The risk of osteoporosis in oral steroid treatment for nasal polyposis: a systematic review.

BACKGROUND: Systemic glucocorticoids are often used in the treatment of chronic rhinosinusitis with nasal polyps (CRSwNP), and osteoporosis is a well-known complication to steroid treatment, associated with significant morbidity. Nevertheless, the burden of steroid induced osteoporosis is unknown in patients with CRSwNP. We aimed to assess the risk of acquiring osteoporosis caused by oral steroids in patients with CRSwNP, and provide recommendations on future research and guidelines. METHODOLOGY: Cochrane Review Database, EMBASE, Ovid Medline, and PubMed were searched for studies including adult patients with CRSwNP treated with oral steroids. Outcomes were Bone Mineral Density (BMD) and prevalence of fractures in relation to dose and duration of oral steroids. In addition, we reviewed general guidelines for treatment with oral steroids. RESULTS: We identified two studies (n=243) that met the inclusion criteria. Doses and durations of oral steroids were over 5 mg/day for more than 3 months and 1 mg/kg body weight/day for 6 to 10 days for 4 or more courses/year. The prevalence of low bone mass was 39% and 61%, respectively. It was not possible to quantify the overall risk of osteoporosis induced by oral steroids from the studies. No studies evaluated prevalence of fracture. CONCLUSIONS: Registry studies and randomized controlled trials would be needed to assess the risk of osteoporosis in CRSwNP patients and future guidelines should include recommendations regarding preventive treatment and recommendations on doses and durations of oral steroids.

Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line.

A novel basic heparin-binding monocyte chemotactic factor (MCF) was purified to homogeneity from the conditioned media of human myelomonocytic cell line THP-1 based on its in vitro monocyte chemotactic activity. The purified MCF was homogenous and estimated to be 15 kD on SDS-PAGE. Purified MCF stimulated normal human monocytes to be growth inhibitory in vitro at 2-3 d for several human tumor cell lines. This represents the first report of the identification and purification of a chemoattractant cytokine that also activates monocytes but is distinct from interferons and other known cytokines.

The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes.

T lymphocyte chemotactic factor (TCF) was purified to homogeneity from the conditioned media of phytohemagglutinin-stimulated human blood mononuclear leukocytes by a sequence of chromatography procedures. The amino-terminal amino acid sequence of the purified TCF showed identity with neutrophil-activating protein (NAP-1). Both TCF and recombinant NAP-1 (rNAP-1) were chemotactic for neutrophils and T lymphocytes in vitro supporting the identity of TCF with NAP-1. Injection of rNAP-1 into lymphatic drainage areas of lymph nodes in Fisher rats caused accelerated emigration of only lymphocytes in high endothelial venules. Intradermal injection of rNAP-1 caused dose-dependent accumulation of neutrophils and lymphocytes.

IL-8 induces T cell chemotaxis, suppresses IL-4, and up-regulates IL-8 production by CD4+ T cells.

Interleukin-8 (IL-8), a neutrophil-activating cytokine, also activates certain T cell functions such as chemotaxis. We additionally find (n = 6) that recombinant (rIL-8; 1-100 ng/ml), when added to 24 h culture of human CD4+ T cells, suppressed the spontaneous production of IL-4 (50-85%). Steady state production of Il-4 was typically around 30 pg/ml, determined by use of a solid- phase immunoabsorbant assay. De novo synthesis of IL-4 from CD4+ T cells cultured for 3 days was also evaluated by use of detection of [35S]methionine incorporation, as visualized by autoradiography of 2-D gels, and showed that IL-8 suppressed IL-4 production. This suppression of IL-4 production was confirmed in the cytosol fraction by use of Western blotting. The effect of IL-8 (100 ng/ml) was comparable to that of 10 ng/ml recombinant interferon-gamma, both strongly suppressing IL-4 production. The regulatory effect of IL-8 on IL-4 production was also indicated by the fact that addition of a neutralizing monoclonal anti-IL-8 antibody (WS.4) enhanced the spontaneous IL-4 production when added to the culture of CD4+ T cells, thereby probably inactivating the effect of IL-8 originating from the cultured T cells. Also, we observed that IL-4 mRNA expression was down-regulated when the CD4+ T cells were cultured for 12 h in the presence of 100 ng/ml IL-8. The suppression of IL-4 mRNA expression could be prevented by adding anti-IL-8 (20 microgram/ml) or IL-10 (100 ng/ml) l h before adding rIL-8. Thus, IL-8 may be an important regulator of CD4+ T cell-derived IL-4, thereby possibly regulating the balance between humoral and cellular T cell-dependent responses.

