Elevated free tyrosine in rhinoceros erythrocytes.

Red blood cells of African black rhinoceroses (Diceros bicornis) are highly sensitive to oxidant-induced hemolysis and they possess a number of enzymatic and biochemical features that differ radically from other mammals. Here we show concentrations of free tyrosine in rhinoceros red blood cells which can approach levels as high as 1 mM, 50-fold higher than in human red blood cells. Elevated levels of tyrosine are also observed in red blood cells of other members of the order Perissodactyla such as the horse and zebra. Captive black rhinoceroses have significantly lower levels of red blood cell tyrosine than black rhinoceroses in the wild. Tyrosine transport studies indicate that black rhinoceros red blood cells have lost the ability to transport tyrosine as efficiently as human red blood cells.

Radiometric assessment of hexose monophosphate shunt capacity in erythrocytes of rhinoceroses.

OBJECTIVES: To measure metabolic rates of the hexose monophosphate shunt (HMPS) in erythrocytes of rhinoceroses, and to test the hypothesis that low concentrations of endogenous ATP in erythrocytes impair HMPS capacity, thereby increasing susceptibility to oxidant-induced hemolysis. ANIMALS: 13 black and 3 white rhinoceroses, free-ranging in several regions of southern Africa, and 1 Sumatran rhinoceros in US captivity. PROCEDURE: HMPS fluxes were measured in rhinoceros erythrocytes with carbon-labeled glucose in the presence and absence of known HMPS activators. RESULTS: Compared with values for human erythrocytes, mean basal state HMPS fluxes were appreciably lower (22 to 46%) in all 3 rhinoceros species studied. Shunt activators increased HMPS rates approximately 5-fold over basal rates in rhinoceros erythrocytes, compared with increases in humans of 10-fold with ascorbate and 15-fold with methylene blue. Stimulated HMPS rates in human erythrocytes were quantitatively 5- to 10-times greater than those observed in rhinoceros erythrocytes. Overall HMPS catabolic rates were completely independent of intracellular ATP concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: HMPS glycolytic and recycling rates and responses to activators are inherently low in erythrocytes from 3 species of rhinoceros, likely contributing to (but not solely responsible for) the high susceptibility of black rhinoceroses to oxidant-induced hemolysis. Slow erythrocyte HMPS capacities were independent of intracellular ATP concentrations, invalidating a current hypothesis regarding the pathogenesis of hemolytic anemia in captive black rhinoceroses. Limitations in HMPS capacities emphasize the importance of protecting rhinoceroses from exposure to drugs, chemicals, toxins, foodstuffs, and other conditions known to increase production of oxidizing metabolites, reactive oxygen species, and free radicals.

Ventilatory failure, ventilator management, and outcome in dogs with cervical spinal disorders: 14 cases (1991-1999).

OBJECTIVE: To determine the incidence of and risk factors for ventilatory failure in dogs undergoing surgery for treatment of cervical spinal disorders and to document ventilator management, clinical course, and long-term outcome of dogs that experienced ventilatory failure in association with cervical spinal disorders or their management. DESIGN: Retrospective study. ANIMALS: 14 dogs. PROCEDURE: Dogs with cervical spinal disorders that required positive-pressure ventilation (PPV) were identified, and signalment, concurrent diseases, neurologic status at initial examination, clinical course, pulmonary function before, during, and after PPV, management techniques, complications, and outcome were recorded. Dogs that underwent surgery and required PPV were compared with dogs that underwent cervical spinal surgery during the same period that did not require PPV. RESULTS: 14 dogs with cervical spinal disorders required PPV to treat hypoventilation, including 13 of 263 (4.9%) dogs that underwent surgery for cervical spinal disorders. Lesions between the second and fourth cervical vertebrae and treatment by means of a dorsal decompressive laminectomy were associated with a significantly increased risk of perioperative hypoventilation. Pulmonary gas exchange function was normal or nearly normal throughout the course of PPV in dogs that survived. Ten dogs survived, and 9 of the 10 regained neurologic function. All 9 dogs that regained neurologic function had deep pain perception on initial examination at the veterinary teaching hospital. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a small percentage of dogs with cervical spinal disorders may require perioperative ventilatory support. With prolonged PPV and aggressive management, a good outcome may be achieved in dogs similar to those described in the present study.

