Multicenter clinical trials on a novel polyherbal formulation in allergic rhinitis.

Allergic rhinitis is the most frequently occurring immunological disorder. It affects men, women and children and represents significant cost in terms of suffering and loss of productivity. Allergy is termed as an excessive reaction to an environmental allergen. Pollen, mold, dust, mite and animal allergens that contact the nasal or eye lining cause sneezing, nasal congestion and itchy, watery, swollen, red eyes. Although a broad spectrum of therapeutic options is available, the treatment of allergic rhinitis appears to be far from satisfactory. A novel polyherbal formulation (PF; Aller-7/NR-A2) comprising seven medicinal herbal extracts was assessed in a multicenter clinical trial involving 545 patients (321 males and 224 females) aged 18-59 years for 12 weeks to evaluate its clinical efficacy in patients suffering from allergic rhinitis. A total of 171 patients participated in double-blind, randomized, placebo-controlled studies in three centers, while 374 patients were included in the open-label studies in 11 centers. The three major symptoms (sneezing, rhinorrhea and nasal congestion) of allergic rhinitis were significantly reduced. Significant improvement was also observed in absolute eosinophil count, mucociliary clearance time, peak expiratory flow rate and peak nasal flow rate. No serious adverse events that warranted cessation of treatment were observed. Minor adverse effects were noted in both the treatment and placebo groups. Thus, this study demonstrates that Aller-7/NR-A2 is well tolerated and efficacious in patients with allergic rhinitis.

A novel strategy for in vivo expansion of transplanted hepatocytes using preparative hepatic irradiation and FasL-induced hepatocellular apoptosis.

A strategy for inducing preferential proliferation of the engrafted hepatocytes over host liver cells should markedly increase the benefit of hepatocyte transplantation for the treatment of liver diseases and ex vivo gene therapy. We hypothesized that preparative hepatic irradiation (HIR) to inhibit host hepatocellular regeneration in combination with the mitotic stimulus of host hepatocellular apoptosis should permit repopulation of the liver by transplanted cells. To test this hypothesis, congeneic normal rat hepatocytes were transplanted into UDP-glucuronosyltransferase (UGT1A1)-deficient jaundiced Gunn rats (a model of Crigler-Najjar syndrome type I), following HIR and adenovirus-mediated FasL gene transfer. Progressive repopulation of the liver by engrafted UGT1A1-proficient hepatocytes over 5 months was demonstrated by the appearance of UGT1A1 protein and enzyme activity in the liver, biliary bilirubin glucuronides secretion, and long-term normalization of serum bilirubin levels. This is the first demonstration of massive hepatic repopulation by transplanted cells by HIR and FasL-induced controlled apoptosis of host liver cells.

Modification of hepatic genomic DNA using RNA/DNA oligonucleotides.

The ideal gene therapy is one that repairs the precise genetic defect without additional modification of the genome. Such a strategy has been developed for correcting single nucleotide mutations by using RNA/DNA oligonucleotides, or chimeraplasts. This approach for in situ repair is based on the delivery of exogenous DNA designed to mediate genomic base conversion, insertion, or deletion, thereby, correcting the genetic mutation. Using in vivo delivery systems to hepatocytes via the asialoglycoprotein receptor, we targeted rat liver DNA and successfully modified the genomic sequence by chimeraplasty. The changes in both the hepatic genes, and their associated phenotypes remained stable for 2 years. In addition, we also examined the potential to alter sequence defects in mitochondrial DNA. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeraplast-mediated DNA changes. Using an in vitro DNA repair assay of mutagenized plasmids and an Escherichia coli readout system, we showed that extracts from highly purified rat liver mitochondria have the essential enzymatic activity to mediate precise single-nucleotide changes at a frequency similar to liver nuclear extracts. Moreover, single-stranded oligonucleotides carrying a single nucleotide mismatch with the target sequence were capable of promoting gene conversion using either mitochondrial or nuclear extracts. Several approaches now exist for the precise repair of genetic mutations using either single-stranded or RNA/DNA chimeric oligonucleotides.

Amplification of engrafted hepatocytes by preparative manipulation of the host liver.

