Fetal Liver Tissue Research Articles

Disposition of Polychlorinated Dibenzo-p-dioxins, Dibenzofurans, and non-ortho Polychlorinated Biphenyls in Pregnant Long Evans Rats and the Transfer to Offspring

Pharmacokinetic properties of polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDFs), and non-ortho biphenyls (PCBs) play a critical role in their relative toxicity. The present study examined the transfer of these chemicals to offspring and placenta. Pregnant Long Evans rats received 0.0 (control), 0.05, 0.2, 0.8, and 1.0 μg/kg of dioxin toxic equivalence (TEQ) by oral gavage on the 15th gestational day (GD 15), using a dosing mixture that contained 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), octachlorodibenzofuran (OCDF), 3,3′,4,4′-tetrachlorobiphenyl (PCB 77), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), and 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169) in ratios approximating that in food. Rats were euthanized on GD 16, GD 21, and postnatal day 4 (PND 4). The chemical concentrations in fetus, pup, placenta, and maternal liver, serum, and adipose tissue were determined using gas chromatography/high-resolution mass spectrometry. A dose-dependent increase in hepatic sequestration was seen with TCDD, PeCDD, 4-PeCDF, OCDF, PCB 126, and PCB 169, and the transfer to offspring was reduced at higher doses. 4-PeCDF, PeCDD and PCB 126 showed higher liver affinity than TCDD. TCDF, 1-PeCDF, and PCB 77 were metabolized rapidly. On GD 16, TCDD and the three PCBs reached equilibration between the fetus and placenta, but this did not occur with PeCDD and 4-PeCDF until GD 21, according to the lipid-based concentrations. Offspring compartments received more of the dosed compounds lactationally than transplacentally (7−28% versus 0.5−3%). The behavior of each congener was dose-dependent; therefore, extrapolation of high-dose experimental data should be used with caution.

Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal hepatocytes in primary culture: suggestive molecular mechanisms for in utero fetal growth restriction caused by uteroplacental insufficiency.

Intrauterine growth restriction (IUGR) can be a consequence of decreased uterine blood flow (uteroplacental insufficiency) and maternal and fetal hypoxia. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are key elements in fetal growth. IGF-I is a major growth promoter in utero. IGFBP-1 is primarily made in the liver, and it mostly inhibits IGF actions at the cellular level. IGFBP-1 is elevated in the fetal circulation of human and animal pregnancies complicated by IUGR caused by placental insufficiency and in utero hypoxia and is believed to restrict fetal growth by sequestering IGFs. In this study, we developed a protocol to establish highly pure primary cultures of human fetal hepatocytes in vitro and investigated their expression of IGFBP-1 messenger RNA (mRNA) and protein and the effects of hypoxia on their expression of IGFBP-1 mRNA and protein. Hepatocytes were isolated from second-trimester human fetal livers (n = 7) and purified by Percoll gradient centrifugation. Hepatocyte cultures were characterized by immunocytochemistry and were compared with hepatocytes in situ in human fetal liver tissue, by immunohistochemistry, using specific antibodies and indirect immunofluorescence. Cultures consisted primarily (>90%) of cells positive for cytokeratin 18, fibrinogen, and IGFBP-1, with less than 2% vascular cells and less than 8% macrophages. Identification of isolated hepatocytes was further confirmed by morphology. Hepatocytes were cultured in defined medium, and Northern analysis revealed expression of a 1.5-kb IGFBP-1 mRNA transcript in hepatocytes cultured under normoxic conditions, for 24 h, that did not increase in steady-state levels after 48 h in culture. Under hypoxic conditions (2% O(2)), IGFBP-1 mRNA expression increased 3- to 4-fold, compared with normoxic controls. Cells cultured under 10% O(2) did not demonstrate an increase in IGFBP-1 mRNA levels. IGFBP-1 protein in conditioned medium (CM) was measured by immunoradiometric assay and increased 3- to 4-fold under hypoxic (2% O(2)), compared with normoxic, conditions. Western ligand blot analysis of CM revealed the presence of IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. IGFBP-1 was the most abundant IGFBP in CM, and densitometric analysis revealed a 2.5-fold increase in IGFBP-1 under hypoxic, compared with normoxic, conditions, supporting the immunoradiometric assay results. A 3-fold increase in IGFBP-3 mRNA, but not other IGFBPs, was noted under hypoxic, compared with normoxic, conditions. This study demonstrates that human fetal hepatocytes can be cultured in defined medium, as primary cultures with high purity, and that they express IGFBP-1 mRNA and secrete IGFBP-1 protein in vitro. In addition, the data demonstrate that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, with this being the major IGFBP derived from the fetal hepatocyte. The data support a role for the fetal liver as a source of elevated circulating levels of IGFBP-1 in fetuses with in utero hypoxia and IUGR.

