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The recent emergence and rapid response to severe acute respiratory syndrome coronavirus 2 was enabled by prototype pathogen and vaccine platform approaches, driven by the preemptive application of RNA vaccine technology to the related Middle East respiratory syndrome coronavirus. Recently, the National Institutes of Allergy and Infectious Diseases identified nine virus families of concern, eight enveloped virus families and one nonenveloped virus family, for which vaccine generation is a priority. Although RNA vaccines have been described for a variety of enveloped viruses, a roadmap for their use against nonenveloped viruses is lacking. Enterovirus D68 was recently designated a prototype pathogen within the family Picornaviridae of nonenveloped viruses because of its rapid evolution and respiratory route of transmission, coupled with a lack of diverse anti-enterovirus vaccine approaches in development. Here, we describe a proof-of-concept approach using a clinical stage RNA vaccine platform that induced robust enterovirus D68-neutralizing antibody responses in mice and nonhuman primates and prevented upper and lower respiratory tract infections and neurological disease in mice. In addition, we used our platform to rapidly characterize the antigenic diversity within the six genotypes of enterovirus D68, providing the necessary data to inform multivalent vaccine compositions that can elicit optimal breadth of neutralizing responses. These results demonstrate that RNA vaccines can be used as tools in our pandemic-preparedness toolbox for nonenveloped viruses.

Epstein-Barr virus (EBV) is associated with several malignancies, neurodegenerative disorders and is the causative agent of infectious mononucleosis. A vaccine that prevents EBV-driven morbidity and mortality remains an unmet need. EBV is orally transmitted, infecting both B cells and epithelial cells. Several virally encoded proteins are involved in entry. The gH/gL glycoprotein complex is essential for infectivity irrespective of cell type, while gp42 is essential for infection of B cells. gp350 promotes viral attachment by binding to CD21 or CD35 and is the most abundant glycoprotein on the virion. gH/gL, gp42 and gp350, are known targets of neutralizing antibodies and therefore relevant immunogens for vaccine development. Here, we developed and optimized the delivery of several alphavirus-derived replicon RNA (repRNA) vaccine candidates encoding gH/gL, gH/gL/gp42 or gp350 delivered by a cationic nanocarrier termed LION™. The lead candidate, encoding full-length gH/gL, elicited high titers of neutralizing antibodies that persisted for at least 8 months and a vaccine-specific CD8 + T cell response. Transfer of vaccine-elicited IgG protected humanized mice from EBV-driven tumor formation and death following high-dose viral challenge. These data demonstrate that LION/repRNA-gH/gL is an ideal candidate vaccine for preventing EBV infection and/or related malignancies in humans., (© 2024. The Author(s).)

Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines., Competing Interests: Declaration of interests T.K., J.M.L., A.S., N.L.W., S.P., R.K., J.F.A., B.J.B., T.H., K.N., S.S., A.K., M.S.D., P.B., S.G.R., and J.H.E. have equity interest in HDT Bio. J.H.E. is a consultant for InBios. P.B. is a consultant for Karkinox, and Chimeron. P.B. and J.H.E. are co-inventors on US patent application no. 63/011,860 “Nucleic acid vaccines against COVID-19” pertaining to repRNA-CoV-2S. J.H.E., A.K., and S.G.R. are co-inventors on US patent application no. 62/993,307 “Compositions and methods for delivery of RNA” pertaining to the LION formulation., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mother-to-child transmission is a major route for infections in newborns. Vaccination in mothers to leverage the maternal immune system is a promising approach to vertically transfer protective immunity. During infectious disease outbreaks, such as the 2016 Zika virus (ZIKV) outbreak, rapid availability of vaccines can prove critical in reducing widespread disease burden. The recent successes of mRNA vaccines support their evaluation in pregnant animal models to justify their use in neonatal settings. Here we evaluated immunogenicity of self-amplifying replicon (repRNA) vaccines, delivered with our clinical-stage LION nanoparticle formulation, in pregnant rabbits using ZIKV and HIV-1 as model disease targets. We showed that LION/repRNA vaccines induced robust antigen-specific antibody responses in adult pregnant rabbits that passively transferred to newborn kits in utero. Using a matrixed study design, we further elucidate the effect of vaccination in kits on the presence of pre-existing maternal antibodies. Our findings showed that timing of maternal vaccination is critical in maximizing in utero antibody transfer, and subsequent vaccination in newborns maintained elevated antibody levels compared with no vaccination. Overall, our results support further development of the LION/repRNA vaccine platform for maternal and neonatal settings., Competing Interests: Declaration of interests A.P.K., J.A., S.R., K.K., B.J.B., N.L.W., and J.H.E. have equity interest in HDT Bio and are listed as inventors on patents covering the LION formulation., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Dengue virus (DENV) is a global health problem, with over half of the world's population at risk for infection. Despite this, there is only one licensed vaccine available to prevent infection and safety concerns limit immunization to only a subset of individuals. Most dengue virus vaccine efforts attempt to evoke broadly neutralizing antibodies against structural proteins. However, eliciting antibodies to block the activity of viral proteins involved in pathogenesis could be a useful complementary approach. Studies suggest that non-structural protein 1, which participates in disruption of the endothelial barrier and is hypothesized to play a significant role in the progression to severe dengue, could be a promising target for vaccine efforts. Here, we used an unbiased approach to identify peptide epitopes of dengue virus non-structural protein 1 that could evoke antibodies that bind to NS1 from all 4 serotypes and also bind to DENV-infected cells. DENV-2 NS1 peptides were generated such that 35 overlapping 15 amino acid peptides represented the entire NS1 protein. These peptides were each chemically conjugated to bacteriophage virus-like particles (VLP) and used to immunize mice. Sera were then screened for IgG to cognate peptide as well as binding to recombinant hexameric NS1 from all four DENV serotypes as well as binding to DENV-2 infected cells by microscopy. From these data, we identified several peptides that were able to elicit antibodies that could bind to infected cells as well as DENV NS1. These peptides and their homologues in the corresponding NS1 of other DENV serotypes could be used as potential immunogens to elicit binding antibodies to NS1. Future studies will investigate the functional and protective capacities of antibodies elicited by these immunogens against DENV NS1.

