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.

A case-control study of grass sickness (equine dysautonomia) in the United Kingdom.

A case-control study was performed to investigate the epidemiology of grass sickness in the United Kingdom from 1992 to 1995. Data were collected by means of postal questionnaire when cases of grass sickness were identified. Sets of three questionnaires were posted to owners of, or veterinary surgeons attending, cases of grass sickness, with a request to provide information on the case, on one healthy animal on the same premises as the case and on another healthy animal on other premises. Controls were matched to cases by date of onset. After univariate analyses, the probability of grass sickness in horses was modelled using conditional logistic regression techniques. Young animals were found to be at increased rick of grass sickness and females were less likely to become affected, as were animals that had a history of contact with previous cases of the disease. The probability of grass sickness was higher in animals that were on premises where grass sickness had previously occurred, particularly if this was recent. Animals were at particular risk of disease if they changed fields within the previous 2 weeks; the risk thereafter reduced with time. The disease had a seasonal pattern, with a peak from April to June. More than 95% of cases had access to grazing, and 66% occurred after 2 week periods of predominantly dry weather.

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.

Long-term prospects for horses with grass sickness (dysautonomia).

Responses to questionnaires were received from 31 owners of horses or ponies treated for chronic grass sickness (dysautonomia). Contrary to previous opinions the respondents indicated that the majority of the animals were capable of strenuous work, had regained the weight they had lost and, apart from a few residual problems such as difficulty in coping with dry fibrous food, had returned to a normal life. They had recovered slowly and had involved the owners in considerable extra work, but all the owners indicated that they considered the effort to have been worthwhile.

An evaluation of the use of cisapride in horses with chronic grass sickness (equine dysautonomia)

A clinical trial was carried out to determine the effect of cisapride on rate of passage of digesta and clinical parameters in horses with chronic grass sickness. Sixteen horses were given intramuscular cisapride (0.1 mg kg-1 three times daily) (group I), and 15 received oral cisapride (0.8 mg kg-1 three times daily) (group O). A liquid-phase marker (cobalt-EDTA) and a solid-phase marker (polystyrene pellets) were given by stomach tube at the beginning of each of three consecutive 7 day periods, i.e., before, during and after cisapride therapy. Seven horses in each group completed the rate of passage trial; the remainder provided clinical data only. The rate of passage was found to be significantly faster after cisapride therapy than before. Comparison with data from 20 normal animals showed a trend towards normal rates of passage after therapy. In cases that died during the trial, the caecum and large colon were the main sites of pellet retention. Dry matter intake was significantly higher after therapy than before in group O and dry matter output was higher after treatment than before in both groups. Gut auscultation score increased in both groups in the periods during and after cisapride administration but heart rate was unaffected. Diarrhoea and colic occurred in each group but its occurrence was not associated with cisapride therapy. The results suggest that by increasing gut motility, cisapride is of benefit in the management of selected cases of chronic grass sickness.

Serum thyroxine and triiodothyronine responses of hyperthyroid cats to thyrotropin.

OBJECTIVE: To document circulating total thyroxine (T4) and triiodothyronine (T3) responses after administration of thyrotropin (thyroid-stimulating hormone [TSH]) to hyperthyroid and healthy cats and assess the value of these responses as an additional diagnostic test for hyperthyroidism. DESIGN: Prospective case series. ANIMALS: 21 healthy and 40 hyperthyroid cats. PROCEDURES: Serum total T4 and T3 concentrations were measured by radioimmunoassay before and 6 hours after administration of 0.5 IU of bovine TSH/kg of body weight. RESULTS: In healthy cats, serum total T4 concentration increased after administration of TSH (mean +/- SD, 114.0 +/- 36.4 nmol/L) representing a mean increment 3 times baseline concentration (mean +/- SD, 33.7 +/- 7.6 nmol/L). In hyperthyroid cats, the relative increase in serum total T4 concentration was significantly (P < 0.001) different; baseline values (mean +/- SD, 236.2 +/- 146.0 nmol/L) increased minimally after TSH administration (mean +/- SD, 308.1 +/- 178.9 nmol/L) There was a significant negative correlation (rs = -0.366) between relative increase in serum total T4 concentration after TSH administration and baseline concentration in hyperthyroid cats. In 3 cats with equivocal baseline serum total T4 concentration, the T4 response to TSH administration was indistinguishable from that in healthy cats. Serum total T3 response to TSH administration was significantly (P < 0.001) lower in hyperthyroid, compared with healthy, cats but the T3 response in healthy cats was more variable than that for T4 CONCLUSIONS: Thyrotoxic cats with high baseline serum total T4 concentration have a limited T4 response to TSH stimulation. Hyperthyroid cats with equivocal baseline serum total T4 concentrations have T4 response after TSH stimulation similar to those of healthy cats. Measurement of serum total T3 concentration provides no additional information. CLINICAL RELEVANCE: The TSH response test is of limited value in diagnosing hyperthyroidism in cats.

An attempt to reproduce 'mal seco' in horses by feeding them Festuca argentina.

'Mal seco' is an almost invariably fatal disease of horses in Argentina and Chile, which resembles grass sickness, a dysautonomia of horses in Europe. The aetiology of mal seco remains unknown. An attempt to reproduce the disease was made by feeding horses with Festuca argentina, a plant considered to be toxic to animals and which was consistently found in the diet of nine horses suffering from mal seco. Three horses were fed with F argentina ad libitum for 28 days. The plant was infected with an endophytic fungus, whose morphological characteristics were in agreement with descriptions of Acremonium chlamydosporioides. No clinical abnormalities were observed in two of the horses, but one died on the fifth day of the trial after becoming incoordinated, unsteady and ataxic in the fore- and hindlimbs. No gross changes were observed post mortem in any of the horses, with the exception of a small number of Fasciola hepatica in the liver of the horse which died, and a moderate number of Gasterophilus species in the stomach of all three horses. No histopathological changes were observed in any of the organs examined, including several autonomic ganglia, brain including most brain stem nuclei, spinal cord, liver, kidney, stomach and small and large intestine. The results of this study suggest that F argentina is either not implicated in the aetiology of mal seco or produces its effects only when they are triggered by other unknown factors.

