Infliximab inhibits activation and effector functions of peripheral blood T cells in vitro from patients with clinically active ulcerative colitis.

Many patients with inflammatory bowel disease (IBD) are undergoing therapy with infliximab, an antibody specific for TNF. However, the exact mechanisms of action of infliximab are not completely understood. The aim of this study was to determine the in vitro effects of infliximab on blood T cells derived from anti-TNF therapy-naïve ulcerative colitis (UC) patients with clinically active disease. Peripheral blood mononuclear cells were stimulated polyclonally or by antigen in the presence or absence of infliximab. The T cell phenotype was investigated by flow cytometry, cytokine secretion was determined by ELISA, and cell proliferation was determined by thymidine assay or CFSE dye. Presence of infliximab resulted in reduced expression of CD25 in CD4(+) and CD8(+) T cell populations and inhibited secretion of IFN-γ, IL-13, IL-17A, TNF as well as granzyme A. Infliximab also suppressed CD4(+) and CD8(+) T cell proliferation. These effects of infliximab were recorded both in T cells activated by polyclonal and antigen-specific stimulation. The effects of infliximab on T cell apoptosis and induction of FOXP3(+) CD4(+) T regulatory cells were ambiguous and depended on the originating cellular source and/or the stimulation mode and strength. In conclusion, infliximab is able to reduce T cell activation as measured by CD25, proliferation and cytokine secretion in vitro from UC patients with clinically active disease. These data suggest that suppression of T cell activity may be important for infliximab-mediated disease remission in patients with UC.

Heart rate variability characteristics of patients with irritable bowel syndrome and associations with symptoms.

BACKGROUND: Disturbed brain-gut interactions are assumed to be of importance for symptom generation in patients with irritable bowel syndrome (IBS). The autonomic nervous system (ANS) is part of the bidirectional brain-gut communication, but previous studies in IBS show diverging results. We aimed to identify subgroups of IBS patients with distinct ANS characteristics differentiating them from healthy controls (HC), and to study associations between ANS status and symptoms. METHODS: Heart rate variability (HRV) was measured in IBS patients and HC (Holter monitoring: supine and standing positions with controlled respiration and ambulatory 24-hour period). Frequency (5 minutes, supine, standing) and time domains (24 hours, day, night) were analyzed. Validated questionnaires were used to measure gastrointestinal and psychological symptoms in patients. Patients and HC were compared on a univariate and multivariate level (principal component analysis [PCA] and orthogonal partial least squares discriminatory analysis (OPLS-DA)). KEY RESULTS: We analyzed 158 IBS patients (Rome III) and 39 HC. Patients differed significantly from HC in HRV parameters during daytime and in standing position. In the PCA, a majority of patients overlapped with HC, but the weighted means differed (P < .01). A subset of patients (n = 30; 19%) with an aberrant global HRV profile was identified through PCA and OPLS-DA; these patients reported more severe symptoms of frequent (P < .05) and loose stools (P = .03), as well as urgency (P = .01). CONCLUSIONS AND INFERENCES: Altered ANS function was demonstrated in patients with IBS, and this might be of particular relevance for symptoms in a subset of the patients.

Neither self-reported atopy nor IgE-mediated allergy are linked to gastrointestinal symptoms in patients with irritable bowel syndrome.

BACKGROUND: Among patients with irritable bowel syndrome (IBS), atopic disease has been proposed as a common comorbidity increasing the IBS symptom burden. We therefore assessed the prevalence of self-reported atopy among patients with IBS as compared to non-IBS controls, and whether atopy and higher serum IgE levels were associated with increased IBS symptom severity. METHODS: Levels of total and specific IgE in serum were measured and questionnaires assessing the presence of atopic disease (ie, eczema, asthma, rhinoconjunctivitis, and pollen allergy), gastrointestinal symptom burden, food intolerance, somatic, and psychological symptoms were completed. KEY RESULTS: In total, 223 patients with IBS and 47 controls participated. Presence of atopic disease was reported in 55% of patients with IBS compared to 40% of controls (P = .07). IBS patients with atopic manifestations (N = 123) had higher total serum IgE levels (median 31 vs 16 kUA /L, P < .001) and higher prevalence of self-reported food intolerance (28% vs 9%, P = .002) than non-atopic IBS patients (N = 100), respectively, but no major difference in gastrointestinal or psychological symptom burden was noted. However, severe somatic symptoms were more common among atopic than non-atopic patients with IBS (38% vs 27%, P = .028). We found no associations between self-reported atopy and IBS symptom severity using linear regression models. CONCLUSIONS & INFERENCES: Atopic disease is common in patients with IBS, but that is also true for subjects without IBS. The presence of atopic disease in IBS is associated with self-reported food intolerance and somatic symptom severity, but unrelated to IBS symptom severity.

