Signal transduction via phosphorylated adhesion molecule, LFA-1 beta (CD18), is increased by culture of natural killer cells with IL-2 in the generation of lymphokine-activated killer cells

Anti-human platelet myosin antibodies and two anti-peptide antibodies, anti-peptide IIA and anti-peptide IIB, which recognize macrophage-type (MIIA) and brain-type (MIIB) isoforms of nonmuscle myosin heavy chain, respectively, were used to study expression of nonmuscle myosin isoforms in various tissues of mice during development. Tissue-specific changes in the relative isoform concentrations were observed by performing immunoblots of crude myosin extracts from nonmuscle and muscle tissues. In fetal and neonatal mouse tissues, the anti-peptide IIB antibodies stained a single band, called MIIB2, while the anti-peptide IIA and anti-platelet myosin antibodies stained a band that migrated faster than MIIB2. In brain, a slower moving band, MIIB1, started to appear at 2 weeks after birth, and in the adult cerebellum it was at least as abundant as MIIB2. In thymus, MIIB2 decreased selectively shortly after birth, while in liver both MIIB2 and MIIA rapidly disappeared, but the isoform(s) detected by anti-platelet myosin antibodies (MIIApla) remained constant. The MIIB2 and MIIA as well as MIIApla found in striated muscles from fetal and neonatal mice decreased to levels that were below the limit of detection by 3 weeks of age. In cryosections of skeletal and cardiac muscles, MIIB2 was localized within the muscle cells, while MIIA and MIIApla were primarily in the blood vessels and capillaries.     

Erythropoietin mRNA expression in human fetal and neonatal tissue

Based on animal experiments, a switch of the erythropoietin (EPO) production site from the liver in the fetus to the kidneys in the adult has been postulated. To study the switch in humans, we have quantitated EPO mRNA expression in liver, kidney, spleen, and bone marrow of human fetuses and neonates by means of a competitive polymerase chain reaction (PCR). Tissue samples from 66 routine postmortem examinations were obtained. EPO mRNA was expressed in 97% of the tissue specimen derived from the liver (n = 66) and in 93% of those from the kidneys (17 weeks of gestation until 18 months after birth; n = 59). For the first time the EPO gene was found expressed in vivo in human spleen (96% of 64 samples) and in fetal and neonatal bone marrow (81% of 21 samples). EPO mRNA expression in the kidneys increased significantly beyond 30 weeks of gestation (P < .05). Although there was a slight decrease in EPO mRNA content per g liver tissue towards birth, the liver accounted for about 80% of the total body EPO mRNA. The contribution of the spleen and bone marrow were minor compared with liver and kidneys. Our results indicate that in humans the liver is the primary site of EPO gene expression not only in fetal, but also in neonatal life. A significant increase of renal EPO mRNA expression after 30 weeks of gestation might indicate the beginning switch.     

Changes in brain memory functions as affected by a constant magnetic field]

OBJECTIVE: The preferred route of delivery for breech presentation has been controversial. We compared the birth weight-specific neonatal mortality of vaginal births to cesarean births in singleton births with breech presentation. METHODS: A total of 371,692 singleton live births with breech presentation were selected for the study from the United States birth cohorts for the years 1989-1991. Differences in birth weight specific mortality were compared using a z-statistic for differences in proportions and by logistic regression. RESULTS: Compared to primary vaginal births, primary cesarean births had significantly lower neonatal mortality for all birth weight groups, despite increased prevalence of fetal malformations in the cesarean as compared with vaginally delivered group. This mortality difference was greatest in the first hour of life. Difference in overall neonatal (less than 28 days) mortality rate ranged from a low of 1.6-fold in the 500-749 g group (726.6 per 1000 vaginal births compared with 456.3 per 1000 cesarean births, P < .001) to as high as about three-fold in the 1250-1499 g group (232.9 per 1000 vaginal births compared to 72.5 per 1000 cesarean births, P < .001). In the group with birth weights over 2500 g, neonatal mortality in the primary vaginal births was 5.3 per 1000 and in the primary cesarean births, 3.2 per 1000 (P < .001). Similarly, repeat cesarean births had significantly lower birth weight-specific neonatal mortality, compared with vaginal births after previous cesarean. CONCLUSION: Singleton live births with breech presentation delivered by cesarean had lower birth weight-specific neonatal mortality as compared with vaginal births.     

Basophil count in neonates is not suitable for atopy predictivity

Basophil granulocytes and their mediators are involved in the pathogenesis of allergic inflammation. We evaluated basophil count, blood histamine content, eosinophil count and serum total IgE levels in one hundred-thirteen healthy newborns at birth. 102 children were prospectively studied with a follow up to 18 months of age for development of atopic disorders. No difference was found in newborns with biparental family history of atopy (FHA) in comparison with newborns with monoparental FHA and with newborns without FHA. Children who developed atopic disorders had neonatal basophil counts higher than those who did not develop atopic symptoms (p = 0.03). No significant correlation was found between basophil and eosinophil counts (rs = 0.013), between basophil count and serum total IgE levels (rs = 0.012) and between basophil count and blood histamine content. Positive predictive value and sensitivity of basophil count for allergy up to 18 months of age was only 33% and 27%, respectively. Our data indicate that an increased basophil count at birth is not associated with FHA and is not a good predictive marker of atopy.     

Expression of calcium-binding protein regucalcin mRNA in fetal rat liver is stimulated by calcium administration

The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, beta-actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic beta-actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.     

Does the onset of neonatal seizures correlate with the timing of fetal neurologic injury?

The onset of seizures after birth has been considered evidence of an intrapartum asphyxial event. The present study was undertaken to determine whether the timing of neonatal seizures after birth correlated with the timing of a fetal asphyxial event. Thus, singleton term infants diagnosed with hypoxic ischemic encephalopathy and permanent brain injury had a mean birth to seizure onset interval of 9.8 +/- 17.7 (range 1-90) hours. When these infants were categorized according to their fetal heart rate (FHR) patterns, the acute group (normal FHR followed by a sudden prolonged FHR deceleration that continued until delivery) tended to have earlier seizures than infants did within the tachycardia group (normal FHR followed by tachycardia, repetitive decelerations, and diminished variability) and the preadmission group (persistent nonreactive FHR pattern intrapartum). These seizure intervals were as follows: acute, 6.6 +/- 18.0 (range 1-90) hours; tachycardia, 11.1 +/- 17.1 (range 1-61) hours; and preadmission, 11.8 +/- 17.9 (range 1-79) hours (p < 0.05). But the range varied widely and no group was categorically distinct. In conclusion, the onset of neonatal seizures after birth does not, in and of itself, appear to be a reliable indicator of the timing of fetal neurologic injury.     

A stable RAG1-RAG2-DNA complex that is active in V(D)J cleavage

A well-known characteristic of gamma delta T cells is that they are produced in waves during ontogeny, with cells expressing T-cell receptor V gamma 5 appearing early in fetal thymic ontogeny, followed by V gamma 6, then by other gamma delta T-cell types. In addition, evidence exists to suggest that the potential of haemopoietic precursors to generate different types of gamma delta T cells changes in ontogeny. We have used these observations as the basis for an extensive study of the potential for haemopoietic precursors isolated from fetal liver, neonatal spleen and adult bone marrow to reconstitute severe combined immunodeficient (SCID) mice. Mice that were reconstituted as newborns with fetal liver cells most closely resembled normal C.B-17 mice with respect to both lymphocyte numbers and subsets, while mice reconstituted with adult bone marrow had fewer cells than normal mice. This deficit spanned both T and B cells in all organs examined. Among the gamma delta T-cell subsets examined, the ability to reconstitute V gamma 4+ cells was particularly dependent on the ontogenic age of the reconstituting presursors, with fetal liver cells having the greatest capacity to generate V gamma 4+ cells, and adult bone marrow cells the least. The vast majority of the T cells produced in the reconstituted mice were of donor origin, and the level of reconstitution was found to be dependent upon some factor other than the presursor frequency.     

