Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages.

Little is known about the differentiation capabilities of nonhematopoietic cells of the human fetal liver. We report the isolation and characterization of a human fetal liver multipotent progenitor cell (hFLMPC) population capable of differentiating into liver and mesenchymal cell lineages. Human fetal livers (74-108 days of gestation) were dissociated and maintained in culture. We treated the colonies with geneticin and mechanically isolated hFLMPCs, which were kept in an undifferentiated state by culturing on feeder layers. We derived daughter colonies by serial dilution, verifying monoclonality using the Humara assay. hFLMPCs, which have been maintained in culture for up to 100 population doublings, have a high self-renewal capability with a doubling time of 46 h. The immunophenotype is: CD34+, CD90+, c-kit+, EPCAM+, c-met+, SSEA-4+, CK18+, CK19+, albumin-, alpha-fetoprotein-, CD44h+, and vimentin+. Passage 1 (P1) and P10 cells have identical morphology, immunophenotype, telomere length, and differentiation capacity. Placed in appropriate media, hFLMPCs differentiate into hepatocytes and bile duct cells, as well as into fat, bone, cartilage, and endothelial cells. Our results suggest that hFLMPCs are mesenchymal-epithelial transitional cells, probably derived from mesendoderm. hFLMPCs survive and differentiate into functional hepatocytes in vivo when transplanted into animal models of liver disease. hFLMPCs are a valuable tool for the study of human liver development, liver injury, and hepatic repopulation.

Construction of fetal charts for biparietal diameter, fetal abdominal circumference and femur length in Bangladeshi population.

Infants born for small for date (SFD) fetuses have an increased risk of perinatal mortality and morbidity. Different methods have been applied to identify these fetuses including history, clinical examination and ultrasonography. Ultrasonography has a better predictive value and majority of such fetuses can be identified. Measurements of the fetal biparietal diameter (BPD), abdominal circumference (AC) and femur length (FL) charts are widely used in dating pregnancies and follow-up of pregnant women in assessing fetal growth, identification of small for date (SFD) and growth retarded fetuses. This prospective study was performed to construct fetal chart for BPD, AC and FL at different gestational weeks from the Bangladeshi pregnant women. Seven hundred and ten women had ultrasonic measurements of fetal BPD, AC and FL between 12 to 42 weeks of pregnancy. Centiles, mean and the standard deviation (SD) were calculated for BPD, AC and FL. Mean maternal age was 24.73 +/- 4.48 (Mean +/- SD) and 310 (43.7%) were primigravidae. There was a gradual increase of the BPD (outer-inner), AC and FL measurements of 5th, 10th, 50th and 90th Centiles upto 38th weeks of gestation with a gradual increase of SD showing increasing dispersion of data. In cases of BPD and AC, After 38th weeks of gestation the Centiles showed a slower growth rate towards 42 weeks of pregnancy. This slower growth rate from 38 weeks of pregnancy was not noted in case of femur length. Fetal charts with the raw data for each measurement with superimposed fitted lines derived from polynomial (quadratic) regression were constructed. Quadratic model showed good fit to the data during construction of fetal charts. The new fetal measurement charts of BPD, AC and FL are unique for the Bangladeshi population and have not been found similar in the later weeks of pregnancy to those published for other Caucasian populations. These charts will help the clinicians and sonographers in dating pregnancy, identifying SFD and growth retarded fetuses.

Increased variability of paced finger tapping accuracy following repetitive magnetic stimulation of the cerebellum in humans.

Imaging and lesion studies suggest that the cerebellum is involved in the self-generation of timed motor responses. Using repetitive transcranial magnetic stimulation (rTMS), we studied the effects of transient disruption of the lateral or medial cerebellum on a paced-finger-tapping task (PFT). Results show greater variability on the PFT task following a 5 min train of 1 Hz rTMS to the medial cerebellum. Magnetic stimulation of the lateral cerebellum or motor cortex, and sham stimulation, had no effect on performance. Expanding the results of neuroimaging studies, these data show the causal link between activity in the medial cerebellum and the production of timed movements. This is the first demonstration of the feasibility of transiently disrupting the cerebellum by rTMS and inducing behavioral effects. This method of 'virtual lesions' can expand the study of the role of the cerebellum in motor control and cognition.

