Animal model of conjunctival primary acquired melanosis.

A condition clinically identical to human conjunctival primary acquired melanosis (PAM) was induced in 16 of 20 Dutch (pigmented) rabbits after weekly topical 60-microliters applications of a 1% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in acetone. Pigment stippling appeared in the conjunctiva as early as 5 weeks after the initial carcinogen application. Confluent patches of flat pigmentation appeared over the palpebral conjunctiva 18 weeks after the onset of treatment and showed progressive lateral enlargement and darkening. Histologically, a spectrum of changes from increased melanin production and melanocytic hyperplasia without atypia (resembling the human condition of PAM without atypia) through atypical melanocytic hyperplasia (resembling human PAM with atypia) was identified. The development of this model permits further investigations to explore and explain the clinically observed phenomenon of waxing and waning of PAM and its promotion to conjunctival malignant melanoma.

Spontaneous hepatitis in Long-Evans rats. A potential animal model for fulminant hepatitis in man.

Spontaneous hepatitis associated with severe jaundice occurred in 90% of an inbred strain of Long-Evans rats. The rapidly progressive syndrome was characterized by abrupt onset, hyperbilirubinemia and increased serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, associated with massive and multifocal necrosis of the liver. This strain should provide a useful animal model for analysis of the pathogenesis of fulminant hepatitis in humans.

Treatment of murine lupus with monoclonal antibody to L3T4. I. Effects on the distribution and function of lymphocyte subsets and on the histopathology of autoimmune disease.

Monoclonal antibodies (MoAb) to L3T4 have been used successfully to suppress autoimmunity in murine models for several human autoimmune diseases. To clarify the immunologic and clinical consequences of treatment with anti-L3T4, we examined the effects of chronic administration of anti-L3T4 on the composition of lymphoid organs, the function of lymphocytes, and the histopathology of autoimmune disease in lupus-prone NZB/NZW F1 (B/W) mice. Weekly treatment with anti-L3T4 (2 mg/mouse) from age 5 to 8 months depleted L3T4+ cells from the spleen and lymph nodes, and prevented the development of splenomegaly and lymphadenopathy. The MoAb bound to target cells in the thymus and modulated their expression of the L3T4 antigen but, in contrast to its effect in extrathymic sites, anti-L3T4 did not deplete the target population from the thymus. In fact, after 3 months of therapy, mice that had been treated with anti-L3T4 had much larger thymuses than control mice that had been treated with saline, suggesting that treatment with anti-L3T4 prevented the thymic atrophy that occurs spontaneously in murine lupus. Despite depleting L3T4+ cells from the spleen, treatment with anti-L3T4 did not diminish the response of splenic lymphocytes to T and B cell mitogens, and it augmented splenic natural killer (NK) cell activity. Finally, treatment with anti-L3T4 decreased the diverse histopathologic manifestations of murine lupus. It dramatically reduced glomerular immunoglobulin and complement deposition and diminished lymphocytic infiltration and vasculitis in the kidneys. Treatment also reduced extrarenal immunopathology, including focal hepatitis and salivary gland infiltration. These observations have implications regarding the use of CD4 MoAb in people with autoimmune diseases.

Pericyte response during choriocapillaris atrophy in the rabbit.

The rabbit and rat choriocapillaris atrophies in response to experimental destruction of the retinal pigment epithelium by intravenous injection of sodium iodate. This provides a convenient model of capillary atrophy. We have observed that pericytes are spared during this process; the atrophy is due to loss of endothelium only. Extensive examination of thin sections obtained 1 day to 11 weeks after administration of iodate showed that pericytes retained their normal relationship to the remnant capillary basement membrane left behind as the endothelial tube atrophied. This was most conspicuously manifested in their retention of processes longitudinally disposed along the sleeves of remnant basement membrane. The processes retained bundles of actin filaments that had dense regions along them and inserted into subplasmalemmal densities at basement membrane attachment sites, i.e. they had the characteristics of stress fibers. The pericytes did not phagocytose the debris of endothelial necrosis, in spite of their known phagocytic abilities. Necrotic endothelial cells were eliminated by sloughing into the capillary lumen. The observations support the idea that the function of pericytes in the choriocapillaris, the major source of nutrition for the retinal photoreceptors, resides in their contractility, and that pericytes do not remove necrotic endothelium during capillary atrophy.

Genetic aspects of tumour metastasis.

The application of techniques for the transfer of genetic material between cells when used with recombinant DNA technologies and gene cloning procedures hold exciting possibilities for the genetic analysis of the metastatic behaviour of tumour cells. So far these genetic studies have produced confusing results, but some of the problems have been defined more clearly. For a more complete understanding of the metastatic phenotype a more critical analysis of the models used to mimic the process and a greater appreciation of the deficiencies in the techniques applied to study it are necessary.

Cerebral hemiatrophy--correlation of human with animal experimental data.

We report the neuropathologic findings in a 63-year-old white male with a history of birth asphyxia, cerebral palsy, seizures and mild mental retardation in conjunction with similar brain pathologic findings in animal models of perinatal asphyxia. The human case showed a left cerebral hemispheric hemiatrophy associated with an extensive ulegyria involving all cerebral lobes on that side and a single microscopic focus of cortical atrophy in the right hemisphere. Among a large number of experimental perinatal asphyctic exposures only an occasional animal, like the human case described, showed unilateral hemispheric injury with softening and necrosis if examined early and ulegyria with hemispheric hemiatrophy if examined late. The present paper suggests that perinatal asphyxia under specific pathophysiologic conditions may cause unilateral brain injury. Our experimental studies suggest the specific condition of perinatal asphyxia potentially causing unilateral or asymmetrical brain damage is marked hypoxemia combined with substantial reductions in blood pressure but without circulatory collapse. Given these conditions, the asymmetry of the brain damage likely reflects fetal head position within the gravitational field relative to the heart. With disturbed cerebral blood flow autoregulation from asphyxia, the gravitational field likely accentuates the ischemia of those brain areas most elevated above the level of the heart. Thus, we postulate head position may play a pivotal role in defining brain regions that are damaged in hypotensive perinatal asphyxia. This interpretation may affect the intensive care of hypoxemic, hypotensive newborns aimed at minimizing the risk of brain damage.

Alterations in neuron morphology in mucopolysaccharidosis type I. A Golgi study.

Morphological changes in neurons with inborn defects of the lysosomal hydrolase, alpha-L-iduronidase, and with concomitant storage of glycosaminoglycans, were evaluated by Golgi staining in two animal models and compared to a similar study of a child with the same disease. Cortical pyramidal neurons in feline mucopolysaccharidosis type I often displayed axon hillock enlargements (meganeurites) and/or ectopic, secondary neuritic processes sprouting from this same region of the cell. The latter structures were prominent and often appeared longer than similar neurites reported in other neuronal storage diseases. Although most meganeurites were aspiny, a few were observed which possessed spine-like processes or neurites. Other than these morphological changes in cortical pyramidal neurons, few other cell types displayed abnormalities demonstrable by Golgi impregnation. In the canine model of this disorder, abnormal Golgi-impregnated cortical neurons resembled more closely those seen in human mucopolysaccharidosis. That is, they possessed meganeurites which typically were aspiny in appearance. Ectopic neurite growth was not observed on any Golgi-impregnated neurons in the cases of canine or human mucopolysaccharidosis used in this study. The latter finding, given the advanced ages of these cases, is consistent with the view that ectopic neuritogenesis seen in neuronal storage diseases may be subject to a developmental window, albeit one open well beyond the period of early postnatal maturation.

Ventricular weights in guinea pigs acclimated to cold plus hypoxia.

Weanling male guinea pigs (Cavia porcellus), 2-3 weeks of age, with initial body weights of 207-271 g were exposed for 2-16 weeks to constant cold (6 degrees C) and hypoxia (PO2 = 85 Torr) equivalent to 4800 m above sea level. Their growth rates and body weights did not differ from those of control animals of the same age maintained under normoxic conditions (22 degrees C, PO2 = 133 Torr). After 2, 3, 4, 6, 10, or 16 weeks exposure the animals were sacrificed, the hearts were removed, the ventricles were separated and weighed, and myoglobin concentrations were determined. Total heart weight as well as both right and left ventricular weights increased linearly with age. By the second week of exposure of the guinea pigs to cold plus hypoxia the total heart and right ventricular weights were 25 and 50% greater than those of the normoxic control animals. Both weights increased at greater rates than those of the controls until Week 6 and then remained at 30 and 80% throughout the 16th week. The weights of the left ventricles in these animals were only slightly greater than those of the controls. In spite of the severe right ventricular hypertrophy these animals showed no clinical signs of right heart failure. Myoglobin concentrations were significantly greater in both ventricles for the cold-plus-hypoxic animals than for the controls.

Campylobacter diarrhea in an adult mouse model.

An adult mouse (18-20 g) model was developed for studying the pathogenesis of Campylobacter isolates. Iron-loaded BALB/c mice given 10(8)-10(9) Campylobacter colony forming units by intraperitoneal injection developed a severe mucoid diarrhea within 4 h. Severe diarrhea, consisting of unformed stools containing blood, mucus, and fecal leukocytes, persisted for 24 h. Diarrheal symptoms in surviving mice resolved gradually; no diarrhea was observed 5 days after inoculation. Mice not pretreated with iron developed no diarrheal symptoms, and no severe diarrhea was produced in mice inoculated orally. A transient (less than 24 h) bacteremia occurred in mice inoculated either orally or intraperitoneally. Liver, spleen, and kidney were positive for Campylobacter for 48 h; intestinal contents were positive for 5-7 days. Mice given greater than or equal to 10(10) colony forming units showed symptoms of endotoxemia (ruffled fur, inactivity, shaking, tearing, and hypothermia) and died without diarrheal symptoms. Mice given nonpathogenic Escherichia coli strain HB101, heat-killed C. jejuni cells (greater than 10(10)), C. jejuni lipopolysaccharide extract, or purified lipopolysaccharide from either Vibrio cholerae 569B or Salmonella typhimurium showed no diarrheal symptoms.

Possible origin of synovial lining cell hyperplasia in rheumatoid arthritis.

A perplexing feature of rheumatoid arthritis is the increase in the number of synovial lining cells with no mitotic activity. This feature has been investigated in the rabbit model. Rabbits with the established condition were injected into the affected joint with tritiated thymidine and killed either up to 24 hours later, or at 3 or 7 days. The location of labelled cells, detected by autoradiography, showed the label predominantly in the stroma in the former, and mainly in the lining cells in the latter, indicating that the lining cells were derived by recruitment from active cells deep in the stroma.

Characterization of a slow-growing, transplantable rat mammary tumor (MCR-83): a model for endocrine-related cell kinetic studies.

Out of 24 primary mammary tumors, arising in rats of the WAG/Rij Wistar strain after low dose irradiation, with or without prolonged treatment with estrogen, a slow-growing, well differentiated adenocarcinoma (MCR-83) was selected. This tumor, induced by radiation alone, is independent of estrogen pellets for growth after transplantation into adult female rats, but nontransplantable into males or ovariectomized females. Measurements of tumor growth and contents of both estrogen and progesterone receptors on three successive passages are not indicative of a rapid progression in growth rate or to hormone independency. Ovariectomy and treatment with tamoxifen give a pronounced inhibition of tumor growth, whereas neither methotrexate nor cyclophosphamide is effective. Growth rate is significantly increased when rats are given 17 beta-estradiol. Flow cytometric DNA analysis as well as in situ S-phase cell detection with anti-bromodeoxyuridine antibodies show a 3-fold increase in S-phase fraction cells within 4 days after the onset of estrogen treatment. No spontaneous metastases have been found so far, but lung nodules develop after i.v. inoculation of tumor cells. From one of these nodules a fast-growing, hormone independent subline (MCR-86) has been derived, showing both lymphatic and hematogenous dissemination upon s.c. transplantation. By showing several features of hormone responsive human disease in its early stage of progression the MCR-83 tumor system may be a clinically relevant model for studies on endocrine regulation of tumor growth and its therapeutic manipulation.

Chronic relapsing necrotizing encephalomyelitis produced by myelin basic protein in mice.

Chronic relapsing experimental autoimmune encephalomyelitis is commonly seen in a number of species after a single injection of whole white matter in adjuvant but not after inoculation with myelin basic protein, the major encephalitogen of central myelin. In the present report on large groups of SJL mice, we describe a form of chronic relapsing experimental autoimmune encephalomyelitis with destructive lesions after a single inoculation of myelin basic protein in complete Freund's adjuvant. This condition was studied for up to 19 months postinoculation and was characterized by a relapsing-remitting or a chronic progressive course, usually with a prolonged latent period. Higher doses of 400 and 800 micrograms of myelin basic protein were more effective in inducing this condition than were lower doses of 100 and 200 micrograms. Large lesions were apparent in the white matter. These comprised widespread destruction and Wallerian degeneration with some demyelination towards the margins. Demyelination was an initial, albeit transient, event which was subsequently masked by nerve fiber destruction. Polymorphonuclear leukocytes were early and prominent components of the inflammatory infiltrate and together with macrophages appeared to be involved in the lysis of myelin and axons. Thus, despite the clinical similarities, these features contrast the model with the more purely demyelinative lesions of chronic relapsing experimental autoimmune encephalomyelitis in other species and multiple sclerosis in man.

Modulation of lymphocyte activating factor activity (interleukin 1 like activity) and acute phase proteins in pertussis-induced air pouch inflammation by muramyl dipeptide.

We have studied the effect of the macrophage activator, muramyl dipeptide (MDP) on immune inflammation induced in the rat six day subcutaneous air pouch. Treated animals received either 100 micrograms or 200 micrograms MDP at the time of challenge and twenty four hours before exudate harvest. Using the thymocyte co-mitogenic assay for lymphocyte activating factor (LAF), 100 micrograms MDP enhanced LAF activity whereas 200 micrograms caused inhibition. Increased dilution of 200 micrograms exudate in this assay removed this inhibition. Similarly, at the lower dose, MDP caused enhanced production of the acute phase protein alpha 1 glycoprotein, whereas the higher dose had no effect. The present study suggests that macrophage activity can be manipulated in vivo to produce LAF and naturally occurring inhibitors of LAF. These studies indicate that the stimulation of LAF inhibitors by MDP may be a potential therapeutic action.

Mandibulofacial dysostosis (Treacher Collins syndrome): a new proposal for its pathogenesis.

Acute exposure to 400 mg/kg 13-cis retinoic acid (13-cis RA, isotretinoin, Accutane) on the ninth day postfertilization in mice (a time that corresponds to the fourth week postfertilization in humans) results in malformations that characterize mandibulofacial dysostosis (MFD, Treacher Collins syndrome). Deficiencies in the infraorbital region and in the mandibular ramus and condyle, abnormalities of the secondary palate, and external ear malformations were observed. Light and scanning electron microscopic analyses of affected embryos illustrate that within 12 hours of maternal 13-cis RA treatment, markedly excessive (possibly premature) cell death occurs in regions where some of the cells are normally destined to undergo programmed cell death. Previous studies with retinoids have shown that they labilize lysosomal membranes and expand and strengthen regions of programmed cell death. Of particular interest for this study was cell death occurring in the dorsal (proximal) aspects of the maxillary and mandibular prominences of the first visceral arch, the second visceral arch, and the first visceral cleft, areas that correspond to the locations of the first and second arch ectodermal ("ganglionic") placodes and first closing membrane, respectively. The derivatives of this region are those that are severely affected in MFD. As described in previous reports from this laboratory, 13-cis RA is known to interfere with neural crest cells, resulting in major craniofacial malformations. However, the exposure times involved were earlier than those described herein. It is hypothesized that effects on the first and second arch ectodermal placodal cells at a time following the release from the neural folds of neural crest cells into the developing cranial region are of great significance in the pathogenesis of MFD. This is in contrast to the prevailing hypothesis that these malformations are the direct result of a primary interference with neural crest cells.

