Perinatal changes in serum mannose-binding protein (MBP) levels.

Changes in serum levels of mannose-binding protein (MBP) from day 0 through day 5 after birth were analyzed for normal newborn infants. A considerable amount of MBP (about 1 microgram/ml) was already present in serum at birth. This level started to elevate from day 2 and attained the highest level in a human lifetime (about 2.5 micrograms/ml) within 5 days after birth. Results indicate that MBP can be produced at birth in response to environmental changes.

Influence of spironolactone on neonatal screening for congenital adrenal hyperplasia.

AIM: To determine if the diuretic spironolactone cross reacts with 17alpha-hydroxyprogesterone (17OHP) in an enzyme linked immunosorbent assay (ELISA) kit used for the mass screening of congenital adrenal hyperplasia. METHODS: Concentrations of 17OHP on a blood filter paper disc were measured using an ELISA kit (kit C-7: ENZAPLATE N-17alpha -OHP-7; Chiron, Tokyo, Japan). The cross reactivity of spironolactone and its metabolites with 17OHP was determined. The concentrations of spironolactone and its metabolites in blood were measured using HPLC (high performance liquid chromatography). RESULTS: Spironolactone cross reacted with 17OHP using kit C-7 (0.01%), by increasing 17OHP concentration in a dose dependent manner. The blood concentration of spironolactone and its metabolites was nearly 900 ng/ml, high enough to show an additive effect on the 17OHP concentration. About 12% of the false positive cases screened using the kit were due to the administration of spironolactone. CONCLUSIONS: Spironolactone interferes with 17OHP concentrations, leading to false positive test results for CAH.

A sero-epidemiological survey of gelatin sensitization in young Japanese children during the 1979-1996 period.

Recently, an increasing number of adverse reactions in children inoculated with live attenuated virus vaccines containing gelatin have been reported. However, the distribution, magnitude and rate of gelatin sensitization in the Japanese population have not been established. Here, the purpose was to investigate the distribution of children with positive gelatin immunoglobulin G (IgG) and/or IgE in Japan and to ascertain whether the incidence of positive antigelatin antibody cases among the general population, as reflected in the sample employed here, has been increasing during the period in question. The presence of IgE and IgG antibodies were measured against gelatin in 1600 panel sera collected from different age groups of Japanese children in Hokkaido/Sapporo from 1979 through 1996. Among the subjects, 39 had gelatin IgG- and/or IgE-positive sera, and these were correlated with the time of obtaining the sera as well as with the age of the subjects. The older the subject and the later the period, the higher the sero-incidence. Japanese children have become increasingly sensitized to gelatin, especially since the mid-1990s.

Human serum mannose binding protein (MBP): development of an enzyme-linked immunosorbent assay (ELISA) and determination of levels in serum from 1085 normal Japanese and in some body fluids.

We developed an enzyme-linked immunosorbent assay (ELISA) for human serum mannose binding protein (MBP), an animal lectin known to function as opsonin and complement activator. A total of 1085 normal Japanese sera from individuals ranging in age from 3 to 100 years old and additional body fluids were measured for the MBP level. Individual serum MBP levels ranged from 0.07 to 6.40 micrograms/ml with a frequency distribution profile of the log-normal type. Arithmetic mean +/- standard deviation (M +/- SD) of serum MBP as a whole was 1.72 +/- 1.15 micrograms/ml, whereas those of cerebrospinal fluid and urine were 1/100 and 1/1000 of serum levels, respectively. When the M +/- SD of serum MBP was calculated for each age group (10-year intervals) and compared, the value declined from age group of 3-9 years old (2.42 +/- 1.31 micrograms/ml) to that of 20-29 years old (1.72 +/- 1.03 micrograms/ml), and thereafter it remained almost constant. Comparing the frequency distribution profile among each group, that of young group revealed a distinct bimodal pattern, one peak at around 1 micrograms/ml and the other at 3 micrograms/ml. The latter peak was gradually depressed with advance of age and finally a typical log-normal type appeared after the age of 30. There was a high age dependency in serum MBP level as well as in distribution profile. It should be stressed that care should be taken with age in the evaluation of MBP level.

Human serum mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1): determination of levels in body fluids and identification of two forms in serum.

We developed an ELISA for human serum MASP-1, a C1s-like serine protease which is known to function in C4 and C2 activation. We then determined MASP-1 levels in 1063 sera from normal Japanese subjects ranging in age from 3 to 100 years, as well as in certain body fluids using this assay. Individual serum MASP-1 levels ranged from 1.48 to 12.83 microg/ml, with a normal frequency distribution pattern. The arithmetic mean +/- s.d. of MASP-1 levels in serum was 6.27 +/- 1.85 microg/ml, whereas levels of MASP-1 in cerebrospinal fluid and in urine were almost undetectable. When the mean +/- s.d. of serum MASP-1 was calculated for each age group (10 year range) and values were then compared, the age group consisting of 3-9-year-olds (7.54 +/- 1.39 microg/ml) was found to have the highest value. When MASP-1 was measured in cord blood, it was shown that levels were already as high as those of 3-9-year-olds. The serum MASP-1 level was found to be as strongly dependent on age as is the serum MBL level. MASP-1 and MBL are thought to play an active part in immunity in younger people. It was found that the serum level of MASP-1 was much higher than that of MBL, and the major portion of human serum MASP-1 appeared to exist in the circulation as a form unbound to MBL.

alpha 2-Macroglobulin binds to and inhibits mannose-binding protein-associated serine protease.