Systemic cytokines are elevated in a subset of patients with irritable bowel syndrome but largely unrelated to symptom characteristics.

BACKGROUND: Serum levels of pro-inflammatory cytokines tend to be increased in irritable bowel syndrome (IBS) patients, or subgroups thereof. Still, the link between cytokine levels and IBS symptoms is unclear. We aim to determine systemic cytokine levels in IBS patients and healthy subjects (HS), confirm the presence of a subset of patients with an increased immune activity and to establish if cytokines are linked to IBS symptoms and pathophysiological factors. METHODS: Serum levels of interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF), and IL-10 were measured. All subjects reported IBS symptoms using validated questionnaires and underwent colonic sensorimotor testing. Multivariate supervised orthogonal partial least squares-discriminant analysis (OPLS-DA) and unsupervised principal component analysis (PCA) and hierarchical cluster analysis (HCA) were implemented. KEY RESULTS: Irritable bowel syndrome patients (n = 246) had higher serum levels of IL-1ß, IL-6, IL-8, TNF, and IL-10 compared to HS (n = 21); however, serum cytokine profiles could not discriminate patients from HS. Moreover, cytokine levels were not correlated with symptoms among patients. Supervised OPLS-DA identified 104 patients (40% of patients) and unsupervised HCA analysis identified 49 patients (20%) with an increased immune activity indicated by elevated levels of serum cytokines compared to HS and the other patients. However, irrespective of how patients with increased immune activity were identified they were symptomatically similar to patients with no indication of increased immune activity. CONCLUSIONS & INFERENCES: Serum cytokines are elevated in IBS patients compared to HS. Immune activation characterizes a subset of patients, but modest associations between cytokine profile and symptoms suggest immune activity does not directly influence symptoms in IBS.

Colonic immune cells in irritable bowel syndrome: A systematic review and meta-analysis.

BACKGROUND & AIMS: Increases in mucosal immune cells have frequently been observed in irritable bowel syndrome (IBS) patients. However, this finding is not completely consistent between studies, possibly due to a combination of methodological variability, population differences and small sample sizes. We performed a meta-analysis of case-control studies that compared immune cell counts in colonic biopsies of IBS patients and controls. METHODS: PubMed and Embase were searched in February 2017. Results were pooled using standardized mean difference (SMD) and were considered significant when zero was not within the 95% confidence interval (CI). Heterogeneity was assessed based on I2 statistics where I2  ≤ 50% and I2  > 50% indicated fixed and random effect models, respectively. KEY RESULTS: Twenty-two studies on 706 IBS patients and 401 controls were included. Mast cells were increased in the rectosigmoid (SMD: 0.38 [95% CI: 0.06-0.71]; P = .02) and descending colon (SMD: 1.69 [95% CI: 0.65-2.73]; P = .001) of IBS patients. Increased mast cells were observed in both constipation (IBS-C) and diarrhea predominant IBS (IBS-D). CD3+ T cells were increased in the rectosigmoid (SMD: 0.53 [95% CI: 0.21-0.85]; P = .001) and the descending colon of the IBS patients (SMD: 0.79, 95% CI [0.28-1.30]; P = .002). This was possibly in relation to higher CD4+ T cells in IBS (SMD: 0.33 [95% CI: 0.01-0.65]; P = .04) as there were no differences in CD8+ T cells. CONCLUSIONS & INFERENCES: Mast cells and CD3+ T cells are increased in colonic biopsies of patients with IBS vs non-inflamed controls. These changes are segmental and sometimes IBS-subtype dependent. The diagnostic value of the quantification of colonic mucosal cells in IBS requires further investigation.

CD4+CD25+ regulatory T cells in irritable bowel syndrome patients.

