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Abstract Background The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). Results Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. Conclusions In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.

Statement: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight., (Copyright © 2020 International Society for Cell and Gene Therapy. All rights reserved.)

The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 × 10 6 intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 × 10 3 to 1:1 × 10 6 human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies., (© 2020 The Authors.)

The biomedical field has witnessed remarkable advances in analytical tools and technologies that have expanded our understanding of healthy and diseased human tissue and, at the same time, enable extensive molecular characterization of living cells. The volume of scientific data generated is expanding in an unprecedented manner; however, these data remain scattered across research groups worldwide. Access to various data sources in a systematic fashion could hugely benefit the progress of nascent fields such as stem cell-based therapeutics. We explore here the currently available databases for stem cell research, and we propose creating a common portal to access these different databases that could act as a translational link between basic and clinical research to advance stem cell-based therapeutic development., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

In view of the existing shortage of human donor organs and tissues, xenogeneic cell therapeutics (xCT) offer an alternative for adequate treatment. In particular, porcine pancreatic islets and islet cells have already entered the field of experimental therapy for type-1 diabetes mellitus (T1DM) patients. Thereby, xCT depict challenging products with a glance on medical, ethical, and regulatory questions. With cross-species transplantation (xenotransplantation), the risk of immunological graft rejection as well as the risk of infectious transmission of microbial and viral pathogens must be considered. This includes the bidirectional transmission of microorganisms from graft to host as well as from host to graft. Crossing the border of species requires a critical risk-benefit evaluation as well as a thorough longtime surveillance of transplant recipients after treatment. The international legal and regulatory requirements for xCT are inter alia based on the World Health Organization criteria summarized in the Changsha Communiqué (2008). In the European Union, they were reflected by the European Medicines Agency (EMA) Guideline on Xenogeneic Cell-based Medicinal Products following the implementation of the Regulation on Advanced Therapies (ATMP). On the basis of this regulation, the first non-clinical and clinical experiences were obtained for porcine islets. The results suggest that supportive treatment of T1DM risk patients with xCT may be an alternative to established allogeneic organ transplantation in the future.

For the development of medicinal products animal models are still indispensable to demonstrate efficacy and safety prior to first use in humans. Advanced therapy medicinal products (ATMP), which include cell-based medicinal products (CBMP), differ in their pharmacology and toxicology compared to conventional pharmaceuticals, and thus, require an adapted regime for non-clinical development. Developers are, therefore, challenged to develop particular individual concepts and to reconcile these with regulatory agencies. Guidelines issued by the European Medicines Agency (EMA), the U.S. Food and Drug Administration (FDA) and other sources can provide direction.The published approaches for non-clinical testing of efficacy document that homologous animal models where the therapeutic effect is investigated in a disease-relevant animal model utilizing cells derived from the same species are commonly used. The challenge is that the selected model should reflect the human disease in all critical features and that the cells should be comparable to the investigated human medicinal product in terms of quality and biological activity. This is not achievable in all cases. In these cases, alternative methods may provide supplemental information. To demonstrate the scientific proof-of-concept (PoC), small animal models such as mice or rats are preferred. During the subsequent product development phase, large animal models (i.e. sheep, minipigs, dogs) must be considered, as they may better reflect the anatomical or physiological situation in humans. In addition to efficacy, those models may also be suitable to prove some safety aspects of ATMP (e.g. regarding dose finding, local tolerance, or undesired interactions and effects of the administered cells in the target tissue). In contrast, for evaluation of the two prominent endpoints for characterizing the safety of ATMP (i.e. biodistribution, tumorigenicity) heterologous small animal models, especially immunodeficient mouse strains, are favourable due to their tolerance to the human cell therapy product. The execution of non-clinical studies under the principles of good laboratory practice (GLP) increases the acceptance of the results by authorities and the scientific community.

