Haemolysins of Salmonella, their role in pathogenesis and subtyping of Salmonella serovars

Haemolysin patterns of 175 strains of different Salmonella enterica subspecies enterica serovars isolated from different animal sources and places were determined using 11 different blood agar media made with either non-washed horse/sheep erythrocytes or with washed erythrocytes of cattle, sheep, horse, goat, rabbit, guinea pig, and human A, O and B blood groups. Study on 47 strains belonging to 10 serovars of Salmonella from buffalo meat (buffen), 42 strains of 11 serovars from goat meat (chevon): 16 strains of Salmonella enterica serovar Paratyphi B and 25 of S. enterica serovar Paratyphi B var Java from fish, meat, meat products and clinical cases; 45 isolates of S. Abortusequi from aborted mares (18), fetal contents (21), aborted donkey mares (2) and 4 reference strains, revealed that all host restricted Salmonella namely, S. enterica serovar Gallinarum, S. enterica serovar Anatum, S. enterica serovar Abortusequi and S. enterica serovar Paratyphi B could be divided into different haemolysin types based on their inability to produce haemolysis on one or more types of blood agar, while strains of all zoonotic Salmonella serovars induced haemolysis on all the 9 types of blood agar made of washed erythrocytes. None of 175 Salmonella could produce hemolytic colonies on blood agar made of non-washed horse/ sheep erythrocytes. Haemolysin type I (lysing all types of washed erythrocytes) was the commonest one among all serovars except S. Abortusequi, none of which lysed horse erythrocytes. Salmonella enterica serovar Abortusequi having hemolytic activity against sheep erythrocytes were more invasive but had lesser ability to survive in sheep mononuclear cells than non-hemolytic strains. Multiplicity of haemolysins appeared significant epidemiological tool.     

The density of myocardial and pericardial rat mast cells in isoproterenol-induced heart failure

Myocardial mast cells (MC) respond to cardiovascular pathology. The behavior of MC population in myocardium and pericardium of rats has been studied 24 h, 14, 28 and 60 days after two isoproterenol injections (at 24 h intervals). The extent of heart failure has been estimated by supersonic inspection 28 and 60 days after isoproterenol injections. The density of MCs of different degrees of maturity was estimated on paraffin sections stained with Alcian blue--Safranin. The MC density in myocardium of intact and experimental rats was relatively low: from 4 to 6 cells/mm2. The MC density in pericardium of intact rats was several times higher than in myocardium: 48.6 +/- 13.0 cells/mm2. In 24 h and 14 days after isoproterenol injections the pericardial MC density was 1.5 times higher than in control rats (P < 0.05) at the expense of increase in the number of mature MCs with Safranine-positive granules without the increase in the number of immature cells with Alcian blue-positive granules. In 28 days the pericardial MC density was 2 times higher than in intact rats (P < 0.05) at the expense of increase in number of immature and mature cells. In 60 days after isoproterenol injections the pericardial MC density and the ratio of immature and mature cells compared with control did not reach statistical significance. The changes in pericardial MC population corresponded to severity of heart failure according to functional indices. The findings show active reaction of pericardial MCs on myocardium dysfunction that stimulates the maturation of resident immature MCs in pericardium and migration of immature cells to pericardium of damage heart.     

Are fingernail lead levels a reliable biomarker of lead internal dose?

BackgroundPotentially toxic elements, such as lead, can bioaccumulate and alter human physiology. Human biomonitoring is an essential tool to evaluate chemical exposures in different biological matrices (blood, urine, saliva, nails, and hair). Of these biological matrices, nails are favorable for their ease of sampling, transport and storage. The aim of this study was to investigate possible correlations between blood lead levels (BLL) and washed and non-washed fingernail lead levels (FLL) in 55 adults living in a lead-contaminated area.MethodVenous blood and fingernail (thumbs and forefingers) samples were collected. Nails from the left hand were washed with Triton X-100 (0.5 % m/v) and HNO₃ solution, while nails from the right hand were not submitted to the pre-analytical procedures. Samples were analyzed by graphite furnace atomic absorption spectrometry, and pairwise correlations were used to correlate lead concentrations between BLL and FLL; nails from fingers of the same hand and between washed and unwashed fingernails. Principal component analysis was performed and scatter diagrams were plotted to investigate correlations.ResultsA non-significant positive correlation was found between BLL and washed forefinger nails lead (r = 0.219, p = 0.112) and between BLL and thumbnail lead levels (r = 0.182, p = 0.191). Comparison of fingernails from the same hand (thumb and forefinger), showed that lead concentrations of non-washed nails varied widely, even on analyses of transversal fragments from the same nail. Lead levels in non-washed forefinger nails were not correlated with non-washed thumbnails (r = 0.169, p = 0.219). Conversely, washed thumb and forefinger nails were found to be correlated (r = 0.39, p = 0.003). Washed and non-washed nails were also found to be correlated (p < 0.0001).ConclusionIn conclusion, the results showed that non-washed nails are not a reliable biomarker for lead exposure. Although washing nails before analysis may reduce external contamination, the correlation of lead concentrations between fingers is poor for fingernail lead levels to serve as an internal dose biomarker to lead exposure. In addition, levels in washed nails were not significantly correlated with blood lead levels. Fingernail lead levels seem to serve as an indicator of lead exposure sources in contact with the individual, but not as a reliable biomarker of internal dose.     

