Normal Adult Serum Research Articles

Development, validation and application of a two-site enzyme-linked immunosorbent assay for activin-AB.

Monoclonal antibodies, specific for the beta A and beta B subunits of activin, were used to develop a new two-site ELISA for activin-AB. The assay had a detection limit of 0.19 ng/ml. High concentrations of activin-AB were found in bovine, ovine and porcine follicular fluids (FF), with less in human FF (1310, 1730, 688 and 7 ng/ml respectively). Recovery of spiked activin-AB standard from human, bovine and ovine FFs and from homogenized human placental extracts averaged 91%, 115%, 115% and 94% respectively. Within-plate coefficients of variation for different concentration of activin-AB were between 1.3% and 2.67%. The between-plate coefficient of variation was 5.5%. Cross-reactivity experiments showed the high specificity of the assay for activin-AB, with inhibin-A, inhibin-B, follistatin, activin-A and activin-B all cross-reacting < 0.2%. Incubation with high concentrations of follistatin (500 ng/ml) prior to assay did not affect the recovery of activin-AB. Samples of bovine, porcine, ovine and human FF gave dose responses parallel to that of the standard, as did bovine granulosa cell-conditioned media. In human and porcine FF, levels of activin-A and activin-AB were similar whereas, in bovine and ovine FF, activin-A levels were approximately threefold higher than activin-A, nearly all of the endogenous activin-AB in bovine FF was detected in the eluate from gel permeation chromatography with an M(r) of > 700000 indicating its association with higher molecular weight binding protein(s). By contrast, after denaturation, immunoreactive activin-AB was detected with an M(r) of approximately 25000 consistent with the complete dissociation from binding proteins. Activin-A was detected in relatively high concentrations in human FF (approximately 5 ng/ml), homogenized placental extracts (4.35-95.5 ng/g), sera from pregnant women (> 4 ng/ml) and amniotic fluid (3-13 ng/ml), and in much lower concentrations in postmenopausal serum (500 pg/ ml), normal cycle serum (100-200 pg/ml), serum from gonadotrophin-treated women (200 pg/ml), and normal adult male serum (225 pg/ml). Activin-A was also found in the culture media from explants of human amnion, chorion, maternal decidua and placenta. In marked contrast, activin-AB was undetectable (< 0.19 ng/ml) in all of these samples with the exception of human FF (approximately 7 ng/ml). In conclusion, we have developed a sensitive and specific ELISA to measure total (bound+free) activin-AB. Preliminary results show a more restricted distribution of this isoform compared with activin-A. The presence of high levels of both activin-A and activin-AB in FF suggests a function for both isoforms in the developing ovarian follicle.

Determination of free follistatin levels in sera of normal subjects and patients with various diseases.

We developed an assay system for measuring free follistatin by using an anti-follistatin mouse monoclonal antibody and [125I]activin A. The sensitivity of this assay was 0.5 microgram/l and cross-reactivities with inhibin, luteinizing hormone, follicle-stimulating hormone and growth hormone were all less than 0.5%. The dose-response curves of human sera and follicular fluid were parallel to the standard curve, and the follicular fluid contained a large amount of follistatin (6.4 +/- 0.5 mg/l, mean +/- SEM; N = 13). The within- and between-assay coefficients of variation calculated from the analysis of serum samples of four different concentrations were 3.3-7.8% and 3.9-11.0%, respectively. The recovery rates of free follistatin at five different doses were 86.4 - 102.4%. When activin A was added to the same sample, free follistatin recovery rate declined dose-dependently. Gel filtration analyses of human serum and follicular fluid resulted in a single peak corresponding to authentic follistatin. Using this assay, free follistatin concentrations in sera were measured in normal, pregnant and diseased subjects. The free follistatin level in serum of normal adults was 3.5 +/- 0.2 micrograms/l (N = 60), which was significantly elevated in pregnant women (16.7 +/- 1.3 micrograms/l, N = 56), and in patients with chronic liver disease (8.1 +/- 1.1 micrograms/l, N = 20), chronic renal failure (6.7 +/- 0.9 micrograms/l, N = 42), advanced solid cancer (8.5 +/- 1.0 micrograms/l, N = 39) and hematological malignancies (6.8 +/- 1.0 micrograms/l, N = 18). These data indicated that the free follistatin concentration in serum is detectable and varies during pregnancy and in various diseased states.

