Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia.

We report the inactivation, via homologous recombination, of two of the three active mouse alkaline phosphatase genes, i.e., embryonic (EAP) and tissue nonspecific (TNAP). Whereas expression of the EAP isozyme was abolished in all tissues that express EAP developmentally (such as the preimplantation embryo, thymus, and testis), the EAP knock-out mice show no obvious phenotypic abnormalities. They reproduce normally and give birth to live offspring, indicating the nonessential role of EAP during embryonic development. Mice deficient in the TNAP gene mimic a severe form of hypophosphatasia. These TNAP-/- mice are growth impaired, develop epileptic seizures and apnea, and die before weaning. Examination of the tissues indicates abnormal bone mineralization and morphological changes in the osteoblasts, aberrant development of the lumbar nerve roots, disturbances in intestinal physiology, increased apoptosis in the thymus, and abnormal spleens. Our results indicate that, in the mouse, TNAP appears not to be essential for the initial events leading to bone mineral deposition but that TNAP seems to play a role in the maintenance of this process after birth. The other phenotypic manifestations may be a consequence of the lack of TNAP in the developing neural tube between stages E8.5 and E13.5 of embryogenesis. We hypothesize that the autonomic nervous system is compromised in these TNAP-/- mice.

Haptoglobin-stimulated bone resorption in neonatal mouse calvarial bones in vitro.

OBJECTIVE: To study the in vitro effects of human haptoglobin (Hp) on bone resorption and prostanoid formation. METHODS: Parietal bones were dissected out from neonatal mice that had been injected with 45Ca, and were cultured in chemically defined medium with or without test substances. Bone resorption was assessed by analysis of 45Ca release. Prostanoid formation was quantified by analysis of the amount of prostaglandin E2 (PGE2) in culture medium. RESULTS: Hp phenotype 2-1, in quantities greater than or equal to 0.17 mg/ml, stimulated the release of 45Ca and the biosynthesis of PGE2, in a time- and dose-dependent manner. Hp-induced PGE2 formation was abolished by indomethacin and flurbiprofen, whereas the stimulation of 45Ca release was only partially reduced. CONCLUSION: These observations suggest that the acute-phase reactant Hp may contribute, by a humoral mechanism, to the bone resorption seen in chronic inflammatory processes.

Alkaline phosphatases as reporters of developmental and cancerous events.

Alkaline phosphatases comprise a family of isozymes whose expression is associated with important oncodevelopmental events. This presentation summarizes the relationship between alkaline phosphatase isozyme expression and specific developmental and disease processes, including malignancy. Several experimentally testable hypothesis are proposed that should help clarify the significance of these disease associations and the in vivo function of this interesting group of molecules.

Haptoglobin synergistically potentiates bradykinin and thrombin induced prostaglandin biosynthesis in isolated osteoblasts.

Haptoglobin of two different phenotypes (Hp 1-1 and Hp 2-1) dose-dependently (1-4 mg/ml) stimulated the formation of prostaglandin E2 (PGE2) in osteoblast-like cells isolated from neonatal mouse calvarial bones. The degree of stimulation obtained by haptoglobins (4 mg/ml) on PGE2 biosynthesis was in the same range as that caused by bradykinin (1 mumol/l). Pretreatment of osteoblasts with Hp 1-1 or Hp 2-1 (1-4 mg/ml) resulted in a dose-dependent, synergistic potentiation of the stimulatory effect of bradykinin (1 mumol/l) on PGE2 formation. Thrombin (7 U/ml) stimulated PGE2 formation in the osteoblast-like cells by a mechanism that was also synergistically potentiated by haptoglobin (2 mg/ml). These data show that haptoglobin per se stimulates PGE2 biosynthesis in isolated osteoblasts and, in addition, synergistically potentiates the effect of bradykinin and thrombin. Consequently, the enhanced production of haptoglobin seen in different inflammatory processes may contribute to the destruction of bone by inducing the formation of prostanoids capable of stimulating bone resorption.

Heterozygosity effects in studies of genetic markers and disease.

Examples were discussed where heterozygosity was associated with increased or decreased disease risks and where the apparent mechanism is direct functional involvement of gene products and not linkage disequilibrium. Special attention was paid to the impact of Hp (haptoglobin) heterozygosity on a number of different multifactorial disorders. When phenotype distributions in patients show large deviations from the Hardy-Weinberg equilibrium significant differences between patients and controls may be found concerning phenotype distributions but not with respect to the frequencies of alleles and phenotypic factors. The common method of studying ratios of phenotypic factors by pooling homo- and heterozygotes is in principle a conservative approach which tends to underestimate the strength of associations and to obscure heterozygosity effects. A significant deviation from the Hardy-Weinberg equilibrium in a marker system examined in a group of patients is in itself a sensitive indicator of phenotypic association with the disease in question.

