Experimental Biology and Medicine

Use of radiolabeled monoclonal antibodies (mAb's) that localize and bind to malignant cells continues to be an attractive mechanism for targeting and delivering either an imagable isotope, be It a y-emitter or B+-emitter, or a particle emitter such as a B—emitter or an a-emitter for therapeutic applications.

Shortly after the appearance of bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD) was identified in young patients with nonclassical presentation such as difficulty in balancing and ataxia. The classical CJD in older patients starts with dementia. To distinguish between the two types, CJD in young persons has been termed “new variant” (nvCJD). The distinguishing features of classical CJD include initial presentation with dementia, confluent spongiform changes are very unusual in the cerebellum, and PrP plaques are rarely observed. For nvCJD, initially, difficulty with balancing and ataxia occurs, confluent spongiform changes are seen in the cerebellum, and a large number of PrP plaques are seen. The Icelandic observation of sheep scrapie revealed a predominantly ataxic form of scrapie, termed Type II, rather than the itchy form termed Type I. Both types have been known to exist in Europe. Since the clinical signs of Type II scrapie in sheep with trembling and ataxia are similar to those seen in BSE and nvCJD, this suggests that Type II is the cause of BSE and nvCJD. Over 8 years, from 1989 to 1996, I examined the clinical histories of 33 CJD cases aged between the ages of 18 and 84. Six under the age of 40 and 15 over the age of 40 had leading clinical features such as difficulty in balancing and ataxia similar to those seen in the young cases classified as “nvCJD.” Brains were examined from the six of 15 cases over the age of 40, which revealed similar pathology to that seen in young patients classified as “nvCJD.” These findings suggest that all age groups are susceptible to the strain of the agent derived from BSE cattle.

Bovine spongiform encephalopathy (BSE) is an infectious disease and has been transmitted orally to many other animals, including humans. There is clear evidence of maternal transmission, although disagreement on the source of the BSE agent remains. The current theories link the origin of BSE to common scrapie in sheep. Twenty different strains of the scrapie agent have been isolated from sheep. A search of the literature indicates two distinct clinical syndromes in sheep, both of which have been called scrapie. I have designated these Type I (the common type), which exhibits itchiness and lose their wool, and Type II, which exhibits trembling and ataxia. Sheep inoculated with BSE develop Type II scrapie and they exhibit trembling. When cattle or mink are injected with the Type I strain, only a few will develop a clinical disease. By contrast, no clinical disease has so far been shown in cattle or mink by feeding them with Type I-infected sheep brains. However, either by injecting or feeding with the BSE strain, 100% of calves and mink develop the clinical disease. Evidence suggests that Type II is the cause of BSE. Identical clinical signs of Type II trembling are found in kuru and many of the recent cases of Creutzfeldt-Jakob disease. The BSE agent has caused spongiform encephalopathies (SEs) in domestic cats, tigers, and in some species of ruminants in zoos. The nature of the BSE agent remains unchanged when passaged through a range of species, irrespective of their genetic make up, demonstrating that variations in the host PrP gene are not a major factor in the susceptibility to the BSE agent. Since more than 85 zoo animals of many species have been diagnosed with SEs, from these studies it seems reasonable to conclude that the BSE agent can infect almost all mammalian species, including humans. For eradication of BSE and to reduce the risk of infection to humans, the development of a vaccine against BSE is suggested. Such a possibility should be fully explored.

One of the important functions of the cornea is to maintain normal vision by refracting light onto the lens and retina. This property is dependent in part on the ability of the corneal epithelium to undergo continuous renewal. Epithelial renewal is essential because it enables this tissue to act as a barrier that protects the corneal interior from becoming infected by noxious environmental agents. Furthermore, the smooth optical properties of the corneal epithelial surface are sustained through this renewal process. The rate of renewal is dependent on a highly integrated balance between the processes of corneal epithelial proliferation, differentiation, and cell death. One experimental approach to characterize these three aspects of the renewal process has been to study the kinetics and dynamics of corneal re-epithelialization in a wound-healing model. This effort has employed in vivo and in vitro studies. From such studies it is evident that the appropriate integration and coordination of corneal epithelial proliferation, adhesion, migration, and cell demise is dependent on the actions of a myriad of cytokines. Our goal here is to provide an overview into how these mediators and environmental factors elicit control of cellular proliferation, adhesion, migration, and apoptosis. To this end we review the pertinent literature dealing with the receptor and the cell signaling events that are responsible for mediating cytokine control of corneal epithelial renewal. It is our hope that a better appreciation can be obtained about the complexity of the control processes that are responsible for assuring continuous corneal epithelial renewal in health and disease.

