Diabetes-induced impairment of macrophage cytokine release in a rat model: potential role of serum lipids.

Diabetes (type I and type II) affects approximately 13 million people in the United States. Delayed and incomplete healing of wounds can be a major problem for diabetic patients. Macrophages are an important cell in the complex process of wound repair representing the major source of cytokines throughout the wound healing process. Cytokines mediate many of the cellular responses critical to timely wound repair. It has been suggested that diabetes impairs wound healing through disruption of local cytokine production. We previously demonstrated that platelet-derived growth factor B chain (PDGF-B) levels are deficient at the wound site of diabetic rats. In the present study, we measured the levels of several marker cytokines released from cultured peritoneal macrophages of diabetic, nondiabetic hyperlipidemic, and normal rats. The diabetic condition was associated with a generalized reduction of macrophage cytokine release. Nondiabetic hyperlipidemic animals demonstrated similar cytokine reduction supporting the hypothesis that elevated serum lipids are the primary determinants of diabetes-induced reductions in macrophage cytokine release. Thus, manipulation of serum lipids may be a therapeutically useful modality for controlling macrophage cytokine release in the inflammatory and/or wound environment.

Platelet-derived growth factor levels in wounds of diabetic rats.

Delayed wound healing is a troublesome complication of Diabetes. Results from recent investigations concerning the potential cellular and molecular mechanisms responsible for diabetic wound healing deficiency are preliminary in nature. Some studies have demonstrated that direct application of certain growth factors/cytokines can facilitate wound healing in diabetic models. It is possible that refractory diabetic wounds are the result of deficiencies in growth factors/cytokines important for the normal wound healing process. Platelet-Derived Growth Factor (PDGF) levels were examined by radioimmunoassay in wound tissue of normal and diabetic rats (streptozotocin-induced diabetes). Immunohistochemical analysis was utilized to localize and characterize PDGF immunopositive cells at the wound site of normal and diabetic animals. At the wound site, normal animals demonstrated significantly elevated PDGF levels compared to diabetic animals at 5 days post-wounding (no differences were observed in the spleen or contralateral control tissue). There appeared to be a visible increase in PDGF immunopositive cells at the wound site in both experimental and control groups. By day 10 post-wounding, PDGF levels at the wound site in normal animals were reduced becoming similar to PDGF levels in diabetic animals. This corresponded to an apparent reduction of PDGF immunopositive cells in both groups (similar to baseline levels). PDGF levels in both groups remained stable until day 20 post-wounding when a significant elevation of wound site PDGF levels occurred in the diabetic group. The findings suggest that absence of an initial increase in PDGF may play an important role in poor wound healing observed in diabetic animals. The reduction in PDGF may be related to decreased cellular PDGF production rather than a lack of PDGF-producing cells. Perhaps the diabetic state inhibits cellular PDGF gene expression signaled by wounding or interferes with normal PDGF expression at the wound site.

Diabetes prevents periodontitis-induced increases in gingival platelet derived growth factor-B and interleukin 1-beta in a rat model.

Periodontitis is a chronic inflammatory disease characterized by a progression that is very much dependent on host response. The gingiva can be considered to be in a constant state of wounding (pathologic wounding by bacterial plaque) and a constant state of maintenance/repair. In this context, any metabolic disturbance in the host which compromises tissue repair/wound healing will exacerbate the progression of periodontitis. Diabetes presents an interesting example because two major complications of diabetes are delayed wound healing and periodontitis. Our previous studies indicate that delayed wound healing and periodontitis may be manifestations of a general systemic deficit in diabetes involving alteration of macrophage cytokine gene expression. The present study was designed to determine whether: 1) diabetes-induced metabolic alterations affect gingival cytokine levels; and 2) diabetes-induced metabolic alterations modify the gingival cytokine profile in periodontitis. Sprague-Dawley rats (N=12/group) were injected with streptozotocin (65 mg/kg) into the tail vein to induce diabetes (defined by blood glucose levels > 250 mg/dl) or received the injection vehicle or no treatment as controls. Periodontitis was induced in additional groups of diabetic and control rats by gavage with Porphyromonas gingivalis A7436. After 90 days, serum glucose was analyzed to document diabetes; alveolar bone level was measured to document severity of periodontitis; gingiva was harvested circumferentially from the first and second molars; and cytokines in gingival homogenates were assayed by ELISA using commercial kits. Cytokine levels were expressed as mean+/-SEM pg/microg protein. Diabetes alone did not alter the gingival cytokine profile for platelet-derived growth factor B (PDGF-B), interleukin 1-beta (IL-1beta), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha). Periodontitis alone demonstrated a significant increase (P < 0.05) in levels of PDGF-B and IL-1beta. Diabetes superimposed on periodontitis prevented these increases. Thus, diabetes-induced metabolic alterations do not affect gingival cytokine levels per se; however, they do alter the normal host response to periodontitis through blockage of periodontitis-induced increases in PDGF-B and IL-1beta.