Background Aims: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process., Methods: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production., Results: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs., Conclusions: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

In the European Union, clinical trials for Advanced Therapy Medicinal Products are regulated at the national level, in contrast to the situation for a Marketing Authorisation Application, in which a centralised procedure is foreseen for these medicinal products. Although based on a common understanding regarding the regulatory requirement to be fulfilled before conduct of a clinical trial with an Advanced Therapy Investigational Medicinal Product, the procedures and partly the scientific requirements for approval of a clinical trial application differ between the European Union Member States. This chapter will thus give an overview about the path to be followed for a clinical trial application and the subsequent approval process for an Advanced Therapy Investigational Medicinal Product in Germany and will describe the role of the stakeholders that are involved. In addition, important aspects of manufacturing, quality control and non-clinical testing of Advanced Therapy Medicinal Products in the clinical development phase are discussed. Finally, current and future approaches for harmonisation of clinical trial authorisation between European Union Member States are summarised.

The human immunodeficiency virus type 1 accessory protein Vif is important for viral infectivity because it counteracts the antiviral protein APOBEC3G (A3G). ³²P metabolic labelling of stimulated cells revealed in vivo phosphorylation of the control protein, whereas no serine/threonine phosphorylation was detected for Vif or the A3G protein. These data were confirmed by in vitro kinase assays using active recombinant kinase. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 efficiently phosphorylated its target ELK, but failed to phosphorylate Vif. Putative serine/threonine phosphorylation point mutations in Vif (T96, S144, S165, T188) using single-round infection assays demonstrated that these mutations did not alter Vif activity, with the exception of Vif.T96E. Interestingly, T96E and not T96A was functionally impaired, indicating that this residue is critical for Vif-A3G physical interaction and activity. Our data suggest that Vif and A3G are not serine/threonine phosphorylated in human cells and phosphorylation is not linked to their functional activities.

Gene-therapy medicinal products are currently applied to patients enrolled in authorized clinical trials to demonstrate safety and efficacy. Given a positive outcome, marketing authorization can subsequently be achieved via the centralized procedure coordinated by the European Medicines Agency. With Regulation (EC) No. 1394/2007 in force, advanced therapy medicinal products, including gene- and cell-therapy products, can be excepted from the obligation of obtaining a marketing authorization via the centralized procedure under specific conditions (so-called "hospital exemption"). This hospital exemption allows the application of gene-therapy medicinal products prepared on a non-routine basis for an individual patient and used under the exclusive professional responsibility of a medical practitioner. Here, we explain the requirements to be fulfilled in order to fall under this exemption, the implementation of this regulation into the German national legislation, and its impact on gene-therapy product development in the future.

Background: The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM)., Results: Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6., Conclusions: In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.

SIVagm does not induce disease in its African green monkey (AGM) host. In comparison, the hybrid simian-human immunodeficiency virus SHIV89.6P that carries the HIV env gene induces disease in rhesus macaques more rapidly than the SIVmac parent virus. To address the possibility that this enhancement of disease by HIV env would also occur when present in SIVagm, a full-length SIVagm/89.6Penv chimeric lentivirus genome (termed SHIV-MP) was constructed. SHIV-MP replicated similarly to SIVagm in simian peripheral blood mononuclear cells (PBMCs). In inoculated AGMs, rhesus macaques and pig-tailed (PT) macaques the absolute number of CD4(+) T lymphocytes remained at normal levels. The peak levels of productively infected cells in SHIV-MP-infected monkeys ranged from 10(1) to 10(2) per 10(6) PBMCs, while in SIVagm infected macaques the levels were 10-100-fold higher. The env gene of SHIV89.6P therefore appears insufficient to confer acute pathogenicity to a non-pathogenic primate lentivirus due to poor in vivo replication., (Copyright 2010 Elsevier Inc. All rights reserved.)