Recovery of horses from dysautonomia (grass sickness).

The outcome for 35 horses with chronic dysautonomia which were kept in the hospital at the Royal (Dick) School of Veterinary Studies and subsequently returned to their owners is recorded. They constituted 42.7 per cent of the 82 chronic cases seen between 1991 and 1994; the other 47 horses were euthanased while in hospital. Of the 35 animals returned to their owners four died and 27 were available for follow up; of these 27, 12 were working competitively and six were being trained for future competitive work. It takes at least a year before it is clear whether a horse can compete successfully again but all the surviving animals were capable of being ridden. Some of the horses suffered excessive sweating, had difficulty in swallowing some foodstuffs, or had coat changes for long periods after returning to a normal weight.

Patterns of feeding and behaviour in horses recovering from dysautonomia (grass sickness).

Three horses suffering from grass sickness were treated successfully but showed clinical signs which varied in severity and duration. They were all characterised by marked and often erratic changes in behaviour and appetite.

The equine enteric nervous system--neuron characterization and distribution in adults and juveniles.

A study of myenteric and submucosal plexuses was undertaken in the jejunum and ileum of horses and ponies in which no clinical or pathological evidence of intestinal abnormality was apparent. Complete transverse sections of the intestine, stained by a modified haematoxylin and eosin method, were examined using up to 20 sequential sections per animal. Information was gathered from adult, juvenile and fetal equidae. In adults, the longitudinal muscle layers were thinner than the circular muscle layers and the ileum had thicker layers compared to the jejunum. In adults, the submucosal plexus had more neurons per section than the myenteric plexus by mean ratios of 1:3 in the jejunum and 1:1.9 in the ileum. In juveniles, the ratios were respectively 1:1.8 and 1:1.5 and in the fetus 1:2.5 and 1:1.3. The three-dimensional distribution of neurons in both plexuses varied from animal to animal and no consistent pattern was observed. Groups of neurons contained between one and 42 cells per section examined and their length in a cranio-caudal direction varied from 10 to over 100 microns. There were few statistical differences observed between the cranial, middle and caudal portions of either the jejunum or the ileum when neuron groups or neuron numbers per section were examined in 10 adult animals.

Small intestine and small colon neuropathy in equine dysautonomia (grass sickness).

The number of neurons in the coeliacomesenteric ganglia and the myenteric and submucosal plexuses of the jejunum, ileum and small colon, and the pathological changes induced in them, were studied in various types of equine dysautonomia. In all forms of dysautonomia, severe and extensive neuron loss and damage occurred in the ileum. In acute and subacute dysautonomia, jejunal neuron loss and damage were severe, but in chronic cases significantly less loss or damage occurred. The damage followed the same pattern in the small colon but it was always less obvious than in the jejunum. The distribution of the damage was uniform within a segment of the intestine. In fatal cases of dysautonomia, the clinical severity and duration of illness seems, in most instances, to be related to the amount of neuronal disruption occurring in the jejunum. Severe disruption results in acute/subacute dysautonomia, while milder damage leads to the chronic form. No case of dysautonomia was encountered in which enteric neuron loss and damage occurred without significant neuronal disruption also occurring in the coeliacomesenteric ganglia. Heal neuronal damage and loss are not invariably worse than that in the jejunum, and the possible reasons for this, together with the relationship between neuronal damage and possible causes of dysautonomia, are discussed.

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.

Evidence that the agent of equine grass sickness may reach neurons by retrograde axonal transport.

Sera from acute and chronic cases of natural grass sickness or normal horses were injected into the parotid salivary gland of ponies. This gland receives its sympathetic innervation from the ipsilateral cranial cervical ganglion. None of the ponies showed any local or systemic signs of illness. After one week the cranial cervical ganglia, stellate and coeliaco-mesenteric ganglia were removed for histological study. Pathological changes were found only in the cranial cervical ganglion ipsilateral to a parotid salivary gland which had received an injection of grass sickness serum. Four out of five batches of test sera from cases of acute natural grass sickness were associated with chromatolytic changes in neurons; the remaining batch of serum produced no abnormalities. The most severe chromatolytic changes were induced by two samples obtained from horses whose signs of grass sickness had been present for less than 12 hours. A serum sample from a chronic case of grass sickness of three weeks duration did not produce chromatolysis but was associated with a moderately severe inflammatory infiltrate and neuronophagia in the ipsilateral cranial cervical ganglion. One batch of serum was size fractionated to separate components with molecular weights above or below 30 kDa. Only the fraction containing components above 30 kDa induced chromatolytic changes.

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.

Use of clinical measurements to predict the outcome in chronic cases of grass sickness (equine dysautonomia).

Forty-five cases of chronic grass sickness were given scores for selected clinical measurements when they were first examined, to assess their value in predicting survival. Sixteen cases survived. The clinical scores for the degree of dysphagia, appetite, colic and the degree of reduction in gut sounds were significantly lower in the survivors than in the non-survivors. In addition, only the non-survivors had severe rhinitis. Ponies were significantly less likely to survive than cob types.

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.

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.