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Introduction: The apolipoprotein E (APOE) ε4 allele carries risk for cognitive impairment, but whether the level of circulating apoE4 protein in carriers affects cognition is unclear, as is how health and lifestyle impact circulating apoE4 levels., Methods: We assayed apoE4 protein levels in dried blood spots of 12,532 adults aged 50+. Regression analyses tested the likelihood of cognitive impairment between groups and within those with detected apoE4 protein. Predictors of circulating apoE4 were assessed., Results: We detected protein binding that indicates the presence of an APOE ε4 allele in 28.4% of this group. This group was more likely to have cognitive impairment, and this risk increases with age. However, higher apoE4 levels were associated with less likelihood of cognitive impairment within this group. Antihypertensive medication predicted apoE4 protein levels., Discussion: The apoE4 isoform is associated with a deficient protein and worse cognition. This association is modulated by the level of circulating apoE4 protein in ε4 carriers., Highlights: An assay to quantify apoE4 levels from dried blood spot samples was applied. The apoE4 protein was detected as specific binding at ≥30,000 pg/mL in 28.4% of samples. Having the apoE4 protein was associated with worse cognitive performance. Higher apoE4 protein levels in those who have it were associated with better cognition. Cardiovascular factors influenced levels of apoE4 protein., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Childhood SES has been extensively studied as a predictor for health outcomes in adulthood, though the direct mechanisms remain unclear. The Long Arm of Childhood Model hypothesizes that this process is a chain of events, moderated by numerous factors such as family economic status and environment, health behaviors, as well as biological processes. We expand on this model with objective measures of health in older age, namely C-reactive protein (CRP), as chronic low grade inflammation, which has been found to be connected to both childhood SES as well as a number of cardiovascular diseases in adulthood. Using life history data from SHARE, as well as a novel dried blood spot dataset, we explore the protective role of parent education on the blood level of C-reactive protein in adulthood. Estimating a stepwise linear regression model, we find evidence that years of parental education are negatively associated with CRP in adulthood, with a one-year increase in mother's (father's) years of education decreasing adult CRP by 1.8% (1.1%). Using a modified Sobel test, we measure both the direct and indirect effects, estimating the extent in which later-life mediators significantly alter the relationship between parental education and CRP. While father's education is completely mediated by individual factors such as respondent's education, employment, and health behavior - we observe a lasting association from mother's education, suggesting a direct link between mother's education and CRP in adulthood., Competing Interests: None., (© 2023 The Author.)

Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

The CTLA4-Ig fusion proteins abatacept and belatacept are clinically proven immunosuppressants used for rheumatoid arthritis and renal transplant, respectively. Given that both biologics are typically administered chronically by infusion, a need exists for a next-generation CTLA4-Ig with more convenient dosing. We used structure-based protein engineering to optimize the affinity of existing CTLA4-Ig therapeutics for the ligands CD80 and CD86, and for the neonatal Fc receptor, FcRn. From a rationally designed library, we identified four substitutions that enhanced binding to human CD80 and CD86. Coupled with two IgG1 Fc substitutions that enhanced binding to human FcRn, these changes comprise the novel CTLA4-Ig fusion protein, XPro9523. Compared with abatacept, XPro9523 demonstrated 5.9-fold, 23-fold, and 12-fold increased binding to CD80, CD86, and FcRn, respectively; compared with belatacept, CD80, CD86, and FcRn binding increased 1.5-fold, 7.7-fold, and 11-fold, respectively. XPro9523 and belatacept suppressed human T cell proliferation and IL-2 production more potently than abatacept. XPro9523 also suppressed inflammation in the mouse collagen-induced arthritis model. In cynomolgus monkeys, XPro9523 saturated CD80 and CD86 more effectively than abatacept and belatacept, potently inhibited IgM and IgG immunization responses, and demonstrated longer half-life. Pharmacokinetic modeling of its increased potency and persistence suggests that, in humans, XPro9523 may demonstrate superior efficacy and dosing convenience compared with abatacept and belatacept.

