Legal developments and practical implications of gene patenting on targeted drug discovery and development.

The use of genetic information to design and guide therapies creates novel patent issues. Gene patents have been integral to the introduction of new biologics, but their role in diagnostic testing is controversial. Genotype-phenotype associations are at the heart of personalized medicine. The intellectual property rules by which these biological relationships are governed have profound implications for the growth of this field. Several cases currently before the courts may add reason and clarity to the law in this area.

Protegrin-1 enhances bacterial killing in thermally injured skin.

OBJECTIVE: Septic complications and the emergence of drug-resistant microbes represent serious risks to patients. Recently, naturally occurring peptides have been discovered that possess potent and broad-spectrum antimicrobial activity. Protegrin-1 is particularly attractive for clinical use in human wounds because, unlike defensins, protegrin-1 retains broad antimicrobial and antifungal activity at physiologic salt concentration and in the presence of serum. The objective of this study was to examine the efficacy of protegrin-1 in killing multiple drug-resistant microbes isolated from human burn patients. DESIGN: For thein vitroexperiment, bilayer radial diffusion was performed comparing standard antibiotics with protegrin-1 on multiple-drug-resistant microbial organisms isolated from infected burn wounds. In vivo, rats received a 20% total body surface area partial-thickness burn by immersion in 60 degrees C water for 20 secs followed by wound seeding with 106 colony forming units of Silvadene-resistant Pseudomonas aeruginosa. SETTING: University of Michigan research laboratory. SUBJECTS: Adult, male Sprague-Dawley rats. INTERVENTIONS: Rats were randomized into three groups: those receiving synthetic protegrin-1, acetic acid (carrier), or gentamicin (positive control). Protegrin-1 was administered by topical application or intradermal injection. Wound tissues were harvested aseptically at different time points for quantitative bacterial counts. MEASUREMENTS AND MAIN RESULTS: In vivo and in vitro experiments revealed rapid and significant decreases in bacterial counts for protegrin-1-treated groups compared with controls. CONCLUSIONS: This study shows that protegrin-1 potentially may be used as an alternative or adjunct therapy to standard agents used to treat wound infections.

Feasibility of biolistic gene therapy in burns.

Skin is an especially attractive target for genetic manipulation because it is readily accessible and easily monitored for both the presence and the expression of inserted genes. This study was designed to assess the feasibility of particle mediated gene transfer to burned skin and to compare the transfection efficiency, anatomic distribution, and duration of transgene expression achievable in normal versus burned skin. Two days following scald injury of varying depths in 60 degrees C water (10 s: superficial partial; 20 s: deep partial; 40 s: full thickness) reporter gene (beta-galactosidase) constructs were delivered using a gene gun at various helium pressures (200-600 psi) to normal and burned skin. A time course study was performed to examine the kinetics of transgene expression. Animals received a superficial partial thickness burn and were sacrificed 12 h, 1, 3, 5, 7, 14, or 21 days after gene transfer. India Ink injection and immunohistochemistry were used to assess the depth of the scald injury. Transfection efficiency was measured in skin homogenates 24 h after gene transfer by morphometric and chemoluminescent assays. We found that the extent of tissue damage was directly related to the duration of heat source exposure. Reporter gene activity was significantly higher in superficial partial thickness burns compared to normal controls and gradually declined with increasing tissue injury. No activity was seen in the full thickness burn group. Beta-galactosidase activity reached a maximum level 12 h after gene transfer in both normal and superficial partial thickness burned skin with no levels seen after 5 days post-transfection. These findings indicate that particle-mediated gene transfer in thermally injured skin is feasible and may provide a means of introducing biologic agents into injured tissue capable of enhancing bacterial clearance and improving wound healing.

Aberrant expression of fibroblast growth factor receptor-1 in prostate epithelial cells allows induction of promatrilysin expression by fibroblast growth factors.

Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, and there is evidence that they play a role in tumor cell growth, invasion and metastasis. Matrilysin (MMP-7) is over-expressed in prostate cancer cells and increases prostate cancer cell invasion. Prostate stromal fibroblasts secrete a factor(s), including fibroblast growth factor-1 (FGF-1), which induces promatrilysin expression in the prostate carcinoma cell line LNCaP but not in normal prostate epithelial cells (PrECs). Since FGF-1 is present in the prostate, an altered sensitivity to FGF-1 might explain the up-regulation of matrilysin expression in prostate cancer cells compared to normal prostate epithelium. FGF receptor-1 (FGFR-1) is not normally expressed by normal prostate epithelial cells; however, aberrant expression of this receptor has been reported in prostate cancer cells, including the LNCaP cell line. We hypothesized that aberrant expression of FGFR-1 in PrECs would render them sensitive to induction of promatrilysin expression by recombinant FGF-1. To test this hypothesis, we transiently transfected PrECs with an FGFR-1 expression vector, which resulted in over-expression of FGFR-1 protein in approximately 40% of cells. FGF-1 increased promatrilysin expression in FGFR-1-transfected PrECs 4-fold over mock-transfected cells, and this induction was inhibited by a specific FGFR-1 inhibitor, SU5402, and by co-expression of a dominant negative FGFR-1 protein. Our results demonstrate that aberrant FGFR-1 expression, an epigenetic phenomenon that has been associated with prostate cancer progression, allows induction of promatrilysin expression by FGF-1 in PrECs.

Interleukin-1beta-induced promatrilysin expression is mediated by NFkappaB-regulated synthesis of interleukin-6 in the prostate carcinoma cell line, LNCaP.

Previously, our laboratory showed that interleukin-1beta (IL-1beta) secreted by lipopolysaccharide-activated monocytes induces promatrilysin expression in the prostate carcinoma cell line, LNCaP. We now demonstrate that IL-1beta-induced promatrilysin expression is mediated by an indirect mechanism that requires nuclear factor Kappa B (NFkappaB)-dependent synthesis of IL-6. Inhibition of protein synthesis with cycloheximide blocked IL-1beta-mediated induction of matrilysin mRNA suggesting that synthesis of one or more additional factors is required for IL-1beta-induced promatrilysin protein expression. Blockage of NFkappaB transactivation activity abrogated IL-1beta-induced promatrilysin expression to baseline levels suggesting that NFkappaB transactivation activity is necessary. Inhibition of IL-6 activity attenuated IL-1beta-induced promatrilysin, but not NFkappaB transactivation activity indicating that IL-6 acts downstream of NFkappaB in potentiation of IL-1beta-mediated promatrilysin expression. Inhibition of protein synthesis with cycloheximide did not alter IL-6-induced induction of matrilysin mRNA indicating that, contrary to the mechanism by which IL-1beta regulates promatrilysin expression, IL-6-mediated matrilysin mRNA expression does not require new protein synthesis. Transient transfection with dominant negative STAT3 inhibited IL-1beta- and IL-6-induced promatrilysin. These data provide evidence that NFkappaB-mediated IL-6 synthesis is required for IL-1beta-induced promatrilysin expression, and IL-6 signaling through STAT3 plays a role in IL-1beta-induced promatrilysin expression.

Lipopolysaccharide-binding protein accelerates and augments Escherichia coli phagocytosis by alveolar macrophages.

BACKGROUND: The first step in bacterial clearance by leukocytes is attachment and phagocytosis. Although lipopolysaccharide-binding protein (LBP) is best known for potentiating LPS-induced cytokine production through a CD14-dependent pathway, recent studies suggest that LBP plays a critical role in clearance of gram-negative bacteria and is essential for survival after bacterial challenge. We therefore sought to examine LBP's effect on Escherichia coli phagocytosis by alveolar macrophages (AMs) and to determine if this effect is mediated through CD14. MATERIALS AND METHODS: Phosphatidylinositol-specific phospholipase C (PIPLC)-treated and untreated rat AMs were incubated in the presence of increasing doses of recombinant LBP or negative control protein (choramphenicol acetyltransferase) prior to E. coli-FITC (Ec-F) BioParticle challenge. Phagocytosed bacteria were assayed by fluorescence measurement. A time course study was also performed. RESULTS: LBP potentiated phagocytosis of Ec-F BioParticles by AMs in a dose-dependent fashion. Kinetic studies showed that LBP augmented Ec-F phagocytosis by 76% at 30 min. Treatment of AMs with PIPLC to remove CD14 resulted in only a partial decrease in LBP-mediated enhancement of phagocytosis. CONCLUSION: These results clearly demonstrate that LBP plays an important role in enhancing Ec-F binding and phagocytosis in a time- and dose-dependent manner. This observed increase may not require the presence of CD14 as significant potentiation of phagocytosis still occurred after PIPLC treatment. We postulate that the LBP-mediated increase in Ec-F phagocytosis can occur in the absence of CD14 through the presence of another receptor.

Low-dose genistein induces cyclin-dependent kinase inhibitors and G(1) cell-cycle arrest in human prostate cancer cells.