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT.

Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.

The Haspin gene: location in an intron of the Integrin αE gene, associated transcription of an Integrin αE-derived RNA and expression in diploid as well as haploid cells

Haspin is a serine/threonine kinase, recently identified in mice, that is thought to regulate cell cycle and differentiation of haploid germ cells. Here, the haspin gene is identified within an intron of the integrin αE gene. Transcription occurs from a bi-directional CpG island-associated promoter that also generates an alternatively spliced integrin αE derived RNA. Remarkably, the human and murine haspin genes lack introns, and have features of retroposons. The human haspin cDNA reveals that the human and murine proteins are 83% identical in the C-terminal kinase domain, but only 53% identical in the N-terminal region. The haspin kinase domain has structural features that distinguish it from previously characterized proteins and suggest that haspin is a member of a new family of protein kinases. Although formerly thought to be expressed selectively in the testes, haspin is also transcribed at lower levels in thymus, bone marrow, fetal liver and other fetal tissues, and in all proliferating cell lines tested. Thus haspin is likely to be important in regulation of diploid as well as haploid cell differentiation in a variety of tissues.

Expression of cytochrome P-450 1A1 in human fetal adrenal cells

Objective: To study the expression and inducibility in vitro of adrenal cytochrome P-450 (CYP) 1A1 implicated in precarcinogen and premutagen activation in human fetuses. Methods: CYP1A1 mRNA expression and CYP1A1 activity of fetal adrenals were studied and compared with those of fetal liver using the reverse transcriptase-polymerase chain reaction (RT-PCR) and 7-ethoxyresorufin O-deethylase (EROD) assay. Results: 3-methylcholanthrene (3MC, 25 mg/kg ip, qd×3), a selective inducer of CYP1A1, could enhance adrenal and liver EROD activities in vivo in adult rat. However, EROD activity was undetected in either adrenal microsomes or 3MC (0.5–2 µmol · L−1 for 24 h)-treated adrenal cells in vitro in human fetuses. Using RT-PCR method, the expression of CYP1A1 mRNA was detected in fetal adrenal and fetal liver tissues and the expressional percentage was 78% and 73%, respectively. Conclusion: Human fetal adrenals as well as fetal liver had the lower CYP1A1 expression and possessed the potential for risk of carcinogenesis during fetal development.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 mediated monooxygenase functions and on oxidative state

