Management of Alcohol and Tobacco Use Disorders in a 39-Year-Old Hispanic Male With a Complex Medical Background: A Case Report.

Substance use disorders affect the mental activities of an individual's brain and behavior, leading to a loss of control over their substance use, such as drugs, alcohol, and medication. However, these disorders are treatable. This case report presents and discusses the management of a 39-year-old Hispanic male with a complex medical background and a history of substance use. The patient, who resided with his mother in the Bronx, was admitted to the Outpatient Program (OPD) at the Life Recovery Center (LRC) Addiction Treatment Center for concurrent alcohol and tobacco use disorders. The patient had a history of anemia after bariatric surgery 10 years ago and no significant psychiatric history. Therefore, a comprehensive approach was required for the patient's treatment. The case further highlights the patient's presentation, treatment options, medication, and outcomes, which are essential for managing substance use disorders in individuals with complex medical backgrounds.

Validation of a novel point-of-care ultrasound method to assess diaphragmatic excursion.

INTRODUCTION: Point-of-care ultrasound can assess diaphragmatic function and rule in or rule out paresis of the diaphragm. While this is a useful bedside tool, established methods have significant limitations. This study explores a new method to assess diaphragmatic motion by measuring the excursion of the uppermost point of the zone of apposition (ZOA) at the mid-axillary line using a high-frequency linear ultrasound probe and compares it with two previously established methods: the assessment of the excursion of the dome of the diaphragm (DOD) and the thickening ratio at the ZOA. METHODS: This is a single-centre, prospective comparative study on elective surgical patients with normal diaphragmatic function. Following research ethics board approval and patient written consent, 75 elective surgical patients with normal diaphragmatic function were evaluated preoperatively. Three ultrasound methods were compared: (1) assessment of the excursion of the DOD using a curvilinear probe through an abdominal window; (2) assessment of the thickening fraction of the ZOA; and (3) assessment of the excursion of the ZOA. The last two methods performed with a linear probe on the lateral aspect of the chest. RESULTS: Seventy-five patients were studied. We found that the evaluation of the excursion of the ZOA was more consistently successful (100% bilaterally) than the evaluation of the excursion of the DOD (98.7% and 34.7% on the right and left sides, respectively). The absolute values of the excursion of the ZOA were greater than and well correlated with the values of the DOD. CONCLUSION: Our preliminary data from this exploratory study suggest that the evaluation of the excursion of the ZOA on the lateral aspect of the chest using a linear probe is consistently successful on both right and left sides. Future studies are needed to establish the distribution of normal values and suggest diagnostic criteria for diaphragmatic paresis or paralysis. TRIAL REGISTRATION NUMBER: NCT03225508.

Resolution of hepatic fibrosis after ZFN-mediated gene editing in the PiZ mouse model of human α1-antitrypsin deficiency.

BACKGROUND: α1-antitrypsin deficiency is most commonly caused by a mutation in exon-7 of SERPINA1 (SA1-ATZ), resulting in hepatocellular accumulation of a misfolded variant (ATZ). Human SA1-ATZ-transgenic (PiZ) mice exhibit hepatocellular ATZ accumulation and liver fibrosis. We hypothesized that disrupting the SA1-ATZ transgene in PiZ mice by in vivo genome editing would confer a proliferative advantage to the genome-edited hepatocytes, enabling them to repopulate the liver. METHODS: To create a targeted DNA break in exon-7 of the SA1-ATZ transgene, we generated 2 recombinant adeno-associated viruses (rAAV) expressing a zinc-finger nuclease pair (rAAV-ZFN), and another rAAV for gene correction by targeted insertion (rAAV-TI). PiZ mice were injected i.v. with rAAV-TI alone or the rAAV-ZFNs at a low (7.5×1010vg/mouse, LD) or a high dose (1.5×1011vg/mouse, HD), with or without rAAV-TI. Two weeks and 6 months after treatment, livers were harvested for molecular, histological, and biochemical analyses. RESULTS: Two weeks after treatment, deep sequencing of the hepatic SA1-ATZ transgene pool showed 6%±3% or 15%±4% nonhomologous end joining in mice receiving LD or HD rAAV-ZFN, respectively, which increased to 36%±12% and 36%±12%, respectively, 6 months after treatment. Two weeks postinjection of rAAV-TI with LD or HD of rAAV-ZFN, repair by targeted insertion occurred in 0.10%±0.09% and 0.25%±0.14% of SA1-ATZ transgenes, respectively, which increased to 5.2%±5.0% and 33%±13%, respectively, 6 months after treatment. Six months after rAAV-ZFN administration, there was a marked clearance of ATZ globules from hepatocytes, and resolution of liver fibrosis, along with reduction of hepatic TAZ/WWTR1, hedgehog ligands, Gli2, a TIMP, and collagen content. CONCLUSIONS: ZFN-mediated SA1-ATZ transgene disruption provides a proliferative advantage to ATZ-depleted hepatocytes, enabling them to repopulate the liver and reverse hepatic fibrosis.

