Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing.

BACKGROUND: The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. METHODS: We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. FINDINGS: After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. INTERPRETATION: Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. FUNDING: Foundation for Liver Research, London.

Novel therapeutic strategies for chronic hepatitis B.

The last few years have seen a resurgence of activity in the hepatitis B drug pipeline, with many compounds in various stages of development. This review aims to provide a comprehensive overview of the latest advances in therapeutics for chronic hepatitis B (CHB). We will discuss the broad spectrum of direct-acting antivirals in clinical development, including capsids inhibitors, siRNA, HBsAg and polymerase inhibitors. In addition, host-targeted therapies (HTT) will be extensively reviewed, focusing on the latest progress in immunotherapeutics such as toll-like receptors and RIG-1 agonists, therapeutic vaccines and immune checkpoints modulators. A growing number of HTT in pre-clinical development directly target the key to HBV persistence, namely the covalently closed circular DNA (cccDNA) and hold great promise for HBV cure. This exciting area of HBV research will be highlighted, and molecules such as cyclophilins inhibitors, APOBEC3 deaminases and epigenetic modifiers will be discussed.

A review of alcohol-pathogen interactions: New insights into combined disease pathomechanisms.

Progression of chronic infections to end-stage diseases and poor treatment results are frequently associated with alcohol abuse. Alcohol metabolism suppresses innate and adaptive immunity leading to increased viral load and its spread. In case of hepatotropic infections, viruses accelerate alcohol-induced hepatitis and liver fibrosis, thereby promoting end-stage outcomes, including cirrhosis and hepatocellular carcinoma (HCC). In this review, we concentrate on several unexplored aspects of these phenomena, which illustrate the combined effects of viral/bacterial infections and alcohol in disease development. We review alcohol-induced alterations implicated in immunometabolism as a central mechanism impacting metabolic homeostasis and viral pathogenesis in Simian immunodeficiency virus/human immunodeficiency virus infection. Furthermore, in hepatocytes, both HIV infection and alcohol activate oxidative stress to cause lysosomal dysfunction and leakage and apoptotic cell death, thereby increasing hepatotoxicity. In addition, we discuss the mechanisms of hepatocellular carcinoma and tumor signaling in hepatitis C virus infection. Finally, we analyze studies that review and describe the immune derangements in hepatotropic viral infections focusing on the development of novel targets and strategies to restore effective immunocompetency in alcohol-associated liver disease. In conclusion, alcohol exacerbates the pathogenesis of viral infections, contributing to a chronic course and poor outcomes, but the mechanisms behind these events are virus specific and depend on virus-alcohol interactions, which differ among the various infections.

Dental School Patient Preferences for Receiving and Returning Patient Satisfaction Surveys.

The aims of this study were to investigate dental school patient, parent, and caregiver preferences for receiving and returning patient satisfaction surveys and to determine how modes of receiving and returning surveys impacted return rates. Two studies were conducted, both of which are reported. In Study 1, adult patients, parents of child patients, and caregivers of patients with special needs were asked to indicate their preferred mode of receiving and returning surveys. In Study 2, patients, parents, and caregivers were randomly assigned to different modes of receiving and returning surveys, and return rates were compared between modes and according to whether the individuals had been assigned to their preferred mode or not. The overall response rates were 90.4% for the first study and 48.1% for the second study. In both studies, the preferred mode was receiving and returning the survey in the clinic (chi-square=84.902 and 32.116; df=3; p<0.001). Younger respondents were more likely to prefer receiving and returning the survey by email (K-W statistics=13.406, 10.241; df=3; p=0.004 and 0.017). In Study 2, respondents were significantly more likely to return surveys in the clinic (chi-square=44.994; df=2; p<0.001) and were also significantly more likely to return surveys if they had received them in their preferred mode (binomial test p<0.001). Although receiving and returning the survey in the clinic was the preferred mode, these respondents' preferences were also related to their age. These results suggest that dental school clinics may be able to expect higher return rates if they can follow patients' preferences for receipt and delivery of surveys.

Peg-Interferon Lambda Treatment Induces Robust Innate and Adaptive Immunity in Chronic Hepatitis B Patients.