Insulin Reverses Enhanced Incorporation of 32P into Polyphosphoinositides in Peripheral Nerve of the Streptozotocin Diabetic Rat

The ability of insulin treatment to reverse altered phosphoinositide metabolism in sciatic nerve from streptozotocin diabetic rats was studied. Diabetes was induced in rats by means of a single injection of streptozotocin. Enhanced incorporation of 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) was detectable as early as 8 days following intravenous injection of streptozotocin and was maximal after 4 weeks. Hormone treatment was initiated at this time by daily injections of protamine zinc insulin followed by the implantation of long-acting insulin osmotic minipumps, and 4 weeks later sciatic nerves were removed and incubated in the presence of [32P]orthophosphate. The increased labeling of PIP2 was completely reversed by hormone administration. In contrast, insulin (0.1 and 1.0 mU/ml) added to the incubation medium failed to reverse the altered pattern of 32P incorporation into PIP2. The uptake of 32P into PIP2 was greater than 80% higher into the proximal than into the distal portion of normal sciatic nerve when these were incubated separately. This metabolic difference was abolished in diabetic rats, although the incorporation into both segments was still significantly higher than in controls. These results strengthen the association of altered nerve PIP2 metabolism with the diabetic state and are consistent with the concept that experimental diabetic neuropathy is a distal axonopathy.     

The density of myocardial and pericardial rat mast cells in isoproterenol-induced heart failure

The multifunctional mast cells (MC), located in the myocardium, actively react to the pathology of a cardiovascular system. We investigated MC density in the pericardium and myocardium of rats in intact and experimental heart failure (HF) 24 h and 14, 28, and 60 days after two (at 24 h intervals) injections of Isoproterenol (IP) inducing necrosises in the myocardium. An expressiveness of HF was estimated with the help of ultrasonic scanning of the heart after 28 and 60 days after two IP injections. MC of different degrees of maturity were identified cytochemically on paraffin sections stained with Alcian blue-Safranine. The MC density in the myocardium of intact and experimental rats was relatively low, ranging from 4 to 6 cells/mm², thus the ratio of cells of a different degrees of maturity during HF development was reliably unchanging. In the fibrous layer of pericardium, the MC density was higher than in the myocardium and, for the intact rats, it amounted 48.6 ± 13.0 cells/mm². 24 h to 14 days after IP injections, the MC density in pericardium in contrast to intact sample increased on the average in 1.5 times (P < 0.05) at the expense of increase of density of more differentiated cells stained with Safranine, without density change of less differentiated cells stained with Alcian blue. After 28 days, the MC density in pericardium was 2 times higher than in the intact sample (P < 0.01) and, at the same time, the density of cells of a different maturity degree (P < 0.05) was considerably increased. For 60 days, the MC density and the balance of Alcian-and Safranine-positive cells did not differ reliably from the intact sample. The dynamic of behavior of the MC population of pericardium was corresponding to HF injury on functional parameters. The data obtained indicate the active reaction of MC pericardium on a dysfunction of the myocardium; it stimulates the maturation of resident MCs and the reinforcement of the population at the expense of the migration of cells from the outside, which allows us to propose an intensification of the MC secretory function.     