Genotoxic impact of emerging contaminant amoxicillin residue on zebra fish (Danio rerio) embryos.

The widest-spectrum, most-consumed ß-lactam antibiotic amoxicillin (AMX) is used to treat bovine mastitis that is caused primarily by many bacteria. Excessive use of antibiotics can lead to established residual contamination of the milk even after pasteurization. The amount of antibiotic residue above Maximum Residue Limit (MRL) has a negative impact on both public health and the environment. Therefore, the objective of this study is to determine the concentration of amoxicillin residue (AMXR) in raw and pasteurized milk samples of cow suffered from mastitis, by the standard methods of HPLC compared to pure AMX drug and effect of the said residue on the developmental toxicity and genotoxicity of zebra fish at 72 hpf and 48 hpf embryo, respectively. Results obtained by HPLC showed that AMXR exhibits 574.89 and 250.75 times higher concentration in the raw and pasteurized milk than MRL in compare to pure AMX drug. This current study showed that AMXR decreased the body length and yolk sac region, while the pure AMX drug-treated group showed increased height and length of the yolk sac and shorter body length relative to the other groups. The Comet Assay measured the DNA damage caused by AMXR. The group where AMXR were applied showed the highest percentage of tail DNA and tail moment relative to other groups. So, here AMXR is considered as the genotoxic contaminant that is emerging and affect on public health.

Genes and Pathways Promoting Long-Term Liver Repopulation by Ex Vivo hYAP-ERT2 Transduced Hepatocytes and Treatment of Jaundice in Gunn Rats.

Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment. Here, we repopulated livers of DPPIV- (dipeptidyl peptidase-4) rats and Ugt1a1 (uridinediphosphoglucuronate glucuronosyltransferase 1a1)-deficient Gunn rats (model of Crigler-Najjar syndrome type 1), both models without underlying liver injury, for up to 1 year by transplanting lenti-hYAP-ERT2 (mutated estrogen receptor ligand-binding domain 2)-transduced hepatocytes (YAP-Hc). Yap (yes-associated protein) nuclear translocation/function in YAP-Hc was regulated by tamoxifen. Repopulating YAP-Hc and host hepatocytes were fluorescence-activated cell sorting-purified and their transcriptomic profiles compared by RNAseq. After 1 year of liver repopulation, YAP-Hc clusters exhibited normal morphology, integration into hepatic plates and hepatocyte-specific gene expression, without dysplasia, dedifferentiation, or tumorigenesis. RNAseq analysis showed up-regulation of 145 genes promoting cell proliferation and 305 genes suppressing apoptosis, including hepatocyte growth factor and connective tissue growth factor among the top 30 in each category and provided insight into the mechanism of cell competition that enabled replacement of host hepatocytes by YAP-Hc. In Gunn rats transplanted with YAP-Hc+tamoxifen, there was a 65%-81% decline in serum bilirubin over 6 months versus 8%-20% with control-Hc, representing a 3-4-fold increase in therapeutic response. This correlated with liver repopulation as demonstrated by the presence of Ugt1a1-positive hepatocyte clusters in livers and western blot analysis of tissue homogenates. Conclusion: Tamoxifen-regulated nuclear translocation/function of hYAP-ERT2 enabled long-term repopulation of DPPIV-/- and Gunn rat livers by hYAP-ERT2-transduced hepatocytes without tumorigenesis. This cell transplantation strategy may offer a potential therapy for most of the inherited monogenic liver diseases that do not exhibit liver injury.

Radiation-primed hepatocyte transplantation in murine monogeneic dyslipidemia normalizes cholesterol and prevents atherosclerosis.

BACKGROUND & AIMS: Inherited abnormalities in apolipoprotein E (ApoE) or low-density lipoprotein receptor (LDLR) function result in early onset cardiovascular disease and death. Currently, the only curative therapy available is liver transplantation. Hepatocyte transplantation is a potential alternative; however, physiological levels of hepatocyte engraftment and repopulation require transplanted cells to have a competitive proliferative advantage of over host hepatocytes. Herein, we aimed to test the efficacy and safety of a novel preparative regimen for hepatocyte transplantation. METHODS: Herein, we used an ApoE-deficient mouse model to test the efficacy of a new regimen for hepatocyte transplantation. We used image-guided external-beam hepatic irradiation targeting the median and right lobes of the liver to enhance cell transplant engraftment. This was combined with administration of the hepatic mitogen GC-1, a thyroid hormone receptor-ß agonist mimetic, which was used to promote repopulation. RESULTS: The non-invasive preparative regimen of hepatic irradiation and GC-1 was well-tolerated in ApoE-/- mice. This regimen led to robust liver repopulation by transplanted hepatocytes, which was associated with significant reductions in serum cholesterol levels after transplantation. Additionally, in mice receiving this regimen, ApoE was detected in the circulation 4 weeks after treatment and did not induce an immunological response. Importantly, the normalization of serum cholesterol prevented the formation of atherosclerotic plaques in this model. CONCLUSIONS: Significant hepatic repopulation and the cure of dyslipidemia in this model, using a novel and well-tolerated preparative regimen, demonstrate the clinical potential of applying this method to the treatment of inherited metabolic diseases of the liver. LAY SUMMARY: Hepatocyte transplantation is a promising alternative to liver transplantation for the treatment of liver diseases. However, it is inefficient, as restricted growth of transplanted cells in the liver limits its therapeutic benefits. Preparative treatments improve the efficiency of this procedure, but no clinically-feasible options are currently available. In this study we develop a novel well-tolerated preparative treatment to improve growth of cells in the liver and then demonstrate that this treatment completely cures an inherited lipid disorder in a mouse model.

Hepatocyte Transplantation: Quo Vadis?

Orthotopic liver transplantation (OLT) has been effective in managing end-stage liver disease since the advent of cyclosporine immunosuppression therapy in 1980. The major limitations of OLT are organ supply, monetary cost, and the burden of lifelong immunosuppression. Hepatocyte transplantation, as a substitute for OLT, has been an exciting topic of investigation for several decades. HT is potentially minimally invasive and can serve as a vehicle for delivery of personalized medicine through autologous cell transplant after modification ex vivo. However, 3 major hurdles have prevented large-scale clinical application: (1) availability of transplantable cells; (2) safe and efficient ex vivo gene therapy methods; and (3) engraftment and repopulation efficiency. This review will discuss new sources for transplantable liver cells obtained by lineage reprogramming, clinically acceptable methods of genetic manipulation, and the development of hepatic irradiation-based preparative regimens for enhancing engraftment and repopulation of transplanted hepatocytes. We will also review the results of the first 3 patients with genetic liver disorders who underwent preparative hepatic irradiation before hepatocyte transplantation.

Drug- and Drug Abuse-Associated Hyperbilirubinemia: Experience With Atazanavir.

Hyperbilirubinemia is a common finding in individuals with a history of substance abuse. Although this may indicate a serious disorder of liver function, this is not always the case. An understanding of bilirubin formation, metabolism, and transport can provide a helpful approach to dealing with these patients. This is typified by studies of patients treated with the antiretroviral drug atazanavir. Atazanavir has been associated with hyperbilirubinemia in as many as one-third of individuals for whom it has been prescribed, evoking concerns of hepatotoxicity. The studies in this report were designed to determine mechanisms by which this occurs. The data show that this drug inhibits the enzyme UDP-glucuronosyl transferase-1A1, responsible for conjugating bilirubin with glucuronic acid. This conjugation step is required for bilirubin excretion into bile, and when it is inhibited, bilirubin refluxes from the liver into the circulation, causing unconjugated hyperbilirubinemia. Other parameters of bilirubin formation, binding to albumin in the circulation, uptake into hepatocytes, and intracellular protein binding in hepatocytes were unaffected by atazanavir. The effect of atazanavir on serum bilirubin levels is reversible, consistent with lack of structural damage to the liver.

Host conditioning and rejection monitoring in hepatocyte transplantation in humans.

BACKGROUND & AIMS: Hepatocyte transplantation partially corrects genetic disorders and has been associated anecdotally with reversal of acute liver failure. Monitoring for graft function and rejection has been difficult, and has contributed to limited graft survival. Here we aimed to use preparative liver-directed radiation therapy, and continuous monitoring for possible rejection in an attempt to overcome these limitations. METHODS: Preparative hepatic irradiation was examined in non-human primates as a strategy to improve engraftment of donor hepatocytes, and was then applied in human subjects. T cell immune monitoring was also examined in human subjects to assess adequacy of immunosuppression. RESULTS: Porcine hepatocyte transplants engrafted and expanded to comprise up to 15% of irradiated segments in immunosuppressed monkeys preconditioned with 10Gy liver-directed irradiation. Two patients with urea cycle deficiencies had early graft loss following hepatocyte transplantation; retrospective immune monitoring suggested the need for additional immunosuppression. Preparative radiation, anti-lymphocyte induction, and frequent immune monitoring were instituted for hepatocyte transplantation in a 27year old female with classical phenylketonuria. Post-transplant liver biopsies demonstrated multiple small clusters of transplanted cells, multiple mitoses, and Ki67+ hepatocytes. Mean peripheral blood phenylalanine (PHE) level fell from pre-transplant levels of 1343±48µM (normal 30-119µM) to 854±25µM (treatment goal ≤360µM) after transplant (36% decrease; p<0.0001), despite transplantation of only half the target number of donor hepatocytes. PHE levels remained below 900µM during supervised follow-up, but graft loss occurred after follow-up became inconsistent. CONCLUSIONS: Radiation preconditioning and serial rejection risk assessment may produce better engraftment and long-term survival of transplanted hepatocytes. Hepatocyte xenografts engraft for a period of months in non-human primates and may provide effective therapy for patients with acute liver failure. LAY SUMMARY: Hepatocyte transplantation can potentially be used to treat genetic liver disorders but its application in clinical practice has been impeded by inefficient hepatocyte engraftment and the inability to monitor rejection of transplanted liver cells. In this study, we first show in non-human primates that pretreatment of the host liver with radiation improves the engraftment of transplanted liver cells. We then used this knowledge in a series of clinical hepatocyte transplants in patients with genetic liver disorders to show that radiation pretreatment and rejection risk monitoring are safe and, if optimized, could improve engraftment and long-term survival of transplanted hepatocytes in patients.

Gunn Rats as a Surrogate Model for Evaluation of Hepatocyte Transplantation-Based Therapies of Crigler-Najjar Syndrome Type 1.

Liver transplantation has been established as a curative therapy for acute and chronic liver failure, as well as liver-based inherited metabolic diseases. Because of the complexity of organ transplantation and the worldwide shortage of donor organs, hepatocyte transplantation is being developed as a bridging therapy until donor organs become available, or for amelioration of inherited liver-based diseases. The Gunn rat is a molecular and metabolic model of Crigler-Najjar syndrome type 1, which is characterized by lifelong unconjugated hyperbilirubinemia due to the lack of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-mediated bilirubin glucuronidation. Gunn rats are convenient for evaluating the effect of hepatocyte transplantation or gene therapy, because the extent of UGT1A1 replacement can be assessed by serial determination of serum bilirubin levels, and excretion of bilirubin glucuronides in bile provide definitive evidence of the function of the transplanted hepatocytes or the effect of gene therapy. The core techniques involved in hepatocyte transplantation in Gunn rats are discussed in this chapter.

Hepatocyte-like cells derived from induced pluripotent stem cells.

The discovery that coordinated expression of a limited number of genes can reprogram differentiated somatic cells to induced pluripotent stem cells (iPSC) has opened novel possibilities for developing cell-based models of diseases and regenerative medicine utilizing cell reprogramming or cell transplantation. Directed differentiation of iPSCs can potentially generate differentiated cells belonging to any germ layer, including cells with hepatocyte-like morphology and function. Such cells, termed iHeps, can be derived by sequential cell signaling using available information on embryological development or by forced expression of hepatocyte-enriched transcription factors. In addition to the translational aspects of iHeps, the experimental findings have provided insights into the mechanisms of cell plasticity that permit one cell type to transition to another. However, iHeps generated by current methods do not fully exhibit all characteristics of mature hepatocytes, highlighting the need for additional research in this area. Here we summarize the current approaches and achievements in this field and discuss some existing hurdles and emerging approaches for improving iPSC differentiation, as well as maintaining such cells in culture for increasing their utility in disease modeling and drug development.

Oxalobacter formigenes-Derived Bioactive Factors Stimulate Oxalate Transport by Intestinal Epithelial Cells.

Hyperoxaluria is a major risk factor for kidney stones and has no specific therapy, although Oxalobacter formigenes colonization is associated with reduced stone risk. O. formigenes interacts with colonic epithelium and induces colonic oxalate secretion, thereby reducing urinary oxalate excretion, via an unknown secretagogue. The difficulties in sustaining O. formigenes colonization underscore the need to identify the derived factors inducing colonic oxalate secretion. We therefore evaluated the effects of O. formigenes culture conditioned medium (CM) on apical 14C-oxalate uptake by human intestinal Caco-2-BBE cells. Compared with control medium, O. formigenes CM significantly stimulated oxalate uptake (>2.4-fold), whereas CM from Lactobacillus acidophilus did not. Treating the O. formigenes CM with heat or pepsin completely abolished this bioactivity, and selective ultrafiltration of the CM revealed that the O. formigenes-derived factors have molecular masses of 10-30 kDa. Treatment with the protein kinase A inhibitor H89 or the anion exchange inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid completely blocked the CM-induced oxalate transport. Knockdown of the oxalate transporter SLC26A6 also significantly restricted the induction of oxalate transport by CM. In a mouse model of primary hyperoxaluria type 1, rectal administration of O. formigenes CM significantly reduced (>32.5%) urinary oxalate excretion and stimulated (>42%) distal colonic oxalate secretion. We conclude that O. formigenes-derived bioactive factors stimulate oxalate transport in intestinal cells through mechanisms including PKA activation. The reduction in urinary oxalate excretion in hyperoxaluric mice treated with O. formigenes CM reflects the in vivo retention of biologic activity and the therapeutic potential of these factors.

Human Urinary Epithelial Cells as a Source of Engraftable Hepatocyte-Like Cells Using Stem Cell Technology.

Although several types of somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (iHeps), the method for generating such cells from renal tubular epithelial cells shed in human urine and transplanting them into animal livers has not been described systematically. We report reprogramming of human urinary epithelial cells into iPSCs and subsequent hepatic differentiation, followed by a detailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by delivering the pluripotency factors OCT3/4, SOX2, KLF4, and MYC using methods that do not involve transgene integration, such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPSC lines, a three-step differentiation toward hepatocytes was performed. The iHeps expressed a large number of hepatocyte-preferred genes, including nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport, and detoxification. MicroRNA profile of the iHeps largely paralleled that of primary human hepatocytes. The iHeps engrafted into the livers of Scid mice transgenic for mutant human SERPINA1 after intrasplenic injection. Thus, urine is a readily available source for generating human iHeps that could be potentially useful for disease modeling, pharmacological development, and regenerative medicine.

Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes.

Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%-7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%-60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.

Induced pluripotent stem cells model personalized variations in liver disease resulting from α1-antitrypsin deficiency.

