Dynamic expression of the signal regulatory protein alpha and CD18 on porcine PBMC during acute endotoxaemia.

Endotoxaemia elicits a massive inflammatory insult affecting the beta2 integrin CD18. Being an adhesion molecule, CD18 is pivotal in inflammation and, moreover, exiting data suggest that CD18 is a lipopolysaccharide (LPS) receptor. Early LPS-induced inflammation is regulated by the signal regulatory protein (SIRPalpha), which is identical to the porcine panmyelocytic marker swine CD workshop 3 (SWC3), and LPS-induced downregulation of SIRPalpha has been described in vitro. The dynamic SIRPalpha/SWC3 and CD18 expression on peripheral blood mononuclear cells (PBMC) in vivo during LPS-induced inflammation is the focus of this study. Pigs were randomized into LPS (n = 12) or control (n = 6) groups. At start 0 min, LPS infusion was stepwise (2.5-15 mug/kg/h, 30 min) followed by maintenance infusion (2.5 mug/kg/h, 330 min). PBMC were isolated at 0, 60, 240 and 360 min, and two-colour flow cytometry was performed using monoclonal antibodies identifying SWC3 and CD18. Viability was tested using 7-amino-actinomycin D. LPS dramatically changed the relative distribution of circulating myeloid cells. At 60 min monocytes disappeared. This was followed by reappearance of a distinct population with low CD18 and SIRPalpha/SWC3 expression. Cell sorting showed that the appearing population comprised band neutrophils and apoptotic/dead cells. The remaining monocytes expressed less CD18 at 360 min than the controls (P = 0.03). The appearance of a distinct cell population comprising apoptotic cells and band neutrophils consistent with LPS-induced apoptosis, and decreased CD18 expression on monocytes suggests that early CD18 downregulation is profitable for the host in a situation with an intense LPS stimulus.

Renal cytokine profile in an endotoxemic porcine model.

INTRODUCTION: In animals exposed to acute endotoxemia with lipopolysaccharide (LPS), high levels of cytokines are found in the kidney. The objective of this study is to determine whether the high renal content of TNF-alpha, IL-1beta, IL-10 and IL-1 receptor antagonist (IL-1ra) is due to glomerular filtration and reabsorption, or whether the cytokines are produced locally in the kidney. METHODS: Eighteen anesthetized and mechanically ventilated pigs (35-43 kg) were randomized into two groups: Group 1 (n=12) LPS infusion for 360 min and Group 2 (n=6) control pigs, no treatment. At 360 min, the pigs were euthanized and tissue samples from the kidneys were obtained. Localization of the cytokines was determined by immunohistochemistry and double immunofluorescence (dIF). RESULTS: Pigs exposed to endotoxemia showed increased accumulation of leukocytes and increased protein expression of TNF-alpha and IL-1beta when compared with controls. dIF showed that TNF-alpha-positive cells co-localized with both endothelial and mesangial cells in the glomeruli. Furthermore, the endothelial cells of the cortical arterioles were positive for IL-1beta. TNF-alpha and IL-1beta staining were absent in renal tubular cells. A positive signal for IL-10 was detected at the tubular brush border while IL-1ra was detected in the glomerulus and in the tubular cells. CONCLUSION: LPS-induced endotoxemia increased TNF-alpha and IL-1beta protein expression and leukocyte accumulation in the kidneys. The results indicate that the increased levels of the pro-inflammatory cytokines TNF-alpha and IL-1beta are caused by a local production in the kidneys while the anti-inflammatory cytokines IL-10 and IL-1ra are filtrated and reabsorbed in the tubuli.

Ten years of extrapulmonary tuberculosis in a Danish university clinic.

The objective of this study was to describe the symptoms, diagnostic measures and outcomes of extrapulmonary tuberculosis (ex-TB) in a Danish university clinic from 1990 to 1999. 48 patients with ex-TB were identified retrospectively and clinical and laboratory data extracted from the patient files. The majority were immigrants from Africa (71%). A direct connection between symptoms on admission and anatomical localization of TB was found in 83%. The main localizations of ex-TB were peripheral lymph nodes (n = 15) and the abdomen (n = 19). In 73% Mycobacterium tuberculosis could be cultured. One culture was resistant to isoniazide and 1 had decreased sensitivity to isoniazide and etambutol. Two patients relapsed with TB. Some pitfalls in diagnosing TB were found, as 13% had a normal erythrocyte sedimentation rate at presentation, 9% had a negative tuberculin skin test and fever was absent in 31% of the cases. The patients' subjective complaints on admission should guide the diagnostic procedures.

Gene expression of a truncated and the full-length growth hormone (GH) receptor in subcutaneous fat and skeletal muscle in GH-deficient adults: impact of GH treatment.