A new form of polyagglutination related to Cad

Abstract Four phenotypes of Cad (Cad 1–4) have been characterized by a continuum of polyagglutinability and reactivity with lectins, with the strongest Cad+ red blood cells (RBCs) being polyagglutinable because of the presence of anti-Cad (anti-sda) in most normal sera. Over a period of 7 years, a French male blood donor’s RBCs demonstrated polyagglutinability with 50 percent to 70 percent of normal adult sera. The reactivity was characteristic of anti-sda (refractile agglutination at 4°C, 20°C, 37°C, and anti-human globulin test), and was inhibitable by two examples of Sd(a+) urine, but not by Sd(a–) urine or dialysate from Sd(a+) urine. The donor’s RBCs reacted 1+ with Glycine max, but did not react with Dolichos biflorus, Leonurus cardiaca, Salvia horminum, or Arachis hypogaea. The first four of these lectins were reactive with five of five Cad+ RBCs, including one example of Cad 4 RBCs. Polybrene® aggregated the donor RBCs. Dilutions of nine samples of anti-Sda reacted more strongly with the donor RBCs than with normal RBCs. Even though lectin studies failed to classify this donor’s RBCs as Cad, the persistent polyagglutinability and serologic characteristics are consistent with Cad and demonstrate the heterogeneity of this antigen. Immunohematology 1996;12:69–71.

Differential removal of insulin-like growth factor binding proteins in rat serum by solvent extraction procedures

Solvent extraction of serum and other biological fluids at an acidic pH is a convenient method to remove the insulin-like growth factor binding proteins (IGFBPs); however, an incomplete removal of IGFBPs can occur and this can potentially interfere with the radioimmunoassay of insulin-like growth factors (IGFs). This study compared the removal of IGFBPs from normal adult rat serum and 5-day old neonatal rat serum by acid-gel filtration, and three solvent extraction methods, i.e., acid-ethanol (AE), acid-cryo-ethanol (ACE) and formic acid-acetone (FAA) treatments by western ligand blotting and slot-blotting analysis. In adult rat serum all three extraction methods removed nearly 75% of total IGFBPs present. For the neonatal serum, AE and FAA were very inefficient in eliminating the IGFBPs, while ACE was somewhat better, as it removed nearly 30% of IGFBPs. Ligand blots of extracted samples showed that IGFBPs of lower size range, 24 to 32 kDa (IGFBP-4, IGFBPs-1 and -2), were resistant to solvent extraction. Acid-gel filtration, in contrast, eliminated > 95% of IGF-binding components in both sera. Determination of IGF-I concentrations in samples after gel filtration and extraction methods revealed lower IGF-I values in neonatal serum in acid extracted samples. These data caution against using solvent extractions for IGFBP removal in fetal/neonatal serum.

Serum components enhance bacterial lipopolysaccharide-induced tissue factor expression and tumor necrosis factor-alpha secretion by bovine alveolar macrophages in vitro.

We have compared the effect of bacterial lipopolysaccharide (LPS) in combination with normal adult bovine serum (NBS), fetal bovine serum (FBS), or a bovine serum fraction on tissue factor expression and tumor necrosis factor alpha (TNF-alpha) secretion by bovine alveolar macrophages. At a concentration of 1 ng/ml, bacterial LPS alone failed to induce measurable tissue factor expression by the macrophages, but the presence of FBS, NBS, or a fraction of normal pooled bovine serum isolated by ion-exchange chromatography (fraction 2) markedly potentiated the effect of LPS. A protein concentration of 64 micrograms/ml NBS, 192 micrograms/ml FBS, and only 640 ng/ml fraction 2 was required to induce maximal tissue factor expression on the macrophages in combination with 1 ng/ml LPS. Comparison of quantities of added serum protein required to induce maximal potentiating effects indicated that fraction 2 was 100 times more potent than whole NBS and 300 times more potent than whole FBS. We similarly found that TNF-alpha secretion by macrophages exposed to LPS was responsive to serum and was highly responsive to fraction 2. LPS alone (1 ng/ml) induced a relatively low level of TNF-alpha secretion by the macrophages, and the presence of FBS, NBS, or fraction 2 potentiated the effect of LPS. A concentration of 64.0 micrograms/ml NBS, 320.0 micrograms/ml FBS, and 3.2 micrograms/ml fraction 2 serum protein induced near-maximal TNF-alpha secretion by the macrophages. Comparison of the concentration of serum protein required to induce these potentiating effects indicated that fraction 2 was approximately 20 times more potent than whole NBS and 100 times more potent than whole FBS. The stimulatory effect of LPS plus fraction 2 serum proteins was dependent on the CD14 receptor, as monoclonal antibodies directed against CD14 (My4, 60bd; 10 micrograms/ml) inhibited tissue factor expression and TNF-alpha secretion by the macrophages.