Quantification of the 3H-ouabain binding site concentration in human myocardium: a postmortem study.

The 3H-ouabain binding site concentration in the human myocardium was determined by measuring vanadate facilitated binding of 3H-ouabain to necropsy specimens of the left ventricle. The 3H-ouabain binding to samples weighing 4-6 mg was specific and saturable and appeared to take place to only one population of high affinity binding sites. After death the 3H-ouabain binding capacity degraded relatively slowly. From 6 to 24 h after death a mean decrease of 11% was seen in five patients, being significant in only one. In 15 patients aged 64-86 years the concentration of 3H-ouabain binding sites measured 6 h after death varied from 223 to 577 pmol X g-1 wet weight with no obvious relation to age or sex. The mean (SEM) value (413(26) pmol X g-1 wet weight) was 1.7 times higher than that previously reported for human myocardium. The concentrations of 3H-ouabain and 3H-digoxin binding sites were identical, and an excess of unlabelled ouabain completely prevented the specific binding of 3H-digoxin. In necropsy specimens weighing 1-2 mg from the endomyocardium obtained using a biotome the 3H-ouabain binding site concentration was in the same range as that in the myocardium. These findings indicate that it is possible to determine the concentration of Na, K-pumps in the human myocardium by measuring the 3H-ouabain binding capacity of biopsy specimens obtained during heart catheterisation or of specimens obtained within the first 18 h after death. This finding may be of importance for studying conditions in which the Na, K-pump concentration is suspected of undergoing variation.

Epidermis and lymphocyte interactions during an allergic patch test reaction. Increased activity of ETAF/IL-1, epidermal derived lymphocyte chemotactic factor and mixed skin lymphocyte reactivity in persons with type IV allergy.

Recently, we have found an increased activity of epidermal-derived thymocyte-activating factor (ETAF/IL-1) and epidermal lymphocyte chemotactic factor (ELCF) in epidermis overlying a positive tuberculin skin reaction. In the present study, we investigated 20 patients with confirmed or suspected allergic contact dermatitis by using the suction blister technique before and during patch testing. The ETAF/IL-1 was found in epidermis before patch testing. Its presence increased 2.8-fold in epidermis overlying a positive patch test compared with pretesting values. This increase was statistically significant. Interestingly, nontested skin also showed a significant increase of ETAF/IL-1, which was 1.9-fold higher than pretest values. The ETAF/IL-1 activity in patch test areas was significantly correlated with the clinical response. ELCF is not present in epidermis from noneczematous persons. We observed a significant content of ELCF in three of seven patients with eczema prior to patch testing. After patch testing, all patients showed ELCF in epidermis. Nontested skin showed a 1.5-fold higher content of ELCF compared with pretest values, and in the test area ELCF was 1.8-fold higher. The increases were statistically significant. We performed mixed skin lymphocyte reactions in seven patients using epidermal cells from the patch test area. All patients with a positive patch test had an increased mixed skin lymphocyte reactivity compared with epidermis coming from a negative reaction.

A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA+CCR4+ lymphocytes into lesional atopic dermatitis skin.