Background: Sequestration of IgE to prevent its binding to high-affinity IgE receptor FcεRI on basophils and mast cells is an effective therapy for allergic asthma. IgE production requires differentiation of activated IgE(+) B cells into plasma cells upon allergen sensitization. B-cell receptor signaling is suppressed by the inhibitory IgG Fc receptor FcγRIIb; therefore, we reasoned that a therapeutic antibody that coengages FcγRIIb and IgE B-cell receptor would not only sequester IgE but also suppress its production by blocking IgE(+) B-cell activation and differentiation to IgE-secreting plasma cells., Objective: To explore the effects of IgE sequestration versus IgE suppression by comparing omalizumab to FcγRIIb-optimized anti-IgE antibodies in humanized mouse models of immunoglobulin production., Methods: By using a murine anti-IgE antibody as a template, we humanized, increased IgE binding, and modified its Fc domain to increase affinity for FcγRIIb. We next compared effects of this antibody (XmAb7195) versus omalizumab on the secretion of IgE and other isotypes in human PBMC cultures and in PBMC-engrafted severe combined immunodeficiency mice., Results: Relative to omalizumab, XmAb7195 has a 5-fold higher affinity for human IgE and more than 400-fold higher affinity for FcγRIIb. In addition to sequestering soluble IgE, XmAb7195 inhibited plasma cell differentiation and consequent human IgE production through coengagement of IgE B-cell receptor with FcγRIIb. In PBMC-engrafted mice, XmAb7195 reduced total human IgE (but not IgG or IgM) levels by up to 40-fold relative to omalizumab., Conclusion: XmAb7195 acts by IgE sequestration coupled with an FcγRIIb-mediated inhibitory mechanism to suppress the formation of IgE-secreting plasma cells and reduce both free and total IgE levels., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.

Engagement of the low-affinity Ab receptor FcγRIIb downregulates B cell activation, and its dysfunction is associated with autoimmunity in mice and humans. We engineered the Fc domain of an anti-human CD19 Ab to bind FcγRIIb with high affinity, promoting the coengagement of FcγRIIb with the BCR complex. This Ab (XmAb5871) stimulated phosphorylation of the ITIM of FcγRIIb and suppressed BCR-induced calcium mobilization, proliferation, and costimulatory molecule expression of human B cells from healthy volunteers and systemic lupus erythematosus (SLE) patients, as well as B cell proliferation induced by LPS, IL-4, or BAFF. XmAb5871 suppressed humoral immunity against tetanus toxoid and reduced serum IgM, IgG, and IgE levels in SCID mice engrafted with SLE or healthy human PBMC. XmAb5871 treatment also increased survival of mice engrafted with PBMC from a unique SLE patient. Unlike anti-CD20 Ab, coengagement of FcγRIIb and BCR complex did not promote B cell depletion in human PBMC cultures or in mice. Thus, amplification of the FcγRIIb inhibitory pathway in activated B cells may represent a novel B cell-targeted immunosuppressive therapeutic approach for SLE and other autoimmune diseases that should avoid the complications associated with B cell depletion.

CD40 is highly expressed on various B-lineage malignancies and represents an attractive immunotherapy target for neoplastic disease. Previous work showed that engineering the Fc domain of an antibody for increased binding to Fcγ receptors (FcγRs) significantly enhanced Fc-mediated immune effector function and antitumor activity in vitro and in vivo. We developed a humanized anti-CD40 antibody similarly Fc-engineered for increased FcγR binding (XmAbCD40) and compared its efficacy with that of an anti-CD40 native IgG1 analog and the anti-CD20 antibody rituximab. XmAbCD40 increased antibody-dependent cell-mediated cytotoxicity (ADCC) up to 150-fold relative to anti-CD40 IgG1 against B-lymphoma, leukemia, and multiple myeloma cell lines, and significantly enhanced ADCC against primary tumors. XmAbCD40 was also superior to rituximab in enhancing ADCC (both in cell lines and primary tumors) and in augmenting antibody-dependent cellular phagocytosis. XmAbCD40 significantly inhibited lymphoma growth in disseminated and established mouse xenografts and was more effective than the IgG1 analog or rituximab. An anti-CD40 antibody constructed to abrogate FcγR binding showed no reduction of tumor growth, indicating that the in vivo antitumor activity of XmAbCD40 is primarily mediated via FcγR-dependent mechanisms. These data demonstrate that XmAbCD40 displays potent antitumor efficacy and merits further evaluation for the treatment of CD40(+) malignancies.

Objective: To determine the best technique for measuring mesial-distal tooth widths on digital models., Methods: The individual mesial-distal tooth widths were measured (first molar to first molar, maxillary and mandibular) on 32 stone casts and corresponding digital models (emodels, GeoDigm, Chanhassen, Minn). The digital models were measured using five different techniques: occlusal aspect, occlusal aspect zooming in on each individual tooth, facial aspect rotating as needed, facial aspect from three standard positions (R buccal, facial, and L buccal), and qualitatively rotating the model in any position deemed necessary. Measurements were repeated three times at least 1 week apart. The operator time needed to complete each set of measurements was recorded., Results: Four of five digital measurement techniques (except for the facial aspect from three standard positions) showed a slight positive bias (overestimation in measured width) compared with stone cast measurements. Measuring from the occlusal aspect resulted in the greatest Pearson correlation (98.509%), the least Altman-Bland standard deviation of differences value (1.881 mm), and the second fastest measuring time (2 minutes 3 seconds). Qualitatively rotating the model had similar Pearson correlation and Altman-Bland values to the Occlusal technique but took the longest time to measure (7 minutes 1 second)., Conclusions: The Occlusal measurement technique for digital models was the best combination of accuracy, repeatability, and speed of measurement.