The aetiology of the irritable bowel syndrome (IBS) is incompletely understood. A low-grade colonic inflammation is frequently seen, but it is unclear to what extent this phenomenon contributes to the pathophysiology of IBS. CD4(+)CD25(+) regulatory T cells (Treg) are implicated to play an important role in suppressing intestinal inflammation. We, therefore, examined whether the intestinal inflammatory process in IBS patients is the result of an altered function and/or frequency of CD25(+) Treg cells. Patients with IBS (n = 34), fulfilling the Rome II criteria, were compared with controls (n = 26). The suppressive activity of blood CD25(+) Treg cells was determined and the frequency of colonic and blood CD25(+) Treg cells was analysed by flow cytometry. The expression of the Treg marker, FOXP3 mRNA, in colonic biopsies was determined by reverse transcription-polymerase chain reaction. Blood CD25(+) Treg cells from IBS patients suppressed the proliferation of blood CD4(+)CD25(low/-) T cells. Similar frequencies of CD25(+) Treg cells were recorded in mucosa and blood of IBS patients and controls. FOXP3 mRNA was equally expressed in the colonic mucosa of patients with IBS and controls. In conclusion, the low-grade intestinal inflammation recorded in patients with IBS is not associated with an altered function or frequency of CD25(+) Treg cells.

New insights into the pathogenesis and pathophysiology of irritable bowel syndrome.

The pathogenesis and pathophysiology of irritable bowel syndrome is complex and still incompletely known. Potential pathogenetic factors include genes, infectious events, psychological symptoms and other loosely defined environmental factors. Both alterations at the central and peripheral level are thought to contribute to the symptoms of irritable bowel syndrome, including psychosocial factors, abnormal gastrointestinal motility and secretion, and visceral hypersensitivity. Today irritable bowel syndrome is viewed upon as a disorder of dysregulation of the so-called brain-gut axis, involving abnormal function in the enteric, autonomic and/or central nervous systems, with peripheral abnormalities probably dominating in some patients and disturbed central processing of signals from the periphery in others. Lines of evidence also suggest that inflammation within the gastrointestinal tract may be of great importance in at least subgroups of irritable bowel syndrome patients. To conclude, a complex picture of the pathogenesis and pathophysiology of irritable bowel syndrome is emerging, with interactions between several different alterations resulting in the divergent symptom pattern in these patients.

Mixture model analysis identifies irritable bowel syndrome subgroups characterised by specific profiles of gastrointestinal, extraintestinal somatic and psychological symptoms.

BACKGROUND: Current subgrouping of Irritable Bowel Syndrome (IBS) is exclusively based on stool consistency without considering other relevant gastrointestinal (GI), extraintestinal somatic or psychological features. AIM: To identify subgroups based on a comprehensive set of IBS-related parameters. METHODS: Mixture model analysis was used, with the following input variables: 13 single-item scores from the IBS-specific Gastrointestinal Symptom Rating Scale, average stool consistency and frequency from a 7-day Bristol Stool Form diary, 12 single-item extraintestinal symptom scores from the Patient Health Questionnaire-12, and anxiety and depression subscale scores from the Hospital Anxiety and Depression scale. The resulting latent subgroups were compared regarding symptom profiles using analysis of variance followed by pair-wise comparisons. RESULTS: One hundred and seventy-two IBS patients (Rome III; 69% female; mean age 33.7 [range 18-60] years) were included. The optimal subgrouping showed six latent groups, characterised by: (I) constipation with low comorbidities, (II) constipation with high comorbidities, (III) diarrhoea with low comorbidities, (IV) diarrhoea and pain with high comorbidities, (V) mixed GI symptoms with high comorbidities, (VI) a mix of symptoms with overall mild severity. The subgroups showed differences in the distribution of Rome III-subtypes, IBS severity, presence of anxiety and depression, and gender, but not regarding age, IBS duration or reported post-infectious onset of IBS. CONCLUSIONS: This model-based subgrouping of IBS partly supports the distinction of subgroups based on bowel habits, but additionally distinguishes subgroups with or without co-morbid extraintestinal somatic and psychological symptoms. The resulting groups show specific profiles of symptom combinations.

Autonomic nervous system function predicts the inflammatory response over three years in newly diagnosed ulcerative colitis patients.

