Ultrastructural observations on life cycle stages of Pneumocystis carinii.

An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristae that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytes of the lung alveolus.

Analysis of the developmental stages of Pneumocystis carinii, in vitro.

Rat derived Pneumocystis carinii cultured in vitro was analyzed by a variety of light microscopic and staining techniques for identification of developmental stages and changes in these populations over time in culture. Evaluation of culture dynamics based on a limited number of life cycle stages indicated that trophic replication contributed significantly to the observed growth in vitro. Identification of additional life cycle stages, particularly early cyst forms, suggested that the process of maturation from early precyst to mature cyst may also contribute to the net growth. All developmental stages identified from culture were verified by their presence in stained impression smears of infected rat lung. Based on these data, a tentative life cycle for P. carinii in vitro has been proposed.

Techniques for examining Pneumocystis carinii in fresh specimens.

Pneumocystis carinii was examined in fresh preparations of infected rat lung homogenates and tissue culture supernatants by a variety of light microscope techniques, vital dyes, and histologic stains. Phase-contrast microscopy, Nomarski interference-contrast microscopy, and bright-field microscopy with oblique illumination provided excellent views of P. carinii. Erythrosin B, and to a lesser extent trypan blue, were helpful in assessing organism viability. The use of Triton X-100-Giemsa stain permitted differentiation of the developmental stages in the P. carinii life cycle. The techniques developed here are easily adaptable to the microbiology laboratory and thus should have important clinical and research applications.

Pneumocystis carinii: growth variables and estimates in the A549 and WI-38 VA13 human cell lines.

Recent studies indicate that rat Pneumocystis carinii can be propagated in the A549 cell line, an alveolar epithelioid cell line derived from human lung carcinoma. In the present study, growth of P. carinii was compared in the A549 cell line and the WI-38 VA13 subline 2RA, an SV40 transformed derivative of the human fetal fibroblast cell line with epithelioid morphology. Similar P. carinii growth occurred in both cell lines under optimal conditions, but the WI-38 VA13 cell line was usually more sensitive to changes in the culture system. Growth of P. carinii was affected by temperature, environmental gas mixture, motion of the cultures, and source and concentration of serum additives, but not by the presence of antibodies in the medium. A technique was developed for quantitating P. carinii in the lung inoculum which permitted analysis of P. carinii growth during the first 24 hr of culture. Inverted microscope and oil immersion phase-contrast microscopy were very helpful in monitoring the organism's stages of development and viability. Thus, this culture system should be helpful in establishing standard methodology for in vitro work with P. carinii.

Effect of chronic ethanol consumption on the pancreas of the hamster.

The purpose of this study was to determine the effect of chronic ethanol consumption on pancreatic morphology and biochemistry in the hamster, with special attention to lipid changes. A control group of Syrian golden hamsters fed a synthetic liquid diet was compared to an ethanol group pair-fed the same diet with ethanol substituted for 35% of the carbohydrate calories. The animals were sacrificed at 7 weeks and 3, 6, 9, and 12 months. After 12 months of ethanol consumption, a significant decrease in pancreatic triglycerides and a significant increase in pancreatic RNA was seen. These changes were associated with a rise in pancreatic weight and protein content in the ethanol group, reversing a six-month decline in these values. This rise in RNA and protein in the ethanol-treated group corresponded with the appearance of large abnormal zymogen granules. Other ultrastructural features such as lipid droplets, mitochondria, and endoplasmic reticulum were not altered by ethanol. Ethanol did increase the water content of the pancreas. Although ethanol had no effect on the fasting levels of insulin or pancreatic polypeptide, the fasting serum gastrin immunoreactivity was significantly lower in the ethanol animals. This study shows that chronic ethanol consumption produces a metabolic change in the hamster by 12 months which is suggestive of increased protein synthesis with a decrease in pancreatic triglycerides and no lipid droplet formation.

Photochemical lesions in the primate retina under conditions of elevated blood oxygen.

