Spontaneous generation of functional osteoclasts from synovial fluid mononuclear cells as a model of inflammatory osteoclastogenesis

In osteoimmunology, osteoclastogenesis is understood in the context of the immune system. Today, the in vitro model for osteoclastogenesis necessitates the addition of recombinant human receptor activator of nuclear factor kappa‐B ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). The peripheral joints of patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA) are characterized by an immune‐mediated inflammation that can lead to bone destruction. Here, we evaluate spontaneous in vitro osteoclastogenesis in cultures of synovial fluid mononuclear cells (SFMCs) activated only in vivo. SFMCs were isolated and cultured for 21 days at 0.5–1.0 × 10⁶ cells/mL in culture medium. SFMCs and healthy control peripheral blood monocytes were cultured with RANKL and M‐CSF as controls. Tartrate‐resistant acid phosphatase (TRAP) positive multinucleated cells were found in the SFMC cultures after 21 days. These cells expressed the osteoclast genes calcitonin receptor, cathepsin K, and integrin β3, formed lacunae on dentin plates and secreted matrix metalloproteinase 9 (MMP9) and TRAP. Adding RANKL and M‐CSF potentiated this secretion. In conclusion, we show that SFMCs from inflamed peripheral joints can spontaneously develop into functionally active osteoclasts ex vivo. Our study provides a simple in vitro model for studying inflammatory osteoclastogenesis.     

Variability of antigenicity/allergenicity in different strains of Alternaria alternata

Biochemical and immunological properties were studied in crude mould extracts from 10 different strains of Alternaria alternata. The mould strains were grown on completely synthetic medium and harvested under identical conditions. The biochemical and immunological analyses (protein content, carbohydrate content, IEF, RAST-inhibition, CIE/CRIE and the content of a major allergen) showed significant variations between the individual strains of A. alternata. 32 antigens were identified in the CIE pattern of A. alternata and 19 different allergens were identified in CRIE analysis (1 major, 7 intermediate and 6 minor allergens). There was a significant variation in the content of the major A. alternata allergen (Ag-8, partially identical to Alt-I) between the different individual strains. Furthermore, a considerable variation in the content of Ag-8 was observed for a specific strain cultivated at different occasions under identical cultivation conditions during a time period of 3 years.     

Influence of suckling and feeding on insulin, gastrin, somatostatin and VIP levels in peripheral venous blood of lactating sows

Blood samples were collected in peripheral venous blood of seven lactating sows, when their piglets were suckling. In four of the experiments samples were also taken when the sows were fed a meal. Gastrin, insulin, somatostatin and VIP levels were measured by radioimmunoassay. Insulin levels increased by approximately 100% for about 10 min in response to suckling, in some experiments even before the suckling occurred, i.e. when the sows saw, heard and smelled their piglets. In four of the sows suckling caused a biphasic twofold increase in gastrin levels - one immediate peak which lasted for a few min and a second peak of longer duration (about 30-60 min), whereas gastrin levels remained unchanged in three animals. Somatostatin levels usually reflected gastrin levels in a reciprocal way. Thus, a biphasic decrease of somatostatin levels occurred in the high gastrin responders. In contrast, somatostatin levels increased in the experiments, in which gastrin levels did not change. Immediate and short-lasting (a few minutes long) increases of VIP levels were also induced by suckling. Large litters and long suckling periods appeared to be related to greater changes of the levels of all the peptides measured. Feeding influenced insulin, gastrin and somatostatin levels in the same way as did suckling from both a qualitative and a quantitative point of view. In contrast, VIP levels were not increased by feeding. The possible functional effects of the suckling-induced release of gastrointestinal hormones and possible mechanisms of their release are discussed.     

Aspergillus fumigatus specific IgE and IgG antibodies for diagnosis of Aspergillus-related lung diseases

IgE and IgG antibodies against Aspergillus fumigatus were detected by crossed radio immunoelectrophoresis (CRIE) on the sera of seven patients with aspergilloma, six patients with allergic broncho-pulmonary aspergillosis (ABPA) and 25 patients with extrinsic asthma with Aspergillus allergy. IgE-CRIE analysis indicated the presence of A. fumigatus-specific IgE in sera of patients with ABPA and Aspergillus asthma but not of aspergilloma patients. IgG-CRIE showed that both aspergilloma and ABPA patient sera contained high levels of circulating specific IgG antibodies in contrast to sera of Aspergillus asthma patients, which did not show detectable amounts of Aspergillus-specific IgG antibodies. Specific IgE binding could be demonstrated for the major allergens Ag-10 and AG-40 in all ABPA patients, in 80% of Aspergillus asthma patients but not in sera from aspergilloma patients. Specific IgG antibodies directed towards the major allergens could be detected in most of the aspergilloma patients, between 30-70% of the ABPA patients but not in sera from patients with Aspergillus asthma.     

