Timothy C. R. Prickett

Characterization of interstitial dendritic cells in human liver

Sensitive immunofluorescence and immunoperoxidase techniques were used to test an extensive range of monoclonal antibodies for reactivity with Kupffer cells and interstitial dendritic cells (DCs) in cryostat-cut sections of human liver. Leucocytes with a dendritic cell morphology were identified with CD45 (antileucocyte common) reagents in portal tracts, predominantly around bile ducts, and these cells stained strongly for the HLA-DP, DQ, and DR antigens. Kupffer cells stained less intensely with anti-class-II reagents, particularly anti-HLA-DQ. The interstitial DCs expressed the LFA-1 antigen but failed to stain with CD11b, CD11c, and the defined T and B cell CD antibodies; nor did they stain with antibodies to FcR1, FcR11, FcRIII, or the C3b receptor. Of the myeloid monoclonal antibodies available from the 3rd Leucocyte Differentiation Antigen Workshop, only Y2/131, Ki-M7, Ki-M8, and a minority of CD14 antibodies stained DCs, whereas Kupffer cells showed a wider reactivity with antimacrophage antibodies including those of workshop groups 11, 15, 16, and other unique antibodies. A 2nd probable DC population was identified in the liver capsule that had a similar phenotype to portal interstitial DCs. Although some minor phenotypic differences between liver portal DCs and the phenotypes of Langerhans cells and isolated tonsil DCs were noted, our results support the view that there is a unique hemopoietic lineage of DCs. The presence of DCs, which stimulate strong allogeneic T cell responses, in the portal triads is consistent with the fact that the histologic changes of graft-versus-host disease seen in bone marrow transplantation and the lymphocytic infiltrate in a rejecting liver allograft occur predominantly in the periportal region.

Amino-Terminal Pro–C-Type Natriuretic Peptide in Heart Failure

Abstract—The levels and pathophysiological role of amino terminal C-type natriuretic peptide in heart failure are unknown. The potential of plasma amino-terminal C-type natriuretic peptide (N-CNP) as a marker of cardiac function was investigated in symptomatic patients. In 305 patients with recent-onset dyspnea and/or peripheral edema, presenting to primary care, assay of plasma amino-terminal C-type natriuretic peptide and other plasma vasoactive hormones was conducted together with echocardiography. Heart failure was diagnosed in 77 (of the 305) patients by rigorous application of predefined criteria. Plasma amino-terminal C-type natriuretic peptide concentrations were elevated in patients with heart failure, and by univariate analysis were related to age, renal function, and other hormones. On multivariate analysis, tertile of plasma N-CNP interacted with tertile of plasma amino-terminal B-type natriuretic peptide to predict heart failure independent of age, gender, renal function, or echocardiographic left ventricular fractional shortening. N-CNP showed significant relations to concurrent plasma CNP, atrial natriuretic peptide (ANP), N-ANP, B-type (or brain) natriuretic peptide (BNP), N-BNP, endothelin-1, and adrenomedullin but not to echocardiographic indicators of left ventricular systolic function. Plasma amino-terminal C-type natriuretic peptide is elevated in heart failure and is related to other plasma hormones in heart failure. These findings suggest a possible compensatory response from the peripheral vasculature to heart failure by an endothelium-based vasodilator peptide and mandate further exploration of the role of C-type natriuretic peptide in this condition.

Arginine vasopressin is associated with hypercortisolemia and suicide attempts in depression

Activation of the hypothalamic–pituitary–adrenal (HPA) axis is commonly seen in patients suffering from depression (Charlton and Ferrier 1989). Raised plasma concentrations of cortisol may be present (Butler and Besser 1968; Deuschle et al 1997), along with increased 24-hour urinary free cortisol levels (Carroll et al 1976). Detailed analysis of plasma adrenocorticotropic hormone (ACTH) reveals an increase in pulse frequency (Mortola et al 1987; Deuschle et al 1997). In approximately 40–50% of all patients with depression, plasma cortisol is not normally suppressed by dexamethasone (Arana et al 1985). The mechanism of the hypercortisolemia is believed to be related to increased hypothalamic secretion of corticotropinreleasing hormone (CRH) (Charlton and Ferrier 1989; Licinio and Gold 1991), and increased adrenal sensitivity to ACTH (Charlton and Ferrier 1989). Since most studies of the HPA axis in depression concentrate on CRH as the predominant ACTH secretagogue, there are relatively few data concerning the role of arginine vasopressin (AVP), which is also a potent stimulus to ACTH secretion (Gillies et al 1982). The aim of this study was to determine whether there was any relationship between afternoon plasma levels of cortisol, ACTH, and AVP in depressed subjects, particularly in those displaying biochemical hypercortisolemia. We also sought to determine if any relationship existed between severity of depression and suicidal behavior and activation of the HPA axis.