In humans at least two GH receptors are significantly expressed. One is the full-length receptor (GHR); the other is a truncated form (GHRtr), that lacks most of the intracellular domain. This receptor may inhibit the action of the full-length receptor. Circulating GH-binding protein (GHBP) is a proteolytically cleaved product from both of these receptors. The clinical relevance of the different receptor types is unknown. We examined the gene expression of GHR and GHRtr in human adipose tissue and skeletal muscle and the influence of GH treatment on this expression. Furthermore, we studied the relationship of circulating GHBP and body composition to GHR and GHRtr gene expression. Eleven adult GH-deficient patients were studied before and after 4 months of GH substitution therapy. Abdominal fat obtained by liposuction and femoral muscle biopsies were taken at baseline and after 4 months. Gene expression of GHR and GHRtr in adipose tissue and skeletal muscle was determined and expressed relative to the expression of beta-actin. Gene expression of GHR in abdominal sc adipose tissue was not altered, whereas the expression of GHRtr increased significantly. In skeletal muscle inverse changes were seen in the expression of messenger ribonucleic acid (mRNA) levels for the two GH receptor forms: expression of GHR increased significantly, whereas mRNA levels for GHRtr decreased. As expected, body composition changed with reduction of body fat mass after 4 months of GH treatment. Levels of circulating GHBP decreased significantly. We conclude that GH treatment in GH-deficient adults changes the expression of mRNA for GHR and GHRtr in adipose tissue and skeletal muscle. Whether these changes are responsible for the observed changes in body composition in response to GH treatment and the observed changes in levels of circulating GHBP, however, needs further elucidation.

The role of nitric oxide in L-arginine-stimulated growth hormone release.

Nitric oxide (NO) exerts widespread and fundamental physiological effects. It is identical to the so-called endothelium-derived relaxing factor which regulates vascular tone. It has also been demonstrated to act as a neurotransmitter in both the peripheral and central nervous systems. NO is generated from L-arginine catalyzed by the NO synthases (NOS), of which two constitutive and one inducible form exist. NO stimulates the soluble guanylate cyclase which generates cyclic guanosine monophosphate (cGMP), that is believed to mediate the effects of NO. Recently, however, it has also been shown that NO is generated non-enzymatically from both L- and D-arginine by reaction with peroxide. The role of this pathway in the neuroregulation of growth hormone (GH) secretion has not yet been investigated. In rats, NO stimulates secretion of GH-releasing hormone (GHRH) and thus increases secretion of GH. However, it has also been observed that GHRH, in turn, increases production of NO in somatotroph cells, which subsequently blunts GH secretion. In humans, L-arginine stimulates pituitary GH release, but the mechanism is not fully clarified. Most studies suggest that an inhibition of somatostatin secretion is responsible for the effect. Infusion of low doses of the NOS inhibitor N(G)-nitro-L-arginine methyl ester have been shown not to change L-arginine-stimulated GH secretion. The effect of the NO donor molsidomine has also been found to have no influence on GH secretion. We investigated whether intravenous infusion of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) influenced L-arginine-stimulated GH secretion in healthy young men. All subjects were examined twice in random order. On both occasions L-arginine was infused intravenously. This treatment was accompanied by either: L-NMMA co-infused or a saline infusion. Plasma cGMP was unchanged and identical in the two treatment groups, and the urine cGMP/creatinine ratio increased identically during both examinations. GH secretion increased significantly during L-arginine infusion and was not influenced by co-infusion of L-NMMA. There is so far no evidence that L-arginine stimulates GH release via NO production. However, it remains to be elucidated whether the doses of different L-arginine inhibitors/NO donors used in the previous studies were insufficient.

Variety in growth hormone determinations due to use of different immunoassays and to the interference of growth hormone-binding protein.

More than 30 years after their introduction, growth hormone (GH) immunoassays showed the poorest inter-laboratory agreement of the various hormone assays evaluated in 1998 by the UK National External Quality Assessment Scheme, in which different laboratories using different assays reported that analyses of identical samples differed two- to threefold in value. There is therefore an urgent requirement and desire within the scientific community, particularly within centres diagnosing and treating GH deficiency and acromegaly, to resolve this problem and to develop a GH assay(s) that measures solely all of the relevant components of circulating GH immunoreactivity. The main confounders in the estimation of GH levels (now that the use of GH standards other than that recommended by the World Health Organization has largely been eliminated) are GH heterogeneity, anti-GH antiserum binding site specificity and interference from circulating high-affinity GH-binding protein (GHBP). The effects of these factors are closely related. The present study investigates these factors, focussing on the influence of GHBP and antibody binding site specificity on various assays for GH. The findings lead the authors to suggest that a solution to the problem may be to develop a GH assay that measures specifically and solely all serum 22 kDa GH, as this is the major circulating fraction and carries the dominant GH bioactivity.