Imprinted M6p/Igf2 receptor is mutated in rat liver tumors

We have previously shown that inactivation of mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is a common early event in both human liver and breast carcinogenesis. The M6p/Igf2r is imprinted in mice while expression is biallelic in most humans. In this investigation the M6p/Igf2r gene is shown to also be imprinted in the liver of Fischer 344, Lewis and Brown Norway rats. In addition, we have identified mutations in the expressed allele of the M6p/Igf2r in 40% of diethylnitrosamine-initiated rat liver tumors. These results provide further evidence that the M6P/IGF2R functions as a liver tumor suppressor gene. They also suggest that mice and rats would be more sensitive than humans to those hepatocarcinogens in which the M6p/Igf2r is mechanistically involved in transformation since one rather than two alleles would need to be inactivated.     

Liver tissue schistosomiasis mansoni vaccine, as a third generation livestock immunogenes

To assess the effects of cholestasis during pregnancy on fetal and neonatal mRNA expression, protein mass, and function of the Na+/taurocholate cotransporting polypeptide (Ntcp), common bile duct ligation (BDL) was performed in pregnant rats on day 14 of pregnancy (maternal cholestasis [MC] group), and livers were harvested at days 20 and 21 of fetal life, as well as at days 4, 7, 14, 21, and 28 after birth. Sham-operated rats and their litters were used as controls. Ntcp steady-state mRNA levels, protein mass, and function were determined by Northern blotting, immunoblotting, and taurocholate (TC) transport studies in isolated short-term cultured hepatocytes, respectively. In addition, protein mass and function of the organic anion transporting polypeptide (Oatp1), another sinusoidal bile acid transporter, were studied at 4 weeks of age. The majority of pregnant cholestatic rats (94%) were able to carry pregnancy to term. Body and liver weights of the offspring from the MC group were lower than those from sham-operated animals at all postnatal time points. Ntcp steady-state mRNA levels, protein mass, and function were unaffected by MC. The ontogenic pattern of expression was identical in offspring from MC and controls with detection of the Ntcp mRNA at day 21 of fetal life. There was a significant increase in mRNA postnatally, reaching adult levels by 7 days of age. Protein mass and function of Ntcp as well as of Oatp1 were similar in offspring from MC and control groups at 4 weeks of age. In conclusion, maternal obstructive cholestasis during the last third of pregnancy does not affect the fetal/neonatal expression of the basolateral bile acid transporters, Ntcp and Oatp1. This suggests that the impaired bile acid excretion described in this experimental model is not related to altered uptake of bile acids in the affected neonate.     

The genetics of labor

The immunomodulatory effects of the antibiotic sodium fusidate (SF) were tested in a model of T cell-dependent hepatic injury that can be induced in normal mice by a single i.v. injection of Con A. Signs of hepatitis with elevated transaminase activities in plasma, severe infiltration of the liver by neutrophil granulocytes, lymphocytes and monocytes, and necrotic areas were observed in control mice treated intraperitoneally with PBS 24 h and 1 h before Con A challenge. T cell- and macrophage-derived cytokines (IL-2, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha, IL-1beta, IL-6) were released with different kinetics in the circulation of these mice. SF, 20, 40 or 80 mg/kg, administered 24 h and 1 h before Con A challenge, protected the mice against the hepatitic effects of Con A. The protective effects of SF were dose-dependent and accompanied by profound modifications of blood levels of cytokines induced by Con A, so that, relative to control mice, SF (80 mg/kg)-treated animals showed markedly diminished plasma levels of IL-2, IFN-gamma and TNF-alpha, along with augmented levels of IL-6. These results suggest that SF might be useful in the treatment of immunoinflammatory liver diseases in humans.     

