Physicochemical analysis of pasteurized human milk and commercial premature formulas fortified with phosphorus solution or commercial fortifier / Análise físico-química do leite humano pasteurizado e de fórmula comercial para prematuros suplementados com solução de fósforo ou fortificante comercial

A study was conducted to determine: the most appropriate proportion (1% or 10% v/v) of a phosphate solution (PS), containing 39mg/mL phosphorus, to be added to pasteurized human milk (HM) or commercial premature formula (FM); the final osmolality of such products, and whether precipitation occurs between calcium (Ca) and phosphorus (P) when commercial fortifier (FOR) or PS is added. A significant increase was observed in the concentrations of Ca in the samples of HM and FM containing FOR and a decrease in the samples of HM containing 10% (v/v) PS. The phosphorus levels increased significantly, in both HM and FM, when FOR or PS (1 and 10%) were added. Osmolality showed a significant increase in the solutions of HM with FOR or 10% PS added, and in the solution of FM containing 10% PS. Qualitative analysis of the precipitate formed on addition of 10% PS to FM revealed that it consisted of dicalcium phosphate. It was concluded that the addition of 10% PS to FM causes a fall in the Ca content, due to its precipitation with phosphate, promoting a reduction in the availability of both ions. On the other hand, the addition of 1% PS was demonstrated to cause no alteration in the Ca concentration, but a significant increase in P. These results suggest that the use of 1% PS is a potential means of supplementation of P after these patients are discharged.

Foi realizado um estudo para verificar: a concentração, 1 ou 10% (v/v), mais apropriada de solução de fósforo (SP), para ser adicionada nos produtos lácteos; a osmolalidade final de tais produtos suplementados; a ocorrência de precipitação entre o cálcio (Ca) e o fósfforo (P) com a adição de um fortificante comercial (FOR) ou SP. Verificou-se aumento significante nas concentrações de Ca nas amostras de leite materno pasteurizado (LM) e fórmula comercial (FL) contendo FOR e diminuição nas amostras de FL contendo 10% (v/v) de SP. Quanto aos níveis de fósforo, houve aumento significante tanto no LM quanto na FL, adicionados de FOR ou SP a 1 e 10%. Em relação à osmolalidade, houve aumento significante nas soluções de LM contendo FOR e de SP a 10% e na solução de FL a 10% de SP. A análise qualitativa do precipitado formado pela adição de 10% SP na FL revelou que o mesmo é constituído de fosfato bicálcico. Concluiu-se que a adição de SP na concentração de 10% em FL acarreta diminuição do Ca devido à precipitação do mesmo com o P, promovendo uma baixa na oferta de ambos os íons para o prematuro. Por outro lado, a adição de SP a 1% demonstrou que não houve diminuição dos níveis de Ca, mas aumento significante nos níveis de P. Estes resultados sugerem que o uso da SP a 1% é uma alternativa de suplementação de fósforo na alta hospitalar destes pacientes.

Requirement of Xmsx-1 in the BMP-triggered ventralization of Xenopus embryos.

Signaling triggered by polypeptide growth factors leads to the activation of their target genes. Several homeobox genes are known to be induced in response to polypeptide growth factors in early Xenopus development. In particular, Xmsx-1, an amphibian homologue of vertebrate Msx-1, is well characterized as a target gene of bone morphogenetic protein (BMP). Here, using a dominant-negative form of Xmsx-1 (VP-Xmsx-1), which is a fusion protein made with the virus-derived VP16 activation domain, we have examined whether Xmsx-1 activity is required in the endogenous ventralizing pathway. VP-Xmsx-1 induced a secondary body axis, complete with muscle and neural tissues, when overexpressed in ventral blastomeres, suggesting that Xmsx-1 activity is necessary for both mesoderm and ectoderm to be ventralized. We have also examined the epistatic relationship between Xmsx-1 and another ventralizing homeobox protein, Xvent-1, and show that Xmsx-1 is likely to be acting upstream of Xvent-1. We propose that Xmsx-1 is required in the BMP-stimulated ventralization pathway that involves the downstream activation of Xvent-1.

The potential angiogenic role of macrophages in the formation of choroidal neovascular membranes.