MACROPHAGE CANDIDACIDAL ACTIVITY IN HUMAN NEONATES: DECREASED SENSNIVITY TO MODULATION BY IFN-γ

In an attempt to understand the mechanisms of host defense in the term neonate against candida, we studied interaction between Calbicans and monocyte-derived macrophages (MDM) cultured for 5 days from the cord blood. In the presence of 25% normal adult serum, the extent of phagocutosis of candida was equivalent with cord or adult MDM. In contrast to candida, MDM did not phagocytose non-opsonized group B Streptococcus type III, Mannosylated BSA (Man BSA, molar ratio of mannose to BSA = 23:1) inhibited non-opsonic ingestion of candida by cord and adult MDM 74 and 87 inhibition by 64 and 320 pg mannose/ml, respectively, after 60 min incubation means, n=6), suggesting a role for the mannose receptor. Cord MDM killed both opsonized and unopsonized Calbicans as effectively as did adult MDM (n=7) However, exposure of adult and cord MDM to IFN-γ (10-500 U/ml) gave quantitativel different results in candida killing and O release maximal increase in these functions with adult MDM were achueved with 100 U/ml IFN-γ, whereas no enhancement with cord MDM could be detected after treatment with 100 Uml, and at 500 Uml there was still a significantly lower killing and O. release by and MDM compared to adult cells. These data suggest that: 1. clearance of candida both the blood stream may occur in the absence of full opsonization a condition that might exist in the newborn; 2 resistance of cord MDM to the effect of IFN-γ may represent a developmental immaturity of human Phagocytes.

Delta‐5‐androstenediol and its sulphate in serum and urine of normal adults and patients with endocrine diseases

We evaluated the role of delta-5-androstenediol (adiol) and its sulphates in health and endocrine diseases. Serum and urine samples from healthy adult men and pre and post-menopausal women were analysed by gas chromatography-mass spectrometry to establish reference values. In patients who were either evaluated or treated for endocrine diseases, sequential serum samples were collected and analysed. Reference values were obtained from 24 healthy male, 23 premenopausal and 30 post-menopausal female volunteers. Adiol and delta-5-androstenediol-3-sulphate (adiol-3S) concentrations were determined in combination with other relevant steroids in patients with either pituitary (n = 5), adrenal (n = 2) or gonadal dysfunction (n = 1), or testicular carcinoma (n = 19). After addition of deuterated adiol as internal standard, serum and urine samples were extracted. Steroid sulphates were hydrolysed. The extracts and hydrolysates were purified on HPLC, adiol was derivatized and finally quantified by gas chromatography-mass spectrometry. The calculated reference ranges for adiol and adiol-3S concentrations in serum are respectively: in men 1.78-7.24 and 123-579 nmol/l, in premenopausal women 0.65-6.93 and 21.2-298 nmol/l and in post-menopausal women 0.29-2.90 and 6.1-184 nmol/l. Urinary values varied considerably. In the population with endocrine abnormalities serum adiol and adiol-3S concentrations were compared with other relevant steroids. The wide concentration range of adiol and adiol-3S in urine makes analysis of these steroids in urine of little clinical value. Serum concentrations of adiol and adiol-3S are higher in men than in women. Premenopausal values are higher than post-menopausal. Adiol and adiol-3S in serum are significantly correlated in pre and post-menopausal women, r = 0.51 and r = 0.69 respectively, but not in men. In endocrine patients the serum concentrations of adiol show an ACTH or LH dependency in women; adiol correlates with cortisol, dehydroepiandrosterone or androstenedione and, in males, additionally with testosterone. However, in several situations adiol correlates with none of these steroids. Although adiol secretion can be stimulated by ACTH and LH, the level of serum adiol is also determined by other factors. Finally, in adrenal carcinoma serum adiol and adiol-3S may be used as tumour markers.