Dengue virus (DENV) is a major global health problem, with over half of the world's population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

Identifying the specific epitopes targeted by antibodies elicited in response to infectious diseases is important for developing vaccines and diagnostics. However, techniques for broadly exploring the specificity of antibodies in a rapid manner are lacking, limiting our ability to quickly respond to emerging viruses. We previously reported a technology that couples deep sequencing technology with a bacteriophage MS2 virus-like particle (VLP) peptide display platform for identifying pathogen-specific antibody responses. Here, we describe refinements that expand the number of patient samples that can be processed at one time, increasing the utility of this technology for rapidly responding to emerging infectious diseases. We used dengue virus (DENV) as a model system since much is already known about the antibody response. Sera from primary DENV-infected patients (n = 28) were used to pan an MS2 bacteriophage VLP library displaying all possible 10-amino-acid peptides from the DENV polypeptide. Selected VLPs were identified by deep sequencing and further investigated by enzyme-linked immunosorbent assay. We identified previously described immunodominant regions of envelope and nonstructural protein-1, as well as a number of other epitopes. Our refinement of the deep sequence-coupled biopanning technology expands the utility of this approach for rapidly investigating the specificity of antibody responses to infectious diseases.

Introduction: Alcohol use disorders are major risk factors for the development of and susceptibility to acute respiratory distress syndrome. Although these risks of alcohol consumption on the lung are well described, mechanisms by which alcohol abuse promotes acute lung injury are poorly understood. These gaps in our understanding are due, at least in part, to limitations of animal models to recapitulate human alcohol consumption. Recently, a new model of chronic plus binge alcohol exposure was developed that is hypothesized to better model drinking patterns of individuals with alcohol use disorders. Specifically, this paradigm models chronic consumption coupled with periodic bouts of heavy drinking. The impacts of this alcohol-exposure regimen on the lung are uncharacterized. Therefore, the goal of this study was to examine lung injury and inflammation in a well-characterized experimental model of chronic + binge alcohol exposure., Methods: 10-week-old male C57Bl6/J mice were administered ethanol-containing (or isocaloric control) liquid diet for 10 days, followed by a single ethanol gavage (5 g/kg). Lung inflammation and pulmonary function were assessed., Results: Ten days of ethanol-containing liquid diet alone (chronic) did not detectably affect any variables measured. However, ethanol diet plus gavage (chronic + binge) caused neutrophils to accumulate in the lung tissue and in the bronchoalveolar lavage fluid 24 h post-binge. This inflammatory cell recruitment was associated with airway hyper-responsiveness to inhaled methacholine, as indicated by elevated resistance, Newtonian resistance, and respiratory resistance., Conclusions: Taken together, the novel findings reveal that ethanol alone, absent of any secondary inflammatory insult, is sufficient to produce inflammation in the lung. Although these changes were relatively mild, they were associated with functional changes in the central airways. This animal model may be useful in the future for identifying mechanisms by which alcohol abuse sensitizes at-risk individuals to lung injury., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mammarenavirusesare single-stranded RNA viruses with a bisegmented ambisense genome. Ingestion has been shown as a natural route of transmission for both Lassa virus (LASV) and Lymphocytic choriomeningitis virus (LCMV). Due to the mechanism of transmission, epithelial tissues are among the first host cells to come in contact with the viruses, and as such they potentially play a role in spread of virus to naïve hosts. The role of the intestinal epithelia during arenavirus infection remains to be uncharacterized. We have utilized a well-established cell culture model, Caco-2, to investigate the role of intestinal epithelia during intragastric infection. We found that LCMV-Armstrong, LCMV-WE, and Mopeia (MOPV) release infectious progeny via similar patterns. However, the reassortant virus, ML-29, containing the L segment of MOPV and S segment of LASV, exhibits a unique pattern of viral release relative to LCMV and MOPV. Furthermore, we have determined attachment efficacy to Caco-2 cells is potentially responsible for observed replication kinetics of these viruses in a polarized Caco-2 cell model. Collectively, our data shows that viral dissemination and interaction with intestinal epithelia may be host, tissue, and viral specific., Competing Interests: The authors declare no conflict of interest.