PURPOSE: To investigate the distribution of inflammatory mediators such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha and angiogenic cytokines such as vascular endothelial growth factor (VEGF) and to identify their cellular source in surgically excised choroidal neovascular membranes (CNVMs) of various origins. METHODS: Immunoperoxidase staining was performed on paraffin-embedded sections of 11 surgically excised CNVMs to identify cellular distribution and localization of cytokines. Immunofluorescent double staining was performed to detect the cellular source of cytokines. RESULTS: Cytokeratin-positive cells were detected in the RPE layer, in stromal cells, and around neovascular vessels. Macrophages identified by their cellular marker CD68 showed almost the same distribution as cytokeratin-positive cells, although they were most prominent in the stroma. A substantial number of neovascular vessels were also immunoreactive to IL-1beta and TNF-alpha. Immunofluorescent double staining revealed that the RPE layers immunopositive for cytokeratin were also immunopositive for all cytokines, whereas stromal cells immunostained for CD68 were positive for IL-1beta and TNF-alpha, but not for VEGF. CONCLUSIONS: These results indicate that IL-1beta and TNF-alpha secreted by macrophages may promote, at least in part, angiogenesis in CNVMs by stimulating VEGF production in RPE cells.

A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta superfamily, have been identified by their ability to induce cartilage and bone from nonskeletal cells and have been shown to act as a ventral morphogen in Xenopus mesoderm. We isolated a murine homeobox-containing gene, distal-less 5 (mDlx5), as a BMP-inducible gene in osteoblastic MC3T3-E1 cells. Stable transfectants of MC3T3-E1 that overexpress mDlx5 mRNA showed increase in various osteogenic markers, a fourfold increase in alkaline phosphatase activity, a sixfold increase in osteocalcin production, and appearance in mineralization of extracellular matrix. Furthermore, mDlx5 was induced orthotopically in mouse embryos treated with BMP-4 and in fractured bone of adult mice. Consistent with these observations, we also found that injection of mDlx5 mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos. These findings suggest that mDlx5 is a target gene of the BMP signaling pathway and acts as an important regulator of both osteogenesis and dorsoventral patterning of embryonic axis.

Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo.

In early development of Xenopus laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins. In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early Xenopus embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.

Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor.

Increased vascular permeability and excessive neovascularization are the hallmarks of endothelial dysfunction, which can lead to diabetic macular edema and proliferative diabetic retinopathy in the eye. Vascular endothelial growth factor (VEGF) is an important mediator of ocular neovascularization and a known vasopermeability factor in nonocular tissues. In these studies, we demonstrate that intravitreal injection of VEGF rapidly activates protein kinase C (PKC) in the retina at concentrations observed clinically, inducing membrane translocation of PKC isoforms alpha, betaII, and delta and >threefold increases in retinal vasopermeability in vivo. The effect of VEGF on retinal vascular permeability appears to be mediated predominantly by the beta-isoform of PKC with >95% inhibition of VEGF-induced permeability by intravitreal or oral administration of a PKC beta-isoform-selective inhibitor that did not inhibit histamine-mediated effects. These studies represent the first direct demonstration that VEGF can increase intraocular vascular permeability through activation of PKC in vivo and suggest that oral pharmacological therapies involving PKC beta-isoform-selective inhibitors may prove efficacious for the treatment of VEGF-associated ocular disorders such as diabetic retinopathy.

Neurofibromatosis complicated with XXX syndrome and renovascular hypertension.

A 25-year-old woman with neurofibromatosis was admitted to our hospital for evaluation of hypertension. When she was 6 years old, she was diagnosed as having neurofibromatosis and XXX syndrome because of multiple café-au-lait spots, neurofibromas of the skin and mental retardation. Chromosome analysis revealed that her karyotype was 46, XX/47, XXX. Renal arteriography disclosed aneurysmal change and stenosis of the right renal artery. After right-side nephrectomy and aneurysmectomy, the kidney was autotransplanted in the left iliac fossa. Surgical procedure resulted in marked amelioration of the hypertension without medical treatment. Thus, aortorenal bypass and renal autotransplantation have emerged as the preferred revascularization operations. This is the first report of a chromosomal linkage between neurofibromatosis which is thought to be an autosomal dominant disease and the XXX syndrome.

Enhanced expression of beta-adrenergic receptor kinase 1 in the hearts of cardiomyopathic Syrian hamsters, BIO53.58.