Role of excessive maternal iron in the pathogenesis of congenital leukoencephalomalacia in captive black rhinoceroses (Diceros bicornis).

OBJECTIVE: To investigate the possibility that excessive maternal iron (overload) may contribute to development of congenital leukoencephalomalacia in captive black rhinoceroses. SAMPLE POPULATION: Tissue specimens and serum samples from 18 rhinoceroses in 2 kindreds harboring 4 (possibly 5) affected female calves. PROCEDURE: Fresh and archival sera and necropsy tissue specimens were evaluated to determine the nature and extent of iron overload in captive and wild black rhinoceroses as well as other rhinoceros species. RESULTS: Quantitative serum and tissue assays of iron and iron analytes, corroborated by histopathologic findings, indicated that these kindreds carried the greatest body burdens of iron yet found among captive black rhinoceroses. Fourteen of 18 rhinoceroses had the highest serum ferritin concentrations measured among 64 black rhinoceroses in captivity in the United States. Dams of affected calves had serum ferritin concentrations 2 orders of magnitude higher than clinically normal humans, equids, or free-ranging rhinoceroses. A neonatal serum sample from 1 affected female calf had a high ferritin concentration (approx 100-fold increase), but a male sibling of another affected female did not, suggesting a possible sex disparity in fetal response to maternal iron overload. Morphologic hallmarks of hemochromatosis were prominent in dams and grandams of affected calves. CONCLUSIONS AND CLINICAL RELEVANCE: Excessive maternal iron may affect female fetuses more than males, possibly inducing leukoencephalomalacia by catalyzing production of highly toxic hydroxyl free radicals during crucial periods of in utero development. Reduction of maternal iron overload may decrease the probability of developing leukoencephalomalacia and some other disorders commonly affecting rhinoceroses in captivity.

Acute lymphoblastic leukemia in a juvenile southern black rhinoceros (Diceros bicornis minor).

A 21-mo-old female southern black rhinoceros (Diceros bicornis minor) developed acute upper respiratory dyspnea in association with lymphadenopathy and marked immature lymphocytosis. A diagnosis of acute lymphoblastic leukemia was reached on the basis of the morphologic and cytochemical characteristics of peripheral lymphoblasts. Antineoplastic chemotherapy included administration of cytarabine, cyclophosphamide, vincristine, and doxorubicin, with clinical remission achieved 19 days after initiation of treatment. The rhinoceros died, however, of congestive heart failure, presumably secondary to doxorubicin cardiotoxicity and a particular sensitivity of rhinoceros myocardial tissue to free hydroxyl radicals. The pharmacologic effects of any therapeutic agent need to be carefully considered before use in the black rhinoceros, especially within the context of the unique physiology of this species.

Idiopathic hemorrhagic vasculopathy syndrome in seven black rhinoceros.

Idiopathic hemorrhagic vasculopathy syndrome (IHVS) was diagnosed in 7 black rhinoceros; this newly described syndrome is characterized by severe body swelling in conjunction with a rapid and profound decrease in Hct. The disorder may be acute or chronic, may recur, and is potentially fatal. Five of the rhinoceros survived an initial episode of IHVS, and 2 of these 5 survived a recurrent episode of IHVS. Two rhinoceros died during treatment of IHVS. Treatment protocols varied, but all 7 rhinoceros received broad-spectrum antibiotics, because an infectious cause was suspected. All rhinoceros also received nonsteroidal antiinflammatory drugs and supportive care. Idiopathic hemorrhagic vasculopathy syndrome has many similarities to other vasculopathies of domestic animals, such as equine purpura hemorrhagica, but it also appears to have unique identifying features. It has been hypothesized that IHVS may be an immune response to an as yet unidentified infectious agent. Thorough and extensive testing has not identified the potential causative agent, nor the factors that predispose some black rhinoceros to developing IHVS. Further research into the rhinoceros immune system is ongoing and should help elucidate the mechanisms through which IHVS develops.