Genetic variation of haptoglobin and transferrin in relation to DNA content and stage in renal cell carcinoma.

Four genetic marker systems were investigated in 102 patients with renal cell carcinoma. The previously observed excess of the transferrin (TF) variant C3 among male patients was confirmed. Interestingly, an excess of TFC3 and a deficit of the haptoglobin heterozygote, HP2-1, were associated with diploid tumor DNA content and Stage I, particularly in male patients. The results are discussed in terms of a possible genetic influence on tumor progression.

Association between haptoglobin groups and hereditary predisposition for bronchial asthma.

Haptoglobin (Hp) groups were investigated in 148 patients with bronchial asthma. A significant (p less than 0.005) deviation from the expected Hardy-Weinberg equilibrium, with a decreased frequency of heterozygotes, was observed among patients with a family history of asthma. This deviation was more pronounced among patients with adult onset of asthma. The presence or absence of atopy had no significant influence on the phenotype distribution.

Haptoglobin groups in acute myocardial infarction.

In patients with acute myocardial infarction, the haptoglobin (HP) groups were found to be associated with serum cholesterol levels. Heterozygotes (HP 2-1) showed serum cholesterol levels significantly higher (p less than 0.0001) than those of patients with HP 1-1 or HP 2-2. Furthermore, an association between HP type and infarction size was observed, HP 2-2 being associated with smaller infarctions.

Growth hormone and somatomedin C during post-menopausal replacement therapy with oestrogen alone and in combination with an antioestrogen.

Serum concentrations of growth hormone (GH) and somatomedin C were monitored in 14 women during post-menopausal replacement therapy with oestrogen alone and in combination with a specific antioestrogen. During 3 mth of treatment with ethinyl oestradiol (10 micrograms daily), the mean serum GH level rose from 2.8 +/- 0.78 mU/l (mean +/- S.E.M.) to 6.5 +/- 0.39 mU/l, whereas the concentration of somatomedin C fell from 22.4 +/- 0.89 to 15.4 +/- 0.43 nM. These changes during unopposed oestrogen treatment were clearly reversed by the addition of tamoxifen (20 mg daily), following which GH concentrations fell to pre-treatment levels. It is suggested that oestrogens inhibit somatomedin C production in the liver and that GH secretion may play an important role in regard to certain liver effects induced by oral oestrogen therapy.

Haptoglobin groups in motor neuron disease.

Haptoglobin (Hp) groups were investigated in 81 patients with motor neuron disease. A significant excess of heterozygotes was observed, accentuated among males and in the progressive spinal muscular atrophy subgroup. The results are discussed in terms of a possible influence of Hp in the immunological response.

Haptoglobin groups in ovarian carcinoma.

Haptoglobin groups were determined in 182 patients with primary ovarian carcinoma. Previously reported associations could not be confirmed. A significant excess of HP2-1 was observed among patients with a family history of cancer.

Continuous 24-hour secretion of growth hormone during late pregnancy. A regulator of maternal metabolic adjustment?

A dramatic change in growth hormone secretion was demonstrated during the 3rd trimester of human pregnancy, when compared with the non-pregnant state. The pulsatile pattern, with intermittent high peaks, and low or undetectable levels between peaks, characteristic of normal men and non-pregnant women, was completely abolished. All the 10 pregnant women investigated had the same stable basal circulating growth hormone concentration. Values were in the range 6-10 mU/l and there was no evidence of pulsatile activity. Previously, in animal experiments, a continuous secretion of growth hormone has been shown to 'feminize' hepatic steroid metabolism, hepatic prolactin receptors, hepatic sulphatase activity and to stimulate pregnancy protein synthesis. The same biological principle could be valid also during human pregnancy and be related to maternal metabolic adjustment.

Effects of oestrogen and human growth hormone on pregnancy-associated plasma proteins in the rat.