We cloned an entire encoding sequence of beta-adrenergic receptor kinase 1 (beta ARK1) cDNA from the hearts of Syrian hamsters through reverse transcription and subsequent polymerase chain reaction. The cloned cDNA contained 2067 nucleotides coding 689 amino acids. The sequence had 95% homology to rat beta ARK1 and 90% homology to human homologue. Cardiomyopathic Syrian hamster, BIO53.58, has been used as a model animal of congestive heart failure. M-mode echocardiography confirmed that left ventricular contractility of 20-week-old BIO53.58 was markedly reduced. The expression of beta ARK1 mRNA in the hearts of BIO53.58 was significantly increased compared to control hamsters, F1b, suggesting that the enhanced beta ARK1 expression is acting as a negative feedback mechanism in order to maintain intracellular homeostasis against accelerated stimulation by catecholamines via phosphorylation of beta-adrenergic receptor.

[Unconsciousness due to hyponatremia in a patient with short stature with panhypopituitarism].

An unconscious woman of short stature (141 cm) was admitted to our hospital in March, 1994. She had hyponatremia (120 mEq/l) and had experienced massive bleeding during delivery. No increment of either plasma ACTH or cortisol levels was observed after insulin-induced hypoglycemia. However, urinary 17OHCS levels gradually increased after repeated intramuscular injections of ACTH. Plasma free T3 and free T4 levels were low. Neither plasma TSH nor prolactin (PRL) levels increased after an intravenous injection of TRH. Basal plasma LH, FSH and growth hormone (GH) levels were low and there were no observable responses to any of the stimulation tests. A magnetic resonance image (MRI) of her pituitary gland showed an empty sella. These results showed that she had a panhypopituitarism with primary empty sella. Replacement therapy with glucocorticoid was started and serum sodium levels normalized immediately. Levothyroxine was also administered. The possibility of pituitary dwarfism during her youth and a gradual postpartum reduction of other pituitary hormones may have caused an impairment of the hypothalamo-pituitary-adrenal axis.

[Complete remission induced by combined treatment with all-trans retinoic acid (ATRA) and granulocyte colony-stimulating factor (G-CSF) in a patient with relapsed acute promyelocytic leukemia].

In February, 1990, a 49-year-old man was admitted with petechia and gingival bleeding. The peripheral blood showed 5,200 leukocytes/microliters including 73% abnormal promyelocytes and 24,000/microliters platelets. Bone marrow puncture revealed that nucleated cell count was 331,250/microliters including 85.4% abnormal promyelocytes with 46XY, i(17q) chromosome. Coagulation tests revealed DIC. He was diagnosed as having acute promyelocytic leukemia, and he was treated with the BHAC-DMP protocol. He achieved complete remission, and received consolidation therapy and maintenance therapy. However, he relapsed in May, 1991 with 46XY, 16q-, i (17q) chromosome. He was treated with BHAC-MV protocol and again achieved complete remission. In June, 1992, he re-relapsed and 3.6% blasts and 10% abnormal promyelocytes was found in his bone marrow. He was treated for 14 days with 15 mg Aclarubicin without any change. Then he was treated with 60 mg All-trans retinoic acid (ATRA). After administration of ATRA, his peripheral blood leukocyte count increased temporarily but bone marrow suppression continued. Then he received continuous subcutaneous infusion of 24 micrograms/day granulocyte colony-stimulating factor (rhG-CSF). After treatment with ATRA and rhG-CSF, he entered a third complete remission.

[A case of Cushing's disease associated with a non-functioning adrenal tumor].

A 52-year-old woman was admitted to our hospital for further examination of central obesity, hypertension and hirsutism suggesting Cushing's syndrome. Hirsutism had been remarkable for two years, and muscle weakness of the lower extremities gradually developed during the past year. CT scan revealed a tumor in the left adrenal gland which was 1 cm in diameter, round, well-circumscribed, homogeneous and not enhanced. Endocrine data disclosed increased urinary 17-OHCS (11.5-16.4 mg/day) and elevated plasma ACTH (125 pg/ml) and cortisol (19 micrograms/dl) with a lack of diurnal rhythm. Administration of the single-dose dexamethasone (1mg) did not suppress plasma cortisol. However, consecutive administration of either 2mg or 8mg of dexamethasone for 2 days suppressed both plasma cortisol and urinary 17-OHCS. Administration of metyrapone raised both urinary 17-OHCS and plasma ACTH levels. Rapid ACTH test resulted in a hyperresponse of plasma cortisol. CRF injection raised plasma ACTH and cortisol. Bilateral adrenal glands were well demonstrated by 19-iodocholesterol (I-131) scintigraphy during the administration of dexamethasone. MRI with Gd-contrast revealed a microadenoma in the sella turcica. With the diagnosis of Cushing's disease, the microadenoma was removed by the transsphenoidal approach and adrenal function was normalized. However, the left adrenal tumor remained on CT scan but was not demonstrated by scintigraphy. These findings indicate that this is a very rare case of Cushing's disease which was associated with an unilateral non-functioning adrenal tumor.