Apoptosis in the pancreatic islet cells of the neonatal rat is associated with a reduced expression of insulin-like growth factor II that may act as a survival factor

Islet cell ontogeny will define adult beta-cell mass and will consist of a balance of islet cell birth and death. We have investigated the ontogeny of factors that may be related to developmental apoptosis in the islets, insulin-like growth factor II (IGF-II) and inducible nitric oxide synthase (iNOS), in pancreata of young Wistar rats. Pancreata were collected from rats of 21 days gestation to 29 days postnatal age. In situ hybridization and immunohistochemistry showed that IGF-II was expressed and present in fetal and neonatal islet cells, but declined rapidly 2 weeks after birth. Little IGF-I was associated with fetal or postnatal islets. Apoptosis in islet cells was visualized by molecular histochemistry for DNA breakage in tissue sections. Apoptosis was low in the fetus, but increased in incidence postnatally so that 13% of islet cells were undergoing apoptosis on postnatal day 14, with the incidence declining thereafter. Immunohistochemistry for iNOS showed that it was expressed within beta-cells and was most abundant 12 days after birth. When islets were isolated from rat pancreata 20-22 days after birth, islet cell viability, DNA synthetic rate, and insulin release were reduced after incubation with interleukin-1beta, tumor necrosis factor, or interferon-gamma. An increased rate of islet cell survival was found after simultaneous incubation with IGF-I or -II. Cytokine-mediated islet cell death involved the induction of apoptosis. Islets isolated from neonatal rats were not killed after exposure to these cytokines at the same concentrations, but cytokine-induced cell death was seen when neonatal islets were incubated with a neutralizing antibody against IGF-II. These experiments show that a peak of islet cell apoptosis that is maximal in the rat pancreas 14 days after birth is temporally associated with a fall in the islet cell expression of IGF-II. IGF-II was shown to function as an islet survival factor in vitro. The induction of islet cell apoptosis in vivo may involve an increased expression of iNOS within beta-cells.     

Transferrin is an early marker of hepatic differentiation, and its expression correlates with the postnatal development of oligodendrocytes in mice

Transferrin (Tf), the iron transport protein, is essential for the growth and differentiation of cells. Therefore, it provides an excellent model to analyze the regulatory mechanisms controlling the expression of a eukaryotic gene in different cell types and during fetal and adult life. In this study, the tissue-specific and developmental regulation of the Tf gene in vivo were analyzed. Human Tf mRNA was detected mainly in fetal and adult liver. A weaker expression was observed in adult and fetal brain and in fetal spleen. By in situ hybridization the presence of mouse Tf mRNA was detected in the hepatic primordia. This is the first observation pointing out Tf as an early marker of hepatic differentiation, prior to the formation of the liver. Thus, TF may be an important tool to follow the hepatic specification of the gut endoderm. Mouse Tf mRNA was also detected in the liver bud and subsequently in the liver throughout fetal life, and in newborn and adult animals. No expression of the Tf gene was observed in the mouse fetal central nervous system (CNS). In contrast, Tf mRNA was detected from the 5th day after birth in the derivatives of the caudal part of the neural tube and subsequently in the derivatives of the rhomboencephalon and that of the prosencephalon. These results indicate that Tf gene expression correlates with the postnatal development of oligodendrocytes in the mouse CNS. To test whether the control elements of the human gene previously found in ex vivo experiments were also active in vivo during fetal and adult life, we fused the -4000/+395' flanking region of the human gene to the coding region of the lacZ gene and generated transgenic mice. The expression of the reporter gene during development was analyzed.     

Effects of maternal ethanol consumption in rats on basal and rhythmic pituitary-adrenal function in neonatal offspring