Anthracycline cardiotoxicity in a black rhinoceros (Diceros bicornis): evidence for impaired antioxidant capacity compounded by iron overload.

Two weeks before dying of congestive heart failure, a juvenile black rhinoceros (Diceros bicornis minor) received a single low dose of doxorubicin as part of combination chemotherapy for acute lymphoblastic leukemia. Diffuse hemosiderosis was present at necropsy in a pattern indicative of dietary iron overload, but unique iron-positive degenerative lesions were found in isolated myocardiocytes. Serum analyses revealed hyperferremia, 87% transferrin saturation, and 5- to 10-fold elevations in ferritin concentration, reflecting markedly increased tissue iron stores. Since both toxic and therapeutic effects of anthracyclines are mediated by formation of reactive free radicals via iron-catalyzed reactions, these observations suggest that iron overload may have enhanced myocardial susceptibility to cardiotoxic effects of doxorubicin. Impairments in other myocardial antioxidant defenses, such as deficiencies in catalase and glutathione S-transferase that are known to exist in rhinoceros erythrocytes, may have been underlying factors contributing to an inherent sensitivity of rhinoceros tissues to oxidant-induced injury.

Differential effects of low-level lead exposure on the natural isozymes of erythrocyte 5'-nucleotidase.

OBJECTIVE: Erythrocyte pyrimidine 5'-nucleotidase (Pyr-5'-N) is highly sensitive to heavy metal inactivation in vitro and in vivo, and a number of studies have verified its usefulness as a biomarker of acute and chronic lead exposures. Retrospective and prospective studies attempted to determine whether the known linearity of Pyr-5'-N inhibition by lead concentrations above 40 microg/dL whole blood might continue into the lowest range of exposures now considered to be toxic in children (<10-20 microg/dL), thereby extending its value as a biomarker of lead exposure. DESIGN: Activities of Pyr-5'-N and a lead-insensitive isozyme, deoxyribonucleotidase (d-5'-N), were compared to blood lead and free erythrocyte protoporphyrin (FEP) concentrations. RESULTS: Pyr-5'-N activities in erythrocytes from 70 children displayed an inverse linear correlation with whole blood lead of 1-35 microg/dL, whereas d-5'-N did not correlate. There was no apparent minimum threshold for Pyr-5'-N inhibition by lead. CONCLUSIONS: Linearity of Pyr-5'-N inhibition by lead extends throughout the range of clinical concern in pediatric cases. Pyr-5'-N/d-5'-N activity ratios may provide an even more sensitive, internally controlled biomarker of low-level lead overburden, since both isozymes vary comparably in activity as a function of reticulocytosis and mean red cell age.

Cyclosporin inhibits intercellular adhesion molecule-1 expression and reduces mast cell numbers in the asebia mouse model of chronic skin inflammation.

