Increased level of compleasomes in cerebrospinal fluid of patients with herpes simplex encephalitis.

Herpes simplex encephalitis (HSE) is a common cause of viral encephalitis (HSV-1) characterised by pronounced inflammation and elevated intracranial pressure. We have shown in a rat model that HSV-1 infection causes an interaction between complement factors and proteasomes, leading to formation of proteasome/complement complexes (compleasomes). Exposure of the proteasome regulatory subunit antisecretory factor 1 (AF1) leads to a decrease in intracranial pressure. The aim of this study was to evaluate the acute and prolonged formation of compleasomes in cerebrospinal fluid (CSF) from patients with HSE. Cerebrospinal fluid samples (n = 55) from 24 HSE patients were analysed for compleasome complexes. Samples from healthy controls (n = 23) and patient controls (n = 27) served as baseline information. Sandwich enzyme-linked immunosorbent assay (ELISA) for proteasomes and their complex formation with complement factor 3 or 4, and Western blot for C3 activation were performed on CSF samples. Increased compleasome formation, both presenting as an initial formation and showing exposure of subunit AF1 in the compleasomes, was found in CSF samples drawn from patients with HSE compared with samples from the control groups (p < 0.0005). The total protein CSF concentration was equal in all groups. The levels were higher in the acute phase compared with late in the disease course (p < 0.0005). Complement 3 breakdown product iC3b was detected in CSF samples of the HSE patients. The early increased formation of compleasomes in CSF suggests that this complex may be involved in host defence against HSE.

Reaction of complement factors and proteasomes in experimental encephalitis.

Herpes simplex virus type 1 (HSV-1) encephalitis causes a deleterious inflammation and elevated intracranial pressure. As a step towards examining the origin of the inflammation, we here report the response of circulating proteasomes and complement factors in blood and cerebrospinal fluid (CSF) in rats infected with HSV-1. Infection was via the nasal route, with 1.1 × 104 plaque-forming units of HSV-1 strain 2762 given in one or both nostrils. A sandwich enzyme-linked immunosorbent assay was used to study the level of 26S proteasomes and their complex formation with complement factors 3 and 4. HSV-1 infection in the rat causes a complex formation between complement factors and proteasomes, which we designate compleasomes. In the first experiment, with HSV-1 given in both nostrils, compleasomes containing complement factors 3 and 4 increased significantly in both blood plasma and CSF. The concentration of proteasomes in plasma was similar in controls and infected rats (320 ± 163 vs. 333 ± 125 ng/ml). In the second experiment, with HSV-1 given in one nostril, CSF levels were 1 ± 1 ng/ml in controls and 56 ± 22 ng/ml in the HSV-1 group, whereas the total protein concentration in CSF remained the same in the two groups. The compleasome response was limited to CSF, with a highly significant difference between infected rats and controls (n = 11, p < 0.001). It was possible to mimic the reaction between proteasomes and complements 3 and 4 in vitro in the presence of ATP.

Interaction of Proteasomes and Complement C3, Assay of Antisecretory Factor in Blood.

Antisecretory factor (AF) is a protein complex which inhibits inflammation and regulates fluid transport. In this article, two new immunoassays (ELISA) are developed. The first ELISA establishes a 26S proteasome concentration of 0.41±0.03 µg/mL in normal plasma; the second ELISA discloses the binding of proteasomes to complement factor C3. The latter test values increased about tenfold following intake of processed cereals, paralleling with the old AF ELISA. The proteasome/C3 complex is purified and shown to expose hidden antisecretory peptide sequence and contain the inactive C3c protein. These findings might explain the antisecretory and anti-inflammatory effect during AF complex formation.

Uptake of the antisecretory factor peptide AF-16 in rat blood and cerebrospinal fluid and effects on elevated intracranial pressure.