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with different cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ], carbon tetrachloride [CCl4], or thioacetamide [TAA]) or the respective solvents 24 or 48 hours before sacrifice. Effects of the cytotoxins on P450 mediated monooxygenase functions in liver and spleen 9,000 g supernatants were assessed by measuring the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), pentoxyresorufin O-depentylation (PROD), and ethylmorphine N-demethylation (EMND). Additionally, the influence on the oxidative state was investigated by assessing the liver and spleen tissue content of lipid peroxidation (LPO) products and of reduced and oxidized glutathione (GSH;GSSG). The livers of both solvent treated transplant recipients and control rats displayed regular EROD, ECOD, PROD and EMND activities. After AAL treatment EROD and EMND activities within the livers were not affected, but ECOD and PROD activities were increased. BBZ administration caused a decrease in EROD and EMND activities, ECOD activity remained unaffected, and PROD activity was even increased. CCl4 and TAA administration caused a strong reduction in the activity of all four model reactions. Spleens of control rats displayed almost no P450 mediated monooxygenase functions, independent whether the rats had been treated with the cytotoxins or not. In the transplant containing spleens, however, significant EROD and ECOD, but hardly any PROD or EMND activities were seen. After AAL administration EROD activity was not affected in the transplant containing spleens, but ECOD activity was increased. BBZ treatment led to a decrease in EROD and an elevation in ECOD activity. CCl4 and TAA strongly reduced the activity of both of these model reactions. The tissue content of LPO products within livers and transplant containing spleens was significantly increased after BBZ and CCl4 treatment. An elevation in LPO products was also seen in the spleens of the control rats due to CCl4 administration. Tissue GSH and GSSG content in both livers and transplant containing spleens were strongly reduced after BBZ treatment. After CCl4 administration only a significant decrease in liver GSSG contents was seen. TAA treatment caused a reduction in the GSH and GSSG content in the spleens of both transplant recipients and control rats, but not in the livers. From these results it can be concluded, that the effects of cytotoxins like AAL, BBZ, CCl4 or TAA on P450 dependent monooxygenase functions and on oxidative state are exerted in the ectopic intrasplenic liver cell transplants in a similar way as in normal orthotopic liver.

Expression of Bisecting N-Acetylglucosaminyltransferase-III in Human Hepatocarcinoma Tissues, Fetal Liver Tissues, and Hepatoma Cell Lines of Hep3B and HepG2

In this paper, uridine diphosphate (UDP)-N-acetylglucosamine/β-D-mannoside β-1,4 N-acetylglucosaminyltransferase III (GlcNAc-transferase-III C 2.4.1.144) activity was determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. GlcNAc-transferase-III enzymes of Hep3B and HepG2 were mainly detected in the membrane fraction. When GlcN,GlcN-biant-PA and UDP-GlcNAc were used as substrates, the Km values (4.7 mM for UDP-GlcNAc and 1.1 mM for GlcN,GlcN-biant-PA) of Hep3B GlcNAc-transferase-III were distinguishable from those of HepG2 GlcNAc-transferase-III (6.8 mM for UDP-GlcNAc and 3.4 mM for GlcN, GlcN-biant-PA). Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1423 pmol/hr/mg) than that of HepG2 (1066 pmol/hr/mg). Normal liver cells and primary adult hepatocytes are characterized by a very low level of GlcNAc-transferase-III activity, whereas human hepatoma cells exhibited high activities. These data were supported by reverse transcription-polymerase chain reaction results, showing that expression of the GlcNAc-transferase-III mRNA increased in proportion to the enzymatic activities. Although the mechanism underlying the induction of this enzyme is unknown, lectin blot analysis showed that oligosaccharides in many glycoproteins were observed in hepatoma cells. By treating hepatocarcinoma cultures that express GlcNAc-transferase-III with inhibitors (tunicamycin, deoxymannojirimycin, and swainsonine) of different steps of the glycosylation, we provide evidence that expression of GlcNAc-transferase-III mRNA is dependent on glycosylation of cellular proteins.

An investigation into the feasibility of fetal lung maturity prediction using statistical textural features.

Fetal lung and liver tissues were examined by ultrasound in 240 subjects during 24 to 38 weeks of gestational age in order to investigate the feasibility of predicting the maturity of the lung from the textural features of sonograms. A region of interest of 64 x 64 pixels is used for extracting textural features. Since the histological properties of the liver are claimed to remain constant with respect to gestational age, features obtained from the lung region are compared with those from liver. Though the mean values of some of the features show a specific trend with respect to gestation age, the variance is too high to guarantee definite prediction of the gestational age. Thus, we restricted our purview to an investigation into the feasibility of fetal lung maturity prediction using statistical textural features. Out of 64 features extracted, those features that are correlated with gestation age and less computationally intensive are selected. The results of our study show that the sonographic features hold some promise in determining whether the fetal lung is mature or immature.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 isoforms expression and on glycogen content