The asebia mouse represents a spontaneous mutation in BALB/c mice leading to hyperplasia of the epidermis and chronic inflammatory dermal changes including enhanced cellularity, oedema and elevated mast cell numbers. We demonstrated that asebia mice have constitutively elevated intercellular adhesion molecule-1 (ICAM-1) mRNA expression, which is not detectable in the wild type, and that dermal mast cell numbers were 3.1-fold higher than the wild type (P < 0.001). We utilized this model to explore the anti-inflammatory effects of cyclosporin A (CsA). After 3 weeks subcutaneous injection with 5 or 10 mg/kg CsA the expression of ICAM-1 mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and was found to be decreased 2.7-fold (P < 0.001) and three-fold (P < 0.001) relative to controls for 5 and 10 mg/kg treatments, respectively. Dermal mast cell counts were dose-dependently decreased by CsA. Mast cells, visualized by toluidine blue staining, decreased 4.5-fold with 10 mg/kg CsA (P < 0.001) bringing them down to numbers typical of the wild type. CsA also appeared to stabilize mast cell histamine content. Histological examination of haematoxylin-eosin-stained sections revealed that CsA treatment restored the wild-type skin phenotype decreasing epidermal hyperplasia, dermal cellularity and oedema. Thus, CsA exhibits a wide range of anti-inflammatory effects including reduction of ICAM-1 expression and mast cell numbers, and may be useful in the treatment of mast cell-mediated dermatoses.

Endogenous natural killer enhancing factor-B increases cellular resistance to oxidative stresses.

Natural killer-enhancing factor (NKEF) was identified and cloned on the basis of its ability to increase NK cytotoxicity. Two genes, NKEF-A and -B, encode NKEF proteins and sequence analysis presented suggests that each belongs to a highly conserved family of antioxidants. To examine the antioxidant potential of NKEF, we transfected the coding region of NKEF-B cDNA into the human endothelial cell line ECV304. The stable transfectant, B/1, was found to overexpress NKEF-B gene transcript and protein. We subjected B/1 to oxidative stress by either culturing them with glucose oxidase (GO), which continuously generates hydrogen peroxide, or by direct addition of hydrogen peroxide. We found that B/1 cells were more resistant than control cell lines. Resistance to hydrogen peroxide was originally thought to be mediated mainly by catalase and the glutathione cycle. Therefore, we used inhibitors to block the two pathways and found that B/1 cells were more resistant to oxidative stress than control cells when we used inhibitors to preblock either pathway. We also examined the cellular inflammatory responses to oxidized low-density lipoprotein (LDL) and bacterial lipopolysaccharide (LPS) by measuring monocyte adhesion to endothelial cells in vitro and found that B/1 cells were resistant to such responses. Lastly, we found that B/1 cells were more resistant to a novel chemotherapeutic agent CT-2584, which appears to kill tumor cells by stimulating production of reactive oxygen intermediates in mitochondria. These results demonstrate that the NKEF-B is an antioxidant that protects cells from oxidative stress, chemotherapy agents, and inflammation-induced monocyte adhesion. Furthermore, its expression may mediate cellular responses to proinflammatory molecules.

"Biospheric medicine" as viewed from the two-year first closure of Biosphere 2.

Biosphere 2 is a 3.15-acre, 7-million ft. enclosed ecological space near Tucson, AZ. It contains five wilderness and two domestic biomes (rain forest, savanna, desert, ocean, marsh; agricultural station, living quarters), an original introduction of 3,800 species (approximately 20% extinctions have occurred), and a large basement "technosphere." Sealed inside Biosphere 2 in September 1991, four women and four men, including two of the authors, maintained themselves and the various systems for 2 yr, the longest-sustained "isolated confined environment" period on record. MMPI psychological profile scores for Biosphere 2 crewmembers correlated closely with those reported for astronauts and shuttle applicants. Major medical problems encountered during the 2 yr included adaptation to a low-calorie (1800-2200 kcal.d-1 per person) but otherwise nutritionally adequate diet, with substantial weight loss (18% for men, 10% for women), and a declining oxygen atmosphere (down to 14.2%). Life in a miniworld such as Biosphere 2 may differ substantially from life in a space station or temporary planetary base. These differences include multiple, shifting, sometimes opposing post-launch objectives; complete self-sustenance with recycling of virtually all materials within a highly complex biologic system; retooling of some areas of practical medicine; an attention to "culture" as a social dynamic and how that may influence crew and leadership selection in a societal rather than a quasi-military community. Assuming that long-term planetary colonies must be largely self-sustaining (due to costs of supply over great distances), they must of necessity approach the condition of biospheres. Subject to chaos dynamic (nonlinear dynamic) perturbations, the behavior of complex biospheres will be inherently non-predictable--as opposed to the linear dynamic situation of most space missions--and will require of the inhabitants, including the medical team, a wide range of coping abilities. Under the circumstances, and while strong similarities exist, important differences may serve to distinguish "biospheric medicine" from "space medicine."