Under conditions of nonthermal radiant exposure to blue light (440 nm) the primate retina can suffer photic injury by a mechanism that must be photochemical in nature. We have examined the effects of elevated blood oxygen (pO2 of 270 mmHg) on the retinal photosensitivity to blue light in two macaque monkeys by histologic analysis of 12 lesions at 1 to 57 days after irradiation. The retinal image diameter from a xenon arc lamp source was 1 mm, the duration of exposure was 100 sec, and the radiant exposures ranged from 11 to 36 J/cm2. When blood oxygenation is not elevated experimentally, the threshold radiant exposure for a blue light lesion to be visible funduscopically at 2 days postexposure is about 30 J/cm2. At a high blood pO2 level, a radiant exposure of only 11 J/cm2 gave a funduscopically visible lesion at 1-day postexposure. This large increase in retinal sensitivity to blue light damage appears to be due to photodynamic action. The only direct effect of elevated blood pO2 on the retina observed histologically was the presence of numerous granules in the cells of the retinal pigment epithelium (RPE). However, there was no apparent histopathology associated with the elevation of blood pO2 alone. Analysis of the various photic lesions showed only moderate damage to the neural retina, but a strong response was seen in the RPE. This is the histopathologic pattern of a typical blue light lesion shown in previous studies but more severe. So the effect of elevated blood O2 is to increase retinal sensitivity to photic damage, to lower the damage threshold, and to increase the severity of damage at a given radiant exposure. The status of lesions at 23 and 57 days postexposure suggests that such injuries are repairable.

Preservation of retinal function in the RCS rat by laser treatment.

The Royal College of Surgeons (RCS) rats have been used as a model for human retinitis pigmentosa. Studies on these animals have shown that the degeneration of the retina is associated with a buildup of debris produced by shed rod outer segment discs. It has been reported that localized laser lesions can increase phagocytosis in these rats. This study examined the effect of laser burns on the function of the retina of the RCS rats. One eye of 19-day-old RCS rats was treated with laser and the other eye used as control. The retinal function was measured by electroretinography at 5, 10, 15, 20, 25, 30, and 40 days after lesioning. The morphology of the retina was examined at 24 and 43 days after laser treatment. The results show that the retinal function in the treated eye was improved at all intervals and that this improvement was significant at 15, 20, and 25 days. Morphologic examination showed a significant reduction in debris accumulation in the area of the laser spot. However, at sites distal to the laser burns, no morphologic difference between the treated and untreated eyes was noted. It is concluded that the progress of retinal dysfunction in the RCS rats can be retarded by laser treatment.

Basic mechanisms underlying the production of photochemical lesions in the mammalian retina.

Extended exposure (100s) of the macaque retina to blue light (400-500nm) produces a photochemical type or types of lesion. The basic mechanisms responsible for such photic damage are unknown but the toxic combination of light and oxygen leading to the free radicals O-.2, H2O2, OH., and O2(1 delta) have been suggested as a possible source of the phototoxicity. To test this hypothesis, the radiant exposure (J. cm-2) to short wavelength light (435-445nm) required for minimal damage in the macaque retina is under investigation as a function of oxygenation and after administration of substances known to either inhibit/scavenge radicals or act as anti-inflammatory/anti-oxidant agents. Substances under study include beta-carotene, steroids, catalase and SOD. Here we report radiant exposure in J.cm-2 needed to produce a minimal lesion vs oxygenation as measured by partial pressure of O2 in arterial blood (Po2). There is a sharp drop in the radiant exposure threshold with increase in the partial pressure of O2 in arterial blood, e.g. 30 J.cm-2 at 75 torr to 10 J.cm-2 at 271 torr, a factor of 3. Methylprednisolone injected intravenously one hour before exposure (125 mg) has been shown to raise the threshold for retinal damage in two macaques by a factor of approximately 2. Another animal fed beta-carotene (7.5 mg daily) over a period of 3 months has been exposed to blue light at several levels of oxygenation. The results suggest a protective effect.

Action spectrum for retinal injury from near-ultraviolet radiation in the aphakic monkey.

We found that the action spectrum for retinal damage (determined by the fundus photographic appearance of a minimal lesion immediately after exposure) extends into the near-ultraviolet by exposing three aphakic eyes from rhesus monkeys to 405-, 380-, 350-, and 320-nm wavelengths produced by a 2,500-W xenon lamp equipped with quartz optics and 10-nm interference filters. Exposure times were 100 and 1,000 seconds and the spot diameter on the retina was 500 micrometers. The retina was six times more sensitive to 350- and 325-nm wavelengths than to blue light (441 nm). Both ophthalmoscopic and histologic data showed that near-ultraviolet lesions differed in important respects from blue-light lesions. Near-ultraviolet produced irreparable damage to rod and cone photoreceptors.

Histologic analysis of photochemical lesions produced in rhesus retina by short-wave-length light.

The photopathology of retinal lesions produced by extended exposure (1000 sec) to low corneal power levels (62 microW) of blue light (441 nm) was investigated by light microscopy in 20 rhesus eyes over an interval ranging from 1 hr to 90 days after exposure. Results indicate a nonthermal type of photochemical lesion originating in the retinal pigment epithelium and leading to a histological response with hypopigmentation which requires 48 hr to appear. This type of lesion helps to explain solar retinitis and eclipse blindness and has significance for aging and degenerative changes in the retina.