Management of infants born to mothers with chorioamnionitis: A retrospective comparison of the three approaches recommended by the committee on fetus and newborn.

BACKGROUND: Based on the most recently published recommendations from the Committee on the Fetus and Newborn (COFN), three approaches currently exist for the use of risk factors to identify infants who are at increased risk of early-onset sepsis (EOS). Categorical risk factor assessments recommend laboratory testing and empiric antibiotic therapy for all infants born to mothers with a clinical diagnosis of chorioamnionitis. Risk assessments based on clinical condition recommend frequent examinations and close vital sign monitoring for infants born to mothers with chorioamnionitis. The Kaiser Permanente EOS risk calculator (SRC) is an example of the third approach, multivariate risk assessments. The aim of our study was to compare the three risk stratification approaches recommended by the COFN for management of chorioamnionitis-exposed infants. METHODS: Retrospective study of 1,521 infants born ≥35 weeks to mothers with chorioamnionitis. Management recommendations of the SRC were compared to the recommendations of categorical risk assessment and risk assessment based on clinical condition (CCA). RESULTS: Hypothetical application of SRC and CCA resulted in 79.6% and 76.8-85.1% respectively fewer infants allocated empiric antibiotic therapy. While CCA recommended enhanced observation for all chorioamnionitis-exposed infants, SRC recommended routine care without enhanced observation in 44.3% infants. For the six infants (0.39%) with EOS, SRC and CCA recommended empiric antibiotics only for three symptomatic infants. CONCLUSION: The SRC and CCA can reduce antibiotic use but potentially delay antibiotic treatment. The SRC does not recommend enhanced observation with frequent and prolonged vital signs for >44% of chorioamnionitis-exposed infants.

Production of a monoclonal antibody to human liver alkaline phosphatase.

A monoclonal antibody to human liver alkaline phosphatase (ALP) has been produced by the mouse-hybridoma method using a partially purified enzyme preparation as antigen. The particular hybridoma secreting the antibody was detected by a screening procedure based on the retention of enzyme activity by the enzyme/antibody complex. The antibody cross-reacts strongly with human kidney and bone ALPs but not with human placental or intestinal ALPs. It also cross-reacts with liver and kidney ALPs from gorilla, chimpanzee and orangutan. It shows no significant reaction, under the conditions used, with liver or kidney ALPs from several lower primates. An antibody affinity column was prepared and shown to be effective for the final stages of liver ALP purification.

Soluble exopeptidases of bovine and human lens: characterization by electrophoresis.

Soluble exopeptidases present in bovine and human lenses were identified and characterized using starch gel electrophoresis separation followed by activity staining with an L-aminoacid oxidase/peroxidase system or a naphthylamine fluorescence system. Sixteen peptide and twelve naphthylamide substrates were used. The profile of substrate specificities for each electrophoretically separated exopeptidase was determined. Characterization also included the effects on activity of pH, EDTA, puromycin, and divalent cations. In addition, molecular weight determinations by gel filtration were made. Six bovine lens peptidases were identified including leucine aminopeptidase and dipeptidylpeptidase III and six human lens peptidase including dipeptidylpeptidase III. Strong homology in terms of substrate specificity and molecular weight was seen between bovine leucine aminopeptidase and one of the human peptidases previously designated peptidase "S". The findings indicate the diversity of exopeptidase available for polypeptide degradation in lens.

Immunochemistry of human Lp[a]: characterization of monoclonal antibodies that cross-react strongly with plasminogen.

Forty different monoclonal antibodies were produced from hybridomas that were raised against human Lp[a]. Of these, 14 strongly cross-reacted with plasminogen on ELISA screening assays while 16 clearly did not and 10 were only marginally cross-reactive. We took advantage of the homology between plasminogen and apo[a] to define the epitopes of 8 strongly cross-reacting monoclonal antibodies. We were able to subdivide these into four general categories based upon site competition assays (using both plasminogen and Lp[a]), and their reactivity with elastolytically derived plasminogen fragments. Group A monoclonal antibodies (F1 1E3, F2 3A3) recognized epitopes within the kringle 5 and protease domains (miniplasminogen) of plasminogen. The group B monoclonal antibody (F6 1A3) reacted solely with plasminogen kringle 4-like domains and appeared to recognize a limited number of sites on Lp[a]. Group C monoclonal antibodies (F6 1B5, F6 1G9) recognized a second, more frequently distributed site within these kringle 4-like domains. The final group, D, monoclonal antibodies (F6 2C3, F6 2G2, F6 3F4) reacted with a cluster of sites found associated with kringle 4-like domains but also reacted with the miniplasminogen domain. Interestingly, only the members of this group were able to interfere with the proteolytic activity of plasmin. Neither periodate treatment of Lp[a] nor incubation of Lp[a] with epsilon-aminocaproic acid affected the binding of any of our monoclonal antibodies.