Chronic alcohol exposure is a clinically important risk factor for the development of acute respiratory distress syndrome, the most severe form of acute lung injury (ALI). However, the mechanisms by which alcohol sensitizes the lung to development of this disease are poorly understood. We determined the role of the antifibrinolytic protein plasminogen activator inhibitor-1 (PAI-1) in alcohol enhancement of experimental endotoxin-induced ALI. Wild-type, PAI-1 -/- , and integrin β 3 -/- mice were fed ethanol-containing Lieber-DeCarli liquid or a control diet for 6 weeks, followed by systemic LPS challenge. LPS administration triggered coagulation cascade activation as evidenced by increased plasma thrombin-antithrombin levels and pulmonary fibrin deposition. Ethanol-exposed animals showed enhanced PAI-1 expression and pulmonary fibrin deposition with coincident exaggeration of pulmonary inflammatory edematous injury. PAI-1 deficiency markedly reduced pulmonary fibrin deposition and greatly reduced inflammation and injury without impacting upstream coagulation. Interestingly, pulmonary platelet accumulation was effectively abolished by PAI-1 deficiency in ethanol/LPS-challenged mice. Moreover, mice lacking integrin α IIB β 3 , the primary platelet receptor for fibrinogen, displayed a dramatic reduction in early inflammatory changes after ethanol/LPS challenge. These results indicate that the mechanism whereby alcohol exaggerates LPS-induced lung injury requires PAI-1-mediated pulmonary fibrin accumulation, and suggest a novel mechanism whereby alcohol contributes to inflammatory ALI by enhancing fibrinogen-platelet engagement.

Vinyl chloride (VC) is a ubiquitous environmental contaminant for which human risk is incompletely understood. We have previously reported that high occupational exposure to VC directly caused liver damage in humans. However, whether VC may also potentiate liver injury from other causes is not known. C57Bl/6J mice were administered chloroethanol (CE), a major metabolite of VC, and lipopolysaccharide (LPS) 24 h after CE. Samples were harvested for determination of liver damage, inflammation, and changes in carbohydrate and lipid metabolism. In mice, CE exposure alone caused no detectable liver damage. LPS exposure caused inflammatory liver damage, oxidative stress, lipid accumulation, and glycogen depletion; the effect of all of these variables was potentiated by CE pre-exposure. In vitro experiments suggest that VC metabolite chloroacetaldehyde (CAA) directly damages mitochondria, which may explain the sensitization effect observed in vivo Moreover, co-exposure of cells to CAA and TNFα caused increased cell death, supporting the hypothesis of sensitization by VC metabolites. Taken together, these data demonstrate that exposure to VC/metabolites at levels that are not overtly hepatotoxic can potentiate liver injury caused by another hepatotoxicant. This serves as proof-of-concept that VC hepatotoxicity may be modified by an additional metabolic stress such as endotoxemia, which commonly occurs in acute (eg, sepsis) and chronic (eg, NAFLD) diseases., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: It is well known that liver and lung injury can occur simultaneously during severe inflammation (e.g., multiple organ failure). However, whether these are parallel or interdependent (i.e., liver-lung axis) mechanisms is unclear. Previous studies have shown that chronic ethanol (EtOH) consumption greatly increases mortality in the setting of sepsis-induced acute lung injury (ALI). The potential contribution of subclinical liver disease in driving this effect of EtOH on the lung remains unknown. Therefore, the purpose of this study was to characterize the impact of chronic EtOH exposure on concomitant liver and lung injury., Methods: Male mice were exposed to EtOH-containing Lieber-DeCarli diet or pair-fed control diet for 6 weeks. Some animals were administered lipopolysaccharide (LPS) 4 or 24 hours prior to sacrifice to mimic sepsis-induced ALI. Some animals received the tumor necrosis factor-alpha (TNF-α)-blocking drug, etanercept, for the duration of alcohol exposure. The expression of cytokine mRNA in lung and liver tissue was determined by quantitative PCR. Cytokine levels in the bronchoalveolar lavage fluid and plasma were determined by Luminex assay., Results: As expected, the combination of EtOH and LPS caused liver injury, as indicated by significantly increased levels of the transaminases alanine aminotransferase/aspartate aminotransferase in the plasma and by changes in liver histology. In the lung, EtOH preexposure enhanced pulmonary inflammation and alveolar hemorrhage caused by LPS. These changes corresponded with unique alterations in the expression of pro-inflammatory cytokines in the liver (i.e., TNF-α) and lung (i.e., macrophage inflammatory protein-2 [MIP-2], keratinocyte chemoattractant [KC]). Systemic depletion of TNF-α (etanercept) blunted injury and the increase in MIP-2 and KC caused by the combination of EtOH and LPS in the lung., Conclusions: Chronic EtOH preexposure enhanced both liver and lung injury caused by LPS. Enhanced organ injury corresponded with unique changes in the pro-inflammatory cytokine expression profiles in the liver and the lung., (Copyright © 2015 by the Research Society on Alcoholism.)