L-arginine-induced growth hormone secretion is not influenced by co-infusion of the nitric oxide synthase inhibitor N-monomethyl-L-arginine in healthy men.

In animals, it has been demonstrated that nitric oxide (NO) is a potent neuroregulatory substance. By intravenous infusion, L-arginine is converted to NO and citrulline, but it is unknown whether NO is responsible for the GH stimulating effect of L-arginine in humans. We investigated whether intravenous infusion of the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA) influenced L-arginine stimulated GH secretion. Ten healthy men, aged 28.6 +/- 1.9 (mean +/- SEM) years were examined twice. L-arginine was infused intravenously in a dose of 0.5 g/kg, max 35 g, from 0 to 30 min, accompanied by either: (1) L-NMMA from -5 to 0 min, in a dose of 3 mg/kg, max 250 mg, and in a dose of 3.5 mg/kg, max 250 mg from 0 to 60 min; or (2) a saline infusion. Heart rate increased (P = 0.032), and diastolic blood pressure decreased (P < 0.001) in the two situations. Plasma cGMP was unchanged and identical in the two situations (P = 0.679). Urine cGMP/creatinine ratio increased during both examinations (P = 0.041). Growth hormone secretion increased significantly during L-arginine infusion (P = < 0.001) without any effect of L-NMMA (P = 0.848). We did not find evidence that NO influences GH secretion. It remains to be tested, however, whether a higher dose of L-NMMA may influence L-arginine stimulated GH secretion.

Influence of growth hormone binding protein on growth hormone estimation in different immunoassays.

Growth hormone (GH) quantitation in biological fluids varies depending on the assays employed, and factors which may interfere in the assays include the high affinity GH-binding protein (GHBP). To evaluate this potential effect on GH estimates, we studied the influence of adding increasing amounts of high affinity glycosylated GHBP to normal, acromegalic and GH-deficient sera, which were then processed in four different immunoassays. Two commercial immunometric assays, Delfia and Nichols (assays 1 and 2), and two RIAs, one using a polyethylene glycol (PEG) precipitation (assay 3) and one using wick-chromatography (assay 4) for separation of free and bound 125I-GH, were employed. In the Delfia assays, GH estimates of 11 sera decreased (p < 0.05) to 87.2 +/- 2.6%, 73.0 +/- 2.7% and 60.1 +/- 2.5% (mean +/- SEM) of basal GH estimates with the addition of GHBP in concentrations of 0.54, 2.14 and 6.42 nmol/l, respectively. In the Nichols assay, GH estimates were not significantly reduced (93.4 +/- 2.6%, 83.8 +/- 4.5% and 83.9 +/- 3.9%) with the applied GHBP concentrations. In assay 3 (RIA), the addition of GHBP increased GH estimates to 122 +/- 10.0% and 167 +/- 19.1% (both p < 0.05) with the addition of GHBP in concentrations of 2.14 and 6.42 nmol/l, respectively, whereas an increase in GHBP concentration of 0.54 nmol/l did not change the estimates from basal levels (99.0 +/- 4.8%, p > 0.05). In assay 4 (RIA), the addition of GHBP induced decreased GH estimates. With this varying influence of GHBP on GH estimates, binding protein interference should be taken into consideration when comparing GH estimates obtained with many currently utilized GH immunoassays. The present results demonstrate that GHBP levels within physiological range may interfere with the results of GH assays, giving either spuriously high or low values depending on the GH assay methodology.

Distribution and characterization of mosquitocidal toxin genes in some strains of Bacillus sphaericus.

The binary toxin of Bacillus sphaericus strains forms a crystal in sporulating cells, while the mosquitocidal toxin is located in the cytoplasm of vegetative cells. The distribution of binary toxin (btx) and mosquitocidal toxin (mtx) genes in 53 strains of B. sphaericus was determined by hybridization of specific gene probes to chromosomal DNA in Southern blots. btx genes were found in all strains of serotype 5a5b examined and in some strains of serotypes 1a, 3, 6, 25, and 48, while mtx genes were detected in strains of serotypes 1a, 2a2b, 5a5b, 6, 9a9c, 25, and 48. Serotype 26a26b strains lacked both toxin genes, as did some strains of serotypes 2a2b, 3, 6, and 48. Partial DNA sequences of btx genes from five strains, together with published sequences, revealed four types of toxin among mosquitocidal B. sphaericus strains. most of the 42-kDa toxin gene of btx was identical in strains from serotypes 1a, 3, 6, and 48, and the gene is here classified as a type 1 btx gene. A serotype 3 strain isolated in Singapore possessed a unique 42-kDa toxin gene, here designated type 4; while the btx genes from strains of serotypes 5a5b and 25 are referred to as types 2 and 3, respectively.