BACKGROUND: The autonomic nervous system (ANS) modulates intestinal inflammation in animal models. Human evidence confirming such modulating influence is limited. We aimed to investigate whether ANS function is associated with inflammatory parameters at disease onset, and whether it predicts the evolution of inflammation in patients with ulcerative colitis (UC). METHODS: We prospectively monitored 51 patients from onset of UC for 3 years. Upon remission of the onset flare, ANS activity was assessed by heart rate variability analysis and compared with healthy controls. Inflammatory parameters in blood, stool, and colonic biopsies obtained at onset and during follow-up visits were analyzed. Generalized linear models were used to test cross-sectional associations between ANS activity and inflammatory parameters at onset; linear mixed models were used to test whether ANS function at onset predicted the evolution of inflammation over the following 3 years. KEY RESULTS: Sympathovagal balance was different in UC patients compared to healthy controls, and cross-sectional associated with higher levels of systemic (erythrocyte sedimentation rate [ESR], CRP, TNF-α, IFN-γ) and mucosal inflammation (interleukin-8, IFN-γ) at onset. Conversely, a negative cross-sectional association with parasympathetic activity was found for ESR & TNF-α. Longitudinally, parasympathetic activity at onset predicted systemic (ESR, WBC), but not mucosal inflammation during follow-up. CONCLUSIONS & INFERENCES: This study further strengthens the association between the ANS system and intestinal inflammation previously found in animal models and recently in patients with inflammatory bowel disease. These results may have important implications for the pathogenesis and treatment of UC.

Macrophage and dendritic cell subsets in IBD: ALDH+ cells are reduced in colon tissue of patients with ulcerative colitis regardless of inflammation.

Disruption of the homeostatic balance of intestinal dendritic cells (DCs) and macrophages (MQs) may contribute to inflammatory bowel disease. We characterized DC and MQ populations, including their ability to produce retinoic acid, in clinical material encompassing Crohn's ileitis, Crohn's colitis and ulcerative colitis (UC) as well as mesenteric lymph nodes (MLNs) draining these sites. Increased CD14(+)DR(int) MQs characterized inflamed intestinal mucosa while total CD141(+) or CD1c(+) DCs numbers were unchanged. However, CD103(+) DCs, including CD141(+)CD103(+) and CD1c(+)CD103(+) DCs, were reduced in inflamed intestine. In MLNs, two CD14(-) DC populations were identified: CD11c(int)HLADR(hi) and CD11c(hi)HLADR(int) cells. A marked increase of CD11c(hi)HLADR(int) DC, particularly DR(int)CD1c(+) DCs, characterized MLNs draining inflamed intestine. The fraction of DC and MQ populations expressing aldehyde dehydrogenase (ALDH) activity, reflecting retinoic acid synthesis, in UC colon, both in active disease and remission, were reduced compared to controls and inflamed Crohn's colon. In contrast, no difference in the frequency of ALDH(+) cells among blood precursors was detected between UC patients and non-inflamed controls. This suggests that ALDH activity in myeloid cells in the colon of UC patients, regardless of whether the disease is active or in remission, is influenced by the intestinal environment.

Seven days of euglycemic hyperinsulinemia induces insulin resistance for glucose metabolism but not hypertension, elevated catecholamine levels, or increased sodium retention in conscious normal rats.

Epidemiological studies have suggested an association among chronic hyperinsulinemia, insulin resistance, and hypertension. However, the causality of this relationship remains uncertain. In this study, chronically catheterized conscious rats were made hyperinsulinemic for 7 days (approximately 90 mU/l, i.e., threefold over basal), while strict euglycemia was maintained (approximately 130 mg/dl, coefficient of variation < 10%) by using a modification of the insulin/glucose clamp technique. Control rats received vehicle infusion. Baseline mean arterial pressure and heart rate were 125 +/- 5 mmHg and 427 +/- 12 beats/min and remained unchanged during the 7-day infusion of insulin (127 +/- 7 mmHg; 401 +/- 12 beats/min) or vehicle (133 +/- 4 mmHg; 411 +/- 10 beats/min). Baseline plasma epinephrine (88 +/- 15 pg/ml), norepinephrine (205 +/- 31 pg/ml), and sodium balance (0.34 +/- 0.09 mmol) remained constant during the 7-day insulin or vehicle infusion. After 7 days of insulin or vehicle infusion, in vivo insulin action was determined in all rats using a 2-h hyperinsulinemic (1 mU/min) euglycemic clamp with [3-3H]glucose infusion to quantitate whole-body glucose uptake, glycolysis, glucose storage (total glucose uptake minus glycolysis), and hepatic glucose production. Compared with vehicle-treated rats, 7 days of sustained hyperinsulinemia resulted in a reduction (P < 0.01) in insulin-mediated glucose uptake, glucose storage, and glycolysis by 39, 62, and 26%, respectively. Hepatic glucose production was normally suppressed after 7 days of hyperinsulinemia. Neither insulin-stimulated glucose uptake nor glucose storage correlated with blood pressure or heart rate. In conclusion, 7 days of euglycemic hyperinsulinemia induces severe insulin resistance with respect to whole-body glucose metabolism but does not increase blood pressure, catecholamine levels, or sodium retention. This indicates that hyperinsulinemia-induced insulin resistance is not associated with the development of hypertension in rats who do not have a genetic predisposition for hypertension. Because hyperinsulinemia was initiated in normal rats under euglycemic conditions, additional (inherited or acquired) factors may be necessary to observe an effect of hyperinsulinemia and/or insulin resistance to increase blood pressure.