Atopic dermatitis is an inflammatory skin disease in which the inflammation is characterized by the influx of lymphocytes into the dermis. It is generally believed that atopic dermatitis is a Th2-type disease, i.e., the T lymphocytes produce interleukin-4, interleukin-5, interleukin-10, and interleukin-13, although it has become evident in recent years that the cytokine profile in the skin changes during the course of the disease towards a Th1-Th2 mixed cytokine profile (interferon-gamma, tumor necrosis factor alpha, and interleukin-2). The lymphocytes that home into the skin express cutaneous lymphocyte-associated antigen, and it has recently been shown that most of the lymphocytes in this population express the chemokine receptor CCR4. CCR4 is the receptor for the CC chemokine TARC (thymus and activation regulated chemokine), and this chemokine is expressed predominantly by keratinocytes in the basal layer of the epidermis of lesional atopic dermatitis skin in mice. In humans, however, it was shown to be expressed in the endothelial cells of the dermis. We have examined the peripheral blood mononuclear cells of atopic dermatitis patients for the expression of cutaneous lymphocyte-associated antigen and CCR4 and compared them with peripheral blood mononuclear cells from normal controls. We found that the proportion of CLA+CCR4+ lymphocytes is upregulated in atopic dermatitis patients. In addition we have examined skin biopsies of lesional and non-lesional skin from atopic dermatitis patients and found that the keratinocytes, but not the endothelial cells, produce TARC in the lesional but not in the nonlesional skin. To gain insight in the stimulatory mechanisms for TARC production in keratinocytes, as previously observed in mice, we cultured HaCaT cells and found that interferon-gamma and tumor necrosis factor alpha work synergistically to induce TARC production. These observations suggest that the induction of TARC production in keratinocytes plays an important role in the late phase skin invasion by CCR4+CLA+ Th2-type lymphocytes in atopic dermatitis.

Quantitative determination of IL-1 alpha-induced IL-8 mRNA levels in cultured human keratinocytes, dermal fibroblasts, endothelial cells, and monocytes.

The presence of the leukocyte chemotactic cytokine interleukin 8 (IL-8) in psoriatic scales and in epidermal tissue overlying allergic patch test reactions suggests a role for this cytokine in certain inflammatory skin diseases. IL-8 can be produced by several cell types present in the skin. Their relative potentials for IL-8 expression has, however, not yet been studied, due to the lack of convenient methods for quantitative comparison of specific mRNA amounts in different cell types. Using a new method for quantification, we compared specific IL-8 mRNA amounts in cultures of keratinocytes, dermal fibroblasts, endothelial cells, and monocytes, stimulated with interleukin 1 alpha (IL-1 alpha). Endothelial cells produced very high, fibroblasts and monocytes intermediate, and keratinocytes low amounts of IL-8 mRNA. We also studied the time course of IL-8 mRNA levels in the four cell types following IL-1 alpha stimulation, and found a clear difference both in onset and stability of the response. We discuss the different strength of the response at different time points in the cell types analyzed in relation to their possible role in regulation of the normal response to stimulation.

Recombinant human interleukin-8 is a potent activator of canine neutrophil aggregation, migration, and leukotriene B4 biosynthesis.

Interleukin-8 (IL-8), formerly known as NAP-1, is formed by a variety of cells upon stimulation with IL-1 or tumor necrosis factor (TNF). The biologic activity of the cytokine involves activation of almost every neutrophil function studied so far in different species. In the present study, we compared the effects of recombinant human IL-8 (rIL-8) and the lipid mediators, leukotriene B4 (LTB4) and platelet-activating factor (PAF), on neutrophil functions in dogs. All three chemotactic factors induced neutrophil aggregation and chemotaxis, with rIL-8 being far more potent than LTB4 and PAF. The migration induced by rIL-8 was significantly greater than that observed towards LTB4 and PAF. In the aggregation assay, rIL-8 was shown for the first time to be a potent stimulant. The aggregation response was more persistent than that obtained with LTB4 and PAF and the potency of rIL-8 was greater. An intradermal dose-response study showed that rIL-8 is an extremely potent inducer of selective neutrophil infiltration in canine skin. The infiltration was more pronounced than following injection of LTB4 or PAF. It was proposed that the superior effect of rIL-8 was caused by a synergistic effect between injected rIL-8 and LTB4, which was shown to be produced in biologically active amounts by canine neutrophils stimulated with rIL-8. From a therapeutic point of view, the simultaneous presence of rIL-8 and LTB4 in inflammatory skin diseases highlights the need to develop drugs that inhibit the production and/or effect of both mediators.

