The germless theory of allergic disease: revisiting the hygiene hypothesis.

Rising rates of allergic disease accompany the healthier benefits of a contemporary westernized lifestyle, such as low infant mortality. It is likely that these twinned phenomena are causally related. The hygiene hypothesis states that allergy and increased longevity are both consequences of reducing infectious stressors during early childhood for millennia. Mechanistic explanations for the hygiene hypothesis have typically invoked the T-helper-type 1/2 (T(H)1/T(H)2) model. Here, we discuss why we favour a broader 'counter-regulatory' model--one that might also explain the increasing incidence of autoimmune disease in westernized countries.

IL-21 receptor signalling partially mediates Th2-mediated allergic airway responses.

BACKGROUND: Interleukin-21 (IL-21) has been implicated in the development of Th2-mediated immune responses; however, the exact role it plays in allergic diseases is not well understood. OBJECTIVE: To elucidate the contribution of IL-21 receptor signalling to Th2-dependent immune responses in the lung. METHODS: We compared allergic airway responses in wild-type BALB/c and Il21r-deficient mice exposed to local airway challenge with house dust mite (HDM). RESULTS: We demonstrate that IL-21R-deficiency reduces HDM-driven airway hyperresponsiveness (AHR) with only partial effects on airway inflammation. Concomitant with the reduction in AHR in Il21r-deficient mice, significant suppression was observed in protein levels of the Th2 cytokines IL-4, and IL-13. In contrast, IL-21R-deficiency was associated with an increase in PBS- and allergen-driven IgE levels, while IgG1 and IgG2a levels were decreased. Moreover, our results suggest that IL-21 may contribute to AHR through its ability to both directly induce Th2 cell survival and to impair regulatory T-cell suppression of Th2 cytokine production. Importantly, we show that IL-21-positive cells are increased in the bronchial mucosa of asthmatics compared with non-asthmatics. CONCLUSION: These results suggest that IL-21 plays an important role in the allergic diathesis by enhancing Th2 cytokine production through multiple mechanisms including the suppression of Treg inhibitory effects on Th2 cell cytokine production.

Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigen-induced airway hyperresponsiveness and mucus production.

The pleiotropic cytokine interleukin 4 (IL-4) has been shown to regulate many processes thought to be important in the allergic diathesis. To determine the mechanism(s) by which IL-4 mediates allergic airway responses to inhaled allergens, we compared the effects of antigen sensitization and challenge on the development of allergic airway responses in mice in which the gene for the signal transducer and activator of transcription factor 6 (Stat6) was disrupted to those of their wild-type littermates. Strikingly, Stat6-deficient mice failed to develop airway hyperresponsiveness (AHR), which was observed in their wild-type littermates after allergen provocation. Moreover, antigen-induced increases in mucus-containing cells were found to be completely Stat6 dependent. Consistent with the lack of Th2 cytokine responses in Stat6-deficient mice, no ovalbumin-specific immunoglobulin (Ig)E was detected in their serum. In contrast, Stat6 signaling only partially mediated antigen-induced eosinophilia with no role in vascular adhesion molecule 1 expression. These results indicate that Stat6 signal transduction is critical in the development of allergen-induced AHR and that agents that specifically inhibit this pathway may provide a novel strategy for the treatment of allergic disorders.

Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice.

Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen-induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma.

Interleukin-13: central mediator of allergic asthma.

The worldwide incidence, morbidity, and mortality of allergic asthma are increasing. The pathophysiological features of allergic asthma are thought to result from the aberrant expansion of CD4(+) T cells producing the type 2 cytokines interleukin-4 (IL-4) and IL-5, although a necessary role for these cytokines in allergic asthma has not been demonstrable. The type 2 cytokine IL-13, which shares a receptor component and signaling pathways with IL-4, was found to be necessary and sufficient for the expression of allergic asthma. IL-13 induces the pathophysiological features of asthma in a manner that is independent of immunoglobulin E and eosinophils. Thus, IL-13 is critical to allergen-induced asthma but operates through mechanisms other than those that are classically implicated in allergic responses.

A new label technology for the detection of specific polymerase chain reaction products in a closed tube.

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5'-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5'-->3' exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10-10(7)initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5'-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.

Expression of bacterial luciferase genes from Vibrio harveyi in Bacillus subtilis and in Escherichia coli.