Viral hemorrhagic fevers (VHFs) encompass a group of diseases with cardinal symptoms of fever, hemorrhage, and shock. The liver is a critical mediator of VHF disease pathogenesis and high levels of ALT/AST transaminases in plasma correlate with poor prognosis. In fact, Lassa Fever (LF), the most prevalent VHF in Africa, was initially clinically described as hepatitis. Previous studies in non-human primate (NHP) models also correlated LF pathogenesis with a robust proliferative response in the liver. The purpose of the current study was to gain insight into the mechanism of liver injury and to determine the potential role of proliferation in LF pathogenesis. C57Bl/6J mice were infected with either the pathogenic (for NHPs) strain of lymphocytic choriomeningitis virus (LCMV, the prototypic arenavirus), LCMV-WE, or with the non-pathogenic strain, LCMV-ARM. As expected, LCMV-WE, but not ARM, caused a hepatitis-like infection. LCMV-WE also induced a robust increase in the number of actively cycling hepatocytes. Despite this increase in proliferation, there was no significant difference in liver size between LCMV-WE and LCMV-ARM, suggesting that cell cycle was incomplete. Indeed, cells appeared arrested in the G1 phase and LCMV-WE infection increased the number of hepatocytes that were simultaneously stained for proliferation and apoptosis. LCMV-WE infection also induced expression of a non-conventional virus receptor, AXL-1, from the TAM (TYRO3/AXL/MERTK) family of receptor tyrosine kinases and this expression correlated with proliferation. Taken together, these results shed new light on the mechanism of liver involvement in VHF pathogenesis. Specifically, it is hypothesized that the induction of hepatocyte proliferation contributes to expansion of the infection to parenchymal cells. Elevated levels of plasma transaminases are likely explained, at least in part, by abortive cell cycle arrest induced by the infection. These results may lead to the development of new therapies to prevent VHF progression.

Immunotherapy using regulatory T cells (Treg) has been proposed, yet cellular and molecular mechanisms of human Tregs remain incompletely characterized. Here, we demonstrate that human Tregs promote the generation of myeloid dendritic cells (DC) with reduced capacity to stimulate effector T cell responses. In a model of xenogeneic graft-versus-host disease (GVHD), allogeneic human DC conditioned with Tregs suppressed human T cell activation and completely abrogated posttransplant lethality. Tregs induced programmed death ligand-1 (PD-L1) expression on Treg-conditioned DC; subsequently, Treg-conditioned DC induced PD-L1 expression in vivo on effector T cells. PD-L1 blockade reversed Treg-conditioned DC function in vitro and in vivo, thereby demonstrating that human Tregs can promote immune suppression via DC modulation through PD-L1 up-regulation. This identification of a human Treg downstream cellular effector (DC) and molecular mechanism (PD-L1) will facilitate the rational design of clinical trials to modulate alloreactivity., Competing Interests: The authors have declared that no competing interests exist.

Wnt and Decapentaplegic cell signaling pathways act synergistically in their contribution to macrochaete (sense organ) patterning on the notum of Drosophila melanogaster. The Wingless-signaling pathway was ectopically activated by removing Shaggy activity (the homologue of vertebrate glycogen synthase kinase 3) in mosaics. Proneural activity is asymmetric within the Shaggy-deficient clone of cells and shows a fixed "polarity" with respect to body axis, independent of the precise location of the clone. This asymmetric response indicates the existence in the epithelium of a second signal, which we suggest is Decapentaplegic. Ectopic expression of Decapentaplegic induces extra macrochaetes only in cells which also receive the Wingless signal. Activation of Hedgehog signaling generates a long-range signal which can promote macrochaete formation in the Wingless activity domain. This signal depends upon decapentaplegic function. Autonomous activation of the Wingless signal response in cells causes them to attenuate or sequester this signal. Our results suggest a novel patterning mechanism which determines sense organ positioning in Drosophila., (Copyright 1999 Academic Press.)

A spontaneously arising murine plasmacytoma, HPC-202, derived from a BALB/c.H-2b congenic mouse that lacks any detectable H-2 determinants on its cell surface is described. However, the expression of H-2 determinants is inducible by interferon-gamma. The H-2 negative cell surface phenotype permits the HPC-202 tumor to escape H-2 allospecific cytotoxic cell lysis but not NK cell lysis, as well as to grow, to varying degrees, in some H-2 incompatible hosts. In those strains which exhibit a resistance to HPC-202 growth, resistance does not map to a single gene within the major histocompatibility complex of the mouse. Resistance is also radiosensitive and is therefore presumably due to a rapidly dividing cell population. The utility of this tumor as a model system to study both the non-H-2-restricted natural resistance to tumor growth, and the mechanism by which H-2 genes are regulated by cells is discussed.