IFN-lambda (IFNλ) is a member of the type III IFN family and is reported to possess anti-pathogen, anti-cancer, and immunomodulatory properties; however, there are limited data regarding its impact on host immune responses in vivo. We performed longitudinal and comprehensive immunosurveillance to assess the ability of pegylated (peg)-IFNλ to augment antiviral host immunity as part of a clinical trial assessing the efficacy of peg-IFNλ in chronic hepatitis B (CHB) patients. These patients were pretreated with directly acting antiviral therapy (entecavir) for 12 weeks with subsequent addition of peg-IFNλ for up to 32 weeks. In a subgroup of patients, the addition of peg-IFNλ provoked high serum levels of antiviral cytokine IL-18. We also observed the enhancement of natural killer cell polyfunctionality and the recovery of a pan-genotypic HBV-specific CD4+ T cells producing IFN-γ with maintenance of HBV-specific CD8+ T cell antiviral and cytotoxic activities. It was only in these patients that we observed strong virological control with reductions in both viral replication and HBV antigen levels. Here, we show for the first time that in vivo peg-IFNλ displays significant immunostimulatory properties with improvements in the main effectors mediating anti-HBV immunity. Interestingly, the maintenance in HBV-specific CD8+ T cells in the presence of peg-IFNλ is in contrast to previous studies showing that peg-IFNα treatment for CHB results in a detrimental effect on the functionality of this important antiviral T cell compartment. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT01204762.

How are allied health notes used for inpatient care and clinical decision-making? A qualitative exploration of the views of doctors, nurses and allied health professionals.

BACKGROUND: Inpatient care is dependent upon the effective transfer of clinical information across multiple professions. However, documented patient clinical information generated by different professions is not always successfully transferred between them. One obstacle to successful information transfer may be the reader's perception of the information, which is framed in a particular professional context, rather than the information per se. OBJECTIVE: The aim of this research was to investigate how different health professionals perceive allied health documentation and to investigate how clinicians of all experience levels across medicine, nursing and allied health perceive and use allied health notes to inform their decision-making and treatment of patients. METHOD: The study used a qualitative approach. A total of 53 speech pathologists, nurses, doctors, occupational therapists, dieticians and social workers (8 males; 43 females) from an Australian regional tertiary hospital participated in eleven single discipline focus groups, conducted over 4 months in 2012. Discussions were recorded and transcribed verbatim and coded into themes by content analysis. FINDINGS: Six themes contributing to the efficacy of clinical information transference emerged from the data: day-to-day care, patient function, discharge and discharge planning, impact of busy workloads, format and structure of allied health documentation and a holistic approach to patient care. DISCUSSION: Other professions read and used allied health notes albeit with differences in focus and need. Readers searched for specific pieces of information to answer their own questions and professional needs, in a process akin to purposive sampling. Staff used allied health notes to explore specific aspects of patient function but did not obtain a holistic picture. CONCLUSION: Improving both the relationship between the various health professions and interpretation of other professions' documented clinical information may reduce the frequency of communication errors, thereby improving patient care.

Alisporivir inhibition of hepatocyte cyclophilins reduces HBV replication and hepatitis B surface antigen production.

BACKGROUND & AIMS: Cyclophilins are host factors required for hepatitis C virus replication. Cyclophilin inhibitors such as alisporivir have shown strong anti-hepatitis C virus activity in vitro and in clinical studies. However, little is known about whether hepatocyte cyclophilins are involved in the hepatitis B virus (HBV) life cycle. We investigated the effects of 2 cyclophilin inhibitors (alisporivir and NIM811) on HBV replication and hepatitis B surface antigen (HBsAg) production in cell lines. METHODS: Liver-derived cell lines producing full-length HBV and HBsAg particles, owing to stable (HepG2215) or transient (HuH-7) transfection, or infected with HBV (HepaRG cells; Invitrogen [Carlsbad, CA]), were incubated with alisporivir or NIM811 alone, or alisporivir in combination with a direct antiviral (telbivudine). The roles of individual cyclophilins in drug response was evaluated by small interfering RNA knockdown of cyclophilin (CYP)A, CYPC, or CYPD in HepG2215 cells, or CYPA knockdown in HuH-7 cells. The kinetics of antiviral activity were assessed based on levels of HBV DNA and HBsAg and Southern blot analysis. RESULTS: In HepG2215, HuH-7, and HepaRG cells, alisporivir reduced intracellular and secreted HBV DNA, in a dose-dependent manner. Knockdown of CYPA, CYPC, or CYPD (reduced by 80%) significantly reduced levels of HBV DNA and secreted HBsAg. Knockdown of CYPA significantly reduced secretion of HBsAg, leading to accumulation of intracellular HBsAg; the addition of alisporivir greatly reduced levels of HBsAg in these cells. The combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone. CONCLUSIONS: Alisporivir inhibition of cyclophilins in hepatocyte cell lines reduces replication of HBV DNA and HBsAg production and secretion. These effects are potentiated in combination with direct antiviral agents that target HBV-DNA polymerase.