Improved affinity for the neonatal Fc receptor (FcRn) is known to extend antibody half-life in vivo. However, this has never been linked with enhanced therapeutic efficacy. We tested whether antibodies with half-lives extended up to fivefold in human (h)FcRn transgenic mice and threefold in cynomolgus monkeys retain efficacy at longer dosing intervals. We observed that prolonged exposure due to FcRn-mediated enhancement of half-life improved antitumor activity of Fc-engineered antibodies in an hFcRn/Rag1(-/-) mouse model. This bridges the demand for dosing convenience with the clinical necessity of maintaining efficacy.

CD19 is a pan B-cell surface receptor expressed from pro-B-cell development until its down-regulation during terminal differentiation into plasma cells. CD19 represents an attractive immunotherapy target for cancers of lymphoid origin due to its high expression levels on the vast majority of non-Hodgkin's lymphomas and some leukemias. A humanized anti-CD19 antibody with an engineered Fc domain (XmAb5574) was generated to increase binding to Fcgamma receptors on immune cells and thus increase Fc-mediated effector functions. In vitro, XmAb5574 enhanced antibody-dependent cell-mediated cytotoxicity 100-fold to 1,000-fold relative to an anti-CD19 IgG1 analogue against a broad range of B-lymphoma and leukemia cell lines. Furthermore, XmAb5574 conferred antibody-dependent cell-mediated cytotoxicity against patient-derived acute lymphoblastic leukemia and mantle cell lymphoma cells, whereas the IgG1 analogue was inactive. XmAb5574 also increased antibody-dependent cellular phagocytosis and apoptosis. In vivo, XmAb5574 significantly inhibited lymphoma growth in prophylactic and established mouse xenograft models, and showed more potent antitumor activity than its IgG1 analogue. Comparisons with a variant incapable of Fcgamma receptor binding showed that engagement of these receptors is critical for optimal antitumor efficacy. These results suggest that XmAb5574 exhibits potent tumor cytotoxicity via direct and indirect effector functions and thus warrants clinical evaluation as an immunotherapeutic for CD19(+) hematologic malignancies.

Electroporation (EP)-assisted intralesional delivery of Interleukin-2 (IL-2) plasmid (pDNA) has the potential to increase the local concentration of the expressed cytokine for an extended time in the injected tumors while minimizing its systemic concentration, in comparison with systemic delivery of the recombinant cytokine. Nonclinical Investigational New Drug application-enabling studies were performed in mice to evaluate the effect of intratumoral administration of murine IL-2 pDNA on local expression and systemic distribution of IL-2 transgene as well as the inhibition of established tumor growth. The safety of repeated administrations of a human IL-2 pDNA product candidate with EP was evaluated in rats. Following the nonclinical safety and efficacy studies, a human IL-2 pDNA product candidate intralesionally administered with EP to metastatic melanoma patients is currently being investigated in a phase I clinical trial.

The contribution of Bacillus anthracis lethal factor (LF)-specific immune responses to protection against anthrax disease in humans remains incompletely defined due, in part, to a paucity of qualified reagents and a lack of standardized serological assays. Toward this end, we have identified and characterized suitable positive quality control and standard reference sera and developed, optimized, and qualified an enzyme-linked immunosorbent assay (ELISA) to measure LF-binding IgG. Herein we describe the performance characteristics of this ELISA and propose criteria for its use in the detection and quantification of anti-LF IgG in human serum.

In vivo delivery of a cytokine gene to treat a tumor has usually involved either injection of ex vivo transfected cells around the tumor site or direct intratumoral injection of a virus or plasmid DNA (pDNA) vector encoding the cytokine gene (1,2). In this manner, transfected cells in or around the tumor site may secrete cytokine locally and stimulate an antitumor immune response (3,4). Recently, a new method of cytokine gene delivery for treating tumors was described. In this method, a naked pDNA encoding a cytokine, in this case, interferon-α (IFN-α), was injected intramuscularly (im) into C57BL/6 mice bearing solid or metastatic B16F10 melanoma tumors (5). The mice treated in this manner had a striking inhibition of tumor growth.