Effect of extracorporeal membrane oxygenation on neutrophil function in neonates.

To study the effect of long-term extracorporeal membrane oxygenation (ECMO) support on neutrophil function. A prospective, clinical investigation. A pediatric intensive care unit. Four groups of patients: ECMO group 1, newborns after 1 day of ECMO support (n = 10); ECMO group 2, newborns after 5 days of ECMO support (n = 6); group 3, normal newborns (n = 20); group 4, normal adults (n = 30). Two mL of heparinized blood was obtained from patients in each group. A modification of the Smith and Rommel technique was used to measure neutrophil phagocytosis and killing utilizing live Candida tropicalis as the test organism. Neutrophils were incubated for 90 mins in normal adult serum with live Candida. Viability of Candida after phagocytosis was tested by vital fluorochrome staining. Phagocytic index (the number of neutrophils with intracytoplasmic Candida divided by the total neutrophils) and candidicidal ratio (neutrophils with dead Candida divided by total neutrophils with Candida) were determined daily. Neutrophils from ECMO group 1 (day 1) and ECMO group 2 (day 5) patients had significantly higher phagocytosis indices (72.8 +/- 20 and 76 +/- 18) and candidicidal ratios (0.15 +/- 0.1 and 0.16 +/- 0.09) compared with neutrophils from group 3 patients (normal neonates) (64 +/- 7 and 0.06 +/- 0.04). The phagocytosis indices were significantly lower in neutrophils from ECMO group 1 (day 1) and ECMO group 2 (day 5) patients compared with group 4 (adults) patients (86 +/- 9). However, the candidicidal ratios in neutrophils from ECMO groups 1 and 2 (ECMO day 1 and day 5) patients were equal to that value in group 4 (adults) (0.10 +/- 0.04). ECMO support for 5 days (ECMO group 2 vs. group 1) did not significantly change either the phagocytosis index or candidicidal ratio. Phagocytosis and intracellular killing by neutrophils of ECMO-supported neonates were significantly greater than those values found in normal newborns. ECMO support for 5 days produced no significant changes in neutrophil phagocytosis or killing.

Methodological Aspects on Separation and Reaction Conditions of Bone and Liver Alkaline Phosphatase Isoform Analysis by High-Performance Liquid Chromatography

Some methodological aspects and characteristics on analysis of bone and liver alkaline phosphatase (EC 3.1.3.1, ALP) isoforms by high-performance liquid chromatography (HPLC) methods are presented. Factors affecting separation (analytical column type, column temperature, mobile phase buffer, mobile phase salt, gradient elution, flow rate) and reaction conditions (substrate type, substrate concentration, fluorescent substrates, substrate buffer type, substrate buffer concentration, substrate pH, substrate activators, substrate detergent, substrate flow rate, reaction temperature, postcolumn reaction detection) are discussed. Six peaks with ALP activity were separated and quantified by our new HPLC method in normal adult nonplacental serum: one intestinal/bone, two bone, and three liver ALP isoforms. Differences between the six ALP iso-forms with respect to diethanolamine buffer concentration and substrate pH were observed. Although our HPLC method offers an improved possibility to clarify the reason for an increased total ALP in the routine clinical chemistry laboratory further research is needed to clarify the cellular origins of the different bone and liver ALP isoforms.

Iron(III)-binding polypeptide in human cord and adult serum: Isolation, purification and partial characterization