Scarcity of donor livers is a major obstacle to the general application of hepatocytes for the development of bioartificial liver assist devices as well as intracorporeal engraftment of hepatocytes for the treatment of inherited metabolic diseases. The number of hepatocytes that can be transplanted into the liver safely in a single sitting also limits the utility of this procedure. These limitations could be addressed by providing preferential proliferative advantage to the transplanted cells. Studies using transgenic mouse recipients or donors have indicated that massive repopulation of the host liver by engrafted hepatocytes requires that the transplanted cells are subjected to a proliferative stimulus to which the host hepatocytes cannot respond. Prevention of host hepatocyte proliferation has been achieved by treatment with a plant alkaloid, retrorsine. Because retrorsine is carcinogenic, we have evaluated preparative irradiation for this purpose. The proliferative stimulus may consist of the loss of hepatic mass (e.g., partial hepatectomy, reperfusion injury or induction of Fas-mediated apoptosis by gene transfer) or administration of stimulants of hepatocellular mitosis (e.g., growth factors or thyroid hormone). Potential applications of these preparative manipulations of the host liver include the treatment of inherited metabolic disorders by transplantation of allogeneic hepatocytes, hepatocyte-mediated ex vivo gene therapy, rescuing liver cancer patients from radiation-induced liver damage, and expansion of human hepatocytes in animal livers.

Hepatocyte-based gene therapy.

Hepatocyte-based gene therapy may be used to replace a missing gene product, confer proliferating ability to cultured hepatocytes, prevent allograft rejection, massively repopulate the host liver, or grow xenogeneic hepatocytes in mammalian liver. Gene transfer into isolated hepatocytes can be accomplished via nonviral or viral vectors, the viral vectors being more useful at this time. Common recombinant viruses that integrate into the host genome include murine leukemia retroviruses and lentiviruses, adenoassociated virus, and the T-antigen-deleted SV40 virus. Episomal viruses, such as adenoviruses, permit efficient gene transfer, but the transgene is lost upon proliferation of the transplanted hepatocyte in the host. Hybrid viruses that combine the high transduction efficiency of adenoviral vectors and the integrative capacity of other vectors, such as adenoassociated viruses, have been designed. Massive repopulation of the liver by transplanted hepatocytes can be achieved if a mitotic stimulus to the transplanted cells is combined with prevention of proliferation of the host hepatocytes. Treatment with a plant alkaloid or retrorsine, or preparative irradiation of the liver can be used to inhibit host hepatocellular proliferation, while partial hepatectomy, expression of Fas ligand, or thyroid hormone administration can be used as a mitotic stimulus to the transplanted cells.

Liver irradiation: a potential preparative regimen for hepatocyte transplantation.

Advances in the understanding of hepatocyte engraftment and repopulation of the host liver have already led to the use of hepatocyte transplantation (HT) with some success in the treatment of inherited and acquired liver diseases. Wider application of HT is severely limited by the unavailability of large number of transplantable hepatocytes and difficulties associated with transplanting an adequate number of cells for achieving therapeutically satisfactory levels of metabolic correction. Therefore, there is a need for preparative regimens that provide a growth advantage to the transplanted (healthy) hepatocytes over the host's own (diseased) hepatocytes so that the former can repopulate the host liver. We have recently shown that when the liver of recipient rats was subjected to radiotherapy and partial hepatectomy before HT, the transplanted hepatocytes engrafted in and massively repopulated the liver, and also ameliorated the adverse clinical and histopathological changes associated with hepatic irradiation. This protocol was then used as a preparative regimen for transplanting normal hepatocytes into jaundice mutant rats (Gunn strain), which lack hepatic bilirubin-uridinediphosphoglucuronate glucuronosyltransferase and is a model of Crigler-Najjar syndrome Type I. The results showed long-term correction of the metabolic abnormality, suggesting that the transplanted hepatocytes repopulated an irradiated liver and were metabolically functional. This strategy could be useful in the treatment of various genetic, metabolic, or malignant diseases of the liver.

Gene therapy for inherited hyperbilirubinemias.

Crigler-Najjar syndrome type 1 (CN-1) is a potentially lethal condition, and is the only inherited disorder of bilirubin metabolism that needs treatment beyond the neonatal period. Currently, orthotopic liver transplantation is the only available cure for CN-1. Because the liver architecture is not disturbed in CN-1 and partial correction of bilirubin-UDP-glucuronosyltransferase (UGT1A1) activity is expected to be sufficient for protection against kernicterus, cell and gene therapies are being developed using the Gunn rat as an animal model of the disease. Ex vivo gene therapy based on the transplantation of genetically manipulated hepatocytes and in vivo gene transfer using recombinant adenovirus and Simian virus 40 (SV40)-based vectors have yielded significant success. The novel strategy of in vivo site-directed mutagenesis has also resulted in modest, but significant, correction of the genetic abnormality. Newer viral and nonviral gene delivery methods are being explored and have been discussed in brief. In summary, effective gene therapy methods have been validated in Gunn rats. Despite considerable remaining hurdles, gene therapy for CN-1 could become a clinical reality by the turn of this decade.