UNLABELLED: In the classical form of α1-antitrypsin deficiency (ATD), aberrant intracellular accumulation of misfolded mutant α1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function, "proteotoxic" mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation, others with the same genetic defect completely escape this clinical phenotype. We investigated whether induced pluripotent stem cells (iPSCs) from ATD individuals with or without SLD could model these personalized variations in hepatic disease phenotypes. Patient-specific iPSCs were generated from ATD patients and a control and differentiated into hepatocyte-like cells (iHeps) having many characteristics of hepatocytes. Pulse-chase and endoglycosidase H analysis demonstrate that the iHeps recapitulate the abnormal accumulation and processing of the ATZ molecule, compared to the wild-type AT molecule. Measurements of the fate of intracellular ATZ show a marked delay in the rate of ATZ degradation in iHeps from SLD patients, compared to those from no liver disease patients. Transmission electron microscopy showed dilated rough endoplasmic reticulum in iHeps from all individuals with ATD, not in controls, but globular inclusions that are partially covered with ribosomes were observed only in iHeps from individuals with SLD. CONCLUSION: iHeps model the individual disease phenotypes of ATD patients with more rapid degradation of misfolded ATZ and lack of globular inclusions in cells from patients who have escaped liver disease. The results support the concept that "proteostasis" mechanisms, such as intracellular degradation pathways, play a role in observed variations in clinical phenotype and show that iPSCs can potentially be used to facilitate predictions of disease susceptibility for more precise and timely application of therapeutic strategies.

Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus and extended spectrum ß-lactamase producing Escherichia coli in bovine mastitis--three cases reported from India.

Emergence of antimicrobial resistance among bovine mastitis pathogens is the major cause of frequent therapeutic failure and a cause of concern for veterinary practitioners. This study describes intra-mammary infection of methicillin-resistant Staphylococcus epidermidis (MRSE), methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum ß-lactamase (ESBL) producing Escherichia coli in two Holstein Friesian crossbred cows with subclinical mastitis and one non-descript cow with clinical mastitis in two different districts of West Bengal, India. In total, three MRSE, one MRSA and three ESBL producing E. coli were isolated from these cases. Both the crossbreds were detected with MRSE (HFSE1 and HFSE2) and ESBL producing E. coli (HFEC1 and HFEC2), whereas, simultaneous infection of three pathogens viz. MRSA (NDSA1), MRSE (NDSE1) and ESBL producing E. coli (NDEC1) was found in the non-descript cow. The methicillin-resistant isolates possessed mecA gene and exhibited resistance to various antibiotics such as amikacin, tetracycline and glycopeptides. The ESBL producers were positive for blaCTX-M and blaTEM genes; in addition, HFEC1 and HFEC2 were positive for blaSHV and possessed the genes for class I integron (int1), sulphonamide resistance (sul1), quinolone resistance (qnrS) and other virulence factors (papC, iucD and ESTA1). All the ESBL producers exhibited resistance to a variety of antibiotics tested including third- and fourth-generation cephalosporins and were also intermediately resistant to carbapenems. This is the first ever report on simultaneous occurrence of MRSE, MRSA and ESBL producing E. coli in bovine mastitis indicating a major concern for dairy industry and public health as well.

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (51Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT + HSV-TK/GCV + Flt3L + CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT + HSV-TK/GCV + Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 1. Flt3 ligand gene transfer increases antitumor effects of a radio-inducible suicide gene therapy in an ectopic tumor model.