A low-molecular-weight, non-transferrin-bound, Fe(III)-binding polypeptide has been isolated and purified from normal human cord and adult sera by gel-filtration and high-performance liquid chromatography. The polypeptide was traced isotopically with 59Fe(III)-chloride and high voltage paper electrophoresis. This Fe(III)-binding polypeptide has been partially characterized, and found to be biochemically distinct from other known Fe(III)-binding proteins, such as transferrin, lactoferrin and ferritin. Furthermore, the electrophoretic mobility and amino-acid composition distinguish the polypeptide from other physiological iron chelators such as a previously described Fe(III)-citrate complex. The polypeptide is highly hydrophilic, rich in lysine residues and phosphorylated. The observed positive charge of the polypeptide is suggested to originate from these lysine residues. The molecular mass of this polypeptide is estimated to be approx. 2500 Da, which is in close agreement with previous reports and is consistent with amino-acid analysis data. Cord serum levels of the polypeptide were significantly higher than adult serum levels. The degree of Fe(III) specificity and the function of the Fe(III)-polypeptide have not been ascertained at the present time. It is possible that the polypeptide may complete for Fe(III) with transferrin and is involved in the mobilization and transportation of iron by some as yet unknown mechanism.

Platelets contribute to circulating levels of bone sialoprotein in human.

Bone sialoprotein (BSP) is a major bone-related protein. Although a few other tissues contain trace amounts of BSP message, bone cells and bone matrix are the major sources of BSP, suggesting that this protein could be a potential marker of bone metabolism. Purified bovine BSP showed a 70% homology of its first 13 amino acid N-terminal sequence with human BSP and was used to raise antibodies in rabbit and to develop a specific radioimmunoassay (RIA). Using this RIA, we have shown that BSP is present in serum with values in the range of 10-30 ngEq/ml in the serum of normal adults. Values obtained in plasma prepared without platelet activation are about one-half of those in matched sera, suggesting that BSP present in serum is in part derived from platelets during the activation process. Using Western blot and RIA techniques, we confirmed that platelets contain immunoreactive BSP and that the protein is released after thrombin stimulation of these cells. In addition to BSP, platelets contain a 45 kD immunoreactive material that has not been precisely identified. Available evidence indicates that this material is not osteonectin or osteopontin and that it may be a BSP-like protein rather than a degradation product of BSP. Platelets from a patient having a gray platelet syndrome, characterized by a deficiency in platelet alpha-granules and in the alpha-granule secretory proteins, did not show any deficiency of BSP, suggesting that immunoreactive BSP present in platelets is not endogenously synthesized by megakaryocytes but rather originates from plasma by endocytosis.

High-performance liquid chromatographic determination of bilirubin distribution on serum proteins

We describe a high-performance liquid Chromatographic method for the determination of bilirubin distribution on serum proteins. The procedure is based on both size-exclusion and adsorptive properties of the column packing material LiChrosorb Diol. The elution of all sample constituents is accomplished by means of an aqueous mobile phase containing phosphate buffered solution of adult serum, supplemented by a small amount of bilirubin. The different distribution of bilirubin on albumin, β-lipoprotein, and as free (loosely bound) pigment fraction is exemplified by assaying normal adult and infant sera.

Mobiluncus species in bacterial vaginosis: aspects of pathogenesis

Mobiluncus is an anaerobic motile rod associated with bacterial vaginosis. In this work, luminol-enhanced chemiluminescence was used to study the ability of Mobiluncus spp. from the vaginas of women with bacterial vaginosis to induce, in the presence of normal adult serum, oxidative metabolism of polymorphonuclear leukocytes, which is an indirect measure of phagocytic activity. M. curtisii induced a significantly (p less than 0.05) lower response than M. mulieris, which indicates that M. curtisii escapes phagocytosis more easily. Indirect immunofluorescence assays showed IgG antibodies to M. curtisii at significantly (p less than 0.01) higher titres than to M. mulieris in women with bacterial vaginosis. The titres were higher in patients with bacterial vaginosis than in women without vaginosis and healthy men. No antibodies to Mobiluncus spp. of secretory IgA type were found in vaginal washings. These results indicate that M. curtisii is a more virulent species than M. mulieris, and agree with reports of M. curtisii found in postoperative and extragenital infections.