Today, sterility of established parenteral drugs including biologicals, such as plasma derived products, is practically guaranteed. Bacterially contaminated products are extremely rare exceptions owing to the efficiency of the manufacturing processes in the pharmaceutical industry. In contrast, the manufacturing processes of cell based medicinal products or tissue preparations show much less defined conditions. The sterility of source materials cannot be guaranteed in many cases. As a rule, these source materials cannot be sterilised, as it holds true for the final products. Furthermore, the established methods for sterility testing are not applicable for cell preparations. Sterility of a restricted sample does not guarantee sterility of the whole preparation. Thus, small amounts of residual bacteria in the product can be overlooked and can grow up to enormous numbers during storage and shipping of cell based medicinal products. Considering these problems, there are some parallels in the warranty of microbial safety of cellular blood components. Therefore, the experiences collected in transfusion medicine in the past decade can be successfully used in the production of cell based medicinal products. Comparable to the situation regarding cellular blood components, there is a need for new principles in rapid bacteria detection.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) pointed out the need to involve authorities throughout the process of validation and legal acceptance of alternatives to animal experiments. The Paul-Ehrlich-Institute (PEI), Federal Agency for Sera and Vaccines, is the national competent authority in Germany which is responsible for the quality and safety of biologicals including blood and cell-based products. This paper is intended to contribute to the discussion concerning the use of alternative methods in safety testing of medicinal products and considers the scientific work of the PEI in this field. From a regulator's perspective, adequate demonstration of safety and quality of medicinal products are of major interest. Additionally, the availability of the products to the patient has to be taken into consideration. It has to be carefully explored whether the respective in vitro method for demonstration of non-clinical safety as part of the non-clinical development programme is able to guarantee safety level comparable to the corresponding experiment in animals. The topics cited above shall be discussed in this paper using the example of the Alternative Pyrogen Test or also called Monocyte Activation Test. The Alternative Pyrogen Test could serve as paradigm to exemplify how an alternative test can provide at least a comparable level of safety estimation in comparison with a conventional animal test. Furthermore, this alternative test creates additional information which cannot be obtained from the animal experiment, and might also open further scientific insight into the mechanisms of pyrogenicity and acute pro-inflammatory reactions in patients. This test method allows the definition of pyrogen limits for medicinal products. Due to its use of relevant cell systems this in vitro test might contribute significantly to safety assessments of advanced medicinal products during the pre-clinical phase.

RNA interference (RNAi) has become a powerful tool for the specific silencing of gene transcription. Especially the targeting of genes in mammalian cells has been greatly improved by generating plasmid based and viral vector-based systems. This permits expression of short hairpin RNA (shRNA) on a longterm basis. However, an inducible expression of shRNA is required, if the target is essential for cell survival. We developed a doxycycline-inducible two-plasmid system for the expression of a ribozyme-processed shRNA. In contrast to other existing systems, we use the highly specific T7 phage RNA polymerase, which does not interact with cellular factors; therefore, interference with cellular functions is limited. One plasmid is responsible for doxycycline-dependent expression of T7 RNA polymerase and a second plasmid expresses a ribozyme-processed shRNA under the control of a T7 promoter. Our results showed that doxycycline- dependent expression of T7 RNA polymerase was tightly controlled and expression of an shRNA against firefly luciferase inhibited 86% of luciferase activity. In conclusion, our plasmid system provides a very useful tool for analyzing essential gene functions in vitro.

The major T cell growth factor interleukin-2 (IL-2) is secreted by activated T cells in response to antigenic stimulation. This requires signal transduction via the CD3/TCR complex and the CD28 coreceptor, leading to activation of mitogen-activated protein kinase (MAPK) and calcineurin/NF-AT signaling pathways. We observed that simian immunodeficiency virus derived from African green monkeys (SIVagm3) is a potent activator of IL-2 gene expression. IL-2 promoter studies in A3.01 T cells demonstrated that SIVagm3 induced an up to 38-fold increased transcriptional activation of the IL-2 promoter. Inhibition of MAPK signaling pathways using inhibitors of MEK, JNK or p38 abolished SIVagm3-induced IL-2 activation in a dose-dependent manner. In contrast, the immunosuppressive drug cyclosporin A (CyA), a classical IL-2 inhibitor that blocks calcineurin activity, had no effect. Consistent with this finding, the nuclear factor of activated T cells (NF-AT), which is activated by calcineurin, was not induced by SIVagm3. Analyzing further transcription factor binding sites located on the IL-2 promoter we found that SIVagm3 did mainly promote transcriptional activation of the CD28/AP-1 and NF-kappaB responsive elements. These DNA elements were also induced by the viral transactivator protein (Tat) and expression of Tat was sufficient to activate IL-2 induction in stimulated cells. Our results show that SIVagm3 is capable of stimulating IL-2 gene expression via molecular mechanisms different from those induced during classical T cell activation.

Despite the ability to infect nonproliferating cells, current lentiviral vectors are inefficient at mediating gene transfer into quiescent primary human cells such as monocytes. Here, a replication-incompetent vector based on a molecular clone of simian immunodeficiency virus strain PBj (SIVsmmPBj1.9) was generated that, in contrast to lenti- and gamma-retroviral control vectors, enabled transfer of heterologous genes into human diploid fibroblasts and cell lines blocked in the G(0) phase of the cell cycle. Moreover, freshly isolated human monocytes refractory to HIV-1-derived vectors were efficiently transduced by the PBj vector independent of the viral Nef protein. Stable chromosomal integration of PBj-derived viral expression vectors was verified in transduced cells. The capability of the PBj vector to transduce quiescent cells such as unstimulated primary human monocytes is an important extension of human gene therapy perspectives.

The CD95/Fas/Apo-1 ligand (CD95L, CD178) induces apoptosis through the death receptor CD95. CD95L was also described as a co-stimulatory receptor for T-cell activation in mice in vivo. The molecular basis for the bidirectional signaling capacity and directed expression of CD95L is unknown. In the present study we identify proteins that precipitate from T-cell lysates with constructs containing fragments of the CD95L cytosolic tail. The determined peptide mass fingerprints correspond to Grb2, actin, beta-tubulin, formin binding protein 17 (FBP17) and PACSIN2. Grb2 had been identified as a putative mediator of T-cell receptor-to-CD95L signaling before. FBP17 and PACSIN2 may be associated with expression and trafficking of CD95L. When overexpressed, CD95L co-precipitates with FBP17 and PACSIN. Protein-protein interactions are mediated via Src homology 3 (SH3) domain binding to the polyproline region of CD95L and can be abolished by mutation or deletion of the respective SH3 domain.

Resting mature T lymphocytes are activated when triggered via their antigen-specific T-cell receptor (TCR) to elicit an appropriate immune response. In contrast, preactivated T cells may undergo activation-induced cell death (AICD) in response to the same signals. along with cell death induced by growth factor deprivation, AICD followed by the elimination of useless or potentially harmful cells preserves homeostasis, leads to the termination of cellular immune responses and ensures peripheral tolerance. T-cell apoptosis and AICD are controlled by survival cytokines such as interleukin-2 (IL-2) and by death factors such as tumor necrosis factor (TNF) and CD95 ligand (CD95L). In AICD-sensitive T cells, stimulation upregulates expression of one or several death factors, which in turn engage specific death receptors on the same or a neighboring cell. Death receptors are activated by oligomerization to rapidly assemble a number of adapter proteins and enzymes to result in an irreversible activation of proteases and nucleases that culminates in cell death by apoptosis. Increased knowledge of the molecular mechanisms that regulate AICD of lymphocytes opens new immunotherapeutic perspectives for the treatment of certain autoimmune diseases, and has implications in other areas such as transplantation medicine and AIDS research.