Cultured blood T-cell responses predict anti-TNF therapy response in patients with ulcerative colitis.

BACKGROUND: Anti-tumour necrosis factor (TNF) therapy is used for treatment of ulcerative colitis (UC). As approximately 30% of patients with UC do not benefit from the treatment, it is of clinical interest to identify biomarkers of response before therapy is initiated. AIM: To identify prognostic biomarkers of anti-TNF therapy response in anti-TNF therapy-naïve patients with UC. METHODS: Peripheral blood cells were obtained from 56 patients with UC before therapy started. Thirty-four patients were included in an exploratory cohort and 22 patients in a validation cohort. Blood cells were stimulated in vitro with influenza vaccine with and without anti-TNF. T-cell surface receptor expression and cytokine release were determined (in total 17 variables). Treatment response was evaluated using the Mayo score 12-14 weeks after the first infusion. RESULTS: In the exploratory cohort, blood cells from the patients showed stronger anti-TNF-dependent suppression of T-cell surface receptor expression and cytokine secretion among therapy responders than nonresponders. In particular, anti-TNF suppressed the expression of CD25 on T cells and secretion of interleukin 5, to a higher degree in responders than in nonresponders. These variables were used to a create model to predict therapy outcome, which was confirmed in the validation cohort. Correct classification of future therapy response was achieved in 91% of the cases in the validation cohort. CONCLUSION: The effects of anti-TNF on cultured blood T cells, obtained before therapy started, predict treatment outcome in patients with UC.

Deviations in human gut microbiota: a novel diagnostic test for determining dysbiosis in patients with IBS or IBD.

BACKGROUND: Dysbiosis is associated with many diseases, including irritable bowel syndrome (IBS), inflammatory bowel diseases (IBD), obesity and diabetes. Potential clinical impact of imbalance in the intestinal microbiota suggests need for new standardised diagnostic methods to facilitate microbiome profiling. AIM: To develop and validate a novel diagnostic test using faecal samples to profile the intestinal microbiota and identify and characterise dysbiosis. METHODS: Fifty-four DNA probes targeting ≥300 bacteria on different taxonomic levels were selected based on ability to distinguish between healthy controls and IBS patients in faecal samples. Overall, 165 healthy controls (normobiotic reference collection) were used to develop a dysbiosis model with a bacterial profile and Dysbiosis Index score output. The model algorithmically assesses faecal bacterial abundance and profile, and potential clinically relevant deviation in the microbiome from normobiosis. This model was tested in different samples from healthy volunteers and IBS and IBD patients (n = 330) to determine the ability to detect dysbiosis. RESULTS: Validation confirms dysbiosis was detected in 73% of IBS patients, 70% of treatment-naïve IBD patients and 80% of IBD patients in remission, vs. 16% of healthy individuals. Comparison of deep sequencing and the GA-map Dysbiosis Test, (Genetic Analysis AS, Oslo, Norway) illustrated good agreement in bacterial capture; the latter showing higher resolution by targeting pre-determined highly relevant bacteria. CONCLUSIONS: The GA-map Dysbiosis Test identifies and characterises dysbiosis in IBS and IBD patients, and provides insight into a patient's intestinal microbiota. Evaluating microbiota as a diagnostic strategy may allow monitoring of prescribed treatment regimens and improvement in new therapeutic approaches.

Increased insulin sensitivity in the high sodium one-kidney, one figure-8 hypertensive rat.