Identification of a low-affinity subset of protamine-reactive IgM antibodies present in normal, deficient in AIDS, sera: Implications for HIV latency

We demonstrate here that the protamine-reactive IgM antibodies previously shown to be present in normal adult sera include two subsets differing in binding affinity. The principal, high-affinity subset was detected in AIDS and ARC as well as normal sera. The secondary, low-affinity subset, however, was absent or markedly deficient in AIDS or ARC sera. Protamine-reactive IgM antibodies were also detected in normal pediatric sera, suggesting that one subset of that class of antibodies may be “natural”, i.e., not antigenically induced. The proportionate titer of the low-affinity protamine-reactive IgM antibodies was determined for HIV-positive males who were asymptomatic or mildly immune deficient at specimen collection. Of those who subsequently remained AIDS free for 18 months to 7 years, more than 90% had titers in the range established for the normal sera, while of those diagnosed with AIDS or ARC within 12 months, more than 80% had titers below the normal range. We propose that the low-affinity subset of adult sera corresponds to the natural antibodies of pediatric sera and that a relationship of those natural antibodies to resistance to progression of HIV pathogenesis is suggested.

An enzyme-linked immunosorbent assay for the determination of human IgG subclass antibodies directed against Branhamella catarrhalis

An ELISA procedure to determine the distribution of human IgG subclass antibodies directed against the gram-negative bacterium Branhamella catarrhalis has been developed using commercially available monoclonal anti-IgG subclass antibodies. Using whole bacteria as coating antigen the specificity of the assay was determined and showed minimal cross-reactivity with a range of other bacteria. Estimations of IgG1, IgG2, IgG3, IgG4 and total IgG antibodies directed against this antigen were performed. All normal adult sera tested had measurable antibody levels of specific IgG1, IgG2, IgG3 and total IgG. Specific IgG4 was undetectable in the majority of adult sera. These assays will be of value for investigation of both children and adults with suspected immunodeficiency and recurrent upper respiratory tract infection.

Increase in serum concentration of keratan sulfate after treatment of growth hormone deficiency with growth hormone

Keratan sulfate is a glycosaminoglycan; in man, most KS is present in cartilage proteoglycans, 1 but small amounts have been identified in blood.l, 2 Recent evidence indicates that this KS is almost exclusively derived from catabolism of cartilage proteoglycans.l-3 As a result, it has been postulated that the concentration of KS in the blood is directly proportional to the rate of degradation of cartilage proteoglycans.I, 2 Linear growth results from bone formation on a cartilage scaffold (endochondral ossification) and depends on adequate function of the hypothalamic-pituitary-somatomedin-insulin-like growth factor I axis and the genetic ability of cartilage to respond to these secretions. 4, 5 In the growing child, serum levels of KS are elevated, which is not unexpected because growing cartilages are constantly undergoing degradation and replacement by new bone formation. We previously reported that serum levels of keratan sulfate rise from low levels in infancy and reach a plateau by the age of 4 to 5 years. Serum concentrations of KS appear to remain high until age 12 years, when they rapidly decline toward the levels found in normal adult sera. Serum levels of KS in a growing child correlate with the individual child's age-adjusted percentile height. 6 In our investigation, we measured the serum concentrations of KS in two groups of children with short stature: one group with Constitutional delay of maturity and the other with growth hormone deficiency. In the latter group, we compared KS levels before and after treatment with growth

Multiple Osteocalcin Fragments in Human Urine and Serum as Detected by a Midmolecule Osteocalcin Radioimmunoassay*

Reliable markers of bone formation are essential to the investigation of metabolic bone disorders. In this regard, evidence indicates that circulating levels of human osteocalcin (OC) correlate with the skeletal isoenzyme of alkaline phosphatase and can be used as an index of bone formation. A disadvantage of using serum OC as a marker of formation is its diurnal variation. To address this problem we carried out our studies to determine the usefulness of urine in the assessment of bone turnover. Using a midmolecule specific human OC RIA, we were able to detect OC in urine of normal adults (42 mugeq/g creatinine), normal children (849 mu/geq/g creatinine), and Paget's disease patients (613 mugeq/g creatinine). Immunoreactive fragments of OC in human urine and human serum were separated by high pressure liquid chromatography. Multiple fragments were found in normal adult urine that were not detected in normal adult serum. Uremic and Paget's disease sera contain several immunoreactive forms of OC, other than the intact molecule, not found in normal adult serum. Additionally, both Paget's disease sera and urine contained a specific peak of immunoreactive material, eluting at 25% acetonitrile, that was not found in any other serum or urine tested. Urinary OC (uOC) correlated with both skeletal alkaline phosphatase (r = 0.91) and serum OC (r = 0.83), indices of skeletal formation. While uOC has a diurnal variation similar to that of serum OC, determinations of 24-h uOC give integrated values of daily bone turnover rates. Z-Score analysis indicates that uOC (z = 14.04) is better able to distinguish between normal children with high bone turnover and normal adults than either skeletal alkaline phosphatase (z = 8.87) or serum OC (z = 9.01).