BACKGROUND: AF-16 is a 16-amino-acid-long peptide derived from the amino-terminal part of the endogenous protein, antisecretory factor (AF). AF-16 in vivo has been shown to regulate dysfunctions in the water and ion transport system under various pathological conditions and also to counteract experimentally increased tissue pressure. METHODS: Rats were subjected to a cryogenic brain injury in order to increase the intracranial pressure (ICP). The distribution of AF-16 in blood and CSF after intravenous or intranasal administration was determined in injured and control rats. ICP was monitored in freely moving, awake rats, by means of an epidural pressure transducer catheter connected to a wireless device placed subcutaneously on the skull. The continuous ICP registrations were achieved by means of telemetry. RESULTS: Intranasal administration of AF-16 resulted in a significantly higher CSF concentrations of AF-16 in injured than in control rats, 1.3 versus 0.6 ng/ml, whereas no difference between injured and control rats was seen when AF-16 was given intravenously. Rats subjected to cryogenic brain injury developed gradually increasing ICP levels. Intranasal administration of AF-16 suppressed the increased ICP to normal values within 30 min. CONCLUSION: Optimal AF-16 concentrations in CSF are achieved after intranasal administration in rats subjected to a cryogenic brain injury. The ability of AF-16 to suppress an increased ICP was manifested.

Development of monoclonal antibodies for detection of Antisecretory Factor activity in human plasma.

Antisecretory Factor (AF) is expressed in most tissues and can be demonstrated in plasma and other body fluids. Most of the AF in plasma is in an inactive form and activation of AF occurs after exposure to bacterial toxins or after intake of various dietary components. Patients with chronic diseases involving disturbances in inflammatory and secretory processes may benefit from an AF-inducing diet. The aim of the present study was to develop an in vitro assay for the analysis of AF-activity in human plasma. Monoclonal antibodies were raised against a native form of AF prepared from human placenta. Nine clones of the monoclonal antibodies recognizing AF and AF peptides were identified. With the aid of these antibodies, we developed a sensitive ELISA method for direct detection of AF-activity in human plasma. The AF activity in plasma from five healthy volunteers was low, 0.112+/-0.022 (absorbance at 405 nm), before intake of the AF-inducing diet with the SPC-Flakes, and increased significantly (p<0.05) to 0.444+/-0.068 after >or=6 weeks on the diet. A comparison of the plasma-AF values, obtained by the bioassay and the immunogenic assay (indirect ELISA), shows that there is a significant correlation (r=0.85) between the values from the two methods. The results indicate that the ELISA measures AF-activity and has the potential to be an important tool for the analysis of AF-activity in further clinical studies on AF-therapy.

B 221, a medical food containing antisecretory factor reduces child diarrhoea: a placebo controlled trial.

AIM: We investigated whether egg yolk in the form of B221 (Salovum), a medical food containing antisecretory factor (AF) might be used for treatment of acute and prolonged diarrhoea. METHODS: 240 children 6-24 months of age, half with acute diarrhoea (<7 days) and half with prolonged diarrhoea (> or = 7 days) were randomly given 2 g of B221 or placebo every 5 h for 3 days, added to an oral rehydration salt solution. RESULTS: B221 reduced the number of stools in the acute diarrhoea group compared with placebo (day 3, p = 0.0054). Stools normalizing in consistency (day 3, p = 0.053) and recovery within 3 days was commoner in the B221 group (p < 0.001). A successful outcome was recorded in 82.8% in the B221 group, compared to 54.4% in the placebo group. In the group with prolonged diarrhoea the stool consistency normalized earlier in the patients receiving B221 than in the patients receiving placebo (p = 0.008). A successful outcome was obtained in 90.9% and 63.2%, (p = 0.0011) in the B221 and placebo-treated groups respectively. CONCLUSION: B221, which is a medical food, can be used to significantly improve the condition of children with acute, as well as prolonged diarrhoea caused by a broad range of undefined pathogens.

Differential expression of intestinal membrane transporters in cholera patients.