Autonomic neurone degeneration in equine dysautonomia (grass sickness).

Histological investigations were undertaken on four sympathetic autonomic ganglia and on the myenteric and sub-mucosal plexuses of the jejunum in healthy animals, in naturally occurring cases of acute, sub-acute and chronic equine dysautonomia and in ponies in which neuronal damage had been induced by the injection of acute grass sickness sera. The degree of neuronal damage is related to the type of dysautonomia. The coeliac-mesenteric ganglion reacts differently from other ganglia and is less severely damaged in cases of short duration. Extensive experimentally induced damage to the coeliac-mesenteric ganglion, even when jejunal damage is also present, is not associated with clinical illness. It is proposed that the rate of autonomic neurone loss and the extent of the damage may both influence the clinical manifestations of grass sickness.

Histopathology of the brain-stem nuclei of horses with "Mal seco", an equine dysautonomia.

"Mal seco" is a disease of unknown aetiology affecting horses in Argentina. It is similar to grass sickness, a primary dysautonomia of horses in Europe. A histopathological study of the brain stem nuclei of three horses with "mal seco" was performed. Changes were found that consisted of chromatolysis, cytoplasmic vacuoles, eosinophilic sphaeroids, and pyknotic and eccentric nuclei. These changes were most severe at the oculomotor, vestibular and abducent nuclei. The results provide further evidence to suggest that "mal seco" and grass sickness may be the same disease.

Lactate dehydrogenase and its isoenzymes in the tissues and sera of clinically normal dogs.

The total activity of lactate dehydrogenase (LDH) and the percentage distribution of its isoenzymes in the tissues and sera of clinically normal adult dogs are presented. Total LDH activity was greatest in skeletal muscle followed by heart muscle, kidney, small intestinal mucosa, liver, lung, pancreas and bone. Each tissue had a unique isoenzyme pattern and the proportions of the isoenzymes in serum suggested that liver is the source of normal serum LDH. The tissue isoenzyme patterns were similar to those obtained by other authors in human beings, horses, cattle, sheep and cats although in liver, differences between ruminants and monogastric animals including dogs were evident. The data presented provide a basis for the interpretation of serum LDH isoenzyme patterns in canine disease.

Gamma-glutamyl transpeptidase and its multiple forms in the tissues and sera of normal dogs.

The total gamma-glutamyl transpeptidase activity and the proportions of its multiple forms in canine tissues and serum have been measured. The kidney showed the greatest activity, followed by the pancreas and small intestine. After separation by cellulose acetate electrophoresis, two main areas of activity were observed in tissues, an insoluble fraction which remained at the origin and a soluble fraction which migrated towards the anode. The soluble fraction was divisible into six zones differing in mobility and, with the exception of liver and lung, the tissues differed in the distances migrated by these soluble fractions. Serum gamma glutamyl transpeptidase formed one zone corresponding to the soluble fraction of liver and lung. The separation of the multiple forms is of potential value in determining the source of increased serum activities of the enzyme in the dog.

Quantitative assessment of fat absorption and its diagnostic value in exocrine pancreatic insufficiency.