Genistein, a naturally occurring isoflavone found chiefly in soy products, reportedly has antiprostate cancer effects, but the mechanisms underlying these effects are unknown. We studied the antiproliferative and apoptosis-inducing effects of genistein in the androgen-sensitive human prostate cancer cell line LNCaP. Viable cell number was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay; cell-cycle progression and apoptosis were evaluated by flow cytometry; apoptosis was also assessed by a histone enzyme-linked immunosorbent assay; and the expression of several cell-cycle- and apoptosis-related genes and their gene products was determined by northern blot analysis, western blot analysis, and/or assays based on polymerase chain reaction. Physiologic concentrations of genistein (< or = 20 microM) decreased LNCaP viable cell number in a dose-dependent manner, induced a G(1) cell-cycle block, decreased prostate-specific antigen mRNA expression, and increased p27(KIP1) and p21(WAF1) (mRNA and protein) but had no effect on apoptosis or the mRNA expression of the apoptosis- and cell-cycle-related markers bcl-2, bax, Rb, and proliferating cell nuclear antigen. Higher concentrations of genistein (> 20 microM) did induce apoptosis. We conclude that genistein (at physiologic concentrations) exerts potent antiproliferative effects on LNCaP cells by inducing a G(1) cell-cycle block. The antiproliferative effects of genistein may be mediated by increased levels of p27(KIP1) and p21(WAF1), which are negative cell-cycle regulators that act as cyclin-dependent kinase inhibitors and that have been recently linked with prostate carcinogenesis. These findings may provide insights into the mechanisms underlying the apparent antiprostate cancer effects of soy consumption observed in epidemiologic studies.

Skin lipopolysaccharide-binding protein and IL-1beta production after thermal injury.

In response to a burn injury, skin can have an inflammatory response characterized by the production of inflammatory cytokines, recruitment of immune cells, containment of invading organisms, and clearance of noxious substances from the wound. Lipopolysaccharide-binding protein (LBP) is a molecule that is capable of coordinating all 4 functions; we previously found evidence that suggested that LBP is produced within surgical wounds. Because of the central role of LBP in the response to bacterial infection, as well as in the high rate of infection after burn injuries, we sought to determine whether a thermal injury could affect wound LBP production and thereby affect host responses against bacterial infection. Rats were given either a burn or a sham burn and were killed 24, 48, and 72 hours after the injuries. Wound specimens were assayed for bacterial counts and for the presence of LBP, messenger (m)RNA, and interleukin (IL)-1beta mRNA. Wound LBP mRNA was significantly upregulated at 24 hours in the group with burn injuries (P < .05; burn vs sham burn); this was followed by decreases at 48 and 72 hours. Immunohistochemistry showed LBP protein in the epidermis of animals with burns. Bacterial counts increased in the group with burn injuries (P < .05; burn vs sham burn) and continued to rise for 72 hours. IL-1beta mRNA levels were elevated at all time points in the group with burn injuries (P < .05). These results suggest an inverse correlation between burn wound LBP expression and bacterial wound counts. This failure to maintain local LBP production after severe thermal injury despite localized inflammation shown by high IL-1beta levels may predispose local wounds to bacterial invasion.

ARS sequences in homologous and heterologous ADE2 loci are capable of promoting autonomous replication of plasmids in Schwanniomyces occidentalis.

We have analyzed ARS elements linked to homologous and heterologous ADE2 loci functioning in Schwanniomyces occidentalis. We have identified a region of the ADE2 locus of S. occidentalis which promotes autonomous replication of plasmids in S. occidentalis cells. This region is within 385 bp preceding the ATG codon of the S. occidentalis ADE2 gene. It contains sequences similar to ARS core consensus sequences, ARS boxes, and a potential transcription activator binding site characterized in Saccharomyces cerevisiae. The ADE2 gene of S. cerevisiae was found to complement the ade2 mutation in S. occidentalis cells and the 5' UTR region of this gene is capable of supporting autonomous replication of plasmids in S. occidentalis. Furthermore, we confirmed that the origin of replication of the 2 microm plasmid and the ARS1 sequence of S. cerevisiae are also functional in S. occidentalis cells. Plasmids carrying either ARS, the SwARSA element of S. occidentalis, the ARS linked to the ADE2 gene of S. cerevisiae, and the ARS1 sequence or the 2 microm ori, were found to be maintained in S. occidentalis cells as episomal monomers or oligomers. However, their stability was low as already reported for the ARS in S. occidentalis.