For gene expression and cell physiology studies of Gram-positive Bacillus subtilis, novel shuttle vectors which cause the host organism to produce light have been constructed. These vectors carry genes encoding luciferase from Vibrio harveyi, a selectable kanamycin marker and an origin of replication for Gram-negative and -positive bacteria. The effect of DNA-insert size on light production in Escherichia coli and in B. subtilis was studied by also cloning into the shuttle vector a gene whose product participates in fatty acid metabolism. B. subtilis containing lux genes was found to differ from its Gram-negative counterpart in light emission characteristics. After addition of the substrate, light emission by B. subtilis was rapid but it decayed quickly, showing biphasic kinetics. In E. coli light emission is continuous, reflecting constant regeneration of substrates for the luciferase reaction. A promoter cloning vehicle, pCSS117, was constructed by inserting a transcriptional termination loop in the upstream sequences of luciferase genes. Using this vector, the mode of action of promoters of interest can be studied in E. coli and in B. subtilis.

Isolation of strong promoters from Clavibacter xyli subsp. cynodontis using a promoter probe plasmid.

To isolate promoters from Clavibacter xyli subsp. cynodontis (C. xyli subsp. cynodontis), we constructed a new promoter probe plasmid and made a C. xyli subsp. cynodontis promoter probe library. Two promoters gave over 2500-times stronger expression than the parental plasmid. The promoters were sequenced and compared to other bacterial promoters. These C. xyli subsp. cynodontis promoter regions are GC-rich and do not resemble E. coli promoters, but do resemble a few individual promoters found in streptomycetes.

Cloning, sequencing, expression and characterization of three anti-estradiol-17beta Fab fragments.

In order provide data for a basic understanding of the mechanisms of antibody specificity and for the design of antibodies with desired properties, we have sequence-analysed three high affinity anti-estradiol-17beta monoclonal antibodies. All three monoclonal antibodies to estradiol-17beta had been raised by conjugation of the 6-carboxymethyloxime derivative to protein carrier. The genes encoding heavy (Fd) and light (L) chains of these three antibodies were cloned and sequenced. The sequenced antibody chains were found to be from 46.0 to 89.7% sequence identical to a monoclonal antibody (DB3) binding a related steroid, progesterone. The Fd and L chains were paired with all possible Fd-L combinations and the corresponding proteins were expressed in Escherichia coli and characterized for their binding (immunoreactivity) to estradiol-17beta. Under the lac promoter and using the pelB signal sequences the production levels of the soluble (total) heavy and light chain Fab fragment combinations in periplasm and in supernatant varied from 115 to 2207 microg/l, while the immunoreactivity percentages (IR%) varied from < 1 to 45%. The production levels and IR% were dependent on the first constant domain subclasses of the heavy chain as well as the Fd-L chain combination expressed.

Functional analysis of the human alpha 2C-C4 adrenergic receptor in insect cells expressed by a luciferase-based baculovirus vector.

A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human alpha 2-adrenergic receptor (alpha 2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-alpha 2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the alpha 2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of alpha 2-receptors at a level of 1.370 pmol/mg protein was obtained at 48 h after infection as determined by ligand-binding experiments using the alpha 2-receptor antagonist, [3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [3H]rauwolscine binding with Ki values 12.3 +/- 1.54 microM and 1.23 +/- 0.11 microM, respectively. The recombinant receptors were functionally intact since the agonists inhibited forskolin-stimulated cAMP production. Here, however, the maximal inhibition was obtained at 36 h after the infection. The results presented here, suggest that the baculovirus expression vector system (BEVS) provides a simple method for abundant expression of functional alpha 2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.

Risk factors for development of diabetic nephropathy and retinopathy in Jewish IDDM patients.

Risk factors associated with diabetic microvascular complications, with special reference to ethnic origin, were looked for in 231 young Jewish insulin-dependent diabetes mellitus (IDDM) patients with duration of diabetes greater than or equal to 10 yr. Median age at diagnosis of diabetes was 9.2 yr (range 0.04-26.2 yr), and median duration of the disease was 15.3 yr (range 10.0-37.2 yr). Sixty-three percent of the patients were Ashkenazi Jews, and 37% were non-Ashkenazi Jews. HbA1 was evaluated every 3 mo in the last 10 yr of follow-up, and albumin excretion rate was tested in three 24-h urine collections. Direct and indirect ophthalmoscopy was performed every year since diagnosis of diabetes, and if retinal pathology was suspected, color photographs were taken. Microalbuminuria was detected in 31% and macroalbuminuria in 7% of the patients. Nonproliferative and proliferative retinopathy was found in 44 and 12% of the patients, respectively. On logistic regression analysis, two variables were significantly and independently associated with diabetic nephropathy--non-Ashkenazi origin and mean HbA1 values over the first 5 of 10 yr of follow-up. Variables significantly and independently related to diabetic retinopathy were non-Ashkenazi origin, mean HbA1 values over the last 10 yr of follow-up, and duration of diabetes. Because non-Ashkenazi Jews in Israel are of lower socioeconomic status than Ashkenazi Jews, we stratified our patients according to their socioeconomic parameters, median HbA1 values, and duration of diabetes. Non-Ashkenazi patients were at a higher risk to develop complications in all strata.(ABSTRACT TRUNCATED AT 250 WORDS)