Hepatocellular carcinoma (HCC) often presents as a diffuse or multifocal tumor making it difficult to control by surgery or radiation. Radio-inducible herpes simplex virus thymidine kinase (HSV-TK) gene therapy has been shown to enhance local tumor control after radiation therapy (RT), while limiting the expression of the transgene in the irradiated tumor tissues. To prevent liver tumor recurrence and control systemic disease while limiting the potential bystander toxicity of HSV-TK therapy, we proposed to stimulate endogenous dendritic cell (DC) proliferation with systemic adenovirus Flt3 ligand (Adeno-Flt3L) gene therapy, followed by primary tumor radiation therapy combined with a radio-inducible HSV-TK gene therapy. We hypothesized that adenovirus-expressing Flt3L gene therapy will stimulate DC proliferation, allowing the upregulated DCs to locally harness tumor antigens released from HSV-TK/RT-treated HCC cells, thereby converting irradiated tumors to an autologous in situ tumor vaccine in mice with primary liver tumors. To test this hypothesis, an expression vector of HSV-TK was constructed under the control of a radio-inducible promoter early-growth response (Egr-TK) and a recombinant adenovirus-expressing human Flt3L was constructed. The Adeno-Flt3L [10(9) plaque forming units (pfu)] was administered intravenously on days 1 and 8 after radiation therapy. The murine hepatoma cell line (BNL1ME) was stably transfected by Egr-TK or Egr-Null (encoding no therapeutic gene). Palpable tumors in BALB/c mice were treated with a localized dose of 25 Gy of radiation followed by ganciclovir (GCV, 100 mg/kg, 14 days). Four treatment cohorts were compared: Egr-Null/GCV + RT + Adeno-LacZ; Egr-Null/GCV + RT + Adeno-Flt3L; Egr-TK/GCV + RT + Adeno-LacZ; and Egr-TK/GCV + RT + Adeno-Flt3L. There was no primary tumor regression in the Egr-Null tumors after radiation therapy alone. In contrast, Egr-TK tumors had nearly complete tumor regression for 3 weeks after radiation therapy (P < 0.01), however, long-term follow-up demonstrated primary tumor recurrence and death secondary to pulmonary metastasis. Flt3L expression was confirmed by serum bioassay (mean = 88 ng/mL) in these animals and Western blotting of tissue culture medium in Adeno-Flt3L-infected BaF/huFlt3L cells. Radiation therapy with Adeno-Flt3L gene therapy effectively retarded primary tumor growth when compared to radiation therapy alone. The trimodality therapy (Egr-TK/GCV + RT + Adeno-Flt3L) was the most efficacious with 40% complete tumor regression (>100 days) and <20% pulmonary metastases, indicating the development of sustained antitumor immune response. These studies provide a rationale for triple modality therapies with radiation-inducible HSV-TK gene therapy and Adeno-Flt3L when used in combination with primary tumor radiation therapy for improved local and systemic control of HCC.

Bone marrow-derived stromal cell therapy in cirrhosis: clinical evidence, cellular mechanisms, and implications for the treatment of hepatocellular carcinoma.

Current treatment options for hepatocellular carcinoma (HCC) are often limited by the presence of underlying liver disease. In patients with liver cirrhosis, surgery, chemotherapy, and radiation therapy all carry a high risk of hepatic complications, ranging from ascites to fulminant liver failure. For patients receiving radiation therapy, cirrhosis dramatically reduces the already limited radiation tolerance of the liver and represents the most important clinical risk factor for the development of radiation-induced liver disease. Although improvements in conformal radiation delivery techniques have improved our ability to safely irradiate confined areas of the liver to increasingly higher doses with excellent local disease control, patients with moderate-to-severe liver cirrhosis continue to face a shortage of treatment options for HCC. In recent years, evidence has emerged supporting the use of bone marrow-derived stromal cells (BMSCs) as a promising treatment for liver cirrhosis, with several clinical studies demonstrating sustained improvement in clinical parameters of liver function after autologous BMSC infusion. Three predominant populations of BMSCs, namely hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells, seem to have therapeutic potential in liver injury and cirrhosis. Preclinical studies of BMSC transplantation have identified a range of mechanisms through which these cells mediate their therapeutic effects, including hepatocyte transdifferentiation and fusion, paracrine stimulation of hepatocyte proliferation, inhibition of activated hepatic stellate cells, enhancement of fibrolytic matrix metalloproteinase activity, and neovascularization of regenerating liver. By bolstering liver function in patients with underlying Child's B or C cirrhosis, autologous BMSC infusion holds great promise as a therapy to improve the safety, efficacy, and utility of surgery, chemotherapy, and hepatic radiation therapy in the treatment of HCC.