There are several strategies by which one may deliver a plasmid DNA (pDNA) encoding a therapeutic gene to a tumor. One may transfect cells ex vivo, single cell clone, expand the clone in vitro, and reinject the cells at the tumor site. This is a labor-intensive process and is especially impractical for human tumor therapy. Another method is intramuscular (im) injection of the therapeutic pDNA to achieve circulating levels of the protein (discussed in Chapter 14 by Horton and Parker). A third method is to directly inject the therapeutic pDNA into the tumor. For accessible neoplasms, this is a simple procedure, and can be useful for delivery of a therapeutic gene, such as a cytokine gene, to the tumor site. Using this technique, one may achieve high local levels of a therapeutic protein, yet have low systemic levels, thereby reducing side effects (1,2). In addition, producing a cytokine locally may attract immune cells to the tumor site and promote an antitumor immune response (1-3). Furthermore, certain cytokines may be more effective when delivered locally, rather than systemically (Horton, unpublished results).

We have evaluated whether i.p. murine ovarian tumors could be treated with an IL-2 plasmid DNA complexed with the cationic lipid, (+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2, 3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (DMRIE/DOPE). Reporter gene studies were initially conducted in which mice bearing i.p. murine ovarian teratocarcinoma (MOT) were injected i.p. with reporter gene plasmid DNA (pDNA):DMRIE/DOPE. Histochemical analyses revealed that transfection occurred primarily in the tumor cells of the ascites, with only a minority of other ascitic cells or surrounding tissues transfected. IL-2 levels in the MOT ascites were determined after i. p. injection of either IL-2 pDNA:DMRIE/DOPE or recombinant IL-2 protein. IL-2 was detected in tumor ascites for up to 10 days after a single i.p. injection of IL-2 pDNA:DMRIE/DOPE, but was undetectable 24 h after a single i.p. injection of IL-2 protein. In an antitumor efficacy study, MOT tumor-bearing mice injected i.p. with IL-2 pDNA:DMRIE/DOPE on days 5, 8, and 11 after tumor cell implant had a significant inhibition of tumor ascites (p = 0.001) as well as a significant increase in survival (p = 0.008). A cytokine profile of the MOT tumor ascites revealed that mice treated with IL-2 pDNA:DMRIE/DOPE had an IL-2-specific increase in the levels of IFN-gamma and GM-CSF. Taken together, these findings indicate that i. p. treatment of ovarian tumors with IL-2 pDNA:DMRIE/DOPE can lead to an increase in local IL-2 levels, a change in the cytokine profile of the tumor ascites, and a significant antitumor effect.

Vaccination with a tumour antigen-expressing plasmid DNA (pDNA) is a novel approach to human cancer immunotherapy. Initial results in preclinical rodent tumour models are promising, revealing that pDNA cancer vaccines can elicit both humoral, as well as cell-mediated immunity and, in some cases, protect against tumour growth. Compared to peptide, viral or dendritic cell vaccines, the delivery of tumour antigens using pDNA has the advantages of ease of manufacture, lack of toxicity and broad applicability to large populations. With advances in modern genomics strategies and the identification of an increasing number of tumour antigen genes, pDNA-based cancer vaccines may be used in the future to treat a wide variety of human cancers.

The antitumor effect of the type I IFN, IFN-omega, was evaluated in both in vitro and in vivo studies of human cancer. For these studies, the cDNA for human IFN-omega was cloned into a eukaryotic expression plasmid DNA (pDNA) driven by the cytomegalovirus promoter. Supernatants from UM449 cells transfected in vitro with IFN-omega pDNA had antiproliferative effects on 11 of 13 human tumor cell lines. For in vivo studies, nude mice were implanted s.c. with one of the following human tumors: NIH: OVCAR-3 ovarian carcinoma, A375 melanoma, or A431 epidermoid carcinoma. Direct intratumoral injection of 100 microg of a IFN-omega pDNA DMRIE/DOPE complex (1:1 DNA:DMRIE mass ratio) for 6 consecutive days resulted in a significant reduction in the tumor volume of NIH: OVCAR-3 ovarian carcinoma or A375 melanoma (P = 0.02). IFN-omega pDNA delivered by i.m. injection also had an antitumor effect. Nude mice bearing s.c. A431 epidermoid carcinoma and injected i.m. with 100 microg of IFN-omega pDNA, twice per week for 3 weeks, had a significant reduction in tumor volume (P = 0.009). These results demonstrate for the first time that IFN-omega can have in vivo antitumor effects in several models of human cancer.

Leuvectin is a plasmid DNA/lipid complex comprised of a plasmid DNA expression vector (VCL-1102, 30) encoding human interleukin (IL)-2 complexed in a 5:1 mass ratio with DMRIE/DOPE lipid that has been developed for the treatment of cancer. DMRIE/DOPE is a cationic lipid, which facilitates in vitro and in vivo transfection of plasmid DNA. In vitro transfection with the IL-2 plasmid DNA/DMRIE/DOPE complex results in the expression of sustained levels of biologically active IL-2. