediately after contact lens removal or displacement, and 5 minutes thereafter. The method of lens removal was either sliding displacement (51 eyes) or plucking (51 eyes). Right eyes were used for the test, and left eyes served as controls. Compared with baseline measurements, mean intraocular pressure immediately after plucking the lens was 0.8 mm Hg lower (P less than .01) and after sliding the lens was 0.5 mm Hg higher (P less than .05). Intraocular pressure changes of 4.0 mm Hg or more occurred in eight (15.6%) of 51 eyes in the pluck group vs four (7.8%) of 51 control eyes and in two (3.9%) of 51 eyes in the slide group vs zero of 51 control eyes. Compared with baseline, significant differences (P less than .05) were not found 5 minutes later. Clinicians who wish to minimize the influence of contact lens removal on tonometry readings should wait several minutes after contact lens removal before measuring intraocular pressure.

Comparison of Oculab Tono-Pen readings obtained from various corneal and scleral locations.

When estimating intraocular pressure in patients who are uncooperative or who have central corneal disturbances, the physician may find it either impractical or undesirable to place the small tip of a portable electronic applanation tonometer (Tono-Pen) over the central cornea. To gauge better the usefulness of Tono-Pen readings obtained from various locations, we compared such readings measured through the central cornea, midperipheral cornea, limbal cornea, and sclera of 15 cannulated eye bank eyes. Mean Tono-Pen readings from the midperipheral and clear limbal cornea did not differ significantly from central corneal readings over a 10- to 35-mm Hg range of intraocular pressures and were within +/- 2.4 mm Hg of mean central corneal readings. Mean readings taken from the sclera, however, were 8.8 to 17.0 mm Hg higher than mean central corneal readings over the 10- to 40-mm Hg range. We concluded that multiple noncentral corneal readings with the Tono-Pen provided a useful approximation of intraocular pressure, whereas scleral readings did not.

Tolerance to effects of clonidine and morphine on sulfobromophthalein disposition in mice.

Chronic treatment of mice with clonidine or morphine caused tolerance to the analgesic and thermoregulatory effects of these drugs. After chronic morphine, mice also became tolerant to the analgesic and thermoregulatory effects of clonidine. Cross tolerance to the hypothermic effect of morphine was demonstrated after chronic clonidine administration, but no diminution of morphine-induced analgesia could be shown. Morphine and clonidine acutely increased the retention of sulfobromophthalein (BSP) in plasma and liver. Chronic dosing with morphine or clonidine caused partial tolerance and cross-tolerance to the rise in hepatic BSP caused by an acute challenge with either agonist. However, both drugs elevated plasma BSP levels similarly in tolerant and non-tolerant mice. Thus, regimens which readily induced tolerance to the analgesic and hypothermic effects of morphine or clonidine were only partially effective in modifying the acute hepatobiliary effects of these drugs.

Metabolism of 4-14 C-progesterone in the adult female chimpanzee.

The metabolism of 4- 14-C-progesterone was studied in two adult female chimpanzees (Pan troglodytes). After intravenous injection of 4- 14-C-progesterone, urine was collected for 5 days. The urinary conjugates were hydrolyzed with Glusulase and the extracts purified by chromatography on silica gel, paper and alumina and then crystallized to constant specific activity with or without carrier steroid. The major metabolite in both experiments was pregnanediol which accounted for 39.8% and 49.5% of the recovered dose respectively. Pregnanolone (0.6% and 2.1%) and pregnanetriol (0.1% and 1.5%) were also isolated in smaller quantities. These data suggest that the pattern of progesterone metabolism in the chimpanzee is similar to that of man in that pregnanediol is the major metabolite whereas androsterone is not.

Chimpanzee reproductive senescence: a possible model for evolution of the menopause.

Reproductive senescence was studied in 2 female Pan troglodytes and one Pan paniscus over 40 yr old. Menstrual cycle data for these animals, in the last 3 yr, when compared to that of the same animals in previous years and to records obtained between 1967 and 1980 on 51 Pan troglodytes between 18-39 yr old, demonstrated increased length of menstrual cycles, as shown by decreases in frequency of menses. Oestrone glucuronide and pregnanediol glucuronide were measured in 24-h urine samples by radioimmunoassay. The pattern of excretion differed only slightly from that of younger animals. One Pan troglodytes had reduced oestrogen levels, but normal gonadotropin levels. The single ovulatory peak of LH normally seen in younger animals was replaced by multiple peaks in the Pan troglodytes. Menopause was observed in the Pan paniscus as documented by cessation of menstrual cyclicity, elevation of gonadotropin levels, and exaggerated response to injection of 100 microgram of gonadotropin-releasing hormone. The FSH: LH ratio was reversed in this animal. Histology of the ovaries of the Pan paniscus revealed a paucity of primary and developing follicles, and an increase in fibrous tissue. The Pan troglodytes appear to be peri-menopausal. This data suggests that the chimpanzee may serve as a model for certain phases of reproductive senescence in the human.

Comparison of monoclonal antibody-based competitive and indirect enzyme-linked immunosorbent assays for the diagnosis of swine trichinosis.

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of swine trichinosis has been developed using a biotinylated monoclonal antibody and an avidin-enzyme conjugate. The assay is based on competitive binding between swine serum antibodies and a monoclonal antibody specific for an antigenic determinant present on proteins from Trichinella spiralis excretory-secretory products with molecular weights of 45,000, 49,000, and 53,000. The competitive ELISA reliably detected pigs infected experimentally with T. spiralis and eliminated false-positive reactions in pigs infected with other swine nematodes, particularly Trichurus suis. When the competitive ELISA and an indirect ELISA using affinity-isolated antigen were compared using serum from pigs with naturally-acquired infections of T. spiralis, both tests were highly effective in detecting infected animals.

Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory--secretory antigen.

An excretory--secretory (ES) antigen was used in a serodiagnostic enzyme-linke immunosorbent assay (ELISA) for swine trichinosis. ELISA procedures included a double-antibody test, using either an anti-swine IgG or a protein A enzyme conjugate, and a triple-antibody test using a pig IgG heavy-chain specific second antibody with a conjugated third antibody. The ES antigen was effective in eliminating all false-positive reactivity in sera from farm-raised hogs. The triple-antibody procedure was more sensitive and demonstrated a greater efficiency in detecting positive animals and early seroconversions. Naturally-infected pigs with worm burdens as low as 0.01 larvae per gram (LPG) of diaphragm were seropositive using these procedures. Seroconversion in experimentally-infected animals receiving low doses of muscle larvae (500) occurred considerably later than in animals receiving high doses (10000). This might account for false-negative reactions in naturally-infected animals with very low (less than 0.1 LPG) worm burdens.

Postovulatory predecidual development in the baboon, chimpanzee, and human.

The development of predecidual tissue during the late postovulatory phase was evaluated by light microscopic study in the baboon, chimpanzee, and human being. While the predecidual cells of these species appeared to be similar morphologically, the degree of predecidual differentiation was greater in humans and chimpanzees than in baboons. The evidence indicated that the presence of a blastocyst was not required for decidualization to occur in these three primates. Further, decidualization was not dependent on coitus in chimpanzees and baboons, and was probably not essential in humans either.

The interrelationship between sex skin swelling and the urinary excretion of LH, estrone, and pregnanediol by the cycling female chimpanzee.

The interrelationship between sex skin swelling and the urinary excretion of luteinizing hormone and sex steroids was investigated during ovulatory menstrual cycles in adult female chimpanzees. Estrone was excreted in two peaks, one during the preovulatory and the other during the midluteal phase. Maximum swelling of the sex skin was attained several days before the preovulatory estrone peak. The LH surge preceded or accompanied beginning detumescence of the sex skin which, in turn, was closely correlated with a rising excretion of pregnanediol. Urinary measurements provide integrated estimates of the concentration of fluctuating hormones and, in addition, are safer and easier to make than blood measurements in this species.

Shedding of antibody complexes by Strongyloides ratti (Nematoda) larvae.

Antigens on the epicuticular surface of Strongyloides ratti infective third-stage larvae (L3) could be demonstrated by an indirect fluorescent antibody technique under certain conditions. Infective L3 shed anti-antibody complexes at room temperature, but not at 4 C or in the presence of sodium azide or colchicine. Shedding of antibody did not appear to involve epicuticular antigens, and only occurred when anti-rat IgG was complexed to rat anti-larval antibody. However, parasitic L3 removed from rats did not exhibit this shedding reaction, suggesting that an important developmental change in cuticle physiology occurs during the transition from a free-living existence to a parasitic mode. The ability to shed foreign objects from the epicuticle of free-living infective L3 may be a defensive or protective response to soil microorganisms.

Monoclonal antibody-purified antigen for the immunodiagnosis of trichinosis.

Monoclonal antibodies generated from spleen cells of Balb/cJ mice infected with Trichinella spiralis reacted with antigenic determinants present in some other common swine parasites, especially Trichuris suis. An antigenic determinant unique to T spiralis was recognized by one immunoglobulin M monoclonal antibody (7C2C5); this determinant was found on larval T spiralis proteins with molecular weights of 53,000, 49,000, and 45,000. These proteins were components of in vitro-derived worm excretory-secretory products. Immunoperoxidase staining techniques and reactions of the monoclonal antibody with living parasites indicated that the source of the antigen was the stichocyte. Two of the proteins (53,000 and 49,000 molecular weight) possessing this determinant were isolated by monoclonal antibody-affinity chromatography, using hybridoma 7C2C5. When used in a serologic enzyme-linked immunosorbent assay, the affinity-isolated antigen was able to completely eliminate false-positive reactions with serum from healthy swine and to detect all pigs infected with T spiralis.

Evaluation of three immunodiagnostic procedures for porcine strongyloidiasis.

An indirect fluorescent antibody test (IFAT), an indirect radioimmune antibody test (IRAT), and an enzyme-linked immunosorbent antibody assay (ELISA) were compared for their efficacies in detecting antibody to Strongyloides ransomi in pigs. The greatest sensitivity was exhibited by the ELISA, using a soluble, whole larval extract. The 2 indirect antibody assays, IFAT and IRAT, using intact 3rd-stage larvae (L3) were comparable in sensitivities. The use of live L3 in the IFAT and IRAT presented technical problems, because the larvae shed antibody complexes at ambient room temperature. This could be inhibited by the addition of 20 mM of sodium azide to all working solutions or by doing the tests at 4 C. Fixation of the L3 by either formalin or heat also eliminated the shedding problem. The specificities of the ELISA and IFAT were supported by the negative results obtained with serum from pigs infected with Ascaris suum, Stephanurus dentatus, and Trichinella spiralis.

Morton's interdigital neuroma: a comprehensive treatment protocol.

One hundred fifteen patients with signs and symptoms of Mortons' interdigital neuroma were studied in an attempt to evaluate the efficacy of a staged treatment program. The first stage consisted of patient education, footwear modifications, and metatarsal head relief. The second stage consisted of a steroid/local anesthetic injection into the affected interspace. The third stage was surgical excision of the inflamed interdigital nerve. Overall, 97 of 115 patients (85%) believed that they had improved with the treatment program. Twenty-four patients (21%) eventually required surgical excision of the nerve and 23 of 24 patients (96%) had satisfactory results. The results of the staged treatment protocol were very satisfactory and patient satisfaction was high.