Psoriasin: a novel chemotactic protein.

Inflammatory skin disorders such as psoriasis show a preferential epidermal infiltration of neutrophils and T lymphocytes. This observation raises a question as to which factors determine the appearance and composition of leukocyte tissue infiltrations. Previously, we described a low molecular mass calcium-binding protein (psoriasin, molecular mass 11,457 Da, pI 6.77) belonging to the S1OO family that is highly upregulated in psoriatic keratinocytes and whose expression patterns implied a role in the inflammatory response. Here we report that human psoriasin is a potent and selective chemotactic inflammatory protein for CD4+ T lymphocytes and neutrophils at concentrations of about 10(-11) M. Psoriasin is not structurally related to the alpha or the beta chemokine subfamilies or to lymphotactin, a member of a newly described class of chemokines. Thus, we have observed a chemotactic protein outside the chemokine subfamilies that could be an important new inflammatory mediator.

Localization of monocyte chemotactic and activating factor (MCAF/MCP-1) in psoriasis.

The monocyte chemotactic protein-1 (MCAF) also termed MCP-1, a strong chemotactic factor towards monocytes, is produced by several cell types present in the skin. The in situ presence of MCAF/MCP-1 protein in the skin has, however, not yet been established. Using immunohistochemical techniques we have investigated the distribution of MCAF in skin from patients with different types of psoriasis and normal healthy volunteers. We report the novel finding that psoriasis has strong positive immunostaining for MCAF located to all the layers of the epidermis, except the stratum granulosum, in pustular, guttate and chronic plaque psoriasis. In the dermis, infiltrating cells in the perivascular aggregates and the blood vessels stained positive for MCAF. No significant differences were observed between the different subtypes of psoriasis except that strongly positive infiltrating cells were observed in the epidermal pustules in pustular psoriasis. In normals positive staining was observed in all the layers of the epidermis and in a few perivascular cells and blood vessels in the dermis. Where present in normal and diseased skin, eccrine ducts of sweat glands and sebaceous glands stained positive for MCAF. Arrector pili muscles were in all cases negative. These findings are consistent with a role for MCAF in attracting inflammatory cells, including monocytes, into the skin in psoriasis.

IL-10 augments the IFN-gamma and TNF-alpha induced TARC production in HaCaT cells: a possible mechanism in the inflammatory reaction of atopic dermatitis.

The CC-chemokine TARC is known to be a ligand for the CCR4 receptor which in turn is known to be expressed selectively on the Th(2)-subset of lymphocytes. Atopic dermatitis is generally believed to be a Th(2)-type disease, and TARC has been shown to be expressed in the skin lesions of a murine model of AD. IL-10 is an interleukine generally known for its ability to inhibit cytokine production, however it has been found to be highly expressed in the skin from AD patients. We show in this report that IL-10 is able to augment the TARC inducing effects of TNFalpha and IFNgamma in HaCaT cells, a property that may be important in the determination of the composition of the cells of the inflammation in the skin of AD patients. In addition, we show that the IL10 agonist IT 9302, a nona-peptide from the carboxylic end of IL-10, has the same effect on TARC production from HaCaT cells.

Monocyte chemotactic and activating factor (MCAF/MCP-1) has an autoinductive effect in monocytes, a process regulated by IL-10.

