Long-term infusion of nonphysiologic solutions into brain parenchyma: effects of pH, osmolarity, and flow rate.

The effects of long-term (3-day) infusion of nonphysiologic solutions into brain parenchyma were investigated in male Fischer (F344) 344 rats. Two weeks prior to infusion, a guide cannula was placed into the striatum, substantia nigra, or hippocampus. Solutions were infused continually for 3 days at flow rates of 0.03 (129.6 microl total) or 0.10 (432 microl total) microl/min. Four days after infusion, rats were euthanized and the brain was removed and processed for histologic evaluation. Rats that received cannula implants alone had the usual mechanical damage induced by implantation of the cannula. The brain regions that received 0.9% saline, pH 5.0 or pH 9.0 buffer at the two aforementioned flow rates had only minor evidence of tissue damage adjacent to the infusion site that was similar to that attributable to mechanical damage from the cannula implants. Brain tissue infused with distilled water or 1.8% saline also had modest effects of the solutions similar to the usual mechanical damage induced by the infusion cannulae. In contrast, contamination of the infusion sites was seen to induce inflammation. Data from these studies support the hypothesis that nonphysiologic solutions can be used to deliver compounds into brain parenchyma, without the infusion solutions themselves causing excess damage to brain tissue.

Caloric restriction enhances evoked DA overflow in striatum and nucleus accumbens of aged Fischer 344 rats.

Previous studies have shown deficits in DA neuronal systems in senescence. Other studies indicate that prolonged dietary restriction can attenuate many of the detrimental effects of age. We have shown previously using in vivo electrochemistry that K+-evoked striatal DA overflow decreases as a function of age. This was a regional effect that appeared to be due to functional changes in DA neurons, rather than a decrease in the storage and synthesis of DA. In the present studies, we used in vivo electrochemistry to investigate the effects of caloric restriction on age related decreases in K+-evoked DA overflow along a dorsal to ventral axis in the striatum of aged female Fischer 344 rats. Aged (26-28-month-old) diet restricted animals (DRF) showed evoked DA overflow that was significantly greater in amplitude and duration compared to aged (26-28-month-old) ad lib fed animals (ALF). These results provide additional evidence that decreased DA neuronal function resulting from age is improved by caloric restriction.

Gene therapy for B-cell lymphoma in a SCID mouse model using an immunoglobulin-regulated diphtheria toxin gene delivered by a novel adenovirus-polylysine conjugate.

Despite advances in conventional therapy, many lives continue to be lost to common forms of B-cell cancers, including leukemias, lymphomas and multiple myeloma. We propose a novel approach to therapy of such cancers using controlled expression of a diphtheria toxin gene (DT-A) to kill malignant cells. We have previously demonstrated selective killing of various cell types, in vitro and in vivo, by cell-specific, transcriptionally controlled expression of this gene. Organ-specific ablation in otherwise healthy transgenic mice has convincingly demonstrated the exquisite specificity achievable by this technique. In the studies now described, DT-A was delivered in vitro and in vivo using a novel gene delivery system employing DNA physically attached to the exterior of adenovirus. After demonstrating the efficacy of gene delivery to Epstein-Barr virus transformed human B-cells in vitro, in vivo work was performed using a SCID mouse model for B-cell lymphoma, in which protection against tumor was observed. The concepts of tissue-regulated toxin gene therapy, and this novel adenovirus gene delivery system are discussed.

Comparison of fetal rabbit brain xenografts to three different strains of athymic nude rats: electrophysiological and immunohistochemical studies of intraocular grafts.

Interest in the use of neural tissue transplantation for the study of CNS development and maturation and the potential use of this technique for the treatment of certain degenerative CNS disorders has led to our use of transplantation of neural tissue across species lines. Prior to extensive transplantation studies using athymic rats as recipients, we wished to evaluate the currently available strains of athymic rat for their suitability as host animals for xenografts of neural tissue. Fetal cerebellar and cerebral cortex tissue from rabbit brain of gestational age 20-25 days was dissected and transplanted to the anterior chamber of the eye of Harlan Wisconsin, Fisher 344 Jnu, or NCI-Harlan athymic nude rat strains. The brain tissue grafts were allowed to mature for 3 mo during which time the size and vascularity of each graft was monitored through the cornea of anesthetized hosts. In each group all of the transplants survived and grew to varying extents in the anterior chamber of the eye. Following the growth study in vivo extracellular recording of single neuronal activity was performed. Spontaneous neural activity was found in most transplants in all three groups with no difference in the viability or discharge rates of neurons between the groups. Illumination of the ipsilateral eye increased the firing rate of neurons in all three groups, suggesting excitatory cholinergic innervation of the grafted neurons from the host parasympathetic iris ground plexus. Antibodies directed against neurofilament protein, glial fibrillary acidic protein, synapsin, and tyrosine hydroxylase were used to characterize the transplants immunocytochemically and revealed no differences between the grafts in the three groups of recipients. All transplants contained significant numbers of glial and neuronal elements with the distribution resembling that in adult brain tissue. Some of the transplants contained a sparse innervation of tyrosine hydroxylase-positive fibers from the sympathetic plexus of the host iris. Furthermore, synapsin-immunoreactivity suggested that synaptogenesis had taken place within the grafts. Histological examination of the grafts revealed that 67% of the grafts had been infiltrated, to varying extents, by lymphocytes which led to areas of cell lysis and necrosis. All host animals had populations of T-cell receptor positive cells, most of which also expressed the T-cell surface antigens CD4 and CD8. However, no transplants were overtly rejected over the 15 wk period of study.(ABSTRACT TRUNCATED AT 400 WORDS)

Malignant fibrous histiocytoma in a bonnet macaque (Macaca radiata).

Spontaneous sarcomas, not associated with an underlying disease, appear to be relatively rare in nonhuman primates. Since 1970, there have been few reported cases of naturally-occurring sarcomas of any kind in these species. A malignant histiocytoma and malignant fibrous histiocytoma have been described in a rhesus macaque and baboon, respectively. A malignant fibrous histiocytoma is defined as a sarcoma of varied pattern consisting of a mixture of histiocytic and fibroblastic elements. It is thought that the two cells types arise from a common precursor or that the fibroblastic elements are derived from the histiocytes. These tumors are relatively common in humans. Here we report a case of spontaneously-occurring malignant fibrous histiocytoma in an adult bonnet macaque.

Myocardium and cortex cerebri xenografts transplanted into the anterior chamber of the eye of athymic rats: a morphologic and electrophysiologic profile.

Heart atria and cortex cerebri from fetal rabbits (E14 and E18, respectively) were grafted into the anterior eye chamber of anesthetized athymic nude rats and allowed to mature for 2-11 weeks. All grafts received a rich vascular supply from the host iris. Atrial transplants survived well but showed no significant growth while cortex grafts increased in size an average of 320%. Spontaneous action potentials were recorded from cellular elements in both tissues and, in the case of the atria, were accompanied by observable contractions. Functional cholinergic innervation from the autonomic ground plexus of the iris was elicited in both types of grafts by phasic retinal illumination. No evidence of immunologic rejection was found by histological analysis. Taken together, these data suggest that athymic rats may provide an appropriate host environment to study transplants of the central nervous and peripheral tissue from immunologically otherwise incompatible mammalian species.

An animal model of antibody binding in cutaneous lupus.

To study antibody binding in cutaneous lupus, we used human skin grafted onto nude mice. By immunofluorescence examination, mice injected with anti-Ro (SS-A) sera from subacute cutaneous or neonatal lupus erythematosus patients showed evidence of human IgG deposited in the skin, while mice injected with anti-native DNA or normal sera did not. We present evidence that there is specific binding of anti-Ro (SS-A) antibodies to Ro (SS-A) antigen in the skin, and we propose that these antibodies may be directly involved in cutaneous disease.

Hormonal, metabolic and morphologic studies of aged C57BL/6J obese mice.

Genetically obese mice (C57BL/6J-ob/ob), fed ad libitum, demonstrated a precipitous increase in the spontaneous death rate after 50 weeks. The first signs of morbidity were a ruffled hair coat and a progressive motor ataxia. Necropsy revealed that obese mice had pale and fatty livers, urolithiasis and grossly distended bladders. Microscopically, the hepatocellular changes observed in all aged obese mice included: a loss of orientation of hepatocytes, an enormous variability in the size of both hepatocytes and their nuclei, and an extensive deposition of both large and small lipid droplets, confirmed by an increase content of triacylglycerols. A subacute-to-chronic, multifocal, necrotizing hepatitis was also present. Kidneys from aged obese mice contained hypertrophied glomeruli and increased PAS-stained material. Tubular dilation with compaction of the tubular cells was also seen. There were no significant alterations in the microanatomy or mineralization of femurs from obese mice, yet there was a significant increase in plasma alkaline phosphatase activity. In obese mice at 62-63 weeks of age, hyperglycemia was present even in spite of hyperinsulinemia. Pituitary immunoreactive ACTH and its molar ratio to pituitary immunoreactive beta-endorphin were also increased in obese mice at this age. Even though the etiology of the decreased lifespan of genetically obese mice remains uncertain, the possibility is discussed that an overall defect in the central nervous system may be involved.