A long-term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag-2-deficient mice.

Development of new therapies for human hepatitis B virus infection (HBV) would be greatly facilitated by the availability of a suitable small-animal model for HBV virus production in vivo. To develop a murine model for HBV production, we established an immortalized, cloned liver cell line by transferring the Simian Virus 40 Large T-Antigen into primary human hepatocytes. These cells were stably transfected with a full-length HBV genome to generate a clone that expresses HBV genes and replicates HBV. The HBV-producing cells were transplanted into the livers of mice with combined immunodeficiency (Rag-2 deficient) by intrasplenic injection. Survival of the engrafted human hepatocytes was shown in several ways: fluorescent in situ hybridization (FISH) with a human-chromosome-specific DNA probe (human alpha satellite), dot-blot hybridization of the genomic DNA extracted from liver biopsy specimens with a human-specific Alu repetitive DNA probe, Blur-8, as well as with an HBV DNA probe, and secretion of human proteins into plasma. Histological examination of mouse liver up to 8 months following human cell transplant shows completely normal architecture. Determination of plasma HBV DNA levels indicated that engrafted cells secreted 3x10(7) to 3x10(8) virions per mL into the blood, and HBsAg was detected in plasma. This new murine model of HBV viremia should be useful for in vivo HBV studies.

Strong, long-term transgene expression in rat liver using chicken beta-actin promoter associated with cytomegalovirus immediate-early enhancer (CAG promoter).

For successful gene therapy in hepatic enzyme deficiencies, it is essential to use promoters that can maintain strong transcriptional activity for the long term in the liver. Using Gunn rats, a model animal for Crigler-Najjar syndrome type I, the long-term transcriptional function of the CAG promoter (a combination of chicken beta-actin promoter and cytomegalovirus immediate-early enhancer) was evaluated in the rat liver. We constructed a plasmid pCAGGHUGT, containing expression cassettes of human bilirubin UDP-glucuronosyltransferase (BUGT) and hygromycin phosphotransferase, under the control of the CAG promoter and murine phosphoglycerate kinase promoter, respectively. Conditionally immortalized Gunn rat hepatocytes (IGRH), which had been established using mutant SV40 large T antigen ((TS)T), were transfected with pCAG-GHUGT. A stably transfected clone IGRHUGT, expressing a high level of BUGT, was obtained after selection with hygromycin. At 33 degrees C, the cells doubled in number in approximately 72 h; however, at 37 degrees C, cell proliferation stopped, indicating that the characteristic of temperature-dependent proliferation was retained in this clone. Ten million cells were injected into the spleen of syngeneic Gunn rats five times at 10-day intervals. Serum bilirubin levels were reduced by 45-50% at 70 days after the first transplantation and remained so throughout the duration of the study (120 days). These results suggested that the CAG promoter was able to maintain strong transcriptional activity in rat liver for at least 120 days.

Amelioration of radiation-induced liver damage in partially hepatectomized rats by hepatocyte transplantation.

Hepatic tumors often recur in the liver after surgical resection. Postoperative radiotherapy (RT) could improve survival, but curative RT may induce delayed life-threatening radiation-induced liver damage. Because RT inhibits liver regeneration, we hypothesized that unirradiated, transplanted hepatocytes would proliferate preferentially in a partially resected and irradiated liver, providing metabolic support. We subjected F344 rats to hepatic RT and partial hepatectomy with/without a single intrasplenic, syngeneic hepatocyte transplantation. Hepatocyte transplantation ameliorated radiation-induced liver damage and improved survival of rats receiving RT after partial hepatectomy. We further demonstrated that transplanted hepatocytes extensively repopulate and function in a heavily irradiated rat liver.

Long-term amelioration of bilirubin glucuronidation defect in Gunn rats by transplanting genetically modified immortalized autologous hepatocytes.