Leukaemia inhibitory factor is expressed by normal human keratinocytes in vitro and in vivo.

Leukaemia inhibitory factor (LIF) is a pleotropic cytokine, regulating differentiation, cell growth, cachexia and inflammation. Using the reverse transcription-polymerase chain reaction (RT-PCR) we found that, in culture, normal human keratinocytes (KC) expressed mRNA transcripts for both LIF and the LIF receptor. In the conditioned medium (CM), constitutive LIF protein production was barely detectable but stimulation of KC with 10 ng/ml of either interleukin (IL)-1 alpha, or IL-8, for 24 h, resulted in small but significant increases (P < 0.05) in LIF protein, as measured by enzyme-linked immunosorbent assay. After culture in media containing 1.5 mmol/l calcium, a time-dependent increase in LIF mRNA was seen up to 72 h (an 8.5-fold increase), over levels in cells cultured in 0.05 mmol/l calcium. A large increase in LIF protein in the CM (from 1.15 +/- 0.15 pg/ml to 178.7 +/- 75.7 pg/ml) was seen 72 h after a switch to media containing 1.5 mmol/l calcium (P = 0.05). Twenty-four hours after stimulation of human KC in culture with 10 ng/ml recombinant LIF, a twofold increase in both IL-1 alpha and IL-8 protein in the CM (P < 0.05) was observed. In normal human scalp and foreskin, the epidermis was shown to contain LIF protein by immunostaining. LIF staining was found throughout the epidermis, and in the cells of the outer layer of the root sheath. Thus, KC synthesize LIF in vitro and in vivo.

A novel endogenous mediator of cutaneous inflammation: leukemia inhibitory factor.

Keratinocytes produce a variety of cytokines, including leukemia inhibitory factor. We hypothesised that this cytokine may play a pro-inflammatory role in the skin and tested this hypothesis by injecting recombinant leukemia inhibitory factor (1-100 ng) into the ear pinnae of C3H/HeJ mice. To other groups of animals, we injected boiled leukemia inhibitory factor or phosphate-buffered saline (negative control) or 0.4 ng human interleukin-1 alpha as a positive control. Following injection of 100 ng leukemia inhibitory factor, ear thickness, measured by micrometer, increased 66% over controls at 12 h and 100% at 24 h (overall p = 0.041 by analysis of variance). Injection of 0.4 ng interleukin-1 alpha caused greater ear swelling. Compared with controls, swelling increased by 67% at 6 h, 100% at 12 h and 340% after 24 h (overall p < or = 0.00001). Leukemia inhibitory factor (100 ng only) stimulated a 3.5-fold increase in leukocytes after 6 h. After 12 h, a 14-fold increase was seen in ears injected with 10 ng leukemia inhibitory factor and a 12-fold increase with 100 ng leukemia inhibitory factor, which remained elevated (17-fold) at 24 h (overall p = 0.0001). Injection of interleukin-1 alpha led to a 3.4-fold increase in leukocytes (mean per 20 high-power fields) after 6 h, a 14-fold increase at 12 h and a 25-fold increase at 24 h (overall p < or = 0.00001). These results demonstrate that leukemia inhibitory factor appears to be a mediator of cutaneous inflammation.

Increased obese mRNA expression in omental fat cells from massively obese humans.