Determination of biomass dry weight of marine microalgae

Total biomass dry weight (DW) and ash free dry weight (AFDW) of five species of marine microalgae, Dunaliella sp., Isochrysis galbana, Nannochloropsis sp., Nitzschiaclosterium and Porphyridium cruentum, retained on filter paper, were determined. Dunaliella and Isochrysis cells have no cell wall; Nannochloropsis, Nitzschia and Porphyridium possess a cell wall and Nitzschia and Porphyridium cells are covered by silica and mucilage coating, respectively. In all these algae, DW of non-washed samples was at least 1.2 times higher than those washed by distilled water,0.9% sodium chloride, 0.5 M ammonium formate or 0.5 M ammonium bicarbonate. DW of 0.9% sodium chloride washed samples was more than 0.8 times higher than the other three washed samples. In most of the cases, there was no significant difference between DW of samples washed by ammonium formate and ammonium bicarbonate solutions (p>0.05). The AFDW of the non-washed algal samples was about twice that washed samples, and could be accounted for by volatile component in the sea water medium. Isotonic solution of ammonium bicarbonate is a satisfactory washing agent for algal cells for dry weight determination. This revised version was published online in September 2006 with corrections to the Cover Date.     

Micronucleus test and bone-marrow chromosome analysis: A comparison of 2 methods in vivo for evaluating chemically induced chromosomal alterations

The sensitivities of 2 cytogenetic tests, chromosome analysis and the micronucleus test, were compared by using mice exposed to the substances methyl methanesulfonate (MMS), mitomycin C (MC) and procarbazine (Natulan®). The lowest dose at which a significant effect could be observed in bone-marrow cells of mice was determined. Both test systems proved equally sensitive for MC and procarbazine. Doses as low as 0.16 mg of MC per kg and 3.12 mg of Natulan® per kg significantly increased both the aberration rates and the micronucleus rates above those of the controls. In contrast, after exposure to MMS, chromosomal aberrations were elevated above control levels at 5 mg/kg, and the micronucleus rate differed significantly from that of the controls after a dose of 10 mg/kg. With the present protocol and sample size one can conclude that the micronucleus test is generally comparable in sensitivity to the chromosome analysis. However, the MMS data indicate that there might be chemicals for which the resolution of the chromosome analysis is higher.

When the mutagens were given in 2 single i.p. injections separated by 24 h, the polychromatic erythrocytes were analyzed for the presence of micronuclei 6 or 24 h after the second injection. The double treatment did not increase the micronucleus rates above the single-treatment results at either sampling interval.     

Effect of dietary zinc intake on the hematological profile of the rat

The effect of dietary zinc deficiency (imposed at weaning) on the hematological profile of the male rat was studied. Lack of dietary zinc resulted in elevated numbers of erythrocytes and segmented neutrophils and decreased reticulocyte number. The hematocrit of zinc-deficient rats was significantly elevated after the first week. The mean lifespan of erythrocytes from zinc-deficient rats (tested in adult control rats) was not significantly altered from that of controls. The lack of dietary zinc did not cause a major change in the erythrocyte density (age) distribution.     

"In situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes

Unsealed membranes from human erythrocytes bind GTP and GTP analogs according to first order kinetics, a single rate constant being observed. With [35S]GTP gamma S this is 0.15 +/- 0.2 min-1. Treatment of the membranes with detergents decreases binding considerably. Scatchard plots reveal uncomplicated patterns of ligand association, with Kd values of 10.2 +/- 2.3 nM [35S]GTP gamma S, of 18.2 +/- 4.3 nM [alpha-32P]GTP and of 28.6 +/- 3.5 nM [alpha-32P]GDP, respectively. The stoichiometry with the three ligands is strictly comparable, i.e. 65 +/- 7 picomoles/mg of membrane protein. Binding of each labeled nucleotide is competitively inhibited by the other two unlabeled ligands, the inhibition constants being very close to the corresponding Kd values. Metabolic depletion and subsequent repletion of intact erythrocytes result in membrane preparations still active in guanine nucleotide binding, with unmodified Kd values. However, the stoichiometry falls to 35 picomoles/mg protein with the "depleted" erythrocyte membranes and regains higher values (50 picomoles/mg protein) with the "repleted" cell membranes. Accordingly, the "in situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes seems to represent a new tool for monitoring the metabolic state of intact erythrocytes.     

A new simple method for determination of erythrocyte filtrability.