Posterior urethral valves: the scenario in a developing center.

We have reviewed 233 patients with posterior urethral valves treated in a single center in Calcutta, India, over the last 20 years: 37 were neonates, 75 were between 1 and 12 months, 88 were between 1 and 5 years, and 33 were more than 5 years old when first seen. The clinical presentation and methods employed in diagnosis and assessment are described. Primary endoscopic valve ablation was performed in 140 patients (60%). One or other form of diversion was done in 100 (43%), 93 before and 7 either during or after valve ablation. The short- and long-term results have been studied. Eleven patients died during the initial hospitalization, 3 died subsequently, 15 are in end-stage renal disease, 17 are in poor health, and 18 have been totally lost to follow-up. The remaining 169 have been in good health for periods between 1 and 20 years. While our results of primary valve ablation in low-risk patients with responsible parents are as good as anywhere else in the world, we are concerned at our relatively high diversion rate and relatively poor long-term follow up; the methods being adopted to reduce these problems are discussed.

The murine Pes1 gene encodes a nuclear protein containing a BRCT domain.

Pescadillo was originally identified in the zebrafish Danio rerio as a site of a retrovirus-insertion mutation that caused severe defects during embryogenesis. In particular, growth of the fetal zebrafish liver was significantly affected by loss of pescadillo function. To begin to understand the role of pescadillo during mammalian hepatogenesis we identified the murine homologue of pescadillo and named it Pes1. A single gene localized to chromosome 11 on the mouse genome encodes Pes1. Although Pes1 mRNA was detected in all tissues examined it was present at the highest levels in both adult and fetal liver. Analysis of the predicted amino acid sequence of Pes1 found it to contain a BRCT domain, which has previously been found in several proteins involved in cell-cycle checkpoints and DNA repair. Consistent with a putative role in these processes we found that when recombinant Pes1 protein was expressed in HepG2 cells it localized to the nucleus.

Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB.

The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated Ser/Thr protein kinase (PKR) plays a role in the antiviral and antiproliferative effects of IFN. PKR phosphorylates initiation factor eIF2alpha, thereby inhibiting protein synthesis, and also activates the transcription factor, nuclear factor-kappaB (NF-kappaB), by phosphorylating the inhibitor of NF-kappaB, IkappaB. Mice devoid of functional PKR (Pkr(o/o)) derived by targeted gene disruption exhibit a diminished response to IFN-gamma and poly(rI:rC) (pIC). In embryo fibroblasts derived from Pkr(o/o) mice, interferon regulatory factor 1 (IRF-1) or guanylate binding protein (Gbp) promoter-reporter constructs were unresponsive to IFN-gamma or pIC but response could be restored by co-transfection with PKR. The lack of responsiveness could be attributed to a diminished activation of IRF-1 and/or NF-kappaB in response to IFN-gamma or pIC. Thus, PKR acts as a signal transducer for IFN-stimulated genes dependent on the transcription factors IRF-1 and NF-kappaB.

Double-stranded RNA-dependent protein kinase activates transcription factor NF-kappa B by phosphorylating I kappa B.

The induction of interferon (IFN) genes by viruses or double-stranded RNA (dsRNA) requires the assembly of a complex set of transcription factors on responsive DNA elements of IFN gene promoters. One of the factors necessary for regulating IFN-beta gene transcription is nuclear factor NF-kappa B, the activation of which is triggered by dsRNA. It has previously been suggested that the dsRNA-activated p68 protein kinase (PKR) may act as an inducer-receptor, transducing the signal from dsRNA to NF-kappa B through phosphorylation of the inhibitor I kappa B. We present direct evidence that PKR can phosphorylate I kappa B-alpha (MAD-3) and activate NF-kappa B DNA binding activity in vitro. We further show that dsRNA induces an unusual phosphorylated form of I kappa B-alpha. The expression of a transdominant mutant PKR is able to perturb the dsRNA-mediated signaling pathway in vivo, suggesting a role for this kinase in IFN-beta gene induction.