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with different cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ], carbon tetrachloride [CCl4], or thioacetamide [TAA]) or the respective solvents 24 or 48 hours before sacrifice. Effects of the cytotoxins on the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers were assessed by immunohistochemistry. Additionally, effects on glycogen content within the hepatocytes were examined. In the livers AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ led to a perivenous necrosis of single cells only, whereas CCl4 and TAA caused complete necrosis of the centrilobular parenchyma. Treatment with each of the four cytotoxins led to necrosis and fatty degeneration of single or groups of hepatocytes within the intrasplenic transplants. This effect was most pronounced with CCl4 and TAA. The orthotopic livers of both solvent treated transplant recipients and control rats displayed only in few lobules a slight P450 1A1, but in all lobules a strong P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. AAL administration led to an increase in the P450 2B1 expression in the perivenous hepatocytes, whereas the staining for P450 1A1 was not affected and that for P450 3A2 in the periportal areas was even decreased. BBZ administration caused a P450 1A1 expression in the periportal hepatocytes but a decrease in this staining of the perivenous cells. The number of hepatocytes positively stained for P450 2B1 and 3A2 in the perivenous and intermediate zones was diminished in comparison to the livers of solvent treated rats. TAA and, more pronounced, CCl4 administration caused a strong reduction in the expression of all three P450 isoforms. Spleens of control rats displayed almost no P450 isoforms expression, independent of the treatment with the cytotoxins. Similar to adult liver, the hepatocytes in the transplant containing spleens showed nearly no P450 1A1, but a noticeable P450 2B1 and 3A2 expression. No staining was observed within the bile duct cells of the intrasplenic transplants. AAL administration slightly reduced the P450 2B1 and 3A2 expression in the transplants. BBZ and, much more pronounced, CCl4 and TAA treatment diminished the staining for all three P450 isoforms. AAL administration led to a marked decrease in the glycogen content of the hepatocytes of the periportal zones of the liver lobules, whereas after BBZ, CCl4 and TAA treatment a strong perivenous reduction in the glycogen content was seen. Similarly, within the intrasplenic transplants a remarkable decline in the glycogen content of the hepatocytes was caused by the treatment with each of the four cytotoxins. Especially after AAL and BBZ treatment the glycogen depletion within both livers and transplants was much more pronounced than the effects on morphology or P450 isoforms expression. It can be concluded that the effects of cytotoxins like AAL, BBZ, CCl4 or TAA seen in normal orthotopic liver are exerted in a similar way also in intrasplenic liver cell transplants.

Effect of cytokines on HIV-induced depletion of thymocytes in vivo.

Cytokines play an important role in the differentiation of thymocytes into mature T cells; consequently, certain cytokines could be useful for immune reconstitution after HIV infection without increasing viral load. To investigate whether cytokines affect immune depletion caused by HIV infection with a CXCR4-tropic strain in SCID-hu mice implanted with human fetal thymus and liver (thy/liv) tissue. The thy/liv implants were either mock infected or infected with HIV-1 NL4-3, a CXCR4-tropic molecular clone. Interleukin (IL)-2, IL-4, IL-7, interferon-gamma (IFN-gamma) or diluent was administered to the mice during the second and third week postinfection. Viral load and immunophenotype were determined in thymocytes. Thymocyte subset distributions at 3 weeks postinfection were significantly influenced by treatment with certain cytokines. In particular, IL-2 caused the infected mice to retain a thymocyte profile that was more similar to that in mock-infected mice than that in diluent-treated infected mice, in that the percentages of immature CD4+CD8+ and CD5+CD1+ cells were slightly higher and much less variable than in diluent-treated infected mice. The effect of IFN-gamma treatment was similar to IL-2 but did not reach statistical significance. However, after IFN-gamma treatment, normal percentages of mature CD3+CD69+ cells were maintained whereas this population was relatively increased in diluent-treated infected mice. Although treatment with IL-4 and IL-7 delayed depletion of immature thymocytes, these cytokines increased viral load. Cytokines such as IL-2 and IFN-gamma maintain immature thymocytes without increasing viral load and may be useful as adjuncts to improve immune reconstitution after HIV infection.