Regional Release and Clearance of C-Type Natriuretic Peptides in the Human Circulation and Relation to Cardiac Function

Production and clearance of plasma C-type natriuretic peptide (CNP) and amino terminal (NT)-proCNP immunoreactivity in the human circulation remain poorly characterized. Accordingly, we have measured arterial and venous concentrations of CNP and NT-proCNP across multiple tissue beds during cardiac catheterization in 120 subjects (age: 64.2±9.0 years; 73% men) investigated for cardiovascular disorders. The heart, head and neck, and musculoskeletal tissues made the clearest contributions to both plasma CNP and NT-proCNP (P<0.05). Net release of NT-proCNP was also observed from hepatic tissue (P<0.001). Negative arteriovenous gradients for CNP were observed across renal, hepatic, and pulmonary tissue (P<0.05), indicating net clearance, whereas no tissue-specific site of NT-proCNP clearance was identified. Age, mean pulmonary artery pressure, left ventricular end diastolic pressure, Brandt score of myocardial jeopardy, and troponin I were independent predictors of circulating CNP levels in multivariable analysis. Sex and kidney function were independently predictive of arterial NT-proCNP. The proportional step-up of CNP (+60%) across the heart was less than for brain natriuretic peptide (+123%) but greater than for NT–pro-brain natriuretic peptide (NT-proBNP) (+36%) and NT-proCNP (+42%; P<0.001 for all). We conclude that cardiac and head and neck tissue are important sources of CNP. Circulating CNP but not NT-proCNP concentrations are related to cardiac hemodynamic load and ischemic burden. Although cardiac release is most evident, multiple additional tissues release NT-proCNP immunoreactivity without evidence for an organ-specific site for NT-proCNP degradation. Taken together, differences in magnitude and direction of transorgan gradients for CNP compared with NT-proCNP suggest net generalized cosecretion with differing mechanisms of clearance.

Amino-Terminal proCNP: A Putative Marker of Cartilage Activity in Postnatal Growth

Recent evidence from rodents and humans shows that C-type natriuretic peptide (CNP) plays an essential role in endochondral bone growth. We recently identified a stable product of proCNP, amino-terminal proCNP (NT-proCNP), which unlike CNP is readily measurable in human and ovine plasma. Hypothesizing that plasma NT-proCNP concentrations reflect in part CNP synthesis within growth plates of rapidly growing cartilage, we studied levels of CNP forms in both children and lambs and related these to age, growth velocity, and biochemical markers of bone turnover. Plasma NT-proCNP levels were elevated at birth and fell progressively with age. Significant associations between plasma NT-proCNP and height velocity, alkaline phosphatase, and type 1 collagen C telopeptide were identified in children (aged 5–18 y). In longitudinal animal studies, elevated plasma concentration of NT-proCNP in 1-wk-old lambs fell progressively to mature adult levels at age 27 wk. Plasma NT-proCNP showed a highly significant association with alkaline phosphatase and metacarpal growth velocity. Glucocorticoids, a treatment known to inhibit cartilage proliferation, reduced metacarpal growth elongation in 4-wk-old lambs and markedly lowered circulating NT-proCNP levels during the treatment period. In summary, NT-proCNP levels in blood show a strong association with growth velocity and markers of bone formation and may well serve as a useful marker of growth plate activity in humans and other mammals.

Adhesion molecules on human tonsil dendritic cells.

Dendritic cells are specialist antigen-presenting cells that have a unique ability to stimulate a primary T cell response. Activation of T cells by DC depends on the formation of cell clusters creating DC-T cell membrane contact that probably involves adhesion molecules. Monoclonal antibodies were used to study adhesion molecules on DC, including members of the integrin and immunoglobulin supergene families. DC expressed LFA-1, ICAM-1, LFA-3, and the Hermes antigen, but no other integrin or immunoglobulin supergene family adhesion molecules were detected using a sensitive immunoperoxidase staining technique. Monoclonal antibodies to LFA-1 alpha and LFA-1 beta inhibited DC-stimulated allogeneic T cell (MLR) responses by 75 +/- 12% and 74 +/- 8%, respectively, as did the anti-LFA-3 (56 +/- 3% inhibition) and anti-LFA-2 (60 +/- 5% inhibition) antibodies. Three different anti-ICAM-1 antibodies inhibited only to a limited degree (mean range 8-24%). The inhibitory effect of the LFA-1 and LFA-3 antibodies was maximal if added early to the MLR. The inhibitory effect of the different antibodies was associated with variable decreases in DC-T cell cluster stability. The simultaneous addition of monoclonal antibodies to MLRs and preincubation washing experiments established that DC have at least 3 independent adhesion ligand interactions (LFA-1-ICAM-1, ICAM-1-LFA-1, and LFA-3-CD2) with T cells. It seems likely that the additional ligand for LFA-1, ICAM-2, is expressed on DC and contributes significantly to DC-T cell adherence and T cell activation. The membrane mobility of these molecules may also be important in the DC-T cell activation process.

The effect of glycerol and desmopressin on exercise performance and hydration in triathletes.