Neonatal corticoid treatment delays development of the circadian rhythm of plasma corticosterone in rats. We therefore sought to determine whether fetal or neonatal exposure to ethanol, a substance which activates the hypothalamo-pituitary-adrenal axis, produces similar effects. Subjects were the offspring of dams fed a 5.0% w/v ethanol-containing liquid diet or pair-fed an isocaloric control diet during gestation weeks two and three or during postnatal week one. At birth (day 1), the fetal ethanol-exposed pups had significantly higher brain and plasma corticosterone levels than the pair-fed or normal controls; brain and body weights were unaffected. By day 3, brain and plasma corticosterone titers in the fetal ethanol-exposed pups declined to the levels of the pair-fed and normal controls, although brain weights were significantly reduced. Significantly higher p.m. than a.m. levels of plasma corticosterone first occurred on day 18 both in the fetal ethanol-exposed pups and in the pair-fed and normal controls. Thus, despite its causing elevated corticosterone levels at birth, fetal exposure to ethanol did not affect the onset of the pituitary-adrenal circadian rhythm. On the other hand, exposure to ethanol during the first neonatal week delayed the onset of the pituitary-adrenal rhythm from day 18 to day 21. However, even greater delays occurred in the neonatal pair-fed controls, suggesting that the delays following neonatal exposure were due to nutritional deficits rather than to alcohol per se. The developmental and long-term influences of elevated corticoid levels at birth in fetal ethanol-exposed rats on other aspects of pituitary-adrenal function remain to be determined.     

Somatostatin is expressed in FRTL-5 thyroid cells and prevents thyrotropin-mediated down-regulation of the cyclin-dependent kinase inhibitor p27kip1

A novel gene, jumonji was identified by a mouse gene trap strategy. The jumonji gene encodes a protein containing a putative DNA binding domain. The mice homozygous for jumonji gene with a BALB/cA genetic background show hypoplasia of the fetal liver and embryonic lethality, suggesting impaired hematopoiesis. In the peripheral blood of jumonji mutant embryos, the number of fetal liver-derived definitive erythrocytes, but not yolk sac-derived primitive erythrocytes, showed a marked reduction, suggesting that jumonji mutants die of anemia. The defects of definitive erythrocytes in jumonji mutants seemed to be caused by a decrease in the numbers of multiple hematopoietic progenitors including colony-forming unit-spleen (CFU-S) in the fetal liver. However, hematopoietic stem cells (HSCs) in the fetal liver of jumonji mutants could reconstitute the hematopoietic system of lethally irradiated recipients. In the fetal liver, the jumonji gene is expressed in fibroblastic cells and endothelial cells, but not in Lin-/c-Kit+/Sca-1(+) cells known to include HSCs. These results suggest that an environmental defect induce the impaired hematopoiesis in the fetal liver of jumonji mutant embryos.     

Ontogeny of the heavy chain immunoglobulin repertoire in fetal liver and bone marrow

We studied the kinetics of maturation of B cell progenitors in the mouse embryo, from day 15 of development to birth, both in liver and bone marrow. The analysis of Ig heavy chain rearrangements at different time points of late fetal development shows that oligoclonal patterns of V(H)-D-J(H) rearrangements are detected by day 15 in fetal liver. The pattern is polyclonal and diverse by day 17; however, 80% of the rearrangements are nonproductive. In bone marrow, the pattern of rearrangements is less diverse at birth, although the percentage of nonproductive rearrangements approaches adult bone marrow levels (35-40%). After day 17 in fetal liver, there is a sudden reversal in the percentage of nonproductive rearrangements that reaches 33% at day 19 (birth). Maturation of B cells, as measured by the fraction of surface Ig+ in total B220+ cells and the presence of N sequence additions in V(H)-D-J(H) joints, occurs in the marrow before fetal liver. These results demonstrate that the lymphopoietic environment in fetal liver and bone marrow of animals at the same stage of development is functionally distinct.     

Hematopoietic remodeling in interferon-gamma-deficient mice infected with mycobacteria

Control of intracellular bacterial infections requires interferon-gamma (IFN-gamma) both for establishing a Th1 T-cell response and for activating macrophages to kill the bacteria. Exposure of mice deficient in IFN-gamma to mycobacterial infection produces an immune response characterized by a Th2 T-cell phenotype, florid bacterial growth, and death. We report here that IFN-gamma-deficient mice infected with mycobacteria also undergo a dramatic remodeling of the hematopoietic system. Myeloid cell proliferation proceeds unchecked throughout the course of mycobacterial infection, resulting in a transition to extramedullary hematopoiesis. The splenic architecture of infected IFN-gamma-deficient mice is completely effaced by expansion of macrophages, granulocytes, and extramedullary hematopoietic tissue. These features coincide with splenomegaly, an increase in splenic myeloid colony-forming activity, and marked granulocytosis in the peripheral blood. Systemic levels of cytokines are elevated, particularly interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF). These results suggest that in addition to its central role in cellular immunity, IFN-gamma may be a key cytokine in coordinate regulation of immune effector cells and myelopoiesis. This model should be valuable for deciphering the cross-talk between the immune response and hematopoiesis during bacterial infection and for improving our understanding of the mechanisms that control chronic infections.     