A new method of quantifying fat absorption based on the estimation of serum triglyceride levels is described. When long chain triglyceride (LCT) was fed to normal dogs a significant elevation of serum triglyceride concentration was recorded which was not observed in dogs with exocrine pancreatic insufficiency. When the test was repeated on the dogs with pancreatic insufficiency using the LCT together with lipase the serum triglyceride concentration increased, suggesting the failure in the initial test was caused by a deficiency in pancreatic lipase. Feeding medium chain triglyceride (MCT) did not raise the serum triglyceride levels in normal dogs.

Serum albumin values from healthy cattle, sheep and horses determined by the immediate bromocresol green reaction and by agarose gel electrophoresis.

Serum albumin concentrations were measured by the immediate reacting bromocresol green (BCG) method and by agarose gel electrophoresis in healthy cattle, sheep and horses. No statistically significant differences were found between the values obtained by the two methods. The immediate reacting BCG method is quicker and cheaper when used under the conditions described.

Lactate dehydrogenase and creatine kinase isoenzyme levels in the tissues and serum of normal lambs.

The normal activities of lactate dehydrogenase and creatine kinase isoenzymes in the tissues and serum of clinically normal five-and-a-half-month-old lambs are presented. The percentage distribution of the lactate dehydrogenase isoenzymes in serum, liver, heart, lung and skeletal muscle were in agreement with previous studies but the distribution in ovine abomasum, small intestine, large intestine and red blood cells has not been previously described. Up to five creatine kinase isoenzymes were detected in ovine tissues and four in serum. One creatine kinase isoenzyme present in serum and tissues was thought to represent a mitochondrial isoenzyme which is absent from the normal serum of other species. Estimations of both lactate dehydrogenase and creatine kinase isoenzymes allowed all the tissues examined to be distinguished and were therefore more likely to allow tissue-specific isoenzyme patterns to be detected in serum than the estimation of the isoenzymes of either enzyme alone.

Serum lipids and lipoproteins in equine colic and grass sickness.

Serum total lipids, lipoprotein fractions, triglycerides, cholesterol, phospholipids and free fatty acids were measured in horses with acute, subacute and chronic grass sickness (equine dysautonomia) and in colic cases. The values were compared with those of normal grazing and stabled horses. A marked individual variation occurred, but total lipids, triglycerides and free fatty acids were significantly higher than normal in grass sickness and colic cases with cholesterol was significantly higher than normal in grass sickness cases only. Pre-beta lipoprotein was significantly increased in colic and subacute grass sickness although all abnormal groups showed this fraction which was absent from normal horses. The percentage of alpha 2b lipoprotein was significantly higher in colic and grass sickness. The changes described are typical of those occurring in fat mobilisation in the horse and are considered to be due to a number of factors including decreased food intake, cortisol and catecholamine release and insulin resistance.

The effects of Eperythrozoon ovis in sheep.

Infection of adult sheep with a single strain of Eperythrozoon ovis led to three different situations. First, the animal resisted the organism and no haematological changes occurred. Second, the host developed a controllable parasitaemia in which erythrocyte values fell shortly after peak parasitaemia and then returned to normal. Third, the host failed to control the parasitaemia and chronic low grade anaemia developed. Dexamethasone sometimes caused a resurgence of parasitaemia in sheep.

Changing morphology of Eperythrozoon ovis.

Light microscopy studies of Eperythrozoon ovis in sheep revealed that Giemsa stain was only less reliable than acridine orange as a means of parasite identification when low parasitaemias were present. The morphology of E ovis altered as the degree of parasitaemia increased.

Colostrum production by ewes and the amounts ingested by lambs.

Studies involving 49 ewes which were well nourished during the last eight weeks of pregnancy indicated that ewe weight loss during pregnancy and lamb birth weights followed the recognised pattern and were within acceptable "normal" limits. Colostrum production ranged from 1238 to 4593 g per ewe during the 48 h following the first suckling and there was evidence that production levels were related to demand by the lambs. Colostrum consumption by individual lambs was related to litter size. The smaller the litter size the greater was the amount ingested during the first 48 h of life. No clear relationship was established between the amount of colostrum consumed and the level of immunoglobulin in a lamb's circulation. Possible reasons for this are discussed.

Immunoglobulin levels in ewe colostrum and in lamb serum.