Obesity presents a significant challenge to the general health of affluent nations in terms of the number of people affected, the serious associated maladies and the lack of effective treatments. While common wisdom has held that obesity results from 'gluttony and sloth', a number of studies have indicated physiological causes of underlying the pathogenesis of obesity, with the degree of adiposity having a strong genetic component. Recently, the obese gene in the ob/ob mouse was cloned, along with its human homologue. The specific production of the obese protein by adipose tissue suggested that it may function in a feedback loop from fat tissue to the hypothalamus to control energy intake and/or energy expenditure, and that it may play a role in the pathogenesis of human obesity. In this study we report that obese mRNA expression is elevated in ex vivo omental adipocytes isolated from massively obese humans in the absence of an identifiable mutation. Therefore, we speculate that this increased expression may suggest that the massively obese are insensitive to the putative regulatory function(s) of the obese gene product.

Expression of leukemia inhibitory factor and interleukin-11 by human melanoma cell lines: LIF, IL-6, and IL-11 are not coregulated.

Dysregulation in cytokines has been associated with melanomas. For example, loss of growth inhibition in advanced melanomas has been associated with interleukin-6 (IL-6) expression. Because IL-6 belongs to the hematopoietic cytokine family, which includes leukemia inhibitory factor (LIF) and interleukin-11 (IL-11), we examined the possibility of coordinate expression of LIF, IL-6, and IL-11 in three human melanoma cell lines derived from primary lesions (early) and in four lines derived from metastatic tumors (advanced). All lines examined produced at least low levels of LIF and IL-11 mRNA as measured by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). By enzyme-linked immunosorbent assay (ELISA), two of three early and three of four advanced lines were found to secrete LIF protein. IL-11 was assayed using growth of the responsive B9/11 cell line, but only one of seven lines made a low but measurable amount of IL-11. Cytokine protein production was not strictly correlated with mRNA abundance, nor was it strongly correlated with tumor staging. Recombinant LIF and IL-11 protein had no effect on the proliferation of any of the seven lines, suggesting that they do not act as autocrine growth factors for these melanomas. Assay of IL-6, IL-11, and LIF protein in conditioned medium from early and advanced melanoma lines gave no evidence of coordinate expression of these cytokines. We conclude that LIF and IL-11 production by melanomas may have some paracrine or endocrine function in the course of melanoma progression.

Hemolytic anemia associated with lead poisoning from shotgun pellets and the response to Succimer treatment.

Symptomatic lead poisoning with severe hemolytic anemia was observed in a patient with retained shot gun pellets. Surgical resection of the retained pellets and the use of a newer chelating agent, Succimer (2,3-dimercaptosuccinic acid) successfully lowered blood lead level. Hemolytic anemia was associated with deficient erythrocyte pyrimidine 5'-nucleotidase, and lowering of the lead level corrected the deficiency, suggesting that the enzyme deficiency is responsible for the hemolysis associated with lead poisoning. This case illustrates that retained lead pellets from shotgun wounds can cause severe lead poisoning.

Acute episodic hemolysis in the African black rhinoceros as an analogue of human glucose-6-phosphate dehydrogenase deficiency.

Sudden episodes of massive hemolysis have become the most common cause of death among captive black rhinoceroses, and there is evidence that they occur in the wild as well. We have observed radically unique enzyme and metabolite profiles in normal rhinoceros erythrocytes compared to humans and other mammals, including marked deficiencies of intracellular adenosine triphosphate (ATP), catalase, adenosine deaminase, and other enzymes involved in glycolysis, glutathione cycling, and nucleotide metabolism. Minimal concentrations of ATP appear to impair effective acceleration of hexosemonophosphate shunt activity in response to oxidants by restricting substrate generation at the hexokinase step. Antioxidant defenses are further compromised by catalase deficiency, which may be a general characteristic of rhinoceros erythrocytes, perhaps related to the common occurrence of severe mucocutaneous ulcerative disease. It is proposed that erythrocyte ATP deficiency in rhinoceroses may be an evolutionary adaptation conferring selective advantage against common hemic parasites, comparable to the role of human glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in falciparum malaria.