Kupffer cell activation by lipopolysaccharide in rats: role for lipopolysaccharide binding protein and toll-like receptor 4.

Lipopolysaccharide (LPS) binding protein (LBP) is a key serum factor that mediates LPS activation of mononuclear cells. In the presence of LBP, 1/1,000 the concentration of LPS is sufficient to activate peripheral blood monocytes. Previous studies with Kupffer cells have shown a variable effect of serum on LPS activation of these cells and led to the conclusion that, unlike extrahepatic mononuclear cells, Kupffer cells do not respond to LPS in an LBP-dependent fashion. Because there are multiple components in serum other than LBP that might affect LPS activation, these reports with serum are difficult to interpret. To investigate the specific role of LBP in LPS activation of Kupffer cells, we produced a functional recombinant rat LBP using a baculovirus expression system, which we used to selectively examine the role of LBP's on Kupffer-cell function. Isolated Kupffer cells exposed to increasing concentrations of LPS (0, 1, 10 ng/mL) showed a dose-dependent increase in TNF-alpha production, which was augmented and accelerated by the presence of LBP. The effects of LBP on Kupffer cell activation by LPS are dependent on a functional Toll-like receptor 4 (Tlr 4) because Kupffer cells from C3H/HeJ mice failed to respond to LPS in the presence of LBP. LBP plays an important role in mediating Kupffer cell activation by LPS, and these effects are dependent on the presence of functioning Tlr 4.

Promatrilysin expression is induced by fibroblast growth factors in the prostatic carcinoma cell line LNCaP but not in normal primary prostate epithelial cells.

BACKGROUND: It has been determined that prostate cancer cells overexpress the matrix metalloprotease matrilysin (MMP-7), but the factors regulating this expression have not been identified. Fibroblast growth factors (FGF), which are expressed in the prostate, might participate in paracrine regulation of matrilysin expression by prostate cancer cells. METHODS: We tested the ability of recombinant FGF proteins and prostate fibroblast-conditioned media (PFCM) to induce promatrilysin expression in the prostate carcinoma cell line, LNCaP, and in normal prostate epithelial (PrEC) cells. We also characterized prostate fibroblast FGF expression by reverse transcriptase-polymerase chain reaction (RT-PCR). An inhibitor of FGF receptor activation (SU5402) was used to determine the role of FGF proteins in the induction of promatrilysin expression by PFCM. RESULTS: Recombinant FGF-1, FGF-2, FGF-9, FGF-10, and PFCM significantly induced promatrilysin expression in LNCaP cells but not in PrEC cells. Prostate fibroblasts express mRNAs for these FGF proteins, and inhibition of LNCaP cell FGF receptors with SU5402 substantially reduced the induction of promatrilysin expression by PFCM. CONCLUSIONS: Stromally expressed FGF proteins induce promatrilysin expression in a prostate carcinoma cell, and may provide a mechanism for the overexpression of promatrilysin observed in prostate cancer.

Haemonchus contortus: sequence heterogeneity of internucleotide binding domains from P-glycoproteins.

P-Glycoproteins are transmembrane proteins associated with acquired multidrug resistance in mammalian cells and some protozoan parasites by a process of active drug export. P-glycoproteins contain two nucleotide binding domains which couple ATP to the drug transport process. The region between the nucleotide binding domains of P-glycoproteins, termed the internucleotide binding domain (IBD), was PCR-amplified from adult and larval cDNA libraries using degenerate primers. The 11 clones isolated by this method fall into several distinct groups, with one group of alleles displaying between 82 and 99% identity at the nucleotide level. This sets a baseline for sequence variation of transcribed alleles from a parasitic nematode. Northern blotting showed that P-glycoprotein genes are transcribed in a developmentally regulated fashion in Haemonchus contortus. Southern blots of H. contortus drug-resistant isolates with an IBD probe revealed a pattern consistent with the involvement of P-glycoprotein in resistance to avermectin/milbemycin anthelmintics.

Reconstitution of a bacterial/plant polyamine biosynthesis pathway in Saccharomyces cerevisiae.

