Douglas C. Hixson

Liver Regeneration in Rats with Retrorsine-Induced Hepatocellular Injury Proceeds through a Novel Cellular Response

The adult rodent liver contains at least two recognized populations of cells with stem-like properties that contribute to liver repair/regeneration under different pathophysiological circumstances: (i) unipotential committed progenitor cells (differentiated hepatocytes and biliary epithelial cells) and (ii) multipotential nonparenchymal progenitor cells (oval cells). In retrorsine-induced hepatocellular injury the capacity of fully differentiated rat hepatocytes to replicate is severely impaired and massive proliferation of oval cells does not occur. Nevertheless, retrorsine-exposed rats can replace their entire liver mass after 2/3 surgical partial hepatectomy through the emergence and expansion of a population of small hepatocyte-like progenitor cells that expresses phenotypic characteristics of fetal hepatoblasts, oval cells, and fully differentiated hepatocytes, but differ distinctly from each type of cell. The activation, proliferation, and complete regeneration of normal liver structure from small hepatocyte-like progenitor cells have not been recognized in other models of liver injury characterized by impaired hepatocyte replication. We suggest that the selective emergence and expansion of small hepatocyte-like progenitor cells observed in the retrorsine model reflect a novel mechanism of complete liver regeneration in the adult rat. Furthermore, we suggest that these cells may represent a novel progenitor cell population that (i) responds to liver deficit when the replication capacity of differentiated hepatocytes is impaired, (ii) expresses an extensive proliferative capacity, (iii) can give rise to large numbers of progeny hepatocytes, and (iv) can restore tissue mass.

Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription

OBJECTIVE Microvesicles have been shown to mediate intercellular communication. Previously, we have correlated entry of murine lung-derived microvesicles into murine bone marrow cells with expression of pulmonary epithelial cell-specific messenger RNA (mRNA) in these marrow cells. The present studies establish that entry of lung-derived microvesicles into marrow cells is a prerequisite for marrow expression of pulmonary epithelial cell-derived mRNA. MATERIALS AND METHODS Murine bone marrow cells cocultured with rat lung, but separated from them using a cell-impermeable membrane (0.4-microm pore size), were analyzed using species-specific primers (for rat or mouse). RESULTS These studies revealed that surfactant B and C mRNA produced by murine marrow cells were of both rat and mouse origin. Similar results were obtained using murine lung cocultured with rat bone marrow cells or when bone marrow cells were analyzed for the presence of species-specific albumin mRNA after coculture with rat or murine liver. These studies show that microvesicles both deliver mRNA to marrow cells and mediate marrow cell transcription of tissue-specific mRNA. The latter likely underlies the longer-term stable change in genetic phenotype that has been observed. We have also observed microRNA in lung-derived microvesicles, and studies with RNase-treated microvesicles indicate that microRNA negatively modulates pulmonary epithelial cell-specific mRNA levels in cocultured marrow cells. In addition, we have also observed tissue-specific expression of brain, heart, and liver mRNA in cocultured marrow cells, suggesting that microvesicle-mediated cellular phenotype change is a universal phenomena. CONCLUSION These studies suggest that cellular systems are more phenotypically labile than previously considered.

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Correlation between mortality and the levels of inter-alpha inhibitors in the plasma of patients with severe sepsis.

Inter-alpha inhibitor protein (IalphaIp) is an endogenous serine protease inhibitor in human plasma. Circulating IalphaIp levels were lower in 51 patients with severe sepsis than in healthy volunteers. Mean levels were 688+/-295 mg/L in patients with severe sepsis who survived (n=32), 486+/-193 mg/L in patients with sepsis who died (n=19), and 872+/-234 mg/L in control subjects (n=25). IalphaIp levels were lower in patients with shock versus those without (540+/-246 [n=33] vs. 746+/-290 [n=18] mg/L; P=.0102). IalphaIp levels were inversely correlated with 28-day mortality rates and Acute Physiology and Chronic Health Evaluation II scores and directly correlated with antithrombin III, protein C, and protein S levels. The administration of IalphaIp (30 mg/kg body weight intravenously) increased the 50% lethal dose in mice by 100-fold after an intravenous challenge of Escherichia coli. Thus, human IalphaIp may be a useful predictive marker and potential therapeutic agent in sepsis.

Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels.

Dipeptidyl peptidase IV (DPPIV) is a serine peptidase that cleaves N-terminal dipeptides from polypeptides when the second residue is a proline or an alanine. We have recently cloned cDNAs for rat gp110, a membrane glycoprotein with Mr of 110,000 isolated initially from rat liver. Studies reported here establish that the gp110 for which we have cloned cDNAs is DPPIV. Using the antibodies against and cDNA for DPPIV, we have assessed the tissue distribution of DPPIV by molecular approaches. Immunoblot analysis demonstrated that DPPIV is present in the kidney, lung, and small intestine at high levels, in the liver and spleen at moderate levels, and in the heart at low levels. The highest levels of mRNA for DPPIV were detected in the kidney and small intestine as compared to moderate levels found in the lung, liver, and spleen. The lowest levels of DPPIV mRNA were found in the stomach, testis, and heart. No detectable DPPIV protein and mRNA were found in brain or muscle. LDPPIV protein and mRNA are present at much lower levels in fetal livers as compared to the adult liver. Indirect immunofluorescence microscopy demonstrated that DPPIV is localized in the bile canaliculus of hematocytes and in the apical membrane domains of kidney tubule and small intestine. Further studies by Southern blot analysis indicate that DPPIV is encoded by a single gene.