The objective of this study was to find a simple method to preserve cells for subsequent flow cytometry (FACS) analysis. We determined that fixation in 1% paraformaldehyde-0.85% saline solution did not significantly alter the Coulter volume, light scatter of fluorescence properties of the cells. This method was suitable for all cell types analyzed, including mouse, human and rat lymphoid cells, erythrocytes and transformed cell lines. Furthermore, fixed cells, previously stained with fluorescein conjugated antibodies, could be stored at 4 degrees C in the dark for at least a week prior to FACS analysis. This method should prove useful to those who work with in vivo derived specimens or have limited access to flow cytometry facilities.

Previous studies have clearly established that murine plasmacytomas suppress B-cell responses, but no effects on T-cell responses have been noted by most investigators. However, in our investigation of the tumor-associated antigens of plasmacytomas, we have found that immunosuppression plays an important role in the complex interaction between host immune T cells and tumor cells. In these investigations, varying the number of plasmacytoma cells added to in vitro induction systems separated two different effects which these cells had on the generation of cytotoxic T cells. On the one hand, when appropriate numbers were present, tumor cells acted as immunogens and stimulated the generation of specific killer cells against tumor-associated antigen or alloantigen which they expressed. In contrast, greater numbers of tumor cells exerted a profound inhibitory effect on the generation of cytotoxic activity when cells inactivated with irradiation were used. Mitomycin-C inactivation of plasmacytoma cells abrogated this inhibitory activity. In further experiments, MPC-II cells that had been inactivated with Mitomycin C were able to prime T cells in vivo, whereas priming by live tumor cells could not be detected in the same situation. It is suggested that immunosuppression by the live tumor cells may account for their inability to prime T cells in vivo. These results indicate that immunosuppression by plasmacytomas may be one of the important variables which influence the generation of cytotoxic T cells against tumor-associated antigens both in vivo and in vitro.

Cyclic AMP-elevating agents have been shown to increase the expression of Fc receptors on the Abelson virus-induced pre-B cell tumor ABE-8. Such agents included isoproterenol, PGE1, 3-isobutyl-1-methyl xanthine, and 8 BrcAMP. The positive inductive effect produced by these agents was inhibited by 8 BrcGMP and PGF2 alpha, which putatively elevate cyclic GMP levels. Other agents also shown to induce Fc receptor expression were LPS and certain batches of fetal calf sera. In contrast to the inductive effect produced by cyclic AMP elevating agents, 8 BrcGMP and PGF2 alpha were unable to reverse the increased Fc receptor expression produced by LPS and fetal calf sera. Thus, these latter agents may act via a qualitatively different mechanism in producing a change in phenotypic expression. The results of this study are discussed in terms of the use of tumor cells as a model system for studying the pharmacologic control of lymphocyte differentiation.

The study of spontaneous autoimmunity in mouse models affords an opportunity to determine the cellular basis of the immune dysregulation observed in this disease. Recently, a new mouse strain, MRL/Mp-Ipr/Ipr (MRL/l) has been developed which carries an autosomal recessive gene (lpr) that results in massive lymph node enlargement concomitant with the development of several autoantibodies. The interest in this strain lies in the possibility that the defect in T-cell regulation of the immune response is manifested at a different level from that in the NZB mouse. It has been reported that the proliferating population of lymphoid cells in the nodes of these mice are T cells, but that many of them are devoid of Lyt surface antigens. We have accordingly initiated several lines of research with these mice, including quantitative flow cytometry characterization of Lyt antigen expression of cells in the lymph nodes of the mice. In an approach to isolate and study the properties of these cells, we have also established continuous cell lines from the lymph node cells of MRL/l mice, using techniques similar to those used to establish continuous lines of antigen-activated cytotoxic T cells and helper T-cell populations.

A series of H-2d B cell tumor lines and one monocytic tumor cell line were shown to be capable of I region-restricted antigen presentation to I-A-d- and I-Ed- restricted, antigen-specific cloned T cell hybridomas. For the most part, antigen presentation correlated with the present of Ia antigens on the presenting cells, although in a few interesting cases Ia-expression lines failed to present antigen. These T cell hybridomas, together with the B cell and to monocyte tumor cell lines, offer a unique set of tools to study the phenomenon of I region-restricted antigen presentation.

The presence of oncofetal antigens (OFA) on a wide variety of murine tumor cells was demonstrated to a totally in vitro system of cellular immunity. Nonimmune spleen lymphocytes were cocultivated with irradiated syngeneic fetal liver cells and, at various times after initiation of culture, were tested for the presence of cytotoxic lymphocytes (CL) by 51Cr-release assay with labeled tumor target cells. Significant cytotoxic activity was regularly detected after such culture, whereas only minor levels appeared in control cultures of spleen lymphocytes with irradiated syngeneic spleen cells. Specificity of the reaction was assessed by inhibition tests in which nonlabeled cells were admixed to the CL and 51Cr-labeled tumor targets. Fetal liver cells gave significant inhibition; however, no inhibition was found with adult spleen cells. Various tumor types gave inhibition, and fibrosarcomas were more effective than plasmacytomas or lymphomas. The results suggested that all tumor types tested possess such OFA, as well as their unique or virus-associated, tumor-associated transplantation antigens, and that the in vitro system permits a more active response to the tumor-associated OFA than that observed in in vivo studies.

One major issue in studies on natural killer activities centers around the concept of heterogeneity of effector cells in the NK population. In this study Ficoll-Hypaque fractionated PBL from normal adult donors were used as effectors against a variety of tumor targets in in vitro chromium release assays with the goal of substantiation of the existence of NK subsets. Effectors with common or distinct specificities were demonstrated by the cold target inhibition assays as well as by immunoadsorption studies using tumor cell monolayers. In particular, a distinct subset of NK effectors, with the unique ability of killing a myeloma cell line (FRV), hitherto an NK-resistant tumor target, was demonstrated with the PBL of one normal donor (LP). Separation of this unique subpopulation based on differential light scatter property was achieved using flow cytometry (FACS III). The unique FRV killers were larger than the effectors which lyse K562 and they resemble the activated NK cells in the mouse system.

Monoclonal antibody (mAb) anti-Leu M5 reacts with a two-chain molecule composed of a 150-kDa alpha subunit noncovalently associated with a 95-kDa beta subunit and probably is specific for an epitope on the 150-kDa alpha chain. This p150/95 antigen is the third member of a family of polypeptides sharing a common 95-kDa beta chain, which includes the lymphocyte function-associated antigen LFA-1 (p177/95) and complement receptor CR3 (Mo1/MAC-1/OKM1; p165/95) antigens. Sequential immunoprecipitation with anti-p95 beta chain mAb specifically removed the antigens detected by anti-LFA-1, anti-CR3 and anti-Leu M5 mAb. Certain patients with recurrent bacterial infections are genetically deficient in expression of the LFA-1 and Mo1 antigens, and have impaired granulocyte function. Granulocytes from a patient with this disease also failed to react with anti-Leu M5. Stimulation of normal granulocytes with f-Met-Leu-Phe, C5a-desArg, or calcium ionophore resulted in increased expression of Mo1 and Leu M5 antigens on the cell surface, but did not significantly increase expression of LFA-1 antigen. In functional assays, anti-Leu M5 did not inhibit T cell-mediated or natural killer cell-mediated cytotoxicity. In addition, anti-Leu M5 neither inhibited the binding of complement-coated particles to CR1 or CR3 nor did it affect the binding of EC3dg to neutrophils (CR4). These studies clearly indicate that the p150/95 antigen recognized by the anti-Leu M5 antibody is a structurally distinct member of the LFA-1/CR3 family.

Various murine lymphoid tumor cell lines were examined for their alterations in intracellular levels of cyclic AMP following exposure to several pharmacologic agents. The agents tested in the present study included 3-isobutyl-1-methylxanthine (MIX), isoproterenol (ISO), prostaglandin E2 (PGE), histamine, and cholera toxin (CT). B cell tumors were generally found to be less responsive to beta-adrenergic and PGE-induced increases in cyclic AMP than were T cell tumors. An exception to this general finding was the murine B lymphoma cell line WEHI-231, which demonstrated marked sensitivity to ISO and PGE. Macrophage tumors were generally found to be even less responsive to PGE than were the B cell tumors, again with some notable exceptions (P388.D1). Cholera toxin produced differential effects in B, T, and macrophage tumors, both in terms of the absolute magnitude of the cyclic AMP response and the kinetics of this response. A T lymphoma cell line (EL-4) was identified that seems to be totally unresponsive to cell surface receptor-mediated increases in intracellular levels of cyclic AMP. The results of this study are discussed in terms of the usefulness of murine lymphoid tumors for the study of pharmacologic modulation of the immune response by cyclic AMP-elevating agents. These results demonstrate the high degree of cellular heterogeneity that can exist within normal lymphoid populations of cells, and suggest that cloned cell lines may be useful in the biochemical characterization of subsets of lymphoid cells.

Recently, antibodies reactive with T cell subpopulations have been reported to exist in children with active juvenile arthritis (JRA). In an attempt to verify and extend these observations, we have studied children with JRA for the presence of anti-T cell antibodies by flow cytometry and cytoadherence rosette techniques. T cells were isolated from peripheral blood mononuclear cells (PBL) by two methods: 1) Differential sedimentation of PBL rosetted with neuraminidase-treated sheep erythrocytes, and 2) removal of immunoglobulin positive PBL by rosetting with rabbit anti-human F(ab')2 coated bovine erythrocytes and differential sedimentation. Utilizing these methods to detect lymphoreactivity of JRA sera to either population of T cell isolates, we observed the binding of ultracentrifuged normal human sera (NHS) to be comparable to JRA sera (active and quiescent). NHS reacted with 15-25% of T cells. Further studies demonstrate that monomeric IgG was chiefly responsible for lymphoreactivity. The results of these studies are discussed in the context of previous observations.