Hypersensitivity pneumonitis in nonhuman primates. I. Studies on the relationship of immunoregulation and disease activity.

We investigated the relationship of immunoregulation to disease activity in a nonhuman primate model of pigeon breeder's disease. Two Macaca arctoides monkeys developed classical symptoms of hypersensitivity pneumonitis after sensitization and prolonged bronchial challenge, whereas 2 other monkeys remained asymptomatic after in vivo challenge. There were no differences in the percentages of T cells, B cells, monocytes, or FC gamma-bearing T cells between symptomatic and asymptomatic animals. Nonetheless, we found a population of concanavalin A-induced, pigeon serum- (PS) induced, and spontaneous T cells that functioned as suppressor cells in autologous in vitro co-cultures in asymptomatic animals that were missing or nonfunctional in symptomatic animals. Monocyte suppressors functioned in both groups. We used low-dose total body irradiation (TBI) to inactivate T suppressor cells. Fifteen radiation units of TBI caused no change in the physical activity, routine chemistries, or blood counts of the 4 animals. After TBI, however, the previously asymptomatic animals developed fever, tachypnea, and signs of pulmonary congestion after in vivo challenge with PS. There was no change in the response to challenge in the symptomatic group. This altered response to in vivo challenge in the previously asymptomatic group persisted for 2 wk after TBI. During this period the difference in vitro immunoregulatory activity between Con A-induced, PS-induced, and spontaneous T cells in symptomatic and asymptomatic animals disappeared. Monocyte suppressors, however, continued to function in both groups after TBI. These data suggest that the monkey is an appropriate model for studies of human HP and that T cell immunoregulation may be an important element in the pathogenesis and disease activity of HP.

The detection of clinically significant erythrocyte alloantibodies using a human mononuclear phagocyte assay.

Red blood cell (RBC) allo- or autoantibodies, which markedly reduce the survival of transfused or autologous RBC, are considered to be clinically significant antibodies. The occurrence of antibodies against high-incidence antigens, which are occasionally associated with clinically significant RBC destruction or are of unknown clinical significance, often creates delays in providing blood to patients. In the majority of cases these antibodies are benign; however, clinically significant examples of these antibodies have been reported. An in vitro homologous human mononuclear phagocyte assay (MPA) was used to study antibodies directed against specificities associated with variable clinical significance. Two antibodies reported to be clinically significant and 25 antibodies known to be clinically insignificant were tested by MPA. The results indicate that clinically significant antibodies have a significantly higher score than do clinically insignificant antibodies, with no overlap observed between the two groups. An additional eight antibodies with unknown clinical significance were tested. None of these antibodies had scores in the clinically significant range.

Cell-mediated immunity in the lungs of rabbits using conjugated proteins.

A study using direct macrophage migration inhibition of cells from the lower respiratory tract of rabbits immunized with conjugated proteins, incorporated into a killed BCG-oil emulsion, revealed that the reaction was largely carrier specific. The only exception was when bronchoalveolar cells (BAC) from animals immunized with dinitrophenol coupled to ovalbumin (DNP-OA) were significantly immobilized with DNP coupled to bovine gamma-globulin (BGG) in which the ratio of DNP/BGG was high [DNP-BGG(H)]. This result could not be explained by a toxic effect or by a high net negative charge on DNP-BGG(H). In addition, inhibition of BAC from DNP-OA-immunized rabbits was not observed when the ratio of DNP to BGG was reduced approximately 5-fold.

Strain variation in BCG-induced chronic pulmonary inflammation in mice. I. Basic model and possible genetic control by non-H-2 genes.

C57BL/6 mice (haplotype H-2b) responded in a dose-dependent fashion to killed BCG by marked enlargement of the spleen and lung. Neither CBA nor C3H mice (haplotype H-2k) responded to such treatment. Pulmonary inflammation in responder B6 animals was characterized by a marked chronic interstitial and alveolar granulomatous process, and was accompanied by occasional granulomata, hyperemia, and loss of architecture in the spleen. Inflammation in non-responder CBA and C3H animals was minimal in both the lung and spleen. The response does not appear to be controlled by genes within the major histocompatibility complex, but is associated with a C57 background. B10.BR mice (responder background, H-2k) were responder animals and C3H.SW mice (nonresponder background, H-2b) were nonresponders. In addition, all animals tested with a C57 background were responders even though two of these strains were not H-2b (C57BL/Ks, H-2d and C57Br/cd, H-2k). The resolution of the mechanism of genetic control of this response in mice may provide information relevant to possible genetic control of chronic pulmonary inflammation in man.