Survival of docetaxel-resistant prostate cancer cells in vitro depends on phenotype alterations and continuity of drug exposure

Abstract: We evaluated in vitro the effect of paclitaxel and docetaxel on PC-3 and DU-145 prostate cancer cell lines to understand better the downstream events in drug-induced tumor cell death. Taxane treatments of DU-145 cells induced rapid cell death by apoptosis, but in PC-3 cells, treatments achieved growth arrest, followed by extensive karyokinesis resulting in multinucleation, giant-cell formation and delayed cell death. To determine if the giant multinucleated cells were able to produce proliferating and drug-resistant survivors, we first delineated the kinetics of drug activity and cytotoxic dose range. Analysis of both lines by colorimetric and cell viability assays demonstrated improved cytotoxicity of taxanes applied continuously. Selected doses and schedules of docetaxel were used to induce giant multinucleated cells that gave rise to docetaxel-resistant survivors, which remained sensitive to paclitaxel and other chemotherapeutics. Growth and morphology of the recovered clones was similar to parental cells. The resistant phenotype of these clones determined by immunofluorescence and immunoblot was associated with transient expression of the β-tubulin IV isoform and was independent of P-glycoprotein, bcl-2 and bcl-xL. Resistant clones will be useful to model progression of resistance to taxanes and to identify unknown and clinically important molecular mechanisms of cell death and resistance.

Long-term culture and characteristics of normal rat liver bile duct epithelial cells

BACKGROUND A method was established for isolation and long-term culture of bile duct epithelial cells (BDEC) of normal adult rat liver that does not require the preparation of highly purified BDEC. METHODS After dissociation of the liver parenchyma by collagenase perfusion, the liver remnant containing the intact biliary tree was minced into small fragments, embedded in a rat tail collagen gel, and cultured for 6 days in hormonally defined serum-free Dulbeccos Modified Eagle Medium/F12 medium (SFDM). BDEC cultures were subsequently subcultured and maintained on rat tail collagen gels in SFDM medium containing 5 mumol/L forskolin and 5%-10% Nu Serum IV (Collaborative Research, Bedford, MA). RESULTS Established BDEC lines continued to express ductal specific markers including gamma-glutamyl transpeptidase, cytokeratins 7 and 19, and a number of monoclonal antibody-defined bile duct antigens, such as OC.2, OC.3, and OV6. CONCLUSIONS The availability of a method to establish normal BDEC lines will allow further investigation of the function of bile duct cells and their role in normal liver differentiation and carcinogenesis.

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine.

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast‐like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6‐hydroxydopamine‐lesioned Parkinsons model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult‐derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.

Inter-α inhibitor proteins in infants and decreased levels in neonatal sepsis

Objective Adjunctive tests are needed to predict sepsis in the newborn and to lower the rate or duration of unnecessary antibiotic use. We evaluated the normal Inter-α inhibitor protein (IaIp) values in infants and the association of plasma levels of IaIp with sepsis in term and preterm newborns. Methods Plasma IaIp levels were measured by enzyme-linked immunosorbent assay in samples from 135 newborn infants at a wide range of gestational ages (24–42 weeks). IaIp levels were also determined in 19 infants undergoing prospective evaluation for sepsis. ResultsIaIp levels in umbilical cord blood and circulating peripheral blood of healthy newborn infants (525±66 mg/L) were not significantly different from the level in healthy adults (691±80 mg/L). IaIp levels were similar in infants between 24 and 42 weeks gestational age. There was a significant reduction in IaIp levels in infants with sepsis compared with nonseptic controls (169±126 mg/L vs 613±286 mg/L, P<.0001). ConclusionsIaIp levels in the blood of newborns are independent of gestational age and similar to adults. IaIp levels are significantly reduced in infants with bacterial sepsis and might serve as an adjunctive diagnostic marker to allow prospective reduction of antibiotic use.

Application of immunomagnetic beads in combination with RT-PCR for the detection of circulating prostate cancer cells

Recently published protocols using Reverse Transcriptase Polymerase Chain reaction (RT‐PCR) for prostate specific antigen (PSA) provide a sensitive means for detecting circulating prostate cancer cells. Attempts to use these assays for staging of prostate cancer have produced conflicting results. As a first step towards rectifying these discrepancies, a modified immunobead‐RT‐PCR assay capable of detecting as few as 10 prostate cancer cells in 8cc of blood was developed. This 10 fold increase in sensitivity was achieved in part by introducing two target cell enrichment steps. As a model system to assess sensitivity of the modified assay, template RNA was extracted from PSA positive human carcinoma cells suspended in human blood and isolated with immunomagnetic beads following incubation with an epithelium specific antibody. After 45 cycles of PCR, product from as few as 10 target cells could be readily detected when displayed on a 2% agarose gel stained with SYBR Green fluorescent dye. The identity of amplified DNA fragments was confirmed by Southern blot hybridization.