Vibrio cholerae causes the cholera disease through secretion of cholera toxin (CT), resulting in severe diarrhoea by modulation of membrane transporters in the intestinal epithelium. Genes encoding membrane-spanning transporters identified as being differentially expressed during cholera disease in a microarray screening were studied by real-time PCR, immunohistochemistry and in a CaCo-2 cell model. Two amino acid transporters, SLC7A11 and SLC6A14, were upregulated in acute cholera patients compared to convalescence. Five other transporters were downregulated; aquaporin 10, SLC6A4, TRPM6, SLC23A1 and SLC30A4, which have specificity for water, serotonin (5-HT), magnesium, vitamin C and zinc, respectively. The majority of these changes appear to be attempts of the host to counteract the secretory response. Our results also support the concept that epithelial cells are involved in 5-HT signalling during acute cholera.

Broad up-regulation of innate defense factors during acute cholera.

We used a whole-genome microarray screening system (Affymetrix human GeneChips covering 47,000 different transcripts) to examine the gene expression in duodenal mucosa during acute cholera. Biopsies were taken from the duodenal mucosa of seven cholera patients 2 and 30 days after the onset of diarrhea, and the gene expression patterns in the acute- and convalescent-phase samples were compared pairwise. Of about 21,000 transcripts expressed in the intestinal epithelium, 29 were defined as transcripts that were up-regulated and 33 were defined as transcripts that were down-regulated during acute cholera. The majority of the up-regulated genes characterized were found to have an established or possible role in the innate defense against infections; these genes included the LPLUNC1, LF, VCC1, TCN1, CD55, SERPINA3, MMP1, MMP3, IL1B, LCN2, SOCS3, GDF15, SLPI, CXCL13, and MUC1 genes. The results of confirmative PCR correlated well with the microarray data. An immunohistochemical analysis revealed increased expression of lactoferrin in lamina propria cells and increased expression of CD55 in epithelial cells, whereas increased expression of the SERPINA3 protein (alpha1-antichymotrypsin) was detected in both lamina propria and epithelial cells during acute cholera. The expression pattern of CD55 and SERPINA3 in cholera toxin (CT)-stimulated Caco-2 cells was the same as the pattern found in the intestinal mucosa during acute cholera, indicating that the activation of the CD55 and SERPINA3 genes in intestinal epithelium was induced by CT. In conclusion, during acute cholera infection, innate defense mechanisms are switched on to an extent not described previously. Both direct effects of CT on the epithelial cells and changes in the lamina propria cells contribute to this up-regulation.

Detection of elafin as a candidate biomarker for ulcerative colitis by whole-genome microarray screening.

The cause of ulcerative colitis (UC) is largely unknown. Microarray studies are an efficient way of investigating the various genes involved. Here, we have used whole-genome microarrays to clarify the clinical picture and to identify new biomarkers for improved diagnosis. Rectal biopsies were taken from five UC patients and five matched controls, and RNA transcripts were prepared. After labeling, each sample was individually applied to the microarray chips. All transcripts that were more than 10-fold up-regulated in all five patients were analyzed further in seven additional patients and seven controls using quantitative polymerase chain reaction. Of 47,000 transcripts examined, 4 were highly up-regulated in all patients: those encoding elafin, a secreted protease inhibitor, the ion and amino acid transporter B (SLC6A14), and the metabolic enzyme aldolase B, as well as a recently identified transcript named similar to numb-interacting homolog. The up-regulation of these transcripts appears to follow the progression of the disease because elevated expression was detected in the proximal part of the colon in patients with total colitis but not in patients with left-sided colitis. Immunohistologic examination showed very distinct differences in the expression of elafin. Extensive expression was detected in enterocytes and goblet cells of the affected mucosa, whereas there was no detectable expression in unaffected mucosa and in healthy controls. The results implicate four transcripts and proteins of special interest as possible targets for pharmacologic interference and as biomarkers in UC. Of these, elafin may be of special interest because it is a secreted protein that may be measured in body fluids.

Cholera toxin induces a transient depletion of CD8+ intraepithelial lymphocytes in the rat small intestine as detected by microarray and immunohistochemistry.