Identification of serum cytokine profiles associated with HBeAg seroconversion following antiviral treatment interruption.

The major shortcoming of nucleos(t)ide analogue therapy for chronic hepatitis B (CHB) is the frequent requirement for indefinite therapy. Withdrawal of treatment can result in viral rebound with an alanine aminotransferase (ALT) flare leading to hepatic decompensation, while in others this can lead to hepatitis B e antigen (HBeAg) seroconversion. The aim of the study was to identify host immune profiles associated with these different outcomes. Eighteen HBeAg(+) patients, enrolled on a phase III trial with the nucleoside analogue adefovir dipivoxil, were followed for up to 128 weeks. For the first 48 weeks, all patients received continuous therapy. Subsequently, patients experienced cycles of treatment interruptions due to random drug/placebo misallocations. Host immune profiles were characterized by measuring a panel of serum cytokines before, during, and after each cycle of treatment withdrawal. Virus-specific T-cell responses were also determined at these time points in a subset of patients to elucidate the mechanisms utilized to control hepatitis B virus (HBV) replication post-treatment. Significantly, elevated levels of IFN-γ, IP-10, and IL-2 on-treatment were associated with HBeAg seroconversion after treatment withdrawal. In these patients, treatment interruption induced further increases in serum IFN-γ levels and marked increases in virus-specific T-cells producing IFN-γ, but minimal alterations in viremia and ALT. In HBeAg(+) patients with low on-treatment levels of serum IFN-γ, the interruption of therapy induced significant elevations in HBV-DNA, ALT, IP-10, and increases in virus-specific T-cells producing IL-10. Evaluating on-treatment serum cytokines in concert with virologic and clinical parameters may help to identify CHB patients who can successfully discontinue nucleos(t)ide analogue therapy.

Truncus arteriosus 5th decade transesophageal and transthoracic echocardiogram features.

Persistent truncus arteriosus in the 5th decade is a rare entity as the only physical anomaly. Truncus arteriosus consists of a single great arterial trunk with the systemic, pulmonary, and coronary circulation, with no atretic aortic or pulmonary vessel. The complex cardiac anatomy can be delineated with transesophageal and transthoracic echocardiography, as is illustrated the case of our patient, who was diagnosed in infancy, at 5 months of age. This is the oldest documented case report of Type 3 truncus arteriosus with balanced biventricular anatomy, whose survival was secondary to an innate severe branch pulmonary artery stenosis. .

Novel mechanism of antibodies to hepatitis B virus in blocking viral particle release from cells.

UNLABELLED: Antibodies are thought to exert antiviral activities by blocking viral entry into cells and/or accelerating viral clearance from circulation. In particular, antibodies to hepatitis B virus (HBV) surface antigen (HBsAg) confer protection, by binding circulating virus. Here, we used mathematical modeling to gain information about viral dynamics during and after single or multiple infusions of a combination of two human monoclonal anti-HBs (HepeX-B) antibodies in patients with chronic hepatitis B. The antibody HBV-17 recognizes a conformational epitope, whereas antibody HBV-19 recognizes a linear epitope on the HBsAg. The kinetic profiles of the decline of serum HBV DNA and HBsAg revealed partial blocking of virion release from infected cells as a new antiviral mechanism, in addition to acceleration of HBV clearance from the circulation. We then replicated this approach in vitro, using cells secreting HBsAg, and compared the prediction of the mathematical modeling obtained from the in vivo kinetics. In vitro, HepeX-B treatment of HBsAg-producing cells showed cellular uptake of antibodies, resulting in intracellular accumulation of viral particles. Blocking of HBsAg secretion also continued after HepeX-B was removed from the cell culture supernatants. CONCLUSION: These results identify a novel antiviral mechanism of antibodies to HBsAg (anti-HBs) involving prolonged blocking of the HBV and HBsAg subviral particles release from infected cells. This may have implications in designing new therapies for patients with chronic HBV infection and may also be relevant in other viral infections.

CD8(+) T cell control of hepatitis B virus replication: direct comparison between cytolytic and noncytolytic functions.

Resolution of hepatitis B virus (HBV) infection was believed to be attributed to the cytotoxic T cell-mediated killing of infected hepatocytes. However, studies in HBV transgenic mice and HBV-infected chimpanzees revealed that T cell control of HBV replication also involves cytokine-mediated noncytolytic mechanisms. The relative role of cytolytic and noncytolytic functions of virus-specific CD8(+) T cells during interaction with HBV-producing hepatocytes is not well understood. By using HLA-A2 matched effector cells (CD8(+) T cell line or clone) and target cells supporting full HBV replication, we demonstrate that virus-specific CD8(+) T cells can inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. Although CD8(+) T cells kill a fraction of infected cells, this effect is minimal, and most of the viral inhibition is mediated by noncytolytic mechanisms. CD8(+) T cells produce an array of cytokines, among which IFN-gamma and TNF-alpha are responsible for HBV inactivation in the target cells. Blockade of IFN-gamma and TNF-alpha abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines mediate the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN-gamma and rTNF-alpha resulted in an efficient suppression of viral replication without cytotoxicity. In contrast, coculture of the same target cells with activated HLA-mismatched mitogen-activated lymphomononuclear cells caused a marked cytolytic effect and was less effective in HBV control. These results provide direct evidence that virus-specific CD8(+) T cells efficiently control HBV replication by noncytolytic mechanisms, and this effect is mediated by IFN-gamma and TNF-alpha.

Expression of Acidothermus cellulolyticus E1 endo-beta-1,4-glucanase catalytic domain in transplastomic tobacco.

As part of an effort to develop transgenic plants as a system for the production of lignocellulose-degrading enzymes, we evaluated the production of the endo-beta-1,4-glucanase E1 catalytic domain (E1cd) of Acidothermus cellulolyticus in transplastomic tobacco. In an attempt to increase the translation efficiency of the E1cd cassette, various lengths of the N-terminus of the psbA gene product were fused to the E1cd protein. The psbA gene of the plastid genome encodes the D1 polypeptide of photosystem II and is known to encode an efficiently translated mRNA. Experiments in an Escherichia coli expression system indicated that the fusion of short (10-22 amino acid) segments of D1 to E1cd resulted in modest increases in E1cd abundance and were compatible with E1cd activity. Plastid expression cassettes encoding unmodified E1cd and a 10-amino-acid D1 fusion (10nE1cd) were used to generate transplastomic tobacco plants. Expression of the E1cd open reading frame in transplastomic tobacco resulted in very low levels of the enzyme. The transplastomic plants accumulated a high level of E1cd mRNA, however, indicating that post-transcriptional processes were probably limiting the production of recombinant protein. The accumulation of 10nE1cd in transplastomic tobacco was approximately 200-fold higher than that of unmodified E1cd, yielding 10nE1cd in excess of 12% of total soluble protein in the extracts of the lower leaves. Most importantly, the active recombinant enzyme was recovered very easily and efficiently from dried plant material and constituted as much as 0.3% of the dry weight of leaf tissue.

Higher copy numbers of the potato RB transgene correspond to enhanced transcript and late blight resistance levels.

Late blight of potato ranks among the costliest of crop diseases worldwide. Host resistance offers the best means for controlling late blight, but previously deployed single resistance genes have been short-lived in their effectiveness. The foliar blight resistance gene RB, previously cloned from the wild potato Solanum bulbocastanum, has proven effective in greenhouse tests of transgenic cultivated potato. In this study, we examined the effects of the RB transgene on foliar late blight resistance in transgenic cultivated potato under field production conditions. In a two-year replicated trial, the RB transgene, under the control of its endogenous promoter, provided effective disease resistance in various genetic backgrounds, including commercially prominent potato cultivars, without fungicides. RB copy numbers and transcript levels were estimated with transgene-specific assays. Disease resistance was enhanced as copy numbers and transcript levels increased. The RB gene, like many other disease resistance genes, is constitutively transcribed at low levels. Transgenic potato lines with an estimated 15 copies of the RB transgene maintain high RB transcript levels and were ranked among the most resistant of 57 lines tested. We conclude that even in these ultra-high copy number lines, innate RNA silencing mechanisms have not been fully activated. Our findings suggest resistance-gene transcript levels may have to surpass a threshold before triggering RNA silencing. Strategies for the deployment of RB are discussed in light of the current research.

Programmed death 1 expression during antiviral treatment of chronic hepatitis B: Impact of hepatitis B e-antigen seroconversion.

UNLABELLED: Hyperexpression of the programmed death 1 (PD-1) molecule is a hallmark of exhausted T-cells, having a negative impact on T-cell activation and function. We studied longitudinally 18 hepatitis B e antigen (HBeAg)-positive patients undergoing treatment with direct antivirals (telbivudine or lamivudine) to determine the relationship between treatment-induced viremia reduction and HBeAg seroconversion with respect to PD-1 levels and T-cell reactivity. PD-1 expression was assessed by (1) flow cytometry and (2) quantitative real-time polymerase chain reaction; hepatitis B virus (HBV)-specific CD8+ T-cells were quantitated by pentamer staining; T-cell reactivity to HBV antigens was determined by interferon gamma (IFNgamma) and interleukin 10 (IL-10) enzyme-linked immunosorbent spot (ELISPOT) assays; and central/effector memory phenotypes were defined by phenotypic markers. PD-1 expression correlated closely with viremia levels. On therapy, PD-1 decreased significantly on total CD8+ T-cells, HBV-specific CD8+ T-cells, and CD3+/CD8- T-cells both as the percentage of positive cells (P < 0.01) and as the mean fluorescent intensity (P < 0.05), and this was paralleled by a marked reduction of PD-1 messenger RNA levels (P = 0.001). HBeAg serocoversion (in 6/18 patients) resulted in a further PD-1 decrease with a 50% reduction in the frequency of PD-1+/CD8+ T-cells, which was not observed in patients remaining HBeAg-positive. The decrease in PD-1 expression was associated with increased frequencies of IFNgamma-producing T-cells and decreased frequencies of IL-10 producing T-cells. At baseline, PD-1 expression correlated directly with the frequency of hepatitis B core antigen (HBcAg) central and effector memory phenotypes, whereas an inverse correlation was observed between PD-1 expression and HBcAg-specific effector phenotypes. CONCLUSION: These results demonstrate that in chronic HBV infection, both viremia levels and HBeAg drive PD-1 expression and resulting T-cell impairment. Treatment-induced suppression of HBV replication reduces PD-1 expression; however, additional immunotherapeutic interventions are needed for restoration of T-cell functions.

The mitochondrial protease AFG3L2 is essential for axonal development.

The mitochondrial metalloprotease AFG3L2 assembles with the homologous protein paraplegin to form a supracomplex in charge of the essential protein quality control within mitochondria. Mutations of paraplegin cause a specific axonal degeneration of the upper motoneuron and, therefore, hereditary spastic paraplegia. Here we present two Afg3l2 murine models: a newly developed null and a spontaneous mutant that we found carrier of a missense mutation. Contrasting with the mild and late onset axonal degeneration of paraplegin-deficient mouse, Afg3l2 models display a marked impairment of axonal development with delayed myelination and poor axonal radial growth leading to lethality at P16. The increased severity of the Afg3l2 mutants is explained by two main molecular features that differentiate AFG3L2 from paraplegin: its higher neuronal expression and its versatile ability to support both hetero-oligomerization and homo-oligomerization. Our data assign to AFG3L2 a crucial role by linking mitochondrial metabolism and axonal development. Moreover, we propose AFG3L2 as an excellent candidate for motoneuron and cerebellar diseases with early onset unknown etiology.

Sgt1, but not Rar1, is essential for the RB-mediated broad-spectrum resistance to potato late blight.

BACKGROUND: Late blight is the most serious potato disease world-wide. The most effective and environmentally sound way for controlling late blight is to incorporate natural resistance into potato cultivars. Several late blight resistance genes have been cloned recently. However, there is almost no information available about the resistance pathways mediated by any of those genes. RESULTS: We previously cloned a late blight resistance gene, RB, from a diploid wild potato species Solanum bulbocastanum. Transgenic potato lines containing a single RB gene showed a rate-limiting resistance against all known races of Phytophthora infestans, the late blight pathogen. To better understand the RB-mediated resistance we silenced the potato Rar1 and Sgt1 genes that have been implicated in mediating disease resistance responses against various plant pathogens and pests. The Rar1 and Sgt1 genes of a RB-containing potato clone were silenced using a RNA interference (RNAi)-based approach. All of the silenced potato plants displayed phenotypically normal growth. The late blight resistance of the Rar1 and Sgt1 silenced lines were evaluated by a traditional greenhouse inoculation method and quantified using a GFP-tagged P. infestans strain. The resistance of the Rar1-silenced plants was not affected. However, silencing of the Sgt1 gene abolished the RB-mediated resistance. CONCLUSION: Our study shows that silencing of the Sgt1 gene in potato does not result in lethality. However, the Sgt1 gene is essential for the RB-mediated late blight resistance. In contrast, the Rar1 gene is not required for RB-mediated resistance. These results provide additional evidence for the universal role of the Sgt1 gene in various R gene-mediated plant defense responses.

The WiscDsLox T-DNA collection: an arabidopsis community resource generated by using an improved high-throughput T-DNA sequencing pipeline.

We have developed a new community resource, called the WiscDsLox collection, for performing reverse-genetic analysis in arabidopsis. This resource is composed of 10,459 T-DNA lines generated using the Arabidopsis thaliana ecotype Columbia. The flanking sequence tag for each T-DNA insertion has been deposited in public databases, and seed for each line is currently available from the Arabidopsis Biological Resource Center. The pDsLox vector used to create this new population contains a Ds transposon and Cre/Lox recombination sites. Each WiscDsLox line therefore has the potential to serve as a launch-pad for performing local saturation mutagenesis by mobilization of the Ds element. In addition, Cre-Lox recombination between the T-DNA and a transposed Ds element should enable targeted deletion of specific genomic regions. We generated the WiscDsLox collection using an improved high-throughput pipeline that streamlines analysis of large numbers of independent Arabidopsis thaliana (L.) Hyenh. lines. In this paper we describe the details of this novel method and also provide potential users of WiscDsLox T-DNA lines with useful background information about this collection. Experiments to characterize the utility of the Ds transposon and Cre/Lox elements present in the WiscDsLox lines are in progress and will be reported in the future.

Alfalfa (Medicago sativa L.).

A protocol for rapid, highly efficient transformation of alfalfa is described. Leaf explants from growth chamber-grown plants of a highly regenerable genotype are surface-sterilized, the margins are removed, and explants are inoculated with Agrobacterium tumefaciens strain LBA4404 carrying the T-DNA vector of interest. The explants and bacteria are cocultured for 7 to 8 d. Bacteria are removed by rinsing explants in sterile distilled water and by culture on regeneration medium containing the antibiotics carbenicillin or ticarcillin. Transformed callus is selected using kanamycin. Somatic embryos are induced by culture of callus on medium lacking plant growth regulators. As mature cotyledonary stage embryos arise, they are transferred to a fresh medium for shoot development and finally to a medium lacking kanamycin for continued shoot and root development. Transgenic plants can be produced in 9 wk with this protocol. Typically 60 to 80% of inoculated explants produce transgenic plants, and escapes are rare.