This study examines the relation between sympathetic activity and in vivo insulin-mediated glucose metabolism in a rat model of acquired hypertension. Two groups of conscious, unrestrained rats were studied in the postabsorptive state: sham-operated normotensive rats (n = 10) and renal-wrapped hypertensive rats (n = 10). Mean arterial pressure was increased in the hypertensive compared with the normotensive group in the fed (184 +/- 9 versus 144 +/- 6 mm Hg; p less than 0.01) and in the fasting (147 +/- 8 versus 112 +/- 7 mm Hg; p less than 0.01) state. After a 24-hour fast, hepatic glucose production, plasma glucose, insulin, and norepinephrine concentrations were similar in the two groups. Blood pressure did not change in either group during the 3-milliunits/kg.min euglycemic insulin clamp study; however, plasma norepinephrine concentration rose significantly in hypertensive (207 +/- 24 versus 329 +/- 11 pg/ml; p less than 0.05) but not in normotensive rats (229 +/- 23 versus 267 +/- 27 pg/ml; p = NS). During the insulin clamp study, the hepatic glucose production was similar in the hypertensive (3.8 +/- 0.8 mg/kg.min) compared with the normotensive (4.0 +/- 0.3 mg/kg.min) rats. Insulin-mediated glucose uptake was significantly higher in hypertensive than in normotensive rats (33.0 +/- 0.7 versus 25.8 +/- 0.8; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of lateral parabrachial nucleus lesions in chronic renal hypertensive rats.

Neuroanatomic studies describing forebrain projections to the lateral parabrachial nucleus suggest a central integrative role in cardiovascular regulation. We performed this study to examine the role of this pontine nucleus in the maintenance of one-kidney, figure-8 renal-wrap hypertension. Bilateral ibotenic acid ablation of the lateral parabrachial nucleus was performed 4 weeks after induction of hypertension or sham operation. In hypertensive rats, ablation produced a significant reduction in mean arterial pressure from 160 +/- 4 to 118 +/- 2 mm Hg and a transient but significant increase in heart rate from 381 +/- 5 to 408 +/- 8 beats per minute on the first day after ablation; arterial pressure returned to preablation values by day 5 after ablation. In sham-operated, normotensive animals, arterial pressure was not altered by ablation, and a transient but significant increase in heart rate from 384 +/- 8 to 419 +/- 7 beats per minute was again observed. Before ablation, trimethaphan administration produced a significantly greater drop in arterial pressure in hypertensive (delta-72.8 +/- 4.6 mm Hg) versus normotensive (delta-55.7 +/- 4.1 mm Hg) animals. This effect was eliminated on day 1 after ablation yet returned on day 4 after ablation. In blood samples obtained before ablation and on days 1 and 4 after ablation, circulating plasma catecholamine concentrations in both groups remained unchanged. These observations suggest that, because of possible alternate neural compensatory mechanisms, lateral parabrachial nucleus ablation produces a significant yet transient reversal of renal-wrap hypertension. Thus, the lateral parabrachial nucleus may contribute to the increased sympathetic nervous system function associated with this model.

Lateral parabrachial nucleus and angiotensin II-induced hypertension.

The objective of this study was to determine if ablation of the lateral parabrachial nucleus (LPBN) would prevent angiotensin II-induced hypertension in rats. Thirteen male Sprague-Dawley rats were studied. Bilateral electrolytic lesions in the LPBN were produced in six rats; the remaining seven rats were subjected to sham lesion surgery only. All rats were instrumented with vascular catheters and housed in metabolism cages. Daily measurements during the 16-day protocol included arterial pressure, heart rate, water intake, urine output, and urinary sodium excretion. Periodically throughout the protocol depressor responses to ganglion blockade and to blockade of V1-type vasopressin receptors also were measured. The protocol was divided into three control-period days, 10 days of continuous (24 hr/day) angiotensin II infusion (10 ng/min i.v.), and three recovery-period days. There were no significant differences between the two groups of rats for any variable during the control period. During angiotensin II infusion, sham-lesion rats exhibited a progressive increase in arterial pressure and the depressor response to ganglion blockade and a decrease in urinary sodium excretion. No other variable was significantly changed. In rats with LPBN lesions, arterial pressure was significantly increased only on days 1 and 3 of angiotensin II infusion. No other variable was affected. It was concluded that ablation of the LPBN in rats prevented sustained hypertension during intravenous infusion of angiotensin II by interfering with neurogenic pressor mechanisms normally activated by the peptide.

Metabolic engineering of animal cells.