Preparation and properties of thyroid cell membranes.

Calf and human thyroids have been disrupted by nitrogen microcavitation, and the thyroid membranes prepared by repeated centrifugation in low ionic strength buffers. Two classes of membranes were prepared by centrifugation on a discontinuous gradient of ficoll. A lighter fraction was comprised of somewhat larger vesicles; they were higher in Na(+)-K(+)-activated ATPase, phosphodiesterase, and 5'-nucleotidase than was the heavier fraction. The heavier fraction had a higher nicotinamide adenine nucleotide dehydrogenase-diaphorase activity. Thus the lighter fraction appears to have been enriched in fragments derived from the plasma membrane.

Different missense mutations at the tissue-nonspecific alkaline phosphatase gene locus in autosomal recessively inherited forms of mild and severe hypophosphatasia.

Hypophosphatasia is a heritable form of rickets/osteomalacia with extremely variable clinical expression. Severe forms are inherited in an autosomal recessive fashion; the mode of transmission of mild forms is uncertain. The biochemical hallmark of hypophosphatasia is deficient activity of the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Previously, we demonstrated in one inbred infant that an identical missense mutation in both alleles of the gene encoding TNSALP caused lethal disease. We have now examined TNSALP cDNAs from four unrelated patients with the severe perinatal or infantile forms of hypophosphatasia. Each of the eight TNSALP alleles from these four individuals contains a different point mutation that causes an amino acid substitution. These base changes were not detected in at least 63 normal individuals and, thus, appear to be causes of hypophosphatasia in the four patients. (Two additional base substitutions, found in one allele from each of the four patients, are linked polymorphisms.) Twenty-three unrelated patients (of 50 screened), who reflect the entire clinical spectrum of hypophosphatasia, possess one of our of the above eight mutations. In two of these additional patients, mild forms of the disease are also inherited in an autosomal recessive fashion. Our findings indicate that hypophosphatasia can be caused by a number of different missense mutations and that the specific interactions of different TNSALP mutant alleles are probably important for determining clinical expression. Severe forms, perinatal and infantile disease, are largely the result of compound heterozygosity for different hypophosphatasia alleles. At least some cases of childhood and adult hypophosphatasia are inherited as autosomal recessive traits.

Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase.

Alkaline phosphatases (ALPs) [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] isolated from human liver, bone, and kidney (L/B/K) exhibit very similar biochemical and immunologic properties that differentiate them from other human ALPs, such as those characteristically found in placenta and intestine. Despite their similarities, the L/B/K ALPs produced in different tissues show slight physical differences. To examine structural and evolutionary relationships between the various ALPs, a cDNA corresponding to L/B/K ALP mRNA has been isolated. A lambda 11 cDNA expression library was constructed using poly(A) RNA from the osteosarcoma cell line Saos-2 and screened with anti-liver ALP antiserum. The 2553-base-pair cDNA contains an open reading frame that encodes a 524 amino acid polypeptide with a predicted molecular mass of 57.2 kDa. This ALP precursor protein contains a presumed signal peptide of 17 amino acids followed by 37 amino acids that are identical to the amino-terminal sequence determined from purified liver ALP. In addition, amino acid sequences of several CNBr peptides obtained from liver ALP are found within the cDNA-encoded protein. The deduced L/B/K ALP precursor polypeptide shows 52% homology to human placental ALP and 25% homology to Escherichia coli ALP precursor polypeptides. Sixty percent nucleotide homology exists between the human L/B/K and placental cDNAs over the protein coding regions. The 5' and 3' untranslated regions of the L/B/K ALP cDNA, 176 and 805 base pairs, respectively, show no homology to the corresponding regions of placental ALP cDNA.

First identification of a gene defect for hypophosphatasia: evidence that alkaline phosphatase acts in skeletal mineralization.