MCAF (MCP-1) a member of the chemokine-beta-family known to be chemotactic for monocytes is believed to play a significant role in several inflammatory processes, both immuno-pathological disorders, such as atherosclerosis, psoriasis, chronic inflammatory diseases of the liver and lungs, and during the normal immune response against microorganisms. This chemokine is produced spontaneously by monocytes, and in the present article we also demonstrate that MCAF induces its own production in monocytes. The methods used are two dimensional SDS-PAGE gel electrophoresis. Western-blotting and ELISA quantification of supernatant from monocyte cultures stimulated with MCAF (1, 10, 100 ng ml). Also, we found that this process is regulated by IL-10 (100 ng ml). Our results suggest that monocytes migrating to a site of inflammation due to the local production of the chemokine MCAF/MCP-1 further enhance the focal accumulation of monocytes by producing and releasing bioactive MCAF MCP-1.

Production and characterization of recombinant human neutrophil chemotactic factor.

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.

IT 9302, a synthetic interleukin-10 agonist, diminishes acute lung injury in rabbits with acute necrotizing pancreatitis.

BACKGROUND: Proinflammatory cytokines (eg, tumor necrosis factor [TNF]-alpha, interleukin [IL]-1 and Il- 8) are believed to play an important role in the pathogenesis of acute necrotizing pancreatitis (ANP) and its systemic complications. Recently, IL-10 has emerged as a major anti-inflammatory cytokine, inhibiting the secretion and activities of inflammatory cytokines. Further, a protective effect of IL-10 has recently been shown in experimental acute pancreatitis. The purpose of this study was to test the potential role of a newly developed IL-10 agonist, IT 9302, in a model of ANP in rabbits. METHODS: ANP was induced in 18 rabbits by retrograde injection of 5% chenodeoxycholic acid in the pancreatic duct, followed by duct ligation. The rabbits were allocated to pretreatment with intravenous physiologic saline solution or IT 9302 (200 micrograms/kg) 30 minutes before the induction of ANP. RESULTS: Injection of IT 9302 resulted in a significant reduction in the blood levels of TNF-alpha and IL-8 from 3 to 6 hours. IT 9302 also reduced the amount of ascitic fluid and significantly inhibited neutrophil infiltration and margination, as well as the number of CD11b- and CD18-positive cells in the lung tissues. By contrast, the local pancreatic necrosis, as well as the biochemical changes such as serum amylase, lipase, and calcium, was sever and similar in both groups. Survival was improved significantly after treatment with IT 9302. CONCLUSIONS: As expected, IT 9302 cannot change the degree of ANP induced by 5% bile acid but does reduce mortality rates and the development of acute lung injury, probably through the inhibition of circulating levels of TNF-alpha, IL-8, and the expression of the adhesion molecule complex CD11b/CD18.

Graded experimental acute pancreatitis: monitoring of a renewed rabbit model focusing on the production of interleukin-8 (IL-8) and CD11b/CD18.

OBJECTIVE: To establish and monitor a rabbit model of graded severity of acute pancreatitis to test the hypothesis that interleukin-8 (IL-8) and the adhesion molecule complex CD11b/CD18 are involved in the development of systemic complications in severe acute pancreatitis. METHODS: Acute pancreatitis induction in rabbits by duct ligation with or without infusion of 5.0% or 0.5% chenodeoxycholic acid or 0.9% saline. Control animals underwent laparotomy. The animals were monitored biochemically, histologically and immunohistochemically. RESULT: Increased serum levels of IL-8, tumour necrosis factor alpha (TNF-alpha), amylase and lipase were found in the chenodeoxycholic acid groups when compared with the saline, duct-ligated or control groups. Leukopenia, hypocalcaemia, and hyperglycaemia were marked in the 5.0% chenodeoxycholic acid group as compared to the saline, duct-ligated and control groups. Histologically, the 5.0% chenodeoxycholic acid group manifested a significant degree of pancreatic necrosis and neutrophil infiltration. The lungs of these animals showed acute lung injury and a significant up-regulation of CD11b/CD18. IL-8 was produced in pancreatic acinar and ductal cells. A significantly large output of ascitic fluid was seen in the 5.0% chenodeoxycholic acid group. CONCLUSION: The rabbit models of acute pancreatitis are reliable in that enzymatic and histological evidence of acute pancreatitis with or without systemic complications developed. IL-8 is produced locally in pancreatic acinar and ductal cells and significantly increased in peripheral blood during severe but not mild pancreatitis. The expression of the adhesion molecule complex CD11b/CB18 is significantly increased in lung tissue during severe acute pancreatitis with acute lung injury. IL-8 and CD11b/CB18 are involved in the pathogenesis of severe acute pancreatitis but not of mild oedematous pancreatitis.