The immunoglobulin content of ewe colostrum falls rapidly after the first suckling and reaches a low level 36 h after the first feed. The whey content of colostrum will rise by more than 50 per cent during the same period. The production of colostral immunoglobulin during the 24 h following the first feed was related, in this work, to the amount of colostrum produced; the greater the colostrum production, the greater the immunoglobulin production. Colostrum production ranged from 1216 to 4493 g and immunoglobulin production from 22-21 to 86-34 g during the eight feeds studied. Colostrum production was related to demand by the lambs. There was a clear positive correlation between the total amount of immunoglobulin the lambs' circulation 30 h after the first feed and the immunoglobulin consumed. Approximately 20 to 25 per cent of the immunoglobulin ingested was present in the lambs' circulation at this time.

Acute phase proteins in grass sickness (equine dysautonomia).

Four acute phase proteins were assayed in the serum of normal horses and those with acute, subacute and chronic grass sickness, colic and inflammatory conditions, in order to investigate their diagnostic value in grass sickness. The grass sickness and inflammation group had a significantly increased haptoglobin concentration (P less than 0.01-P less than 0.001). Orosomucoid was elevated in acute, subacute and chronic grass sickness and inflammation (P less than 0.001, P less than 0.001, P less than 0.05 and P less than 0.05, respectively). Highest concentrations of haptoglobin and orosomucoid were recorded in subacute grass sickness. Ceruloplasmin was significantly higher in acute grass sickness cases than all other groups except the colic group (P less than 0.05-P less than 0.01). alpha 2-macroglobulin was significantly higher in acute grass sickness than normal, colic and chronic grass sickness cases (P less than 0.01, P less than 0.05 and P less than 0.05). The time scale of changes suggests that the stimulus to haptoglobin and orosomucoid synthesis occurs at the onset of clinical signs whereas the increase in ceruloplasmin and alpha 2-macroglobulin is more likely to reflect haemoconcentration.

Characterisation of compounds isolated from the sera of horses with acute grass sickness.

Isolates were prepared from the sera of 12 horses with acute grass sickness, using methods reported to yield serum fractions associated with neurotoxicity, and their components identified by liquid chromatography and spectroscopy. All isolates were found to contain cortisol and six isolates also contained a degradation product of an analgesic drug, dipyrone. However, no recognised neurotoxin was detected.

Clinical equine dysautonomia and autonomic neuron damage.

Damage to the neurons of selected autonomic ganglia was quantified in relation to the severity of the clinical signs shown in acute, subacute and chronic cases of dysautonomia (grass sickness). No connection between the clinical severity of acute or subacute dysautonomia and the amount of neuronal damage in the superior cervical, stellate and coeliaco-mesenteric ganglia could be demonstrated. However, a higher proportion of normal neurons were found in chronic cases. Jejunal submucosal neuronal damage was correlated with clinical severity but further work is required to confirm this finding and to establish how widespread the alimentary neuronal lesions are in dysautonomia of different severities.

The relationship between meteorological features and equine grass sickness (dysautonomia).

Local weather patterns associated with 15 outbreaks of equine grass sickness in eastern Scotland were studied. The majority showed a trend toward cooler drier weather associated with irregular ground frosts. This would not preclude the hypothesis that fungi might be connected with the aetiology of grass sickness.

A comparative study of normal equine populations and those with grass sickness (dysautonomia) in eastern Scotland.

A retrospective survey was made of premises in eastern Scotland on which at least two cases of grass sickness had occurred between 1970 and 1987. For comparison, a further survey of 49 equine establishments, on which no grass sickness had been recorded, was conducted from 1986 to 1988. The results indicated that younger animals are more susceptible, especially those in good physical condition grazing full-time in the spring or early summer. Movement to new grazing increases the risk of grass sickness and identifiable stress may contribute. The nature of the establishment governed the animals' condition during the summer, but it did not appear to influence the prevalence of grass sickness. However, riding schools and livery establishments which experienced the disease kept, on average, significantly more animals than unaffected premises of the same type. No relationship was found between supplementary feeding or stage of pasture growth and grass sickness. The results of the survey support the hypothesis that the causal agent of grass sickness is associated with grazing but multiple factors may influence the expression of illness.