Review: experimental manipulations of microglia in mouse models of Alzheimer's pathology: activation reduces amyloid but hastens tau pathology.

The inflammation hypothesis of Alzheimer's pathogenesis has directed much scientific effort towards ameliorating this disease. The development of mouse models of amyloid deposition permitted direct tests of the proposal that amyloid-activated microglia could cause neurodegeneration in vivo. Many approaches to manipulating microglial activation have been applied to these mouse models, and are the subject of this review. In general, these results do not support a direct neuricidal action of microglia in mouse amyloid models under any activation state. Some of the manipulations cause both a reduction in pathology and a reduction in microglial activation. However, at least for agents like ibuprofen, this outcome may result from a direct action on amyloid production, and a reduction in the microglial-provoking amyloid deposits, rather than from reduced microglial activation leading to a decline in amyloid deposition. Instead, a surprising number of the experimental manipulations which increase microglial activation lead to enhanced clearance of the amyloid deposits. Both the literature and new data presented here suggest that either classical or alternative activation of microglia can lead to enhanced amyloid clearance. However, a limited number of studies comparing the same treatments in amyloid-depositing vs. tau-depositing mice find the opposite effects. Treatments that benefit amyloid pathology accelerate tau pathology. This observation argues strongly that potential treatments be tested for impact on both amyloid and tau pathology before consideration of testing in humans.

Necrotising fasciitis in a bull due to infection with Arcanobacterium haemolyticum.

CASE HISTORY: A 2-year-old Hereford bull was lame for one week before becoming recumbent. CLINICAL AND PATHOLOGICAL FINDINGS: The scrotum and ventral perineal region were cold and blackened caudally. The semimembranosus and semitendinosus muscles were firm on palpation. The bull was subject to euthanasia, and necropsy revealed that the skin and S/C tissues of the caudal half of the scrotum were grey and necrotic. The caudal and distal aspects of the semimembranosus and semitendinosus muscles were grey and necrotic to a depth of approximately 15 cm, and these changes appeared to track along fascial planes. The tissue had an offensive smell, and large amounts of flocculent, watery, brown fluid and some gas were present. Histology of affected muscle and S/C tissues revealed coagulative necrosis, with oedema and large numbers of bacteria that were predominantly Gram-positive rods. Adjacent blood vessels contained thrombi while the epidermis overlying the affected areas appeared diffusely necrotic, suggesting infarction. Culture of the fluid yielded a pure growth of Arcanobacterium spp., which was identified as Arcanobacterium haemolyticum, using an API Coryne biochemical test strip. DIAGNOSIS: Necrotising fasciitis and myositis due to Arcanobacterium haemolyticum. CLINICAL RELEVANCE: Arcanobacterium haemolyticum has not previously been reported as a cause of necrotising fasciitis in any species. Necrotising fasciitis is probably an under-reported condition in cattle due to its clinical similarity to clostridial disease.

Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry.

Functional proteomics provides a powerful method for monitoring global molecular responses following activation of signal transduction pathways, reporting altered protein posttranslational modification and expression. Here we combine functional proteomics with selective activation and inhibition of MKK1/2, in order to identify cellular targets regulated by the MKK/ERK cascade. Twenty-five targets of this signaling pathway were identified, of which only five were previously characterized as MKK/ERK effectors. The remaining targets suggest novel roles for this signaling cascade in cellular processes of nuclear transport, nucleotide excision repair, nucleosome assembly, membrane trafficking, and cytoskeletal regulation. This study represents an application of functional proteomics toward identifying regulated targets of a discrete signal transduction pathway and demonstrates the utility of this discovery-based strategy in elucidating novel MAP kinase pathway effectors.

Structural characterization of the membrane-associated regulatory subunit of type I cAMP-dependent protein kinase by mass spectrometry: identification of Ser81 as the in vivo phosphorylation site of RIalpha.

The mechanism by which the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RIalpha is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane-associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane-associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane-associated and cytosolic forms of RI were the same RIalpha gene product. A single RIalpha phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane-associated and cytosolic RIalpha. Because both R subunit preparations were 30-40% phosphorylated, this post-translational modification could not be responsible for the membrane compartmentation of the majority of RIalpha. Mass spectrometry also indicated that membrane-associated RIalpha had a higher extent of disulfide bond formation in the amino-terminal dimerization domain. No other structural differences between cytosolic and membrane-associated RIalpha were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane-associated and cytosolic RIalpha strongly suggests an interaction between RIalpha and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIalpha membrane association in the heart.