We determined the presence in human serum of a complex consisting of mannose-binding protein (MBP), MBP-associated serine protease (MASP), which is a C1s-like protein with complement activation activity, and alpha 2-macroglobulin (alpha 2M). Binding between these three molecules was in an ascending order of MBP, MASP and alpha 2M, in that alpha 2M bound directly to MASP, possibly through covalent bonds, whereas the binding between MBP and MASP was reversible and Ca(2+)-dependent. Since it was found that alpha 2M can inhibit complement activation by MASP and that MASP in the complex lacks esterolytic activity, it is conceivable that alpha 2M plays a regulatory role in MBP-derived complement activation via a mechanism involving MASP (the lectin pathway).

Differences in titres of IgE, IgG4 and other IgG subclass anti-Der p 2 antibodies in allergic and non-allergic patients measured with recombinant allergen.

BACKGROUND: The mite allergens are recognized as major causes of allergic disease such as bronchial asthma, allergic rhinitis and atopic dermatitis. The functions of allergen-specific IgG subclass antibodies are not defined. OBJECTIVE: In order to clarify the relationship between IgE and IgG subclasses, we examined serum levels of the Dermatophagoides pteronyssinus group 2 (Der p 2)-specific antibodies of IgE, IgG total and IgG subclasses in children with mite allergy. METHODS: We prepared a recombinant Der p 2 fusion protein and examined serum levels of Der p 2 antigen-specific antibodies by enzyme-linked immunosorbent assay (ELISA) systems developed in our laboratory using a recombinant Der p 2 as target antigen. Sera from 240 children with mite allergy and 25 controls were measured. RESULTS: The serum levels of specific IgE and, to lesser degree, IgG4 were higher in allergic children than non-allergic controls, while in the levels of the other IgG subclasses there was no difference between the two groups. There was no correlation between levels of specific IgE and IgG4 or in those between specific IgG4 and other IgG subclasses. CONCLUSION: Results indicate that the induction of Der p 2-specific IgG4 in allergic diseases is independent to IgE as well as other IgG subclasses.

Spontaneous porphyria of the Long-evans cinnamon rat: an animal model of Wilson's disease.

To confirm and extend our previous microspectrophotometric observations of 30-week-old male Long-Evans Cinnamon (LEC) rats, an animal model of human Wilson's disease, we analyzed the porphyrin patterns of the organs, urine, and plasma of LEC rats. Abnormal accumulation of porphyrins, especially highly carboxylated porphyrins (uro- and heptaporphyrin), in the kidneys and liver was seen in male and female LEC rats aged 30 weeks and also in 10-week-old rats, before the onset of spontaneous hepatic dysfunction. Accumulation of copper and iron in the kidneys was not observed in the 10-week-old rats. Massive accumulation of porphyrins was observed only in the kidneys of the 30-week-old male LEC rat, indicating that this symptom is related to sex and age. Renal accumulation of porphyrins was reflected in the rate of urinary porphyrin excretion. Hepatic accumulation of porphyrins appeared to be independent of sex and age. These results indicate that neither renal nor hepatic porphyrin accumulation is the result of renal deposition of metals or of spontaneous hepatic dysfunction and that porphyrinuria in the LEC rat is closely related to the renal accumulation of porphyrins. In contrast to these organs, a reduction in the porphyrin levels was observed in the brain of the LEC rat. This was independent of sex and age. The present work stresses the existence of an abnormal heme metabolism in the LEC rat, and thus, the necessity to study the heme metabolism in human Wilson's disease is strongly suggested.

Proteolytic activities of two types of mannose-binding lectin-associated serine protease.

Mannose (or mannan)-binding lectin (MBL) is an oligomeric serum lectin that plays a role in innate immunity by activating the complement system. In human, two types of MBL-associated serine protease (MASP-1 and MASP-2) and a truncated protein of MASP-2 (small MBL-associated protein; sMAP or MAp19) are complexed with MBL. To clarify the proteolytic activities of MASP-1 and MASP-2 against C4, C2, and C3, we isolated these two types of MASP in activated forms from human serum by sequential affinity chromatography. On an anti-MASP-1 column, MASP-2 passed through the column in the presence of EDTA and high salt concentration, whereas MASP-1 was retained. Isolated MASP-1 and MASP-2 exhibited proteolytic activities against C3 and C4, respectively. C2 was activated by both MASPs. C1 inhibitor (C1 INH), an inhibitor for C1r and C1s, formed equimolar complexes with MASP-1 and MASP-2 and inhibited their proteolytic activities.

Single nucleotide polymorphisms of the mannose-binding lectin are associated with susceptibility to primary biliary cirrhosis.

Although the immunopathogenesis of primary biliary cirrhosis (PBC) remains unknown, familial clustering of patients with PBC suggests an important role for genetic factors. In addition, recent data support the thesis that the mucosal immune response against intraluminal pathogens may be involved with the onset of PBC. Mannose-binding lectin (MBL) is a key factor in innate mucosal defenses and has several key single nucleotide polymorphisms (SNPs). To study whether MBL gene SNPs are associated with susceptibility to PBC, we studied 65 patients with PBC and 218 controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequence specific priming-polymerase chain reaction (SSP-PCR) to examine four polymorphic loci: two (H/L and X/Y) within the promoter region and the other two (P/Q and A/B) within exon-1. We also analyzed serum MBL concentrations. Interestingly, the prevalence of haplotype HYPA, leading to hyper-production of MBL, as well as HYPA/HYPA genotype were significantly increased in PBC compared to controls (0.53 vs. 0.44, P=0.031; 33.9%vs. 17.0%, P=0.003, respectively). Furthermore, individuals homozygous for HYPA had a significantly increased risk for PBC (odds ratio (OR)=2.51, 95% confidence interval (CI)=1.34-4.66). Our results demonstrate that the MBL genotype can be significantly associated with increased risk for PBC, and further, that increased production of MBL plays a critical role in immunopathogenesis.