A new simple filtration technique designed for measuring red cell filtrability in the routine laboratory use was developed. The suspension of the whole blood in saline (1:20,000 dilution) was processed on the Sartorius filter membranes, pore size 8 micron. The percentage of passed erythrocytes indicating red cell filtrability was determined. The suitability and perspective applicability of this method for studying various hematological disorders is proposed.     

The sequestration capacity of "hypersplenic" rat spleens for heat-damaged erythrocytes

The sequestration capacity of the spleens of controls and hypersplenic rats was examined. Hypersplenism was induced by long-term intraperitoneal application of methyl cellulose. The animals were injected single doses of various amounts of heat-damaged 51Cr-labelled erythrocytes (0.1 ml to 1.5 ml); radioactivity in spleen was determined 4 hrs. following application. The amount of red cells sequestrated in the spleens of hypersplenic animals was significantly increased against the controls, after administration of massive volumes of cells. The maximum amount of erythrocytes sequestrated in the spleens of the control rats amounted to an average weight of spleens 1.25 g to 0.158 ml (0.126 ml per g of spleen), and in hypersplenic animals to an average weight of the spleen of 4.87 g to 0.283 ml (0.058 ml/l g of spleen weight).     

Effect of "vilon" on cirrhotically changed rat liver. Liver regeneration, and status of glycogen-forming function of hepatocytes

Effects of a dipeptide preparation "Vilon" on rehabilitation of functional activity of hepatocytes and regeneration of the cirrhotically altered rat liver were studied. The liver cirrhosis was produced by poisoning of rats for 4 months with carbon tetrachloride (CCl4). On the end of the poisoning with CCl4, one group of animals was not submitted to any further actions, whereas animals of the other group were injected "Vilon" (1.7 micrograms/kg) daily for 5 days. On smears of isolated hepatocytes, contents of total glycogen (TG), and its labile and stable fractions (LF and SF) were determined in addition to cell ploidy levels and the total protein content. In liver homogenates, activities of glucose-6-phosphatase (G6P), glycogen synthase (GS), and glycogen phosphorylase (GP) were measured. In 2 weeks after the drug application, G6P activity being reduced in cirrhosis 1.2 times, elevated under effect of "Vilon". In non-treated rats the contents of TG and its fractions and of G6P activity remained at the level characteristic of the cirrhotic liver prior to "Vilon" administration. In both groups of rats, GP and GS activities in the cirrhotically altered liver did not differ from their control values throughout the entire experiment. "Vilon" has been shown to exert a weak stimulating effect on regeneration of the cirrotically altered rat liver: in hepatocytes of the second group of rats the total protein content and ploidy levels were higher than those in the first group by 4.7 and 11.5%, respectively.     

Effect of the melanocortin receptor stimulation or inhibition on ethanol intake in alcohol-preferring rats

It has been recently reported that acute intracerebroventricular injection of 1 nmol/rat of the non-selective melanocortin 3 and 4 receptor (MC3/4) agonist MTII reduces ethanol intake in female AA alcohol-preferring rats and alters opioid peptide levels in the ventral tegmental area of rats. To better understand the role of the MC system in the control of ethanol intake, we tested the acute and chronic effects of lateral ventricular (LV) injections of 0.01-1 nmol MTII, of 0.1-1 nmol of the MC3/4R receptor antagonist agouti related peptide (AgRP), and 0.1-0.5 nmol of the MC3/4R receptor antagonist SHU9119 on food, water, and 10% ethanol intake in Marchigian-Sardinian alcohol-preferring (msP) rats, which spontaneously ingest pharmacologically relevant quantities of ethanol both under short and long term access conditions. The data showed that with 2h/day ethanol access, LV MTII injections reduced intake of food and ethanol intakes. When food, water, and ethanol were available ad libitum and 0.01 nmol MTII was given by daily LV injection, however, ethanol intake was reduced for only the first 2 days, whereas food intake was reduced for all 5 days of treatment. Finally, acute LV injection of neither AgRP nor SHU9119 affected ethanol intake under ad libitum conditions, although both antagonists significantly increased food and water intake. In conclusion, these data fail to support a role for endogenous MC3/4R in the control of spontaneous ethanol intake in the msP rat. MC3/4R agonism, however, reduced ethanol intake in association with reduced food intake, suggesting that MTII might reduce nutrient-related controls of ethanol intake rather than, or in addition to, reward-related controls of ethanol intake.     