Developmentally regulated expression of organic ion transporters NKT (OAT1), OCT1, NLT (OAT2), and Roct.

Several xenobiotic (organic cation and anion) transporters have recently been identified, although their endogenous substrates, if such exist, remain unknown. When we initially identified NKT, also known as OAT1, the first member of the organic anion transporter (OAT) family (Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, and Nigam SK. J Biol Chem 272: 6471-6478, 1997), we noted its expression in the embryonic kidney. We have now demonstrated its transporter function and more fully examined the spatiotemporal expression patterns of representative organic ion transporters, [NKT (OAT1), Roct, OCT1, and NLT, also known as OAT2] during murine development. In the kidney, NKT (OAT1), OCT1, and Roct transcripts appeared at midgestation, coinciding with proximal tubule differentiation, and gradually increased during nephron maturation. A similar pattern was observed for NLT (OAT2) in the liver and kidney, although, in the kidney, NLT (OAT2) transcription did not increase as dramatically. The roughly cotemporal expression of these related transporters in the developing proximal tubule may indicate common transcriptional regulation. Expression during embryogenesis in extrarenal sites could suggest a role in the formation and maintenance of nonrenal tissues. Importantly, all four genes were expressed in unexpected places during nonrenal organogenesis: Roct in the fetal liver (temporally coinciding with the onset of hematopoiesis) and neural tissue; NKT (OAT1) in the fetal brain; OCT1 in the ascending aorta and atrium; and NLT (OAT2) in the fetal lung, intestine, skin, and developing bone. Because these gene products mediate the transport of a broad range of metabolites and toxins, it seems likely that, apart from their known functions, these transporters play a role in transport of organic molecules, perhaps including those with morphogenetic activity. These genes could also play important developmental roles independent of transport function.

The induction of specific pig skin graft tolerance by grafting with neonatal pig thymus in thymectomized mice.

Xenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymus and liver tissue (FP THY/LIV) to thymectomized (ATX), T/NK cell-depleted mice. By using neonatal pig tissue, we hoped to overcome two obstacles that arise with the use of fetal pig tissue: (1) the inability to keep fetal pigs alive after harvesting their thymic tissue, resulting in unavailability of their skin or other organs for grafting; and (2) the limited fetal thymic tissue yield, making application to large animals and humans more difficult. Neonatal pig thymus tissue (NP THY) was grafted into ATX, T/NK cell-depleted, 3Gy whole body-irradiated, originally immunocompetent B6 mice to evaluate the ability of NP THY to reconstitute mouse CD4+ T cells and to induce xenogeneic tolerance to donor pig skin grafts. Repopulation of mouse CD4+ T cells in the peripheral tissues was observed in T/NK cell-depleted, ATX B6 mice that received NP THY with or without neonatal pig spleen (NP SPL), but not in those receiving NP SPL alone, indicating that pig thymus grafting was necessary and sufficient for mouse T cell recovery. Seven of nine NP THY/SPL-grafted ATX mice and two of six NP THY-grafted ATX mice that reconstituted >5% CD4+ cells in PBL accepted donor pig skin long-term without lymphocyte infiltration, whereas they rejected allogeneic BALB/c skin and third party pig skin grafts as rapidly as euthymic mice. NP THY can support the development of mouse CD4+ T cells that are functional and specifically tolerant to donor pig antigens in ATX, T/NK cell-depleted, 3 Gy whole body-irradiated, originally immunocompetent B6 mice. Additional grafting of NP SPL with NP THY improves the efficiency of tolerance induction in this model.