The search for new anticopper drugs for Wilson's disease is culminating in two excellent new drugs: zinc for maintenance therapy and tetrathiomolybdate (TM) for initial therapy. Both are effective and nontoxic. TM is a very potent, fast-acting new anticopper drug and its properties may be useful well beyond Wilson's disease. Angiogenesis (new blood vessel growth) is required for tumor growth, and a sufficient level of copper appears to be required for angiogenesis. We hypothesize that there is a “window” to which the copper level can be reduced that inhibits angiogenesis in tumors, but does not interfere with vital cellular functions of copper. Using TM therapy, this approach has worked to slow or stabilize tumor growth in several animal tumor models, and preliminary results are also very encouraging in human patients with a variety of advanced and metastatic malignancies. A hypothesis is advanced that copper availability has played a fundamental role in growth regulation throughout evolution and that is the reason that so many angiogenic promoters appear to be dependent upon copper levels.

The apical sodium-dependent bile acid cotransporter (ASBT) and the ileal bile acid binding protein (IBABP) are two components of ileal bile acid absorption. During the third postnatal week of the rat, there is a dramatic increase in ASBT and IBABP expression. The goals of this study were to examine the role of hormones on the ontogenic expression of ASBT mRNA and the role of weaning for both ASBT and IBABP mRNA. Administration of various doses of dexamethasone during the second postnatal week induced ASBT mRNA levels, and this effect was significantly increased with concomitant thyroxine treatment. Early weaning and weaning prevention were utilized to investigate the influence of dietary factors. ASBT and IBABP mRNA levels were significantly elevated by early weaning and were decreased by weaning prevention compared with littermate controls. Thus, glucocorticoids and thyroxine appear to play a role in the ontogenic expression of ASBT mRNA and weaning appears to participate in both ASBT and IBABP expression.

To investigate the long-term effects of normal pancreatic islet transplantation on progression of obese type 2 diabetes mellitus (DM), 1500 normal islets (per rat) from Wistar King A rats at 8 weeks of age were transplanted into the liver through the portal vein of Otsuka Long Evans Tokushima Fatty (OLETF) rats, an animal model of obese type 2 DM, at 12 weeks of age. Body weight in the transplanted OLETF (IT) rats 8 and 28 weeks after islet transplantation did not differ from that in the corresponding sham-operated (SO) rats, but was greater than that in lean littermates (LETO rats; P < 0.05 for each group). In the early phase, 8 weeks after transplantation, rats in both IT and SO groups were normoglycemic, but hyperinsulinemic (P < 0.05 for each compared with LETO rats), probably resulting from increased body weight. In the late phase, 28 weeks after transplantation, hyperglycemia in the IT group was greatly attenuated compared with the SO group (P < 0.05), but hyperinsulinemia remained in both the IT and the SO groups compared with that in the LETO group (P < 0.05 for each). Immunohistochemical studies demonstrated that hypertrophic and fibrotic changes in pancreatic islets, together with mesangial proliferation of the glomerular matrix, an indicator for diabetic nephropathy, were attenuated predominantly in the IT group at the late phase after transplantation compared with those in the corresponding phase of the SO group. Islet transplantation into the liver of OLETF rats thus prevented further progression of obese type 2 DM. A possible mechanism is that islet transplantation may prevent development of hyperglycemia by improving abnormal hepatic glucose metabolism and consequently insulin resistance, which may lead to blockade of a vicious cycle between advancing damage to pancreatic islet cells and increased demand for insulin secretion, thus sparing original pancreatic cells from exhaustion induced by increased demand for insulin secretion.

Endothelial cells (EC) that form the inner lining of blood vessels remain quiescent in the normal adult vasculature except during angiogenesis and reendothelialization, which result in EC proliferation and migration. EC placed in culture at subconfluent density also undergo cell multiplication and movement. This report demonstrates that whereas in confluent EC in a compact monolayer, the EC-EC adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1) is strongly expressed at cell borders, little or no PECAM-1 immunostaining is detected in sparse or migrating cultured EC. Consistent with this observation, steady-state PECAM-1 mRNA expression was much lower in subconfluent EC than in confluent EC. The absence of PECAM-1 expression in sparse EC appeared not to be linked to ability to proliferate, since PECAM-1 expression remained low even in the presence of nitric oxide (NO) or mitomycin C, agents that inhibit EC growth. However, another growth-inhibitory agent, TGF-β1, did not alter PECAM-1 staining. Based on these observations, it is hypothesized that cell-associated mechanical forces underlying cell tensegrity regulate PECAM-1 expression.