Polyamine synthesis in most organisms is initiated by the decarboxylation of ornithine to form putrescine via ornithine decarboxylase (ODC). Plants, some bacteria and some fungi and protozoa generate putrescine from arginine, via arginine decarboxylase (ADC) and agmatine ureohydrolase (AUH) or agmatine iminohydrolase. A polyamine-requiring strain of Saccharomyces cerevisiae with a mutation in the gene encoding ODC was transformed with plasmids bearing genes encoding Escherichia coli ADC and AUH. Transformants regained the ability to grow in the absence of exogenous polyamines and contained enzyme activities consistent with the presence of both prokaryotic enzymes. Similar results were obtained when a plasmid containing a gene encoding oat (Avena sativa L.) ADC was substituted for the E. coli gene. These data demonstrate the successful complementation of a yeast biosynthetic polyamine synthesis defect by genes encoding an alternative pathway found in bacteria; they also show that plant ADC can substitute for the bacterial enzyme in this pathway. The recombinant yeast provides a tool for the study of the functional properties of these enzymes and for discovery of compounds that specifically inhibit this pathway.

Mechanism-based screening: discovery of the next generation of anthelmintics depends upon more basic research.

The therapeutic arsenal for the control of helminth infections contains only a few chemical classes. The development and spread of resistance has eroded the utility of most currently available anthelmintics, at least for some indications, and is a constant threat to further reduce the options for treatment. Discovery and development of novel anthelmintic templates is strategically necessary to preserve the economic and health advantages now gained through chemotherapy. As the costs of development escalate, the question of how best to discover new drugs becomes paramount. Although random screening in infected animals led to the discovery of all currently available anthelmintics, cost constraints and a perception of diminishing returns require new approaches. Taking a cue from drug discovery programmes for human illnesses, we suggest that mechanism-based screening will provide the next generation of anthelmintic molecules. Critical to success in this venture will be the exploitation of the Caenorhabditis elegans genome through bioinformatics and genetic technologies. The greatest obstacle to success in this endeavour is the paucity of information available about the molecular physiology of helminths, making the choice of a discovery target a risky proposition.

Pulmonary LPS-binding protein (LBP) upregulation following LPS-mediated injury.

BACKGROUND: The acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality among trauma patients. Although multiple factors have been implicated, pulmonary injury in this population may be due to inflammatory mediators released in response to stimuli such as endotoxin (LPS). LBP plays an integral part in LPS-mediated release of inflammatory cytokines and increased local expression of LBP as the result of a primary injury may prime the lung to secondary LPS-mediated damage. MATERIALS AND METHODS: To determine the magnitude of pulmonary LBP upregulation following LPS injury we challenged rats with either intravenous (IV) or intratracheal (IT) LPS. Animals from each group were euthanized at 1, 2, 4, and 8 h postchallenge. Lung LBP and CD14 mRNA levels were assayed by Northern blot. Serum and bronchoalveolar lavage (BAL) fluid were assayed for inflammatory cytokines (TNF-alpha, MCP-1, IL-1beta, IL-6, and IL-10) by ELISA. RESULTS: LBP and CD14 mRNA levels were found to increase significantly in lung tissue after both IV and IT LPS with the IV LPS animals having a greater increase over 8 h. Serum TNF-alpha was significantly elevated in the IV LPS group whereas very low levels were detected in the BAL. Only BAL TNF-alpha was increased in the IT group at 8 h. CONCLUSION: Local pulmonary LBP and CD14 mRNA are both upregulated after either systemic or local LPS exposure. Such upregulation may render thelung more susceptible to local immune overactivation and injury during subsequent exposures to LPS.

Tissue coexpression of LBP and CD14 mRNA in a mouse model of sepsis.

BACKGROUND: Lipopolysaccharide binding protein (LBP) markedly increases the sensitivity of immune cells to LPS and CD14 expression correlates with cellular responsiveness to LPS. LBP gene expression can be induced in multiple organs following injury and CD14 upregulation on monocytes correlates with the infection and mortality rates in severely injured patients. We sought to determine the time-course induction of LBP and CD14 gene expression following experimental peritonitis. MATERIAL AND METHODS: BALB/c mice were subjected to laparotomy alone or laparotomy with cecal ligation and puncture and treated with Imipenem. At serial time points, animals were sacrificed and tissues harvested for isolation of RNA and protein. LBP, CD14, and cytokine mRNAs were analyzed by Northern blot analysis and TaqMan fluorescent quantitative RT-PCR. RESULTS: LBP and CD14 mRNA levels were significantly increased in all three organs from CLP mice compared to sham-operated mice. IL-1 mRNA levels increased in all three organs following CLP with significantly higher levels found in the lungs compared to the kidney and liver. No significant differences were noted in local TNF mRNA levels. CONCLUSIONS: LBP, CD14, and IL-1 mRNA levels are induced concurrently in the lung, kidney, and liver after cecal ligation and puncture. Given the synergistic affect of LBP and CD14 in potentiating LPS-induced production of inflammatory cytokines and the hypothesized role of such cytokines in the etiology of MSOF following injury and sepsis, our findings suggest a mechanism by which these organs may be rendered more susceptible to a "second hit" from endotoxemia after initial injury.