Human cystathionine γ-lyase: developmental and in vitro expression of two isoforms

Cystathionine gamma-lyase (CGL) is the last enzyme of the trans-sulphuration pathway, which converts methionine into cysteine. To study the possible differences in enzymic activity of the two human cystathionine gamma-lyase isoforms characterized earlier, these were separately expressed in human kidney embryonic 293T cells. Furthermore, developmental changes in the expression of the two mRNA forms as well as the enzymic activity in human liver were studied, as it has been postulated that a change in the relative expression of CGL isoforms causes the postnatal increase in CGL activity. Transfection with the longer isoform increased the CGL activity 1.5-fold, while the activity of the cells transfected with the shorter form did not differ from the basal activity. In human liver samples, CGL activity was only detected in adult tissue (68+/-9 nmol of cysteine/h per mg of protein), whereas activity in fetal, premature and full-term neonatal liver tissue was undetectable. In contrast, strong mRNA expression of both mRNA isoforms was detected from the 19th gestational week onwards and the longer form of CGL appeared to be predominant. The expression of the two mRNA forms varied in parallel. In conclusion, we have shown that only cells overexpressing the longer form of CGL have increased activity, and CGL appears to be regulated at the post-transcriptional level during development.

Mixed Lymphocyte Culture of Human Fetal Liver Cells

Objective: In order to study the immunological function of the human fetus in the first and second trimesters, mixed lymphocyte culture (MLC) of fetal liver and thymic cells was performed. MLC is a functional test to determine human lymphocyte antigen-D incompatibilities. Methods: Human fetal liver and thymic tissue was obtained from abortions in gestational weeks 7–17.5. Forty-seven fetuses were studied with one-way MLC. The cells were stimulated by adding irradiated fetal liver cells, adult bone marrow and peripheral blood lymphocytes. The activity was measured as DNA incorporation of radiolabeled thymidine. Results: The results indicate that the human fetus is competent to react as early as 11–12 weeks of gestation and in some cases even earlier. In very immature fetal livers (< 8 weeks), the MLC seems to be inhibited. Conclusions: Our data suggest that the human fetus can react against foreign transplantation antigens earlier than previous papers have claimed. The onset of reactivity seems to differ considerably among fetuses. The present findings may explain some of the limited success of in utero transplantations of hematopoietic stem cells in human fetuses of normal immunological status.

Human immunodeficiency virus type 1-induced hematopoietic inhibition is independent of productive infection of progenitor cells in vivo.

Human immunodeficiency virus (HIV)-infected individuals exhibit a variety of hematopoietic dysfunctions. The SCID-hu mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues) can be used to model the loss of human hematopoietic precursor cell function following HIV infection and has a distinct advantage in that data can be obtained in the absence of confounding factors often seen in infected humans. In this study, we establish that HIV type 1 (HIV-1) bearing a reporter gene inserted into the viral vpr gene is highly aggressive in depleting human myeloid and erythroid colony-forming precursor activity in vivo. Human CD34(+) progenitor cells can be efficiently recovered from infected implants yet do not express the viral reporter gene, despite severe functional defects. Our results indicate that HIV-1 infection alone leads to hematopoietic inhibition in vivo; however, this effect is due to indirect mechanisms rather than to direct infection of CD34(+) cells in vivo.

An evaluation of the tissue expression of a bovine dodecapeptide bactenecin in the adult and fetal animal

Various antimicrobial cationic peptides have been isolated from the granules of bovine neutrophils. One of these peptides is only 12 amino acids in length and is called a dodecapeptide bactenecin. A monoclonal antibody to the synthetic bovine dodecapeptide bactenecin was produced, characterised and used in an immunofluorescent assay to assess the peptide's expression. When adult bovine peripheral blood and both adult and fetal bone marrow, tracheal lining, liver, spleen and intestinal tissues were examined, the peptide was found only within neutrophils and cells within the bone marrow. These findings both confirm and supplement another report in the literature (Storici et al. 1992), however this is the first report of a bovine antimicrobial peptide detected within the bone marrow of fetal mammals. Our results also suggest the use of immunofluorescence assays to be especially well suited for the quick screening of antimicrobial peptide tissue expression.