Murine B cell lymphomas and myelomas were examined for the expression of a determinant previously found exclusively on normal pluripotent stem cells colony-forming unit-spleen (CFU-s). This determinant(s), which is defined by a rabbit antimouse brain antiserum (R alpha MB), is present on the tumor stem cell population of some but not all B cell neoplasms examined. The determinant is not detected on tumor cells of the macrophage or T cell lineage. Absorption of the activity in R alpha MB with myeloma cells, concomitantly removed reactivity with the normal stem cell, CFU-s, and the myeloma stem cell, plasmacytoma CFU-s. Sorting analysis further showed that the antigen was diminished within a positive tumor population as cells acquired the capacity to secrete immunoglobulin. These studies suggest that this normal stem cell-associated antigen may also be an early differentiation antigen for the B cell lineage, and is expressed on some stem cells of B cell tumors.

Human peripheral blood nylon wool nonadherent lymphocytes were fractionated according to cellular density on discontinuous Percoll gradients. The various fractions of cells obtained by this procedure were then analyzed to correlate morphology, cytotoxicity, light scatter properties and antigenic profile. The majority of natural killer (NK) cell activity was manifested by light density lymphocytes, which demonstrated relatively high light scatter characteristics, large granular lymphocyte morphology, and expressed the phenotypes: Leu 11+(B73.1+), Leu 7-; Leu 11+(B73.1+), Leu 7+, and Leu 11+(B73.1+), Leu 2a+ (low density), Leu 4-. The denser fractions of Percoll (fractions 3-4) tended to show lower NK activity, fewer large granular lymphocytes, lower light scatter profiles, and to selectively localize the subpopulations of lymphocytes with the phenotypes: Leu 11-, Leu 7+, Leu 4+, and Leu 11-, Leu 7+, Leu 2a+ (high density).

This study reports the initial phenotypic and functional characterization of a series of cloned, murine, myelomonocytic tumors and their parent cell line WEHI-3, and a group of murine B lymphoma tumors. The tumor cell lines of the myelomonocytic lineage demonstrated the ability to reconstitute a macrophage-depleted, primary in vitro anti-SRBC PFC response, but only marginally enhanced an accessory cell-depleted, ova-primed, lymphocyte proliferation in vitro response. The B lymphoma tumors displayed exactly the reverse functional profile, being highly efficient in reconstitution of the proliferation response, but not supporting the SRBC PFC response. Detailed analysis of the cell surface phenotype of the various B lymphoma tumors used in this study show they display cell surface markers characteristic of normal B lymphocytes but are heterogeneous in their various stages of differential arrest. Future work will concentrate on the orchestration of Ia-mediated and soluble factor-mediated (IL 1) modalities of antigen-triggering of lymphocytes by these B lymphoma and myelomonocytic tumor cell lines.

Dehydrated Mueller-Hinton agar from five different manufacturers was used to prepare plates containing 4% NaCl and 6 micrograms of oxacillin per ml to screen for oxacillin resistance in Staphylococcus aureus. A total of 137 isolates which included 109 oxacillin-resistant isolates obtained from at least eight geographic areas were examined. Results were compared to MICs obtained by using a standard broth microdilution method. Results obtained with screening plates prepared with dehydrated media from three of the manufacturers showed 100% correlation with MICs in detecting oxacillin-resistant S. aureus, and plates prepared with the remaining two media identified oxacillin resistance in 90 and 99% of the resistant isolates, respectively. None of the oxacillin-susceptible isolates grew on any of the screening plates. This study demonstrated that oxacillin screening plates are reliable for detecting oxacillin-resistant S. aureus; however, the source of Mueller-Hinton agar can influence the results.

This study was designed to answer the question, do molecules with carcinoembryonic antigen (CEA) activity from colon, breast, and ovarian cancer differ? Extracts of two breast and three ovarian cancers with CEA activity were compared to three colon cancer CEA preparations and to the related antigen, colon carcinoma antigen-III, in terms of lectin- and antiserum-binding properties. With the use of Farr-type radioimmunoassays with the lectins, concanavalin A and wheat germ agglutinin, the iodinated colon CEA and CEA-like preparations from breast and ovarian cancer all showed distinctly different patterns of binding. Specificity of binding was confirmed by inhibition studies with the relevant monosaccharides. Similarly, with antisera prepared against colon CEA, colon carcinoma antigen-III, or breast CEA, it was shown that, although all preparations shared some antigens, unique antigenic determinants were also present on all preparations. These data are consistent with the concept of a series of closely related CEA and CEA-like molecules with distinct characteristics for each tissue source of CEA.