In experimental models of acute pancreatitis (AP), acinar cell death occurs by both necrosis and programmed cell death or apoptosis. Apoptosis is an active form of cell death associated with a tightly regulated expression of gene products that are either pro- or antiapoptotic. The aim of this study was to characterize pancreatic mRNA levels by Northern blotting analysis of apoptosis-associated genes used during the course of cerulein-induced AP in mice. Histone H3 mRNA levels were also examined as an indicator of cell proliferation. Acinar cell apoptosis was confirmed histologically. The findings show that AP modifies pancreatic mRNA levels of both pro- and antiapoptotic genes simultaneously. Pancreatic bclXL, bax, and p53 mRNA levels increased significantly in a temporal fashion during induction of AP. Pancreatic bcl-2 mRNA levels were unchanged during AP. Pancreatic mRNA levels of insulin-like growth factor-1 (IGF-1), a mitogen and cell survival factor, and its receptor (IGF-1R) also increased in a temporal fashion during induction of AP. In summary, this study indicates that acinar cell death during cerulein-induced AP in mice can occur by the apoptotic pathway. Since factors promoting and antagonistic for cell survival are activated simultaneously, regulation of acinar cell survival appears complex and dynamic during AP.

Nitric oxide synthetase (NOS), the conversion enzyme for nitric oxide (NO) is localized in the anterior pituitary of female rats, particularly in gonadotrophs and folliculo-stellate cells, suggesting that NO regulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary. The focus of this study was to determine the effect of chronic NO deficiency on the subsequent pituitary release of LH and FSH in vitro and the hypothalamic immunoexpression of GnRH in vivo. NO deficiency was induced by adding the NOS inhibitor, N-nitro-L-arginine (L-NNA, 0.6g/L) to the drinking water of female Wistar rats. After 8 weeks, the animals were euthanized, the pituitaries were removed, and they were incubated in vitro. Pituitaries were perfused for 4 hr in the presence of pulsatile gonadotropin release hormone (GnRH, 500 ng/pulse) every 30 min. S-Nitroso-L-acetyl penicillamine (SNAP, an NO donor, 0.1 mM) or L-nitro-argine methyl ester (L-NAME, a NOS inhibitor, 0.1 mM) was added to the media and perfusate samples were collected at 10-min intervals. LH and FSH levels in the perfusate were measured by double antibody radioimmunoassays. Pituitaries from the NO-deficient rats had a significantly smaller GnRH-stimulated release of LH and FSH compared with proestrous control rats. The addition of S-NAP to the perfusate resulted in decreased LH and FSH secretion in the control group, but increased LH secretion in the NO-deficient group. The addition of L-NAME to the perfusate suppressed LH secretion from control pituitaries, but not in pituitaries from NO-deficient animals. Immunohistochemistry of brain slices demonstrated that NO-deficient rats had a large qualitative decrease of GnRH in the median eminence compared with their controls. This decrease was particularly evident in the external capillary plexus of the median eminence. We concluded that chronic NO deficiency is associated with a decreased GnRH in neurosecretory terminals in the external capillary layer of the median eminence, accompanied by a decrease in LH and FSH release from the pituitaries.

Estrogens exert pro-oxidative effects and have been shown to damage DNA, potentially leading to cancer. Melatonin is a well-known antioxidant, free radical scavenger, and oncostatic agent. Changes in the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), an index of DNA damage, and the levels of malondialdehyde + 4-hydroxyalkenals, an index of lipid peroxidation, were measured in kidneys, liver, and testes from hamsters treated with E2 (75 mg/kg body wt) and were collected 3 or 5 hr later. Other animals were treated with melatonin (15 mg/kg body wt, 30 min before and 120 min after E2 treatment) or were given both compounds. Additionally, lipid peroxidation was measured in liver homogenates exposed to ferrous sulfate (15 μM) in vitro. E2 treatment caused an increase in 8-oxodGuo levels in kidneys collected 5 hr after E2 administration, and in liver 3 hr after estrogen treatment. Melatonin completely prevented E2-induced DNA damage in both organs. Melatonin alone or when given with E2 and examined 3 hr later decreased the base level of 8-oxodGuo in testes. A tendency for a reduction in in vivo lipid peroxidation was observed after treatment of hamsters with either melatonin, E2, or both compounds, with a statistically significant decrease being measured in the liver following E2 administration. In vitro exposure to iron significantly enhanced lipid peroxidation in hepatic homogenates from untreated, melatonin-treated, or E2-injected hamsters; in the hepatic homogenates of hamsters given both E2 and melatonin, ferrous sulfate failed to augment lipid peroxidation. Our results confirm the dual actions of estrogens relative to oxidative damage, i.e., estrogen increases oxidative destruction of DNA while reducing lipid peroxidation. Melatonin had antioxidative actions in reducing oxidative damage to both DNA and to membrane lipids. Melatonin completely prevented the damaging action of E2 on DNA and synergized with the steroid to reduce lipid peroxidation.