Transplantation of CD34 human cells into mice with severe combined immunodeficiency results in functional T cells 4 weeks after transplantation

Objective: Our purpose was to determine whether transplantation of fetal human CD34 + cells into mice with severe combined immunodeficiency results in functional T cells. Study Design: The cells used in this study were isolated from fetal human liver tissue obtained after elective termination of normal 18- to 24-week pregnancies. Women with medical conditions that could confound the outcome were excluded. Cells were labeled with fluorochrome-conjugated antibodies that recognized CD34 or other cell surface antigens. The cells were then sorted with the use of a fluorescein-activated cell sorter. The human sorted cells were injected intraperitoneally in mice with severe combined immunodeficiency. Four groups of mice were studied: group 1, injected with 10 5 CD34 + cells (n = 17); group 2, injected with 10 5 CD34 – cells (n = 14); group 3, injected with 10 6 unsorted cells (n = 19); and group 4, sham-injected with phosphate-buffered saline solution as controls (n = 14). At 1, 2, and 4 weeks after transplantation, the peripheral blood monocytes of the study mice were analyzed for functional T cells. Aliquots of cells (10 5) were incubated for 48 hours with 0, 5, 10, and 20 μg of phytohemagglutinin. Thereafter the cells were treated with 1 μCi of tritiated thymidine. Subsequently the incorporation of tritiated thymidine was determined by liquid scintillation counting. Results: Cells from mice transplanted with either unsorted cells, sorted CD34 + cells, or CD34 – cells showed a response to phytohemagglutinin that varied with time and with the mitogen concentration. Even though unsorted fetal human liver cells had a maximal response at 2 weeks, this posttransplantation response was not statistically significant. CD34 + cell response to phytohemagglutinin was significant at 4 weeks after transplantation. CD34 – cells also had a peripheral blood cell response at 4 weeks after transplantation; however, this response was not statistically significant. In addition, all mice transplanted with fetal human liver cells had some functional T cells at 4 weeks; however, this response was statistically significant only for CD34 + cells. Conclusion: Transplantation of either sorted CD34 (positive or negative) cells or unsorted fetal human liver cell preparations into mice with severe combined immunodeficiency results in functional T cells. However, only the mice with transplanted CD34 + cells demonstrated a statistically significant response. (Am J Obstet Gynecol 1999;181:80-6.)

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of various mitogens and cytotoxins on cytochrome P450 (P450) isoforms expression and on P450 mediated monooxygenase functions

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with various mitogens (fluorene [FEN], fluorenone [FON] and 2-acetylaminofluorene [AAF]) or cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ] and carbon tetrachloride [CCl4]) or the respective solvents 24 or 48 hours before sacrifice. The expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers was assessed by immunohistochemistry and P450 mediated monooxygenase functions in spleen and liver 9000 g supernatants by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (EMND). The orthotopic livers of both solvent treated transplant recipients and control rats displayed only in few lobules a slight P450 1A1, but in all lobules a moderate P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. Correspondingly, regular EROD, ECOD and EMND activities were observed. Each of the three mitogens increased the P450 1A1 expression in the hepatocytes of the perivenous zones of the liver lobules. FON administration caused an additional P450 1A1 immunostaining in the periportal areas, and AAF treatment a P450 1A1 expression in bile duct epithelia. Also the staining for P450 2B1 and 3A2 in the hepatocytes of the perivenous and intermediate zones of the liver lobules was intensified after treatment with any of the mitogens. The three model reactions were significantly increased within the livers after FEN and FON administration, whereas after AAF treatment only ECOD was enhanced, EROD remained unaffected and EMND was decreased. The cytotoxin AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ only produced a perivenous necrosis of single cells, whereas CCl4 caused complete necrosis of the centrilobular parenchyma. Immunohistochemically, AAL administration led to an increase in the P450 2B1 expression in the perivenous hepatocytes, whereas the staining for P450 1A1 was not affected and that for P450 3A2 was even decreased in the periportal areas. Due to AAL treatment EROD and EMND activities were not affected and ECOD activity was increased. BBZ administration caused a P450 1A1 expression in the periportal hepatocytes but a decrease in this staining of the perivenous cells. The number of hepatocytes positively stained for P450 2B1 and 3A2 in the perivenous and intermediate zones was diminished in comparison to the livers of solvent treated rats. After BBZ treatment, EROD and EMND activities were decreased, ECOD activity was not affected. CCl4 administration caused a strong reduction in the expression of all three P450 isoforms and in the activity of all three model reactions. Spleens of control rats displayed almost no P450 isoforms expression and P450 mediated monooxygenase functions, without as well as after treatment with the mitogens or cytotoxins. Similar to adult liver, the hepatocytes in the transplant containing spleens showed nearly no P450 1A1, but a noticeable P450 2B1 and 3A2 expression. No staining was observed within the bile duct cells of the intrasplenic transplants.