Epidermis and lymphocyte interactions during a tuberculin skin reaction. I. Increased ETAF/IL-1 like activity, expression of tissue antigens and mixed skin lymphocyte reactivity.

Forty-one persons were tested for tuberculin skin reactivity. Epidermal cells (ECs) were isolated from the tuberculin reaction and from a contra lateral, non injected skin area. We found a significant increase of epidermal thymocyte activating factor (ETAF) in epidermis overlying a positive tuberculin reaction together with an increase of OKT6 and class II (HLA-DR) positive cells. Allogeneic lymphocytes proliferated significantly more when mixed with ECs from a positive tuberculin skin test. Injection of tuberculin per se or a negative reaction did not induce similar changes. The described model seems useful for functional studies of ECs and lymphocytes in patients with contact dermatitis.

Epidermal lymphocyte chemotactic factor specifically attracts OKT4-positive lymphocytes.

Epidermal lymphocyte chemotactic factor (ELCF) from skin overlying a positive tuberculin reaction was compared with the chemoattractants leukotriene B4 (LTB4), N-formyl-methionyl-leukyl-phenylalanine (FMLP), and complement split product C5a (C5a). The chemotactic assay used is a modified Boyden chamber technique. The lymphocytes were subsets of T lymphocytes from healthy young individuals first separated by flotation of E rosettes on Isopaque Ficoll followed by incubation of T cells with anti-CD4 and anti-CD8 monoclonal antibodies and further separation using fluorescence-activated cell sorting. ELCF specifically attracted OKT4+ lymphocytes, while LTB4, FMLP, and C5a induced significant migration in both OKT4+ and OKT8+ lymphocytes without any clear difference between the various chemoattractants or cell populations. We found no blocking of the chemotactic capacity of ELCF when we added antibodies towards IL-1 alpha and IL-1 beta to the chemotactic assay. Further recombinant IL-1 alpha and Il-1 beta did not induce any chemotactic response. Our observation may be of significance in explaining the predominance of OKT4+ cells in allergic contact dermatitis and certain other skin diseases.

A two-constant equation for multiple albumin-binding isotherms.

It has been found that binding of low molecular weight ligands to human serum albumin is generally nonsaturating. Equations commonly used for describing the binding equilibria, i.e., the Scatchard and Klotz (Adair) equations, are saturation functions. We have accordingly tried to establish an equation which would fit the observed data, i.e., not reach a saturation plateau. An empirical equation, in which the bound ligand is expressed as a function of the free ligand [R(C) = b1 in (b2C + 1)], is shown to give reasonably good fits to observed binding equilibrium data for the binding of several organic ligands to human serum albumin, when the two parameters, b1 and b2, are given suitable values. The curve of bound versus free ligand, as plotted from this equation, has the same slope and curvature as that obtained from the Klotz stepwise binding equation at C = 0, if b1 = K1/[2(K1 - 2K2)] and b2 = 2(K1 - 2K2), where K1 and K2 are the first and second stoichiometric binding constants.

Warfarin-sulfinpyrazone interaction on binding to human serum albumin.

Sulfinpyrazone displacement of warfarin from human serum albumin was studied in-vitro. At low sulfinpyrazone concentrations one molecule of warfarin is displaced on binding by one molecule of sulfinpyrazone. Clinical plasma concentrations of sulfinpyrazone are, however, too low to cause significant displacement.