An Immunoassay to Detect Human Mullerian Inhibiting Substance in Males and Females during Normal Development*

An enzyme-linked immunosorbent assay has been developed to measure human Müllerian inhibiting substance (MIS) in biological fluids. The enzyme-linked immunosorbent assay is specific for MIS, with a sensitivity in human serum to 0.5 ng/ml and does not recognize transforming growth factor-beta 1 or -beta 2, LH, or FSH. It similarly fails to recognize other proteins secreted from the cell type into which the MIS gene was cloned. MIS was detected in the serum of normal newborns, infants, children, and adults. In males the serum level of MIS is 10-70 ng/mL at birth. The level increases slightly after birth, and then decreases to a basal level of 2-5 ng/mL after the first 10 yr of life. Newborn male urine contains minimal amounts of MIS (0.5 ng/mL). In females MIS is barely detectable in serum at birth, but rises to the basal level equal to that seen in males after 10 yr of age. Similar basal levels of MIS were found in adult ovarian follicular fluid. MIS levels were high in the serum of a female patient with a sex cord tumor (3200 ng/mL), but fell to 100 ng/mL after multiple excisional operations. In addition, a serum MIS level of 20 ng/mL was detected in a patient with an ovarian granulosa cell tumor. A sensitive assay for MIS could be useful in the diagnosis of patients with congenital abnormalities of sexual development and patients with Sertoli cell and/or other MIS-producing neoplasms. Other applications may also be recognized as the biology of MIS in both males and females is further elucidated.

Stratum corneum antibodies detected by hemagglutination are not directed against keratin intermediate filaments

Autoantibodies to stratum corneum (SC) occur in virtually all normal adult human sera. These antibodies may be directed against various antigens of the SC. They have been detected by indirect immunofluorescence, passive hemagglutination (HA), immune adherence, and most recently by enzyme immunoassay and immunoblot methods. The purpose of our study was to examine whether antibodies to SC antigens as detected by passive HA are similar to the keratin intermediate filament (KIF) reactive antibodies. SC antigen preparation was prepared from psoriatic scales by the trypsin-phenol-water (TPW) extraction method. KIFs were prepared by 8 M urea extraction of normal callus or psoriatic scales. The anti-SC antibody titers of normal human sera were determined by passive HA before and after absorption with TPW-SC antigen preparation and upon absorption with KIFs. Similarly, titers of anti-KIF antibodies were determined on absorbed and unabsorbed sera by immunoblot assay. The results of this study indicate that the absorption of the sera with KIFs did not affect the titer of antibodies to TPW-extractable SC antigens whereas the titer of KIF antibodies dropped. KIF-reactive antibodies, on the other hand, were not affected by absorption with TPW-SC antigen, whereas the latter absorbed out the corresponding reactive antibodies. These results indicate that antibodies directed against SC antigen are different from the KIF-reactive antibodies.

An infant rat model of bacteremia with Brazilian purpuric fever isolates of Hemophilus influenzae biogroup aegyptius. Brazilian Purpuric Fever Study Group.

Brazilian purpuric fever (BPF) is a newly recognized fulminant pediatric infection caused by bacteremia with Hemophilus influenzae biogroup aegyptius (Hae). Following intraperitoneal inoculation, each of five disease isolates caused bacteremia more frequently than control, conjunctival isolates of Hae in complement-depleted 6-d-old rats. Sustained but self-limited bacteremia was observed in normal infant rats after inoculation with a disease strain. These rats developed meningitis and had depressed hemoglobin concentration and platelet counts. Pathologic examination showed meningitis and contiguous otitis. Pretreatment of infant rats with immune adult rat serum raised against disease isolates protected rats from bacteremia. Normal adult rat serum or immune rat serum against control strains failed to protect infant rats. Thus, strains of Hae isolated from patients with BPF are more virulent than control strains. Antibody against antigens unique to disease isolates protects infant rats from bacteremia.