Substrate-limited fed-batch cultures were used to study growth and overflow metabolism in hybridoma and insect cells. In hybridoma cells a glucose-limited fed-batch culture decreased lactate formation but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation but did not enhance glucose consumption. Instead lactate formation was reduced, indicating that glucose was used more efficiently. The formation of lactate, alanine, and ammonium was negligible in a dual substrate-limited fed-batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose of 100% and for glutamine of 150% and by the change in the metabolic ratios lac/glc, ala/gln, and NHx/gln, in the combined fed-batch culture. Insect cell metabolism was studied in Spodoptera frugiperda (Sf-9) cells. A stringent relation between glucose excess and alanine formation was found. In contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. Simultaneous glucose and glutamine limitation suppressed both alanine and ammonium formation. Alanine formation appears as wasteful as lactate formation because the growth rate of insect cells in substrate-limited cultures was the same as in batch cultures with substrate excess. In batch and fed-batch cultures of both cell lines, mu reaches it maximum early during growth and decreases thereafter so that no exponential growth occurs. The growth rate limiting factor for hybridoma cells was found to be a component of serum, because intermittent serum additions to batch cultures resulted in a high and constant growth rate. Insulin was identified as the main cause, inasmuch as intermittent insulin additions gave the same result as serum.

The kinetics of peritoneal clearance of Escherichia coli and Bacteroides fragilis and participating defense mechanisms.

In a study using pigs, we delineated the dynamic character and the interplay of defense mechanisms that operate in the peritoneal cavity and their effectiveness against Escherichia coli and Bacteroides fragilis. The bacteria were extensively cleared during the first three hours. The concentration of both organisms in the peritoneal fluid decreased by up to 300,000-fold, and the final concentration was a function of the inoculum given. Clearance efficiency did not differ between a bolus dose and prolonged bacterial administration. Peritoneal absorption was a major eliminating mechanism during the first three hours but was limited to bacterial concentrations exceeding approximately 10(9) colony-forming units per milliliter of peritoneal fluid. Intraperitoneal elimination started immediately and continued for approximately six hours. Thereafter, residual bacteria were not eliminated, even though the defense capacity was intact. Prolonged bacterial administration, however, protracted the period for active bacterial destruction.

The melanophore aggregating response of isolated fish scales: a very rapid and sensitive diagnosis of whooping cough.

Pertussis toxin (PT) has been found to block noradrenaline-induced pigment aggregation in fish melanophores, and, based on this, a rapid and highly sensitive assay for PT was developed. Some preliminary results have also indicated that it may be possible to detect PT-like activity in saliva samples from patients with clinically suspected pertussis. In the present study the diagnostic value of the fish melanophore method was evaluated in 70 patients suspected of having pertussis; culture, serology and physician diagnosis were used as reference methods. In 60 of the patients, pertussis was verified by at least one of the reference methods. The melanophore test showed PT-like activity in saliva samples from 58 of the patients. Three patients with reference-verified pertussis showed no PT-like activity in the test; among these, one patient had been immunized and had also been treated with erythromycin during 3 days immediately prior to visiting the hospital. The melanophore test has three major advantages: it allows detection of pertussis in the early and curable stage of the disease; it takes only 2 h to perform; and it requires no sophisticated equipment.

Coagulase-negative staphylococci and sternal infections after cardiac operation.

BACKGROUND: Coagulase negative staphylococci (CoNS) have been recognized as important pathogens in nosocomial infections, especially in connection with implanted foreign materials. In cardiac operation they are among the most common pathogens isolated from infected sternal wounds. The definition of the infection is very important. In this study we focus on deep postoperative chest infections. METHODS: By studying 33 infected patients retrospectively and comparing them to 33 matched uninfected controls, we studied the characteristics and costs of the infections. RESULTS: Typical for these infections is the late and insidious onset, and that the infections initially give only minor symptoms such as pain, redness, and serous secretion. We found the following risk factors for infection: number of preoperative days in a hospital, the total length of the operation, and if the patient had undergone an early reoperation due to causes other than infection. This kind of infection more than doubled the hospital costs for the patients affected. CONCLUSIONS: Coagulase negative staphylococci are the most important pathogens in deep postoperative infections in this material. They cause infections that are difficult to recognize since they give only discrete symptoms and start well after the patients leave the hospital. The risk factors for patients with CoNS infections are mostly associated with a long exposure to the hospital environment. The treatment is often difficult and costly because of multiresistant bacteria and frequent need for repeated surgical revisions.