Prepubertal testicular teratomas.

Testicular teratomas in prepubertal children are distinct from adult testicular teratomas as well as from teratomas located elsewhere in the body, both with respect to their pathologic features and biologic behavior. Over a period of 12 years, testicular teratomas comprised 25% (5 of 20) of all testicular tumors in prepubertal children, treated at our institution. Their clinical presentation and pathologic profile was analysed. Their mean age at presentation was 6.7 m (range 1 m to 18 m) with the mass having been noted at birth in 3 infants. The right testis was involved in all except one patient. Serum AFP was elevated preoperatively in only one case with an immature (grade II) teratoma containing fetal liver tissue. Immunohistochemical staining for AFP and alfa-1-antitrypsin were positive in these fetal liver cells. Serum AFP levels returned to normal after orchiectomy in this patient. While a high inguinal orchiectomy was the standard operative procedure, testicular-sparing surgery was planned in the last two patients. However the tumor had replaced the entire testis in one of these patients, while in the other, the mass was homogeneous, with no visible cystic areas or dermal elements on cut section. Hence both of these patients also underwent orchiectomy. Histologic examination and immunohistochemical staining for AFP were performed on the specimen. Peri-tumor testicular parenchyma was found to be unremarkable and did not stain for placental alkaline phosphatase, a marker for carcinoma in situ. All these children are doing well with no evidence of residual or recurrent disease at a mean follow-up of 4.7 years (range 4 m to 11 yrs).

Free amino acids in human fetal liver and fluids at 12-17 weeks of gestation.

The concentration of 23 free amino acids was measured in homogenates of fetal liver and samples of fetal plasma from 20 pregnancies between 12 and 17 weeks gestation and compared with those found in matched samples of maternal plasma and amniotic fluid. A feto-maternal plasma concentration gradient was observed for 21 amino acids indicating that the feto-maternal amino acid gradient across the placenta is established from very early in pregnancy. The amino acid concentration pattern was similar in fetal plasma and amniotic fluid but different in fetal liver, supporting the concept that it is essentially placental transport and metabolism that provides the fetus with these molecules. The highest amino acid concentration was found for glutamine in fetal plasma and glutamic acid in fetal liver. Very low concentrations of glutamic acid in fetal plasma suggest that this amino acid is actively taken up by the fetal liver. Citrulline, alpha-aminobutyric acid, methionine, arginine and tryptophan were not measurable in fetal liver tissue, indicating that this organ has a limited role in utero in the metabolism of these amino acids. Significant positive correlations were found between fetal plasma and amniotic fluid for the concentration of most amino acids whereas only the concentration of threonine was found to be positively correlated between fetal liver and plasma. These results suggest that during the second trimester passive diffusion through the unkeratinized fetal skin is the main pathway for amino acids between the fetal circulation and the amniotic cavity.