The inflammatory reaction in the airways in an animal model of the late asthmatic response.

The late asthmatic response is defined as airway obstruction that occurs hours after antigen exposure in some atopic asthmatics. The importance of this reaction is that the airway obstruction may be severe, prolonged, and difficult to control unless corticosteroids are employed. In addition, this response may lead to an increase in airway reactivity. To investigate the immunopathogenesis of this disorder, an animal model in rabbits was developed. In this model, antigen-specific IgE was associated with the late asthmatic response and antigen-specific IgG was associated with blunting of the reaction. Antigen challenge of immune rabbits led to edema within the large airways shortly after antigen exposure, with infiltration of inflammatory cells (neutrophils and eosinophils) into the large and small airways during the late response. The infiltrates became more mononuclear with time and resolved over 10 days. As in humans, the late response was associated with an increase in airway reactivity and correlated temporally with infiltration of the airways with neutrophils and eosinophils. The contribution of granulocytic cells to the airway responses to antigen was studied by granulocyte depletion, which prevented both the late response and the heightened airway reactivity. In addition, transfusion of a neutrophil-rich population of white cells into granulocytopenic immune rabbits restored both responses. Thus, in this animal model, the antigen-induced late asthmatic response and subsequent increase in airway reactivity were dependent on the presence of granulocytes at the time of exposure to antigen.

Beige mouse mast cells generated in vitro: ultrastructural analysis of maturation induced by sodium butyrate and of IgE-mediated, antigen-dependent degranulation.

We derived growth factor-dependent mast cell lines from C57BL/6-bgJ/bgJ ('beige') mouse bone marrow cells using techniques previously described for deriving mast cell lines from normal mice. According to examination by transmission electron microscopy, the cytoplasmic granules of cultured mast cells derived from beige mice were larger in size and fewer in number than those in cultured mast cells derived from normal C57BL/6 mice. Mast cells derived from beige mice underwent maturation when exposed to sodium butyrate, as judged by increased content of electron-dense material in the cytoplasmic granules. Cultured mast cells derived from beige mice also underwent IgE-mediated, antigen-dependent anaphylactic degranulation which was similar in its ultrastructural features to that described for cultured mast cells from normal mice. However, in mast cells not stimulated with IgE and antigen, fusion between individual cytoplasmic granules was observed more commonly in mast cells derived from beige mice than in normal mast cells. This might mean that these events are more common in C57BL/6-bgJ/bgJ mast cells, and/or that their resolution is slower and thus more easily captured by electron microscopy.

Immunohistochemical characterization of sialadenitis in NZB X NZW F1 mice.

The spontaneously developing sialadenitis in female autoimmune NZB X NZW F1 (NZB/W) mice has been studied with the help of immunohistochemistry and monoclonal antibodies to cell surface antigens. Semiquantitative assessment of stained cells within the infiltrates disclosed a progressive focal inflammation most pronounced in submandibular and parotid glands. The majority of cells expressed Ly-1 (all T cells) and L3T4 (T helper) phenotype, whereas only few Lyt-2 (cytotoxic/suppressor) expressing T cells were seen. A large proportion of the infiltrating cells stained for Ia antigens, which was also found on salivary gland ductal epithelium in the proximity of lymphoid infiltrates. The phenotypic pattern in sialadenitis of NZB/W mice thus closely resembles the pattern previously described for human Sjögren's syndrome (SS). Accordingly, immunomorphological analysis of the NZB/W sialadenitis may be useful in further studies of pathogenesis and therapy of both experimental and human SS.