Ex vivo gene therapy, in which hepatocytes are harvested from mutants, retrovirally transduced with a normal gene and transplanted back into the donor, has been used for correction of inherited metabolic defects of liver. Major drawbacks of this method include limited availability of autologous hepatocytes, inefficient retroviral transduction of primary hepatocytes, and the limited number of hepatocytes that can be transplanted safely. To obviate these problems, we transduced primary hepatocytes derived from inbred bilirubin-UDP-glucuronosyl-transferase (BUGT)-deficient Gunn rats by infection with a recombinant retrovirus expressing temperature-sensitive mutant SV40 large T antigen (tsT). The immortalized cells were then transduced with a second recombinant retrovirus expressing human B-UGT, and a clone expressing high levels of the enzyme was expanded by culturing at permissive temperature (33 degrees C). At 37 degrees C, tsT antigen was degraded and the cells expressed UGT activity toward bilirubin at a level approximately twice that present in normal rat liver homogenates. For seeding the cells into the liver bed, 1 x 10(7) cells were injected into the spleens of syngeneic Gunn rats five times at 10-day intervals. Excretion of bilirubin glucuronides in bile was demonstrated by HPLC analysis and serum bilirubin levels were reduced by 27 to 52% in 40 days after the first transplantation and remained so throughout the duration of the study (120 days). None of the transplanted Gunn rats or SCID mice transplanted with the immortalized cells developed tumors.

Splice-site mutations: a novel genetic mechanism of Crigler-Najjar syndrome type 1.

Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression.

Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts.

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells.

Transient immunosuppression with FK506 permits long-term expression of therapeutic genes introduced into the liver using recombinant adenoviruses in the rat.

The host immune response limits the duration of expression of adenovirally transduced genes and precludes long-term gene expression upon re-administration of the virus. In this study we wished to evaluate whether short-term immunosuppression of the host, at the time of recombinant virus administration, would allow expression of the therapeutic gene product upon virus reinjection. Gunn rats were used as recipients of recombinant adenoviruses expressing human BUGT (Ad-hBUGT) or E. coli beta-galactosidase (Ad-LacZ). Rats were treated with FK506 (1-1.5 mg/kg, per OS daily) for three days beginning 24 hours before each virus injection. Control groups did not receive any immunosuppressant. The serum bilirubin level was reduced from 7.1 +/- 0.75 mg/dL to 2.0 +/- 0.7 mg/dL within two days of viral injection in both FK506 treated and control groups, and then gradually increased in 6 weeks. FK506-treated rats had low or undetectable antibody titers against the recombinant adenovirus and minimal or no cytotoxic T lymphocyte (CTL) response against adenovirus-infected cells. The tolerized rats received two subsequent injections 42 and 98 days after the first injection, which reduced the bilirubin levels again to 2.0 +/- 0.56 and 2.2 +/- 0.61 mg/dL, respectively. In contrast, control rats developed high titer neutralizing antibodies and a CTL response, and their serum bilirubin levels were not reduced following subsequent injections. We conclude that short-term FK506 treatment around the time of virus administration prevents the host immune response, permitting long-term gene therapy by repeated administration of the recombinant virus.

Massive repopulation of rat liver by transplantation of hepatocytes into specific lobes of the liver and ligation of portal vein branches to other lobes.

An important consideration in application of hepatocyte transplantation is whether the number of engrafted hepatocytes is sufficient to achieve the desired effect. Here we have evaluated the proliferative potential of transplanted primary hepatocytes during regeneration of hepatic lobes. Two million hepatocytes isolated from congeneic normal Wistar-RHA rats were injected into the main portal vein of deficient, jaundiced Gunn rats. The right branch of the portal vein was ligated 24 hr before hepatocyte transplantation (group A) or transiently clamped during hepatocyte injection (group B) or 24 hr after hepatocyte injection (group C). In these groups, the three lobes supplied by the right branch of the portal vein rapidly atrophied and disappeared in 4 days, whereas the remaining lobes proliferated, as shown by size increase and 5-bromo-2-deoxy-uridine uptake. Two control groups received 2 million (group D) or 20 million hepatocytes (group E) without ligation. Hepatocyte engraftment occurred in all groups. The greatest hypobilirubinemic effect was observed in group A, in which serum bilirubin concentrations were reduced to 1.7+/-0.45 mg/dl from pretransplantation levels of 6.9+/-1.2 mg/dl. This effect was even greater than that observed after transplantation of 20 times more hepatocytes without ligation (group E). Specific endonuclease digestion of a polymerase chain reaction-amplified segment of the ugt1 gene from hepatic DNA showed that up to 25% of the DNA was of donor origin. This paralleled the hepatic bilirubin-UDP-glucuronosyltransferase activity, which was above 50% of normal. The results indicate that the transplanted hepatocytes proliferate preferentially within the regenerating lobes, replacing more than 20% of the liver mass with the progeny of the transplanted phenotypically normal hepatocytes.

Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase.

Crigler-Najjar (CN) disease is an inherited disorder of bilirubin metabolism. The disease is caused by a deficiency of the hepatic enzyme bilirubin UDP-glucuronosyltransferase (B-UGT). Patients with CN disease have high serum levels of the toxic compound, unconjugated bilirubin. The only defect in bilirubin metabolism of CN patients is the absence of B-UGT activity. The transplantation of cells able to glucuronidate bilirubin should therefore lower serum bilirubin levels. The Gunn rat is the animal model of CN disease. Primary Gunn rat fibroblasts (GURF) were transduced with a recombinant retrovirus, capable of transferring B-UGT cDNA. A cell line was obtained expressing B-UGT at a level comparable to hepatocytes. Bilirubin added to the culture medium of these cells was glucuronidated and excreted. The B-UGT activities of transduced GURF and freshly isolated Wistar hepatocytes were compared at different bilirubin concentrations. The specific B-UGT activities of these two cell types were comparable when physiological bilirubin concentrations (5-10 microM) were present in the culture media. At higher bilirubin concentrations (20-80 microM) the hepatocytes were more active than the transduced GURF. We conclude that with the addition of only one enzyme (B-UGT) fibroblasts can perform the complete set of reactions necessary for bilirubin glucuronidation. The difference in B-UGT activity between transduced GURF and hepatocytes at 20-80 microM bilirubin can be explained by lower UDP-glucuronic acid and glutathione S-transferase levels in GURF. Our findings also indicate that these cells could be used to develop extrahepatic gene therapy for CN disease.

Molecular basis for the lack of bilirubin-specific and 3-methylcholanthrene-inducible UDP-glucuronosyltransferase activities in Gunn rats. The two isoforms are encoded by distinct mRNA species that share an identical single base deletion.

Gunn rats lack UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. In addition, a UDPGT isoform which is active toward 4-nitrophenol and is induced by 3-methylcholanthrene (3-MC) in normal rats, is produced in a nonfunctional truncated form in Gunn rats due to the deletion of a single guanosine residue in the coding region of its mRNA. The hepatic concentration of bilirubin-UDPGT mRNA was lower in Gunn rats than in congeneic normal rats. However, bilirubin-UDPGT mRNA was of apparently normal length and was induced by clofibrate, a known inducer of bilirubin-UDPGT activity. 3' regions of bilirubin- and 3-MC-inducible UDPGT mRNAs have identical nucleotide sequences; the single base deletion in the 3-MC-inducible UDPGT in Gunn rats occurs within this region. Using oligonucleotide primers corresponding to the identical and unique regions of the two mRNAs, and polymerase chain reaction, we amplified segments of mRNAs for the bilirubin- and 3-MC-inducible UDPGTs from normal and Gunn rat livers. Both amplified DNAs in Gunn rats lacked the restriction site for BstNI. Nucleotide sequence determination revealed that bilirubin- and 3-MC-inducible UDPGT mRNAs in Gunn rats contain an identical frame-shift deletion of a single guanosine residue within the common region of their coding sequences.

Hepatic uptake of bilirubin diglucuronide: analysis by using sinusoidal plasma membrane vesicles.

In order to characterize the mechanism for bilirubin transport in the liver, the uptake of bilirubin diglucuronide (BDG) into purified sinusoidal plasma membrane vesicles was investigated. BDG uptake was saturable, and was inhibited by sulfobromophthalein and unconjugated bilirubin, but was not affected by sodium taurocholate. BDG uptake was sodium-independent and was stimulated by intravesicular bilirubin or BDG (trans-stimulation). BDG transport showed strong potential sensitivity; vesicle inside-negative membrane potential created by different anion gradients inhibited BDG uptake whereas vesicle inside-positive membrane potential generated by potassium gradients and valinomycin markedly stimulated BDG transport. These data suggest that BDG, sulfobromophthalein, and probably unconjugated bilirubin share a common transporter in liver cells which is sodium independent, membrane-potential-dependent and capable of exchange. The direction of transport in vivo may be governed by the intracellular concentration of BDG and of other yet unidentified organic anions sharing this transporter.