Background: Restimulation of T lymphocytes via the TCR/CD3 complex can result in CD95/CD95L-dependent activation-induced cell death (AICD). Although the correlation of AICD sensitivity to the T helper 1 phenotype was confirmed in different studies, the underlying mechanism is still debated. Thus, it has been suggested that in Th2 cells, AICD resistance is controlled by a TCR-induced upregulation of the CD95-associated inhibitory phosphatase, FAP-1. We and others demonstrated that AICD resistance is associated with a reduced surface expression of CD95L upon restimulation., Methods: Utilizing RT-PCR, Western blotting and flow cytometry, we analyzed time-dependent changes in levels of CD95L mRNA, cytosolic protein and surface expression in five long-term human T cell clones and polarized helper populations., Results: We confirm that the inducible CD95L surface expression is lower or absent in all tested AICD-resistant clones as compared to sensitive cells. It is of interest that striking differences with respect to the activation-dependent inducibility of CD95L mRNA expression in individual resistant clones were observed. In addition, alterations in the expression of the inhibitory phosphatase FAP-1 or TCR-dependent changes in CD95 sensitivity in AICD-resistant clones could be ruled out as a mechanism for AICD resistance of human T cell clones., Conclusions: (1) The data presented strongly support the previous notion that AICD resistance of human T cell clones is mainly regulated by a differential expression of CD95L. (2) Differential expression of CD95L on individual resistant clones results from a lack of mRNA induction in one set and from a markedly decreased surface expression of translated protein in another set of clones., (Copyright 2000 S. Karger AG, Basel)

Nonreceptor protein tyrosine kinases and associated substrates play a pivotal role in Ag receptor stimulation of resting cells and in the initiation of activation-induced cell death (AICD) of preactivated T cells. CD4-associated p56lck has been implicated not only in the activation of primary T cells, but also in the inhibition of T cell responses. We have previously shown that CD4+ T cell clones can be rescued from AICD when surface CD4 is engaged before the TCR stimulus. In this study, we show that prevention of AICD is associated with a CD4-dependent inhibition of TCR-triggered tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and Vav. We provide evidence for a SLP-76 interaction with Src homology 3 domains of p56lck and identify amino acids 185-194 of SLP-76 as relevant docking site. In view of the multiple functions of p56lck and SLP-76/Vav in the initiation of TCR/CD3/CD4 signaling, we propose a model for the CD4-dependent inhibition of TCR signaling and AICD of preactivated T cells. Our data suggest that preformed activation complexes of adapter proteins and enzymes in the vicinity of the CD4/p56lck complex are no longer available for the TCR signal when CD4 receptors are engaged before TCR stimulation.

An experimental procedure was devised using the pentobarbital-anesthetized dog that could be used for the comprehensive evaluation of the renal effects of chemicals. After IV or renal arterial administration of 0.9% saline solution (vehicle), 12 renal function determinants were continuously monitored for periods of 2 and 6 hours. At the completion of the 2 or 6 hours of study, the kidneys of a number of dogs (usually between 1 and 7) in each vehicle-treated group were subjected to a modification of the intravascular perfusion-of-fixative technique to evaluate the ultrastructural status of the outer cortical, inner cortical, and outer medullary tissue. The remaining dogs (at least 3) in each vehicle-treated group were given a nonnephrotoxic, but maximally effective, diuretic dose of ethacrynic acid, which enabled an assessment of the functional integrity of the thick ascending limb of Henle's loop. Renal function and glomerular and tubular ultrastructure remained stable in the pentobarbital-anesthetized dog for up to 6 hours after administration of vehicle. Sustained infusion of inulin (included in the procedure to estimate glomerular filtration rate) throughout the duration of the experiments, and pentobarbital anesthesia of various durations did not alter the morphologic status of the canine nephron. The procedure used for the renal perfusion of fixative circumvented any manipulation of the kidneys before fixation and allowed for the acquisition of normal (unaltered) appearing tissue from all areas of the kidneys. The responses of pentobarbital-anesthetized dogs to ethacrynic acid administration were similar when given 2 and 6 hours after the vehicle administration.(ABSTRACT TRUNCATED AT 250 WORDS)