Cloning and characterization of cDNAs encoding beta-tubulin from Dirofilaria immitis and Onchocerca volvulus.

Beta-Tubulin is the target for the benzimidazole anthelmintics. Unfortunately, none of these drugs is clinically useful against adult filariae. However, beta-tubulin has been shown to be a target for antibody-based toxicity to Brugia pahangi. We cloned and characterized cDNAs encoding beta-tubulin from 2 filariae, Dirofilaria immitis and Onchocerca volvulus, to explore possible explanations for benzimidazole insensitivity among adult filariae and the likelihood that epitopes of beta-tubulin could be used as antigens for a broad-spectrum filarial vaccine. The proteins predicted by these cDNAs were almost identical to the beta-tubulin previously reported from B. pahangi but were less similar to a beta-tubulin cDNA from Onchocerca gibsoni. We cloned the genomic locus for the O. volvulus beta-tubulin cDNA and compared its organization to the reported genomic loci for beta-tubulin in B. pahangi and O. gibsoni. The comparison reinforces the conclusion that the published O. gibsoni gene is in a different family, possibly the beta2 family previously described in B. pahangi. The substitution of tyr for phe at position 200 of beta-tubulin is associated with benzimidazole resistance. All 4 filarial beta-tubulins are predicted to encode phe at this position, suggesting that filarial beta-tubulin is not inherently insensitive to the benzimidazoles. A monoclonal antibody that recognizes the COOH terminus of B. pahangi beta-tubulin is lethal to this parasite in culture. The COOH terminal region is the most variable among the different isotypes of beta-tubulin and distinguishes mammalian from nematode tubulins. This region is highly conserved in 3 of the filarial beta-tubulins.

Persephin, a novel neurotrophic factor related to GDNF and neurturin.

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

CD14 and lipopolysaccharide binding protein expression in a rat model of alcoholic liver disease.

Lipopolysaccharide-binding protein (LBP) and CD14 play key intermediary roles in the activation of cells by endotoxin. As endotoxin has been postulated to participate in promoting pathological liver injury in alcoholic liver disease, we investigated the role of LBP and CD14 in alcoholic liver injury. Rats were fed intragastrically ethanol or dextrose and either medium-chain triglycerides, corn oil, or fish oil for 4 weeks. Kupffer cells, endothelial cells, and hepatocytes were isolated. LBP and CD14 mRNA levels were measured in liver and individual cell types. The highest levels of LBP and CD14 mRNA levels in the liver were found in the fish oil/ethanol group, which was also the group with the greatest degree of pathological injury and inflammation. CD14 mRNA levels were also significantly elevated in groups fed unsaturated fatty acids with dextrose. CD14 expression was localized to the Kupffer cells and LBP expression to the hepatocytes. Expression of CD14 mRNA was also found in nonmyeloid cells in the two experimental groups (fish oil/ethanol and corn oil/ethanol) that had liver necrosis and inflammation. Our results suggest that enhanced LBP and CD14 expression correlates with the presence of pathological liver injury in alcoholic liver injury. Furthermore, unsaturated fatty acids may prime cells to respond to endotoxin by enhancing CD14 expression.

Calcium-dependent Ret activation by GDNF and neurturin.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) define a new family of neurotrophic factors that play crucial roles in survival and differentiation of various neurons. Recent studies demonstrated that GDNF and NTN use a multicomponent receptor system in which glycosyl-phosphatidylinositol (GPI)-linked cell surface proteins and Ret receptor tyrosine kinase function as the ligand-binding and signalling components, respectively. In the present study, we investigated the role of Ca2+ ions for biochemical and biological activities of Ret because Ret has a unique structure of the extracellular domain with the cadherin-like motif. The results demonstrated that Ca2+ ions might be required for the complex formation of Ret and GDNF or NTN that induces Ret oligomerization and autophosphorylation. Full morphological differentiation of neuroblastoma cells by these neurotrophic factors was also Ca2+-dependent. These findings thus suggested that, in addition to GPI-linked cell surface proteins, Ca2+ ions are components of the signal transducing complex formed by Ret and GDNF protein family.