BACKGROUND Hydration is an important determinant of athletic performance, and glycerol-containing solutions have been demonstrated to produce a state of hyperhydration. Secretions of arginine vasopressin (AVP) and/or other renal mechanisms may account for reduced urine output following glycerol ingestion. This study examined the effect of glycerol and the AVP analog desmopressin (DDAVP) on hydration and exercise performance in triathletes ingesting routine volumes of prerace fluids. METHODS Eight male triathletes ages 19 to 43 participated. After determination of their VO(2peak), each athlete completed a strenuous exercise protocol three times involving 60 min of exercise at 70% VO(2peak) followed immediately by an incremental increase in workload every 2 min until exhaustion. RESULTS Pretreatment with 1 gxkg(-1) glycerol or 20 microgram of DDAVP intranasally failed to produce hyperhydration or any enhancement of athletic performance. There was a significant difference in reduction in body mass between DDAVP and control (P < 0.05) but no change in sweat volume. No physiologically relevant differences in plasma sodium, renin, or hemoglobin were seen with either active agent. Plasma osmolality did have a different time course in response to exercise following glycerol (P < 0.03) owing to a smaller incremental increase. Urine osmolality was also raised at baseline following glycerol (P < 0.05). Responses to exercise of plasma AVP, cortisol, and indices of carbohydrate metabolism were similar, although AVP was elevated following DDAVP administration (P < 0.01) owing to assay cross-reactivity. CONCLUSION Although maintaining adequate hydration remains important for the endurance athlete, the routine use of either glycerol of DDAVP before athletic training or competition in a thermoneutral environment does not seem to confer any advantage over conventional fluid replacement.

An activating mutation in the kinase homology domain of the natriuretic peptide receptor-2 causes extremely tall stature without skeletal deformities

BACKGROUND C-type natriuretic peptide (CNP)/natriuretic peptide receptor 2 (NPR2) signaling is essential for long bone growth. Enhanced CNP production caused by chromosomal translocations results in tall stature, a Marfanoid phenotype, and skeletal abnormalities. A similar phenotype was described in a family with an activating NPR2 mutation within the guanylyl cyclase domain. CASE Here we describe an extremely tall male without skeletal deformities, with a novel NPR2 mutation (p.Arg655Cys) located in the kinase homology domain. OBJECTIVES The objective of the study was to investigate the functional and structural effects of the NPR2 mutation. METHODS Guanylyl cyclase activities of wild-type vs mutant NPR2 were analyzed in transfected human embryonic kidney 293 cells and in skin fibroblasts. The former were also used to study possible interactions between both isoforms. Homology modeling was performed to understand the molecular impact of the mutation. RESULTS CNP-stimulated cGMP production by the mutant NPR2 was markedly increased in patient skin fibroblasts and transfected human embryonic kidney 293 cells. The stimulatory effects of ATP on CNP-dependent guanylyl cyclase activity were augmented, suggesting that this novel mutation enhances both the responsiveness of NPR2 to CNP and its allosteric modulation/stabilization by ATP. Coimmunoprecipitation showed that wild-type and mutant NPR2 can form stable heterodimers, suggesting a dominant-positive effect. In accordance with augmented endogenous receptor activity, plasma N-terminal pro-CNP (a marker of CNP production in tissues) was reduced in the proband. CONCLUSIONS We report the first activating mutation within the kinase homology domain of NPR2, resulting in extremely tall stature. Our observations emphasize the important role of this domain in the regulation of guanylyl cyclase activity and bone growth in response to CNP.

N-terminal pro-C-type natriuretic peptide, but not C-type natriuretic peptide, is greatly elevated in the fetal circulation.

We have identified recently a new peptide, NT-proCNP(1-50) (N-terminal pro-C-type natriuretic peptide), in the circulation of humans and sheep. A previous report of an elevated fetal-maternal gradient in immunoreactive CNP raised the possibility that processing and metabolism of proCNP may differ in maternal and fetal tissues. We therefore collected matching peripheral maternal and umbilical cord plasma samples at delivery from women with normotensive and pre-eclamptic pregnancies to investigate the presence and concentrations of CNP and NT-proCNP using HPLC and RIA. Plasma concentrations of NT-proCNP in normotensive umbilical cord plasma were 10-fold higher than maternal venous levels (246+/-17 compared with 24.3+/-1.8 pmol/l; P <0.001) and much higher than corresponding levels of CNP (3.6+/-0.4 compared with 1.8+/-0.3 pmol/l in the fetal and maternal plasma respectively; P <0.001). Although there was no significant difference between normotensive and pre-eclamptic plasma CNP concentrations in either maternal or umbilical cord blood, NT-proCNP showed a significant statistical interaction ( F =5.8, P =0.025) between the source (maternal or fetal) and gestational group (normotensive or pre-eclamptic). Maternal NT-proCNP levels were raised in the pre-eclampsia group, whereas the converse was observed in umbilical cord blood. In conclusion, the greatly elevated ratio of NT-proCNP/CNP in fetal compared with maternal plasma suggests that synthesis, as well as clearance, of CNP (but not NT-proCNP clearance) are markedly increased in fetal tissues.

Normal opioid tone and hypothalamic–pituitary–adrenal axis function in chronic fatigue syndrome despite marked functional impairment

Objective  To determine whether the functional impairment seen in chronic fatigue syndrome (CFS) is associated with reduced levels of central opioids and/or deficiency of the hypothalamic–pituitary–adrenal (HPA) axis.