Cholera toxin (CT), besides causing intestinal hypersecretion after intragastric administration or during cholera infection, affects a multitude of regulatory mechanisms within the gut mucosal network, including T cells. By use of microarray screening, real-time PCR, and immunohistochemistry, we demonstrate here a rapid depletion of jejunal CD8(+) intraepithelial lymphocytes (IEL) in rats after intragastric CT challenge. This depletion may depend on CT-induced migration of IEL, since it was associated with a progressive decrease of CD8(+) cells in the epithelium and a contemporary transient increase of such cells, preferentially at the base of the villi, in the lamina propria. A significant decrease in the total number of villous CD8(+) cells at 6 and 18 h after CT challenge was detected; this possibly reflects an efflux from the jejunal mucosa. The kinetics of the CD8(+) IEL demonstrate the return to normal intraepithelial position at original numbers already 72 h after the single CT dose. The induced migration seems to be dependent on the enzymatic A-subunit of CT, since challenge with neither sorbitol nor CT B-subunit did mimic the effects of CT on CD8(+) IEL. Furthermore, a decrease in the level of both RANTES transcript and protein was detected, most likely as a consequence of the CT-induced migration of CD8(+) IEL. These results point to a complex interaction between CT, epithelial cells, and IEL, resulting in a disturbance of the gut homeostasis, which might have relevance for the strong immunomodulatory effects of intragastrically administered CT.

Cholera toxin induces expression of ion channels and carriers in rat small intestinal mucosa.

Cholera toxin causes cyclic adenosine monophosphate (cAMP)-induced electrolyte and water secretion in the small intestine. The toxin-induced change in gene expression in rat small intestine was evaluated with microarray technique and the results were confirmed by semiquantitative polymerase chain reaction (PCR). The transporter CNT2 for nucleosides was upregulated between 6 and 18 h after challenge, whereas the level of GLUT1 transporter for glucose became elevated at 6 h. Both changes probably facilitate uptake of these nutrients in the gut. At 18 h, the major chloride channel in the villus, ClC2, was upregulated. Aquaporin 8 was downregulated at 6 h and two mucin-producing genes were upregulated 18 h after toxin challenge. The expression was back to normal after 72 h, which is the turnover time for intestinal epithelial cells.

Cholera toxin protects against action by Clostridium difficile toxin A. The role of antisecretory factor in intestinal secretion and inflammation in rat.

UNLABELLED: The protein antisecretory factor (AF) inhibits intestinal fluid secretion induced by the cholera toxin (CT) and Clostridium difficile toxin A (CDA). The present work investigated whether CT-induced AF protects against the enterotoxin action by CDA. Rats were pretreated perorally with CT or buffer as control, whereafter CDA-induced fluid secretion and cytotoxicity was tested in vivo in ligated intestinal loops; the mucosal level of AF was estimated using the Western blot technique. Rats given repeated peroral doses of CT became tolerant to CDA, the inhibition of fluid secretion and of cytotoxicity being 79% in eight out of nine animals. The repeated CT-treatment also induced long-lasting rise of AF in the mucosal epithelium. Recombinant AF given either perorally or intravenously inhibited both fluid secretion and cytotoxicity by CDA; similar results were obtained with a truncated 16-mer AF peptide. IN CONCLUSION: peroral CT-treatment induced tolerance to CDA in rat small intestine. The tolerance was probably mediated by AF induced via action of cholera toxin on the enteric nervous and immune system.

Food-induced antisecretory factor activity is correlated with small bowel length in patients with intestinal resections.

Specially processed cereals (SPC) can increase antisecretory factor (AF) activity in humans with an intact intestine. The aim of the present study was to investigate whether AF synthesis could be induced in patients who had been subjected to intestinal resections. Eight patients with varying extents of intestinal resections due to Crohn's disease and six healthy controls participated. All subjects received 54 g SPC daily for 2 weeks. Plasma AF activity was determined before, during and after the treatment period. Baseline diet and medications were kept unchanged. The patients registered the daily number of bowel movements. The SPC diet increased AF activity in all controls. In the patients there was a significant correlation between the length of the remaining small intestine and AF induction (r=0.94, p<0.01) and only those patients with a remaining small intestine of about 3 m reached AF values comparable to those in healthy subjects. It is concluded that small bowel length is related to the ability of humans to induce AF activity by dietary means.