[Effect of subcutaneous administration of interleukin-1 beta on blood platelet count and serum GM-CSF in patients with myelodysplastic syndrome and aplastic anemia].

Subcutaneous administration of recombinant human Interleukin-1 beta (IL-1 beta) in a dose of 1-3 x 10(4) U/day for 14 to 72 days resulted in an increase in circulating granulocytes and bone marrow monocytes in all the 4 patients examined. Circulating platelet count was also increased in two of four patients with myelodysplastic syndrome (MDS) and aplastic anemia (AA). Bone marrow examination revealed an increase in megakaryocyte count in these patients, whereas the percentage of blast was not changed. An increase in blood platelet count was accompanied by an increase in serum GM-CSF in a patient with AA, whereas serum IL-6 level was not changed throughout the treatment with IL-1 beta. These findings suggest that IL-1 beta may be useful for the treatment of a proportion of patients with MDS and AA who are associated with thrombocytopenia.

[Clinical use of serum erythropoietin determination by the recombigen EPO RIA kit].

Serum erythropoietin (EPO) levels were determined by the recombigen EPO RIA kit (DPC) in normal subjects and patients with renal dysfunction, diabetes mellitus, hypothyroidism and a variety of hematological disorders. Mean (+/- SD) serum EPO levels were 18.6 +/- 5.6 mU/ml in 180 normal subjects and no sex difference was obtained. Serum EPO levels in older subjects were slightly greater than those in younger subjects. There was a negative correlation between serum EPO levels and Ht values in anemic patients with normal renal function, whereas serum EPO levels were within the normal range in anemic patients with renal disorders, suggesting that serum EPO levels were relatively low in patients with chronic renal failure. Serum EPO levels were rather increased in patients with diabetes mellitus and hypothyroidism. High serum EPO levels were obtained in patients with a variety of hematological disorders such as acute leukemia, multiple myeloma, myelodysplasia syndrome, aplastic anemia and pure red cell aplasia. In a patient with pure red cell aplasia treated with glucocorticoids, serum EPO levels were lowered before anemia was recovered and reticulocytes were increased. These findings indicate that measurement of serum EPO levels are useful for not only differential diagnosis of anemia but also clinical evaluation of the treatment.

[Acute myelogenous leukemia transformed from myelodysplastic syndrome with tetraploid chromosome constitution].

A 57-year-old female presented with general fatigue. She had neither lymphadenopathy nor hepatosplenomegaly. Laboratory data revealed anemia and leukopenia (1,500/microliters) with a differential count of 4.5% leukemic cells. The myelogram revealed 34.4% leukemic cells, of which diameter ranged from 20 to 28 microns. The diagnosis was acute myelogenous leukemia (FAB: M2) with myelodysplasia. Cytogenetic analysis revealed that the leukemic cells had chromosome abnormalities involving both diploidy and tetraploidy with structural rearrangement. Structural rearrangement included del(5) (q22q33), del(15) (q22q24), and t(3; 12) (q25;p13). Small dose aclacinomycin-A treatment was effective in reducing the number of leukemic cells in bone marrow, and both anemia and leukocytopenia were improved.

Natural killer cell activity against a variety of target cell lines in normal persons: NK-target sensitivity and effect of age and sex on NK levels.

Natural killer (NK) cells have been implicated as first line of defence mechanism for carcinogenesis in humans. A lot of studies of depressed NK activity in patients with malignancies have supported this. Two major problems, however, in these studies are the choice of normal controls and target cells. To study this problem, peripheral mononuclear cells (PBMC) of sixty-six normal subjects from young and elderly, males and females were tested for NK function against twenty target cell lines with a microcytotoxicity assay. The result was shown that no sex or age difference existed with respect to NK function, except for a slight but significant decreased in NK activity of young female to K562 target cells. Target cells were divided into four groups by their NK sensitivity, namely, high, moderate, low and refractory sensitive. In general, NK activity of healthy persons is considered to remain stable and polyspecific in our results.