Thermogenic effects of a "cafeteria" diet on the rat during its reproductive cycle

The heat production and oxygen consumption of intact virgin, pregnant, lactating and postlactating rats has been investigated both in groups fed a "cafeteria" diet as well as in controls. A third group of rats fed the cafeteria diet after parturition has been investigated. Pregnant rats fed a "cafeteria" diet increased their weight faster than controls. During lactation no increases in weight were observed, and in postlactation both "cafeteria" groups attained the same values higher than controls. The ingestion of a "cafeteria" diet resulted in higher heat production in all groups except lactating rats, which--in addition--showed higher heat outputs than all the other groups when the actual data were corrected by metabolic weight according to the surface law. The high lactation heat production (and oxygen consumption) can be a consequence of increased metabolic activity in the rat organism, devoted to milk production. It can be concluded that during lactation the dam energy output through the milk must absorb any increases in energy input due to the more densely-packed energy in the "cafeteria" diet, and this did not result in increased heat production.     

Aortic coarctation induces oxidative stress in rat tissues

The purpose of this study was to evaluate the induction of oxidative stress in heart and erythrocytes from rats with abdominal aorta coarctation (Coa) compared with sham-operated normotensive controls (Sham). The group of Coa animals developed myocardial hypertrophy, showing heart homogenates markedly increased levels of reduced glutathione (48%), lipid peroxidation (148%) and activation of superoxide dismutase and glutathione peroxidase (189% and 37%, respectively), compared with controls. Other oxidative stress indicators were also altered in erythrocytes from Coa rats: increased protein carbonyl content (141%) and total glutathione level (349%) were determined. Inactivation of the antioxidant enzymes catalase (27%), superoxide dismutase (58%) and glutathione peroxidase (25%) was observed in erythrocytes from the Coa group. Taken jointly our results provide strong evidence for the production of oxidative stress in heart and erythrocytes from aortic coarcted rats.     

Induction of Oxidative Stress by Chronic and Acute Glutaric Acid Administration to Rats

1. Glutaric acidemia type I (GA I) is a neurometabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase, which leads to tissue accumulation of predominantly glutaric acid (GA) and also 3-hydroxyglutaric acid to a lesser amount. Affected patients usually present progressive cortical atrophy and acute striatal degeneration attributed to the toxic accumulating metabolites. 2. In the present study, we determined a number of oxidative stress parameters, namely chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), total antioxidant reactivity (TAR), glutathione (GSH) levels, and the activities of catalase and glutathione peroxidase (GPx), in various tissues from rats chronically exposed to GA or to saline (controls). High GA concentrations, similar to those found in glutaric aciduria type I, were induced in the brain by three daily subcutaneous injections of saline-buffered GA (5 μmol/g body weight) to Wistar rats of 5–22 days of life. The parameters were assessed 12 h after the last GA administration in different brain structures, skeletal muscle, heart, liver, erythrocytes, and plasma. The lipid peroxidation parameters chemiluminescence and/or TBA-RS measurements were found significantly increased in midbrain, liver, and erythrocytes of GA-injected rats. The activity of GPx was significantly reduced in midbrain and markedly increased in liver. TAR measurement was significantly reduced in midbrain and liver. Furthermore, GSH levels were reduced in liver and heart. We also investigated the acute in vivo effect of GA administration on the same oxidative stress parameters in cerebral structures and erythrocytes from 22-day-old rats. We found that TBA-RS values were significantly increased in erythrocytes, TAR levels were markedly decreased in midbrain and cerebellum, and GPx activity mildly reduced in the midbrain. 3. These data showing an imbalance between antioxidant defences and oxidative damage, particularly in midbrain, liver, and erythrocytes from GA-injected rats, indicate that oxidative stress might be involved in GA toxicity and that the midbrain, where the striatum is located, is the brain structure more susceptible to GA chronic and acute exposition.     

Ca content of human erythrocytes. What is the true value?

It has been generally accepted that the Ca content of washed erythrocytes is in the range of 1 - 3 X 10(-5) mole/l cells (1-10). These values were obtained after samples were treated in various way for Ca measurement. However, by using a simplified careful preparation technique to avoid Ca contamination together with inductively coupled argon plasma emission spectroscopy, we have found that the Ca content of erythrocytes washed with nominally Ca2+-free solution, is one order of magnitude lower than the reported values, in the range of 1 X 10(-6) moles/l cells.     

Central mineralocorticoid receptor blockade improves volume regulation and reduces sympathetic drive in heart failure

The mineralocorticoid (MC) receptor antagonist spironolactone (SL) improves morbidity and mortality in patients with congestive heart failure (CHF). We tested the hypothesis that the central nervous system actions of SL contribute to its beneficial effects. SL (100 ng/h for 28 days) or ethanol vehicle (VEH) was administered intracerebroventricularly or intraperitoneally to rats with CHF induced by coronary artery ligation (CL) and to SHAM-operated controls. The intracerebroventricular SL treatment prevented the increase in sodium appetite and the decreases in sodium and water excretion observed within a week of CL in VEH-treated CHF rats. Intraperitoneal SL also improved volume regulation in the CHF rats, but only after 3 wk of treatment. Four weeks of SL treatment, either intracerebroventricularly or intraperitoneally, ameliorated both the increase in sympathetic drive and the impaired baroreflex function observed in VEH-treated CHF rats. These findings suggest that activation of MC receptors in the central nervous system plays a critical role in the altered volume regulation and augmented sympathetic drive that characterize clinical heart failure.     

Comparison of life span of erythrocytes in some inbred strains of mouse using 14C-labelled glycine.

The erythrocyte life span in four inbred strains of mice--C57BL/10ScSnPh, B10.LP, BALB/c and CBA/JPh--was determined by means of erythrocyte labelling with 14C-glycine. Experimental data [decrease of 14C-activity of washed erythrocytes], corrected for blood loss, reincorporation of the label and delay in label incorporation during the initial period, were treated by a novel mathematical procedure based on the death probability function of the form: mu [t] = a + btlambda. The mean erythrocyte life span calculated using this function was, in the given sequence of strains, 42.1 +/- 0.6, 41.3 +/- 1.2, 39.3 +/- 0.9 and 38.6 +/- 0.6 days respectively. The rate of "random" destruction of erythrocytes was 1.20, 1.31, 0.70 and 0.63% of the total number of erythrocytes per day and a "mean potential" erthrocyte life span was found to be 58.9, 59.9, 46.2 and 44.5 days respectively. All the given parameters have similar numerical values in related strains and are apparently genetically conditioned. The erythrocyte life span determined simultaneously using DF32P labelling in the C57BL/10ScSnPh strain was 47.4 +/- 1.0 days. The implications of the results yielded by these two techniques is discussed.     

Refractory hypothalamic alpha-mSH satiety and AGRP feeding systems in rats bearing MCA sarcomas

In pre-anorectic tumor-bearing (TB: methylcholanthrene-induced sarcoma) rats, injection of alpha-melanocyte stimulating hormone (alpha-MSH) into the perifornical hypothalamus (PFH) had no significant effect on food intake at a dose (5 microg) that reduced feeding in non-TB control rats. Following the development of anorexia, injection of alpha-MSH MC3/MC4 receptor antagonists, SHU9119 (1 microg) or 4 microg agouti-related protein (AGRP), stimulated feeding in non-TB rats, while having no significant effect in TB rats. Concentrations of alpha-MSH were not altered significantly in ventromedial, dorsomedial or lateral hypothalamic areas of TB rats, and proopiomelanocortin (POMC) messenger RNA was not changed in TB rats in these hypothalamic areas. Determination of cytokines by ELISA in non-operated TB and non-TB rats revealed elevated IL-2 in plasma and hypothalamus as well as increased TNF-alpha in the hypothalamus of anorectic TB rats. IL-1B was not detectable in plasma and was not altered significantly in hypothalamus of TB rats. These results suggest that the POMC alpha-MSH satiety system is refractory in TB rats, even prior to the onset of anorexia. This change in MC3/MC4 receptor response does not appear to be secondary to alterations of endogenous alpha-MSH in TB rats. Cytokine involvement in the altered response to MC3/MC4 receptor stimulation and blockade is a possibility, since TNF-alpha and IL-2 were increased in hypothalamus of anorectic TB rats. Therefore, these results suggest major alterations in POMC neuropeptide systems in TB rats as anorexia progresses. Although these changes do not appear to have occurred due to grossly-altered concentrations of alpha-MSH, elevated cytokine activity in the hypothalamus may be an important factor. Due to the complex multi-factorial nature of feeding control, additional factors are likely to be involved in cancer anorexia.