Histochemical demonstration of neuronal nitric oxide synthase during development of mouse respiratory tract

Several studies, including histochemical ones, have indicated that nitric oxide (NO) of endothelial origin may be related to the pulmonary vasodilation that occurs at birth. Since no histologic studies have been done of the possible parallel perinatal increase in production of neuronal NO synthase (nNOS) by pulmonary nerve plexuses, we investigated the distribution of nNOS in fetal, neonatal, and adult mouse lung. Lungs from mice aged 13 d gestation to 6 d after birth and lungs of adults were studied through histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and immunocytochemistry. Both techniques gave almost similar results in relation to time of appearance, distribution, and frequency of neural structures positive for NADPH-d and NOS. NADPH-d staining was also applied to whole mounts of developing and adult tracheae. Staining was found from gestational days 13 to 15 onward in a small portion of the neuronal population. In all stages studied, NADPH-d/NOS staining was found in neuron cell bodies in the hilar region and bronchiolar wall, as well as in neuronal processes. Labeled terminal nerve fibers with varicosities were more frequent in pulmonary blood vessels than in airways. In tracheae, similar NADPH-d/NOS-positive nerve plexuses were found. The presence of nNOS in fetal and neonatal mouse respiratory tract suggests that neurally derived NO must play a role in developing lung physiology. However, because no perinatal increase in the number or intensity of staining of nNOS-positive nerve structures was seen, no apparent relation between neural NO and vasodilation can be established at birth.     

Further evidence for the presence of cannabinoid CB1 receptors in mouse vas deferens

Basophil granulocytes and their mediators are involved in the pathogenesis of allergic inflammation. We evaluated basophil count, blood histamine content, eosinophil count and serum total IgE levels in one hundred-thirteen healthy newborns at birth. 108 children were prospectively studied with a follow-up to 18 months of age for development of topic disorders. No difference was found in newborns with biparental family history of atopy (FHA) in comparison with newborns with monoparental FHA and with newborns without FHA. Children who developed atopic disorders had neonatal basophil count higher than those who did not develop atopic symptoms (p = 0.03). No significant correlation was found between basophil and eosinophil counts (r = 0.013), between basophil count and serum total IgE levels (r = 0.012) and between basophil count and blood histamine content. Positive predictive value and sensitivity of basophil count for allergy up to 18 months of age was respectively only 33% and 27%. Our data indicate that an increased basophil count at birth is not associated with FHA and is not a good predictive marker of atopy.     

IL-12 is a potent neonatal vaccine adjuvant

Neonatal animals show generally poor responsiveness to foreign antigens and are known to display polarized expression of Th2-like cytokines and antibody responses. We now report that newborn mice display a reduction in peripheral expression of the Th1-inducing cytokine, IL-12. Attempts to overcome this decrease by immunization and treatment with IL-12 within 24 h of birth resulted in elevated levels of IFN-gamma and IL-10 mRNA in the spleens of mice compared to animals exposed to antigen only. Moreover, such animals showed dramatic enhancement of IgG2a and IgG2b antibody levels upon adult challenge compared to mice primed with antigen alone. These effects appeared to be due to induction of neonatal B cell memory. IgG1 antibody levels, a measure of Th2 activity, were unaffected or even somewhat enhanced by neonatal IL-12 treatment. Taken together, these results provide evidence that IL-12 administration induces a Th1-like cytokine response in newborns and causes priming for heightened memory antibody responses in vivo. Our findings suggest the use of IL-12 as a vaccine adjuvant in neonates for inducing protection against common childhood pathogens.