Cultural control of larval mosquito production in a fallow citrus grove used for disposal of secondary-treated sewage effluent.

Larval mosquito production was monitored for 16 months in the furrows of a 13.4-ha citrus grove in east-central Florida used for disposal of secondary-treated sewage effluent. Twenty-one species of mosquito were collected, and the 2 most abundant species were Culex nigripalpus and Aedes vexans. An unplanned removal of all brush and trees from the site during the study resulted in an overall decline in larval production, but species diversity remained the same.

Enhancing clinical efficacy of oral rehydration therapy: is low osmolality the key?

Many empirical clinical trials have used complex carbohydrate as substrate in oral rehydration solutions (ORSs) instead of glucose and have shown a number of important clinical benefits. Foremost among these are reduced stool volumes, shorter duration of diarrheal illness, and lower ORS intake. The underlying mechanisms to explain this clinical advantage have not been fully established, but a number of possible factors have been proposed: (1) increased substrate availability, (2) a "kinetic advantage" for glucose absorption by glucose polymer, (3) differential handling of glucose monomer and polymer by the small intestine, (4) low osmolality, (5) a separate effect of peptides and amino acids on solute-linked sodium absorption, (6) an antisecretory moiety in rice, and (6) enhanced mucosal repair and regeneration by luminal nutrients. In this report, we assess the relative contribution of these factors using evidence from laboratory-based studies, mainly in disease-related intestinal perfusion systems in animals and humans, and the relevant clinical studies available to date. We advance the hypothesis that of all the possible mechanisms proposed to underlie the enhanced clinical efficacy of complex carbohydrate ORSs, their hypotonicity plays the dominant role. If confirmed, this concept could guide future development of glucose and complex carbohydrate-based ORSs.

Jejunal water and electrolyte transport in human cryptosporidiosis.

Cryptosporidiosis may have severe clinical consequences in both immunocompromised and immunocompetent individuals. However, pathophysiological mechanisms that are responsible for diarrhea are poorly understood. We performed jejunal perfusion studies in patients with human immunodeficiency virus-related cryptosporidial diarrhea to measure water and electrolyte transport in vivo. Five patients with human immunodeficiency virus-related cryptosporidiosis and nine healthy volunteers were studied using a triple-lumen steady-state jejunal perfusion technique. Stool volume measurement and distal duodenal biopsy showed that the patients had diarrhea (600-1500 ml/24 hr) and morphological abnormalities of small intestinal mucosa. Net water, sodium, and chloride movement in the jejunum was not significantly different from healthy controls. In these patients with watery diarrhea and morphological mucosal abnormalities, we found no evidence that cryptosporidial diarrhea was due to a secretory state in the proximal small intestine. We conclude that diarrhea may be due to secretion of electrolytes and water efflux more distally or to other abnormalities of gastrointestinal function.

Absorption of a hypotonic oral rehydration solution in a human model of cholera.

The development of oral rehydration solutions (ORSs) has been one of the important therapeutic advances of this century. The optimal formulation, however, of ORSs for both cholera and other infective diarrhoeas is still debated. Part of the problem in developing ORSs has been the lack of adequate test systems for the assessment of new formulations before clinical trial. We have developed a jejunal perfusion, cholera toxin induced, secretory model in humans and have compared net water and solute absorption from a hypotonic ORS (HYPO-ORS: sodium 60 mmol/l, glucose 90 mmol/l, osmolality 240 mOsm/kg) and the British Pharmacopoeia recommended ORS (UK-ORS: sodium 35 mmol/l, glucose 200 mmol/l, osmolality 310 mOsm/kg) in six healthy volunteers. A plasma electrolyte solution (PES) was also perfused in all subjects to confirm a secretory state. Only HYPO-ORS reversed sodium secretion to absorption (p < 0.01). Both ORSs promoted net water absorption but this was greatest with HYPO-ORS (p < 0.01). Glucose and potassium absorption rates were similar for both ORSs whereas chloride absorption mirrored sodium absorption and was greatest from HYPO-ORS (p < 0.05). These results, in a biologically relevant model of secretory diarrhoea, suggest it may be possible to achieve improved rates of rehydration by the use of hypotonic ORS with mid range sodium concentrations.

Decreased contact factor mediated fibrinolysis in cirrhosis.