Hypophosphatasia is a heritable disorder characterized by defective osteogenesis and deficient liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Severe forms of the disease are inherited in an autosomal recessive fashion. We examined cultured skin fibroblasts from twelve patients with severe hypophosphatasia. All were deficient in L/B/K ALP activity, yet produced normal levels of the corresponding mRNA. Sequence analysis of L/B/K ALP cDNA isolated from one of the patient-derived fibroblast lines revealed a point mutation that converted amino acid 162 of mature L/B/K ALP from alanine to threonine. The patient was homozygous and the parents, who are second cousins, heterozygous for this mutation. Introduction of the mutation into an otherwise normal cDNA disrupted the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene resulted in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.

Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase.

A cDNA clone for human adult intestinal alkaline phosphatase (ALP) [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] was isolated from a lambda gt11 expression library. The cDNA insert of this clone is 2513 base pairs in length and contains an open reading frame that encodes a 528-amino acid polypeptide. This deduced polypeptide contains the first 40 amino acids of human intestinal ALP, as determined by direct protein sequencing. Intestinal ALP shows 86.5% amino acid identity to placental (type 1) ALP and 56.6% amino acid identity to liver/bone/kidney ALP. In the 3'-untranslated regions, intestinal and placental ALP cDNAs are 73.5% identical (excluding gaps). The evolution of this multigene enzyme family is discussed.

Products of two common alleles at the locus for human placental alkaline phosphatase differ by seven amino acids.

Amino-terminal amino acid sequences (42 residues) were determined for the products of the three common alleles at the human placental alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] gene locus. The sequences differ at position 3, which is proline in types 1 and 2 but is leucine in type 3. cDNA libraries were constructed in phage lambda gt11 and used to isolate clones covering the coding regions of types 1 and 3 cDNAs. Comparison of the deduced amino acid sequences of the types 1 and 3 proteins showed 7 differences out of 513 amino acids, each due to a single base substitution. cDNA sequence comparisons showed three silent substitutions in the coding regions and three base differences in the greater than 1 kilobase pairs of 3' untranslated sequences.

Analysis of liver/bone/kidney alkaline phosphatase mRNA, DNA, and enzymatic activity in cultured skin fibroblasts from 14 unrelated patients with severe hypophosphatasia.

Hypophosphatasia is a heritable disorder characterized by defective bone mineralization and a deficiency of liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity in serum and tissues. Severe forms of the disease, which are generally lethal in infancy, are inherited in an autosomal recessive fashion. The gene defects that produce hypophosphatasia are poorly understood, but many are likely to occur at the L/B/K ALP locus. To investigate these gene defects, we analyzed L/B/K ALP DNA, RNA, and enzyme activity in cultured dermal fibroblasts from 14 patients with perinatal or infantile hypophosphatasia and from 12 normal individuals. Southern blot analyses of the L/B/K ALP genes from patients and controls revealed identical restriction patterns. Control fibroblast ALP activity correlated with the corresponding L/B/K ALP mRNA levels estimated by blot hybridization analysis and densitometry (r = .94, P less than .0001). In contrast, fibroblasts from the hypophosphatasia patients were deficient in ALP enzyme activity but expressed apparently full-sized L/B/K ALP mRNA at normal levels. Bone specimens from one of the patients were examined and found to be deficient in histochemical ALP but contained immunologic cross-reactive material detected by anti-human liver ALP antiserum. Our results demonstrate that the deficiency of ALP activity in fibroblasts from 14 patients with severe hypophosphatasia is not due to decreased steady-state levels of the corresponding mRNA. The presence of enzymatically inactive L/B/K ALP protein in one of these patients is consistent with a point mutation or small in-frame deletion in the coding region of L/B/K ALP gene.

A missense mutation in the human liver/bone/kidney alkaline phosphatase gene causing a lethal form of hypophosphatasia.

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Clinical severity is variable, ranging from death in utero (due to severe rickets) to pathologic fractures first presenting in adult life. Affected siblings, however, are phenotypically similar. Severe forms of the disease are inherited in an autosomal recessive fashion; heterozygotes often show reduced serum ALP activity. The specific gene defects in hypophosphatasia are unknown but are thought to occur either at the L/B/K ALP locus or within another gene that regulates L/B/K ALP expression. We used the polymerase chain reaction to examine L/B/K ALP cDNA from a patient with a perinatal (lethal) form of the disease. We observed a guanine-to-adenine transition in nucleotide 711 of the cDNA that converts alanine-162 of the mature enzyme to threonine. The affected individual, whose parents are second cousins, is homozygous for the mutant allele. Introduction of this mutation into an otherwise normal cDNA by site-directed mutagenesis abolishes the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene results in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.