A Double-Blind, Multicenter Immunotherapy Trial in Children, Using a Purified and Standardized Cladosporium herbarum Preparation II. In Vitro Results

This double-blind immunotherapy trial in children, using a purified and standardized Cladosporium herbarum allergen preparation, has shown that children with mould asthma and/or rhinoconjunctivitis, responded to immunotherapy with a decrease in specific IgE and a significant increase in specific IgG. There was a marked increase in the ratio specific IgG/specific IgE as a result of active treatment. IgE-CRIE radiostaining patterns showed no pronounced changes after 10 months' active treatment and no "new sensitivities" could be detected in the studied patients. IgG-CRIE radiostaining, primarily directed towards the important allergens, was significantly increased in the active group and particularly towards Ag-12 (partially identical to a previously described major allergen in Cladosporium herbarum, Ag-54). Children treated with histamine placebo showed no change in antibody patterns during 10 months of treatment.     

The major allergen content of allergenic preparations reflects their biological activity

The content of major allergens in biologically standardized allergenic preparations of birch, mite (Der p) , cat, Alternaria (Alt a) and ragweed (Amb e) was determined. It was found fairly constant between species , i.e. varied within a factor of 2, with the exception of Alt a 1 in Alternaria alternata extract. This variation is allowed by authorities between different batches prepared from the same species of allergen. The method for biological standardization (BS) prescribed in the Nordic Guidelines has, for common inhalant allergens, been shown to give reproducible results between regions of Europe. However, it is difficult to define patients suitable for BS of most food allergens as well as less common inhalant allergens. Therefore we propose that, in the future, BS is replaced by determination of well-established major allergens and that 1 ng of major allergen is given the value of 1 Biological Unit.
Clinical aspects
Clinicians have had difficulties in understanding differences and similarities between units used by manufacturers for labelling of allergenic extracts. Biological standardization is time-consuming and expensive. Probably therefore, and to avoid comparison with extracts prepared by other manufacturers, most manufacturers have used their own units and few of them have used the biological units as defined by the Nordic Guidelines or FDA. Determination of the amount of major allergen by ELISA is simple and cheap. However, the biological relevance of major allergen content has not been established. Our results clearly indicate the possibility of replacing biological units by major allergen content, provided the composition of allergens is adequate. The major allergen content can easily be declared by all manufacturers. In the future, manufacturers should be forced to declare the major allergen content, thus making it easier for clinicians to compare extracts from different suppliers.     

Tissue polypeptide antigen-specific (tps) and carcinoembryonic antigen (cea) levels in cancerous and precancerous lesions in human colon

Serum levels of tissue polypeptide antigen specific (TPS), a cytokeratin 18 marker, were determined and compared with serum levels of carcinoembryonic antigen (CEA) in 45 patients with colon adenocarcinoma and in 34 patients with benign diseases (adenomatous polyps and ulcerative colitis) at the time of diagnosis. In colon carcinoma patients 58% had an elevated TPS level (cut-off 100 U/l) and 53% had an elevated CEA level (cut-off 3.0 ng/ml). The sensitivity of the cytokeratin marker TPS was related to the stage of the disease. Significant correlation was observed between TPS and Dukes stages in colon cancer patients and the highest TPS values were achieved in Dukes stage D. The combined use of the two markers increased the sensitivity to 82% compared with the use of only one. Simultaneous raise of both serum markers TPS and CEA was observed in 36% of cases. In the majority of the patients with adenomatous polyps and ulcerative colitis the serum TPS and CEA levels were below the upper reference limit. However the initial high levels in some patients could be considered as a prognostic indicator for identifying a group of patients with increased risk of cancer development. No significant correlation was observed between serum TPS and CEA concentrations in individual patients with benign diseases.