Tumor-associated antigens (TAA) were demonstrated on plasmacytomas derived from BALB/c, NZB, and C3H mouse strains, by in vivo and in vitro techniques. By immunizing the appropriate F1 hybrid mice with these tumors, it was possible to show that all the plasmacytomas expressed cross-reactive tumor-associated transplantation antigens. When cytotoxic lymphocytes (CL) were induced in vitro by the coculturing of syngeneic or F1 hybrid spleen cells with irradiated plasmacytoma cells, "shared" and "unique" plasmacytoma TAA were demonstrable. This was accomplished by inducing CL in vitro against one syngeneic plasmacytoma and assaying for lytic activity on a range of 51Cr-labeled BALB/c, NZB and C3H plasmacytoma cells in vitro. In a second in vitro assay, unlabeled plasmacytoma cells were tested for their ability to inhibit the lysis of a particular 51Cr-labeled plasmacytoma, with the use of CL induced in vitro against it. The possibility that these TAA were "self" antigens was excluded by demonstrating in the inhibition assay that they were not present on T lymphomas and spleen cells of the same strain, and that CL "autosensitized" in vitro could not significantly lyse 51Cr-labeled plasmacytoma cells in vitro. From both in vivo and in vitro studies of immunity to these tumors, it was concluded that any one plasmacytoma line possesses multiple TAA of both shared and unique types.

Simultaneous dual immunofluorescence and flow cytometry was used to study sixteen lymphocyte phenotypes in 209 men including: healthy homosexuals, lymphadenopathy patients (LAN), and AIDS patients. Significant differences between the distribution of lymphocytes in healthy homosexuals and healthy heterosexuals were decreased percentages of helper/inducer T cells (Leu 3), increased cytotoxic/suppressor T cells (Leu 2), and consequently a decreased Leu 3/Leu 2 ratio. The increased Leu 2 cells were identified as functionally cytotoxic subset Leu 2+ 15- phenotype rather than suppressor cells which are Leu 15+. Leu 2 and Leu 3 bearing cells exhibited an excess of membrane-bound immunoglobulins which were easily elutable at 37 degrees C. An increased percentage of an HLA-DR framework determinant bearing T cells were also detected. Within the NK cell family, Leu 7 cells were moderately increased and the functionally unidentified Leu 2+ 7+ population was strikingly elevated. LAN or AIDS patients were compared to healthy homosexual controls. Lower percentages of Leu 3 cells and higher percentages of Leu 2 cells were evident in LAN patients. These subsets were similar in LAN and AIDS patients. The increase in Leu 2+ cells was due to the Leu 2+ 15- cytotoxic subset. Fewer T cells had immunoglobulin in LAN and AIDS. A definite increase in Leu 2+ DR+ cells but not Leu 3+ DR+ cells occurred in AIDS compared to LAN or healthy controls. NK cell changes already present in healthy homosexuals persisted in LAN and AIDS patients. No differences in the distribution of B cells was detected in any intergroup comparisons. Changes in monocytes or pan-T cells were relatively insensitive measures of immunologic alterations among any of the groups. These results indicate many of the changes in lymphocyte subsets seen in AIDS and LAN subjects are already present in a carefully screened population of healthy homosexuals in San Francisco. Many of the changes in Leu 2 and NK family of cells suggest a possible adaptive response to viral or neoplastic challenge. Whether these interesting phenotypic alterations relate to functional changes in response to such challenge of the identified subsets waits further investigation.

Lymphocyte surface antigens from spleen cells of several mouse strains were studied by cell surface radioiodination, extraction with detergent incubation with various antisera, and separation of complexes using protein A-containing staphylococci as a solid phase adsorbent. Complexes were then dissociated and analyzed by polyacrylamide gel electrophoresis in the presence of sodium diodecyl sulfate. Using this technique and an alloantiserum prepared in C57BL mice against CBA spleen cells, four distinct specific peaks of radioactivity were found with CBA spleen cells. These corresponded to H-2 and Ia antigens, immunoglobulin light chain, and a heavy chain previously proposed to be the murine homolog of the human delta chain. With the same serum, B10.BR spleen cells revealed only H-2 and Ia antigens, whereas C57BL.Ige (allotype congenic) spleen cells showed only the light chain and "delta" chain peaks. Depletion of immunoglobulin from the surface-iodinated preparations resulted in removal of the light chain and "delta" chain peaks. The tissue distribution and membrane expression of this "delta" chain antigen was then studied by indirect immunofluorescence with various C57BL derived alloantisera and lymphoid cells from C57.Ige allotype congenic mice. Significant numbers of positive cells were found in spleen, lymph nodes, and Peyer's patches, whereas few if any positive were found in bone marrow or thymus. No reaction was found between this molecule and alloantisera to any of the previously described immunoglobulin allotypes. It is proposed that these alloantisera to spleen cells recognise one allelic form of the murine "delta" chain coded for by a gene locus closely linked to the known structural genes for mouse immunoglobulin heavy chains. The designation Ig-5 is proposed for this new immunoglobulin heavy chain locus.