The incidence of insulin-dependent diabetes mellitus in Israeli children and adolescents 0-20 years of age: a retrospective study, 1975-1980.

A survey of the entire population of Israel revealed 392 newly diagnosed type I diabetic children and adolescents aged 0-20 for the period of 1975-80. The mean annual age specific incidence of type I (insulin-dependent) diabetes mellitus was 3.8/10(5) for the age group 0-14 yr and 4.2/10(5) for the age group 0-20 yr. The incidence among the Jews of Ashkenazi origin was 6.8 X 10(5) and that for Jews of non-Ashkenazi origin was 4.3 X 10(5), whereas that for the Arabs was 1.2 X 10(5). The overall incidence is lower than that reported for similar populations in most European countries, the USA, Canada, and New Zealand; similar to that reported for Arabs in Kuwait; and higher than only that found in Japan. The relative importance of environmental and genetic factors in the interpopulation differences in incidence of type I diabetes remains to be established.

Crisis intervention program in newly diagnosed diabetic children.

A group of 223 insulin-dependent diabetic patients, aged 7-24 yr, who had been under the regular care of our clinic up to 15 yr, were rated by two independent judges on a two-level scale of adjustment and maladjustment. The patients were divided into two groups. Group A (N = 107) comprised those who had been under care from diagnosis of the disease and had been subjected to the special crisis intervention program offered to every family upon referral of a newly diagnosed patient. Group B (N = 116) comprised patients who were diagnosed and treated initially in a clinic that had no crisis intervention program. Significant differences between the two groups were found in respect to three of the four aspects studied, i.e., compliance, familial relationships, and sociability, with group A showing a better adjustment than group B. There was no significant difference in the fourth aspect studied, i.e., school achievement and work performance. It was found that it took three times the effort, i.e., the time invested in counseling and psychotherapeutic measures, to bring group B to a good level of adjustment than it did to achieve similar results with group A. It is suggested that the initial period after diagnosis of diabetes in a child should be considered a period of crisis, requiring special multidisciplinary services to reduce future psychosocial maladjustments and improve compliance.

A multidisciplinary, comprehensive, ambulatory treatment scheme for diabetes mellitus in children.

A study has been carried out on 262 children with juvenile diabetes and their parents, treated up to 10 yr on an ambulatory basis by a multidisciplinary team composed of pediatric endocrinologist, nurse, dietitian, psychologist, and social worker. Comparison of the findings with those of a study performed before inception of the Counselling Center for Juvenile Diabetics revealed the following positive influences: the degree of control attained was both higher and sustained with greater regularity; there were fewer complications with no episodes of coma, brittle diabetes, or severe ketoacidosis and almost no need for hospitalization; the attitude of the affected child, his parents, and his teachers was found to be considerably improved; there was better understanding of the nature of the disease and its requirements; the child's motivation to maintain the diabetic regimen was greater and conflicts within the family circle were markedly reduced; the child's self-concept was much higher; and both scholastic achievements and social adjustment were greater. We concluded that psychological stability is a basic factor in the control of diabetes, and the value of the multidisciplinary approach in the treatment of this chronic disease is indicated.

The prevalence of cutaneous manifestations in IDDM patients and their association with diabetes risk factors and microvascular complications.

OBJECTIVE: The aim of our study was to evaluate the frequency of skin manifestations, including the diabetic hand syndrome, in young IDDM patients. In addition, we studied the relation of the cutaneous manifestations to diabetes duration, glycemic control, and microvascular complications. RESEARCH DESIGN AND METHODS: The frequency of skin manifestations, including the diabetic hand syndrome, were examined in 238 IDDM patients (disease duration > 5 years) and 122 healthy control subjects in a cross-sectional study. In addition, we studied the relation of the cutaneous manifestations with diabetes duration, glycemic control, BMI, microvascular complications, and stratum corneum hydration using a stepwise logistic regression. RESULTS: Diabetic skin manifestations were detected in 168 of 238 (71%) IDDM patients and in 18 of 122 (14%) of the control subjects. Ichthyosiform skin changes of the shins, scleroderma-like skin changes, tinea pedis, and dry scaly palms were detected in 48 vs. 7%, 39 vs. 0%, 32 vs. 7%, and 21 vs. 0.8% of the patients and control subjects, respectively. In the diabetic patients, a significant association was found between ichthyosis of the shins and scleroderma-like skin changes of the hand (P < 0.001) and between scleroderma-like skin changes and the skin dryness of the palms (P < 0.0001). When diabetic risk factors were considered, diabetes duration was significantly associated with scleroderma-like skin changes and ichthyosis of the shins (P < 0.0001). The latter was also found to be related to diabetic retinopathy (P < 0.0001). Keratosis pilaris was present in 21% of the patients versus 9% in control subjects and was found to be exclusively associated with high BMI. CONCLUSIONS: Acquired ichthyosis is a common finding and the most prevalent skin manifestation in young IDDM patients. The development of several skin manifestations in insulin-dependent patients seems to be related to duration of diabetes and to development of diabetic microvascular complications.

Distinct Tlr4-expressing cell compartments control neutrophilic and eosinophilic airway inflammation.

Allergic asthma is a chronic, inflammatory lung disease. Some forms of allergic asthma are characterized by T helper type 2 (Th2)-driven eosinophilia, whereas others are distinguished by Th17-driven neutrophilia. Stimulation of Toll-like receptor 4 (TLR4) on hematopoietic and airway epithelial cells (AECs) contributes to the inflammatory response to lipopolysaccharide (LPS) and allergens, but the specific contribution of TLR4 in these cell compartments to airway inflammatory responses remains poorly understood. We used novel, conditionally mutant Tlr4(fl/fl) mice to define the relative contributions of AEC and hematopoietic cell Tlr4 expression to LPS- and allergen-induced airway inflammation. We found that Tlr4 expression by hematopoietic cells is critical for neutrophilic airway inflammation following LPS exposure and for Th17-driven neutrophilic responses to the house dust mite (HDM) lysates and ovalbumin (OVA). Conversely, Tlr4 expression by AECs was found to be important for robust eosinophilic airway inflammation following sensitization and challenge with these same allergens. Thus, Tlr4 expression by hematopoietic and airway epithelial cells controls distinct arms of the immune response to inhaled allergens.

Demonstration of the predominant urine osteocalcin fragments detectable by two-site immunoassays.

We have isolated and characterized human osteocalcin (OC) fragments from pubertal urine. The fragments were isolated by immunoaffinity chromatography based on monoclonal antibody 6F9 and further purified by reverse phase chromatography. The major isolated forms, which were detectable with two-site immunofluorometric assays for serum OC, span residues 6-30 and 7-30 as determined by mass spectrometry and N-terminal amino acid sequencing. Full-length OC was not detectable in the supernatant fraction of urine but could be extracted with guanidinium hydrochloride from the sediment of urine samples. Urine samples from subjects with different menopausal status were measured by two different two-site assays. Urine OC (uOC) concentrations were 12- to 16-fold higher in the pubertal group than in the adult group. Also, the uOC concentration in a postmenopausal group was significantly higher than in a premenopausal group. The difference was 125% and 75% (values for p < 0.0001), respectively, when measured with the two assays. uOC concentrations in postmenopausal subjects on hormone replacement therapy were indistinguishable from the premenopausal subjects. The fact that uOC can be measured by a noncompetetive two-site assay design offers improved analytical sensitivity. Urine as the sample matrix is also especially interesting because the predominant markers of bone resorption, collagen type I peptides or cross-links, are performed on urine samples. Our results from the technical validation of two-site assays for uOC and from applying these to human pubertal and pre- and postmenopausal samples calls for more extensive clinical validation.

Characterization of serum tartrate-resistant acid phosphatase and development of a direct two-site immunoassay.

Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP) to the circulation, where the amount of TRAP is expected to correlate with the bone resorption rate. We have developed two monoclonal antibodies, O1A and J1B, using purified human bone TRAP as antigen. The antibodies recognized different epitopes, allowing us to develop a two-site fluoroimmunoassay. The immunoreactivity in fresh serum specimens was less than 10% of the concentrations measured from the same specimens after 24 h of storage at 4 degrees C, or after addition of 5 mM EDTA or EGTA to them. When fresh serum was gel filtrated using Sephacryl S-200 column, all of the enzyme eluted in the void volume as a complex with a molecular weight of more than 250 kDa. If the serum was treated with EDTA before the gel filtration, the complex was destroyed and the enzyme eluted in fractions corresponding to a molecular weight of 30 kDa, the size of monomeric purified human bone TRAP. The immunoassay was used to measure TRAP concentrations from serum samples that had been stored at 4 degrees C for 24 h. According to the assay, premenopausal women had 13.1 +/- 3.1, postmenopausal women 17.6 +/- 4.2, and children 32.6 +/- 12.2 microg TRAP/l of serum. We conclude that TRAP circulates in the serum as part of a complex, which also contains Ca2+, and that TRAP-immunoassay is a potentially useful method for determining bone resorption rates, as long as the complex is destroyed before the assay.

Two-site immunoassays for osteoclastic tartrate-resistant acid phosphatase based on characterization of six monoclonal antibodies.

Tartrate-resistant acid phosphatase (TRAP), an enzyme expressed in bone-resorbing osteoclasts, is secreted into the circulation during bone resorption. We used six monoclonal antibodies (MAbs) to optimize direct two-site fluoroimmunoassays for determining serum TRAP concentrations. Four of the MABs, 1F1, 2H1, 4E6, and 5C1, were raised against recombinant human TRAP, and the other two, O1A and J1B, against human bone TRAP. 2H1, J1B, and O1A appeared to be highly specific for TRAP. 1F1 and 4E6 were poor in recognizing bone TRAP and were not useful in the assay. 5C1, while having a good affinity for the bone enzyme, was not specific. Serum TRAP is relatively stable, because 7 days of storage of serum samples at 4 degreesC and -20 degreesC or five thawing-freezing cycles, did not change the TRAP concentration detected using the two-site assays. All studied assays detected an increase in serum TRAP concentrations of postmenopausal women compared with premenopausal women, the difference being highest with MAB pairs 2H1-5C1 and O1A-J1B. These results suggest that serum TRAP may be a useful bone resorption marker, and the MAB pairs 2H1-5C1 and O1A-J1B may be useful in determining the bone resorption rate.

Epitope mapping of nine monoclonal antibodies against osteocalcin: combinations into two-site assays affect both assay specificity and sample stability.

Nine monoclonal antibodies (Mabs) were raised against human recombinant osteocalcin fusion protein (rGST-hOC) or bovine osteocalcin (bOC) and selected to develop two-site immunoassays for human osteocalcin (hOC). The detection system was based on the time-resolved measurement of the fluorescence of europium chelates conjugated to the tracer Mabs. Based on the ability of the Mabs to recognize different forms of hOC (carboxypeptidase Y-digested, alkylated hOC, thermally decarboxylated hOC, recombinant forms of hOC, and tryptic peptides derived from hOC) and the information obtained from combinations of the Mabs in two-site assays, an epitope map was created. The epitope map was useful in understanding the behavior of the two-site combinations of the Mabs with serum samples. The two-site combinations could be divided into subgroups detecting either full-length hOC or full length+large NH2-terminal fragment as stimulated by the carboxypeptidase Y-digested form of hOC (it lacks four COOH-terminal residues), which with intact specific assays showed cross-reactivities ranging from 7 to 14% when compared with full-length hOC. In addition, differences were observed in the ability of the combinations to detect thermally decarboxylated hOC (lacks gamma-carboxylation at residues 17, 21, and 24) with cross-reactivities ranging from 8 to 85% when compared to gamma-carboxylated hOC. The analysis of human serum samples showed considerable differences in the concentration and stability of serum OC. This was attributed as the varying ability of the Mabs to detect different proteolytic fragments derived from hOC and/or differences in the degree of gamma-carboxylation of hOC. The in vitro generation of the large NH2-terminal fragment during incubation of the serum samples at room temperature (RT) and during prolonged storage at -20 degrees C in an undercooled state was detectable as loss of immunoreactivity (ranging from -42 +/- 17 to -50 +/- 15% in 16 h at RT, n = 3) with Mab combinations detecting only full-length hOC. Two-site combinations detecting full-length+large NH2-terminal fragment showed no loss of immunoreactivity after incubation of the serum samples at RT for 16 h. With all assays, an increase of serum OC ranging from 16 to 38% was found in postmenopausal samples (n = 24) when compared with premenopausal samples (n = 17), but the degree of statistical significance varied from not significant to p < 0.01.