Enhanced selectivity screening of GPCR ligands using a label-free cell based assay technology.

Drug discovery efforts advance in step with advancements in assay technologies, as new technologies provide new lenses through which biology can be viewed. The novel information gathered results in the better understanding of drug-target interactions leading to better decision making during the drug discovery process. One area of rapid development is within label-free technologies. Label-free technologies offer many distinct advantages to the drug discovery workflow. One such novel technology is the CellKey System, an impedance-based label-free live cell assay platform. The system is based on impedance technology and is a universal platform for the functional measurement of all classes of G-protein coupled receptors (GPCRs). Data are generated in a kinetic fashion on both endogenously expressed and transfected receptors in a wide variety of cell types. In the studies detailed here, we used the system to perform an enhanced selectivity screen of a small panel of compounds simultaneously against two unrelated GPCR targets signaling through different pathways. Utilizing both the quantitative measures of cellular activation and the qualitative information inherent in the rich output data, we gained knowledge not only about the relative selectivity of each compound across both targets, but also about the character of the interaction of each with the cellular target. In this manner, we successfully demonstrated proof of principal for using an impedance-based technology to perform selectivity analyses and to triage lead compounds in a simplified format.

Effluent quality from 200 on-site sewage systems: design values for guidelines.

The quality of effluent from an on-site sewage treatment system is a critical factor in designing the disposal area and, hence, ensuring the sustained performance of the system. Contaminant concentrations in effluent are typically specified in regulatory guidelines or standards; however, the accuracy of these guideline values are brought into question due to the poor performance of septic tanks and the high failure rates of disposal systems reported here and elsewhere. Results from studies of septic tank effluent quality indicated that the effluent is of poorer quality than currently suggested by guidelines. Aerated wastewater treatment systems were found to perform to accreditation guidelines; however, insufficient nutrient data is presently available to assess nutrient loads. It is proposed that the 80th percentile of system performance be adopted as the design value for sizing effluent disposal areas to minimise failure associated with overloading. For septic tanks this equates to 660 mg L(-1) SS, 330 mg L(-1) BOD, 250 mg L(-1) TN and 36 mg L(-1) TP.

Buffer distances for on-site sewage systems in Sydney's drinking water catchments.

Pathogens and nutrients released from on-site sewage systems represent a risk to surface and ground water quality, particularly where there are sensitive receiving waters such as in drinking water catchments. Buffer zones between on-site systems and waterways are one barrier used to protect water quality. The increased time and distance they provide increases the opportunities for the effluent purification functions of the soil to occur. A risk management model is proposed to assess the efficacy of the buffer zones in Sydney's drinking water catchments. The model is the basis for the development of performance based setback distances for on-site systems from waterways, and incorporates stochastic analysis of pathogen and nutrient transport in the environment and consideration of the effluent quality variability from on-site systems. Catchment-scale integration of contaminant transport is employed to facilitate a risk assessment of on-site systems. The risk management model also allows for the impact of on-site system management and maintenance on catchment water quality to be assessed through scenario building and feedback mechanisms.

Centralised versus decentralised sewage systems: a comparison of pathogen and nutrient loads released into Sydney's drinking water catchments.

Data collected from centralised and decentralised sewage treatment plants throughout Sydney's drinking water catchments was used to calculate the relative catchment loads of Cryptosporidium, enteric viruses, nitrogen and phosphorus for an initial screening assessment. Loads were assessed at median and 90 percentile values for expected and worst-cases scenarios. The expected scenario in the Sydney drinking water catchments is that decentralised systems (servicing 32,800 people) provide similar total loads to centralised systems (serving 70% of the catchment population) for total phosphorus (37,090 kg x y(-1)), Cryptosporidium (10(11) oocysts x y(-1)) and enteric viruses (9.1 x 10(13) y(-1)), but higher loads of total nitrogen (237,610 vs. 136,740 kg x y(-1)). Decentralised systems, however, were predicted to have higher loads in the worst-case scenario with 620,620 kg x y(-1) TN, 82,040 kg x y(-1) TP, 7.3 x 10(13) Cryptosporidium oocysts x y(-1) and 9 x 10(15) enteric viruses per year. Greater load variability was experienced with decentralised systems, which presumably reflects less reliability in their current operation and maintenance. Overall, catchment water quality is therefore not only affected by sewage disposal methods, but also failure issues. Decentralised system disposal to land may afford a degree of mitigation that can be enhanced, if the degree of failure is reduced.

A new-generation stable inducible packaging cell line for lentiviral vectors.