The serum concentrations of pregnancy-associated murine protein-1 (PAMP-1), acute phase alpha 2-macroglobulin, albumin, transferrin, and complement factor 3(C3) were followed in male rats during continuous infusions of oestradiol-17 beta and human growth hormone. Three different patterns of protein response could be distinguished. A distinct acute phase response without any additive influence of the given hormones was recorded for alpha 2-macroglobulin, whereas the levels of albumin, transferrin and C3 were virtually unaffected throughout the experiment. Growth hormone gave a rapid and pronounced increase of PAMP-1 levels, whereas the response to oestradiol of this 'steroid-sensitive' protein was significantly weaker and delayed. It is suggested that the apparent oestrogenic influence on certain pregnancy-associated plasma proteins is mediated via growth hormone.

Effects of growth hormone on pregnancy-associated murine protein-1.

In male mice which normally do not synthesize measurable amounts of the pregnancy-associated murine protein-1 (PAMP-1), synthesis occurred when there was continuous infusion of hGH but not by repeated subcutaneous injections. The decrease in PAMP-1 values after hypophysectomy in female mice was rapidly restored by continuous infusion of hGH, 80 micrograms daily. PAMP-1 has generally been regarded as an 'oestrogen-inducible' protein regulated by the oestrogen/androgen balance. Our results suggest that the apparent effects of sex steroids are mediated via the pituitary and possibly growth hormone secretion.

Haptoglobin groups and serum cholesterol levels.

The serum levels of cholesterol and cholesterol esters were studied in relation to haptoglobin groups in a series of 277 healthy blood donors from northern Sweden. Previous reports of associations between Hp 2-2 and high serum cholesterol levels were not confirmed, though a nonsignificant deviation in the same direction was observed. Possible mechanisms behind an association between haptoglobin groups and serum cholesterol were discussed.

Steroid-sensitive proteins, growth hormone and somatomedin C in prostatic cancer: effects of parenteral and oral estrogen therapy.

The effects of parenteral and parenteral plus oral estrogen therapy on the serum levels of sex hormone binding globulin (SHBG), pregnancy-associated alpha 2-macroglobulin (alpha 2-PAG), growth hormone (GH), and somatomedin C (SmC) were studied in 26 patients with prostatic cancer. Intramuscular polyestradiol phosphate treatment, yielding a mean serum level of estradiol-17 beta of 1,446 pM and a mean testosterone level of 4.5 nM, had no significant effects on alpha 2-PAG, GH, and SmC and increased SHBG levels only marginally. Combined treatment with intramuscular polyestradiol phosphate and oral ethinyl estradiol greatly increased SHBG and alpha 2-PAG levels and caused elevated GH and decreased SmC levels. The route of estrogen administration is probably of major importance for the hormonal effects on hepatic activity as reflected by SHBG and alpha 2-PAG levels. Bypassing the portal circulation might be advantageous with respect to liver-related side effects of estrogen therapy. GH and SmC might act as mediators of estrogen effects on the human liver.

Haptoglobin groups and lung cancer.

Haptoglobin groups were investigated in 309 patients with primary lung cancer divided by sex, smoking habits and tumor type. Patients with squamous epithelial cancer and oat-cell cancer showed no significant difference from normal controls. Among patients with pulmonary adenocarcinoma the frequency of the Hp 2-2 type was significantly (p less than 0.05) lower compared to the controls, with a corresponding increase of the Hp 1-1 and Hp 2-1 types. This difference was more pronounced (p less than 0.025) among females with pulmonary adenocarcinoma.

Serum protein groups in renal cell carcinoma.

Hp, C3, Gc and Tf serum groups were determined in 66 patients with renal cell carcinoma. Previously reported associations between renal cell carcinoma and the C3F and Gc2 genes were not confirmed. Among female patients a significant excess of GcIF was observed whereas male patients showed a significant excess of TfC3.

Haptoglobin groups in dementia of Alzheimer type and multi-infarct dementia.

Haptoglobin (Hp) groups were determined in 65 patients with dementia of Alzheimer type (DAT) and 74 with multi-infarct dementia (MID). The increased Hp1 gene frequency among patients with DAT described by other investigators could not be confirmed in this study. The patients showed no significant difference from the controls with respect to phenotype and gene frequencies, and there were no significant differences between early and late onset cases of DAT.

Haptoglobin and transferrin types in schizophrenia.

Haptoglobin and transferrin types were studied in schizophrenic patients and controls. In the haptoglobin system a significant departure from the Hardy-Weinberg equilibrium with an excess of heterozygotes was found among the patients (p less than 0.01). The distribution of haptoglobin types in the schizophrenic patients was significantly different from that in the controls. The distribution of transferrin types showed a good agreement with the Hardy-Weinberg equilibrium. There was no significant difference between patients and controls with respect to transferrin types.