We studied extrinsic and intrinsic fibrinolysis in 20 patients with cirrhosis (nine mild/moderate, group 1; 11 severe, group 2) and 19 normal controls to define the role of intrinsic (contact factor medaited) fibrinolysis in cirrhosis. Global plasma fibrinolytic activity (fibrin plate lysis) was similar in all groups. Dextran sulphate activated contact factor mediated fibrinolysis was decreased in group 2 (median 95.2%) compared with group 1 (121.0%) and controls (131.7%). Tissue plasminogen activator antigen (t-PA Ag) levels were increased in group 2 (28.2 ng/ml) compared both with group 1 (8.5 ng/ml) and controls (5.9 ng/ml). Plasma t-PA activity was raised in group 2 (5.50 IU/ml) and group 1 (5.25 IU/ml) versus controls (0.82 IU/ml). Plasminogen activator inhibitor-1 (PAI-1 Ag) levels were raised in group 2 (28.0 IU/ml) versus controls (8.5 IU/ml) but PAI activity was similar in all groups. Factor XII activity was decreased in group 2 (48.76 u/dl), but not group 1, versus controls (89.1 u/dl). Prekallikrein activity was decreased both in group 2 (27.27 u/dl) and group 1 (33.01 u/dl) versus controls (108.59 u/dl) and was lower in group 2 than group 1. C1-esterase inhibitor chromogenic activity was decreased in group 1 (102.30 u/dl) and group 2 (58.76 u/dl) versus controls (116.24 u/dl). The normal global fibrinolytic activity despite increased t-PA activity may be due to a concomitant increase in PAI. The decreased intrinsic fibrinolysis in severe cirrhosis, unaccompanied by a rise in C1-esterase inhibitor, may be explained by the decreased factor XII and prekallikrein activity. These changes are probably due to reduced liver cell mass.

Combined percutaneous transhepatic and endoscopic placement of biliary stents.

Combined percutaneous transhepatic cholangiography and endoscopic retrograde cholangiography can be used to stent biliary obstruction when attempts at endoscopic stenting have failed. Between January 1987 and August 1991 we performed 35 combined procedures in 31 patients with malignant obstruction. Post stenting serum bilirubin and serum alkaline phosphatase concentration fell after 33 and 29 procedures, respectively. In six studies there was evidence of infection prior to stenting. In spite of the use of prophylactic antibiotics, septic complications developed after eight procedures (23%). Pseudomonas was the most commonly isolated pathogen (46%). Twenty-three patients were discharged, 30-day mortality was 8 (23%) and median survival was 14 weeks (range 0-75 weeks). Seven patients required eight stent changes because of blockage (median patency time 18 weeks; range 7-75 weeks). Use of this technique allows relief of biliary obstruction. Potential infective and bleeding complications must be anticipated.

Initial response and subsequent course of Crohn's disease treated with elemental diet or prednisolone.

Elemental diet is as effective as corticosteroids in the treatment of previously untreated Crohn's disease. It is unclear whether a poor nutritional state is a prerequisite for efficacy of elemental diet, whether previously treated patients respond as well, or how duration of remission using elemental diet compares with corticosteroid induced remission. Forty two patients with active Crohn's disease were stratified for nutritional state and randomised to receive Vivonex TEN 2.1 l/day for four weeks, or 0.75 mg prednisolone/kg/day for two weeks and subsequent reducing doses. Nine of 22 (41%) patients assigned to nutritional treatment were intolerant of the diet. Thirty patients completed four weeks treatment. Disease activity decreased on elemental diet from mean (SEM) 4.8 (0.9) to 1.7 (0.6), p < 0.05, and on prednisolone from 5.3 (0.5) to 1.9 (0.6), p < 0.05. For each treatment, nourished and malnourished patients responded similarly. Patients with longstanding disease responded as well as newly diagnosed patients. The probability of maintaining remission at six months was 0.67 after prednisolone, 0.28 after elemental diet, and at one year was 0.35 after prednisolone and 0.09 after elemental diet, p < 0.05. When tolerated, elemental diet is as effective in the short term as prednisolone in newly and previously diagnosed Crohn's disease, and its benefit is independent of nutritional state. The subsequent relapse rate after elemental diet induced remission, however, is greater than after treatment with prednisolone.

Recurrent primary liposarcoma of the pericardium: management by repeated resections.

In 1979 we published a report of a patient with primary liposarcoma of the pericardium treated by surgical resection. Since then we have performed a total of seven resections on the same patient for recurrent liposarcoma, which have given excellent symptomatic relief, and her life was prolonged for 14 years. Repeated resection of recurrent liposarcoma of the mediastinum should always be considered as the appropriate management for these patients.