We have successfully generated and characterized a stable packaging cell line for HIV-1-based vectors. To allow safe production of vector, a minimal packaging construct carrying only the coding sequences of the HIV-1 gag-pol, tat, and rev genes was stably introduced into 293G cells under the control of a Tet(o) minimal promoter. 293G cells express the chimeric Tet(R)/VP16 trans-activator and contain a tetracycline-regulated vesicular stomatitis virus protein G (VSV-G) envelope gene. When the cells were grown in the presence of tetracycline the expression of both HIV-1-derived and VSV-derived packaging functions was suppressed. On induction, approximately 50 ng/ml/24 hr of Gag p24 equivalent of vector was obtained. After introduction of the transfer vector by serial infection, vector could be collected for several days with a transduction efficiency similar or superior to that of vector produced by transient transfection both for dividing and growth-arrested cells. The vector could be effectively concentrated to titers reaching 10(9) transducing units/ml and allowed for efficient delivery and stable expression of a GFP transgene in the mouse brain. The packaging cell line and all vector producer clones described here were shown to be free from replication-competent recombinants, and from recombinants between packaging and vector constructs that transfer the viral gag-pol genes. The packaging cell line and the assays developed will advance lentiviral vectors toward the stringent requirements of clinical applications.

T-cell killing of heterogenous tumor or viral targets with bispecific chimeric immune receptors.

We have previously described several novel chimeric immune receptors (CIRs) that redirect human T cells to kill malignant or HIV-infected cells. These CIRs comprise a cancer- or virus-specific ligand or single-chain antibody fused to the signaling domain of the T-cell receptor CD3-zeta subunit. Binding of the ligand- or antibody-based CIR to the target antigen (Ag) triggers T-cell-mediated cytolysis of the tumor- or virus-infected cell independent of target cell major histocompatibility complex class I expression. A new type of CIR was developed to mediate the lysis of cells that expressed one or more distinct viral or tumor Ags; three bispecific CIRs (BCIRs) were generated that recognized the carcinoembryonic Ag (CEA) and TAG-72 tumor Ags or, alternatively, distinct epitopes in the HIV envelope (HIVenv). T cells expressing the antitumoral Ag BCIR lysed both CEA- and TAG-72-expressing targets and did not kill Ag-negative targets or target cells expressing other members of the CEA family. Similarly, T cells expressing the anti-HIVenv BCIR lysed target cells expressing both the wild-type HIVenv and a mutant HIVenv that lacked the epitopes recognized by the monospecific CIRs. This approach permits the generation of T cells with a broader spectrum of activity capable of killing virus-infected cells and malignant cells and reduces the potential of progression of disease due to Ag loss variants.

Anti-tumor activity of human T cells expressing the CC49-zeta chimeric immune receptor.

A chimeric immune receptor consisting of an extracellular antigen-binding domain derived from the CC49 humanized single-chain antibody, linked to the CD3zeta signaling domain of the T cell receptor, was generated (CC49-zeta). This receptor binds to TAG-72, a mucin antigen expressed by most human adenocarcinomas. CC49-zeta was expressed in CD4+ and CD8+ T cells and induced cytokine production on stimulation. Human T cells expressing CC49-zeta recognized and killed tumor cell lines and primary tumor cells expressing TAG-72. CC49-zeta T cells did not mediate bystander killing of TAG-72-negative cells. In addition, CC49-zeta T cells not only killed FasL-positive tumor cells in vitro and in vivo, but also survived in their presence, and were immunoprotective in intraperitoneal and subcutaneous murine tumor xenograft models with TAG-72-positive human tumor cells. Finally, receptor-positive T cells were still effective in killing TAG-72-positive targets in the presence of physiological levels of soluble TAG-72, and did not induce killing of TAG-72-negative cells under the same conditions. This approach is being currently being utilized in a phase I clinical trial for the treatment of colon cancer.

Large-scale manufacturing of safe and efficient retrovirus packaging lines for use in immunotherapy protocols.

BACKGROUND: The use of gene modified T lymphocytes for immunotherapy in a cancer or AIDS clinical trial requires an efficient, safe ex vivo method for modification of these cells at manufacturing scale. Since retroviruses have been shown to be a moderately effective means of stably integrating therapeutic genes into T lymphocytes, we wanted to create packaging and producer cell lines that would produce replication competent retrovirus (RCR)-free supernatants, at large scale (> 200 l), and transduce with high efficiency. METHODS: cDNA expression plasmids containing only coding sequences for gagpol or env were built and sequentially transfected into human 293 cells. Packaging and producer clones were characterized for stability, titer and RCR. A producer clone delivering chimeric immune receptors was scaled-up and supernatants used to transduce patient T lymphocytes for clinical studies. PCR and RT-PCR assays were utilized to evaluate the transmission of HERV-H sequences. Relative infectivity of producer clones pseudotyped with different envelopes was determined by transduction and RT assays. RESULTS: RCR-free, human 293 split-genome packaging lines, pseudotyped with amphotropic, xenotropic, or 10A1 envelopes, were created. A CC49 zeta producer clone was scaled-up to 5 x 54 l lots and supernatants used to safely and efficiently transduce patient T lymphocytes with minimal ex vivo manipulation. While 293 cells express HERV-H mRNA, the transmission frequency in our packaging clones was less than 1 HERV-H sequence per 5 x 10(5) proviral integrations. Additionally, 10A1 and xenotropic packaging lines had higher infectivities than the amphotropic clone. CONCLUSION: These packaging lines represent the safest configuration for the large-scale production of retroviral vectors, and are capable of producing high titer, RCR-free retroviral vector for large scale clinical use. While all three clones efficiently transduce human T lymphocytes, the 10A1 clone has the highest infectivity. These packaging cell lines will be valuable for use in human gene therapy protocols.

Production of antigen-specific human antibodies from mice engineered with human heavy and light chain YACs.

Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies. The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes. This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration. The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation. Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies. Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum. These results suggest the potential value of these mice as a source of fully human antibodies for human therapy. Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy. Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization. The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders. The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

Implantation of sutured posterior chamber intraocular lenses.

We describe a technique for implanting sutured posterior chamber intraocular lenses. Results of a small series using this technique in different clinical situations with at least 12 months follow-up are presented.

Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs.

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and kappa light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen-specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.

Analysis of homozygous mutant chimeric mice: deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production.

Using a recently described method for efficiently deriving homozygous targeted alleles in embryonic stem cells, we produced chimeric mice whose tissues were derived partially from embryonic stem cells bearing homozygous deletion of the mouse immunoglobulin heavy-chain joining (JH) region. Characterization of these chimeric mice indicated that homozygous JH deletion leads to arrest of B-cell development at an early stage, resulting in a total lack of peripheral B cells and serum IgM. These results were confirmed in mice containing the homozygous JH deletion in their germ line. This novel B-cell-deficient mouse strain provides a tool for studying the recombination and expression of exogenous immunoglobulin genes introduced into the mouse germ line.

The cellular immune response to immunization with zona pellucida antigens.

The cellular immune response of mice to porcine and rat zona pellucida and cynomolgus macaques to porcine zona pellucida antigens was evaluated. Mice mounted a vigorous cellular response to both antigens, as determined by the T cell proliferation response in vitro. There was poor cross-reactivity to murine zonae by T cells or serum antibodies from mice immunized with rat zona pellucida. Nevertheless, ovaries from the mice immunized with rat zona had significantly fewer antral follicles than adjuvant-treated controls, suggesting that the immune response to the zona antigen disrupted follicular development. T cells from two macaques that had been immunized with porcine zona pelludica proteins proliferated in vitro in response to this antigen. Both macaques also had strong antibody responses. The patterns of urinary steroid metabolites in these animals provided clear evidence of ovarian malfunction following immunization. The data indicate that a significant cellular immune response is generated upon immunization of animals with zona pellucida antigens regardless of whether the antigens are cross reactive with the host zona antigens. Whether impaired ovarian function and follicular development are related to the cellular response must be determined in future studies.

Immunization of male but not female mice with the sperm-specific isozyme of lactate dehydrogenase (LDH-C4) impairs fertilization in vivo.

The goals of this study were to determine the site at which fertility is impaired in mice immunized with LDH-C4 and to determine whether immunization of both males and females would have a greater antifertility effect than immunization of one sex alone. Mice were immunized with LDH-C4 in two systemic doses in Freund's adjuvants and two gastric doses in bicarbonate buffer. The presence of anti-LDH-C4 antibodies in uterine fluid was confirmed. Male and female mice were assigned to four blocks in which either the males, the females, both, or neither were immunized. Oviducts were viewed directly 1 h after mating for the presence of sperm. No significant effect of immunization on sperm transport to the oviduct could be demonstrated. Fertilization was evaluated 4 h after mating. It was found that immunization of males, but not females, impaired fertilization (19.6% versus 50.8% of the oocytes penetrated in 17 and 19 females, respectively). Orchitis was found histologically in 43.8% of the immunized males and 10% of the control males.

Alport's syndrome and the eye.

Alport's syndrome comprises hereditary deafness, nephritis and ocular abnormalities. The features of Alport's syndrome are illustrated by a family with Alport's syndrome and hereditary oesophageal leiomyomatosis. The evidence that Alport's syndrome is due to a widespread basement membrane disorder is noted. Treatment of anterior lenticonus, the principal ocular abnormality, by lensectomy and intraocular lens insertion is recommended.

Canthaxanthine (orobronze) retinopathy.

A 50-year-old white Australian male was found on routine examination to have small golden particles in the macular region of both eyes. A history of canthaxanthine (Orobronze) ingestion to produce skin bronzing was obtained.