Intraperitoneal haemorrhage from anterior abdominal wall varices.

Patients with oesophageal varices frequently present with gastrointestinal haemorrhage but bleeding from varices at other sites is rare. We present a patient with hepatitis C-induced cirrhosis and partial portal vein occlusion who developed spontaneous haemorrhage from anterior abdominal wall varices into the rectus abdominus muscle and peritoneal cavity.

Water and solute absorption from a new hypotonic oral rehydration solution: evaluation in human and animal perfusion models.

Controversy continues regarding the optimal composition of glucose electrolyte oral rehydration solutions for the treatment of acute diarrhoea. Four perfusion models (normal human jejunum, normal rat small intestine, cholera toxin treated secreting rat small intestine and rotavirus infected rat small intestine) have been developed and used to compare the efficacy of a hypotonic oral rehydration solution with standard United Kingdom British National formulary and developing world oral rehydration solutions (WHO). Despite obvious physiological and pathophysiological differences between these models there was general congruence in the water and solute absorption profiles of the different oral rehydration solutions. Hypotonic oral rehydration solution promoted significantly greater water absorption than other oral rehydration solutions in all rat models (p < 0.001) but apparently increased water absorption failed to achieve significance in human jejunum. British National Formulary-oral rehydration solution was unable to reverse net water secretion in both rotavirus and cholera toxin models. Net sodium absorption from hypotonic and WHO-oral rehydration solutions was significantly greater than from the low sodium British National Formulary-oral rehydration solutions (p < 0.001) except in the rotavirus model when absorption was similar to hypotonic-oral rehydration solutions. These findings show that there is agreement in the apparent efficacy of oral rehydration solutions in these animal and human perfusion models, and that improved water absorption with adequate sodium absorption may be achieved by reducing oral rehydration solution osmolality.

A new model of human secretory diarrhoea using cholera toxin.

Secretory diarrhoea is a major cause of morbidity and mortality worldwide. However, there is no biologically relevant test system in man for assessing new anti-diarrhoeal therapies prior to clinical trial. We have used highly purified cholera toxin in combination with the triple lumen jejunal perfusion technique to establish a subclinical model of cholera in man. Cholera toxin was administered either by mouth with sodium bicarbonate or directly into a 30 cm 'open' or 'closed' (isolated between two inflated balloons) jejunal segment in healthy adult volunteers. Both oral dosing and direct delivery into an 'open' jejunal segment failed to produce consistent secretion of water and electrolytes. In contrast 15 micrograms or 25 micrograms of cholera toxin elicited secretion of water and sodium 3 h after instillation into the balloon occluded 'closed' jejunal segment (P less than 0.05 vs. controls). The rate of secretion was constant over the maximal period studied (4.5 h) and was similar to that reported in human cholera. None of the subjects experienced troublesome diarrhoea. We believe this model offers a relevant test system for assessing anti-diarrhoeal therapy in man.

Water and solute absorption from hypotonic glucose-electrolyte solutions in human jejunum.

While oral rehydration therapy with glucose-electrolyte solutions is highly effective, the optimal formulation has not yet been defined. Recent clinical studies suggest that stool volume, and thus water losses, may be reduced if glucose is replaced by a polymeric substrate which reduces osmolality. It is possible that the efficacy of glucose monomer based oral rehydration solutions (ORS) will also improve if osmolality is decreased. Using jejunal triple lumen perfusion in healthy adult volunteers net water and solute absorption were studied from three hypotonic solutions with different sodium concentrations (46, 60, 75 mmol/l) but identical glucose concentrations (90 mmol/l), thus allowing osmolality to rise (210, 240, and 270 mOsm/kg, respectively). Results from these solutions (ORS 45:210, ORS 60:240, and ORS 75:270) were compared with the World Health Organisation oral rehydration solution (WHO-ORS). Greatest water absorption was seen with ORS 60:240 (p less than 0.01). Sodium absorption from ORS 60:240 and WHO-ORS was similar and greater than sodium absorption from ORS 45:210 (p less than 0.05). Potassium and glucose absorption were greater from ORS 60:240 than from any of the other hypotonic solutions (p less than 0.05) and were equal to absorption from WHO-ORS). These results in a short segment of healthy human jejunum suggest that hypotonic ORS containing monomeric glucose may increase water absorption.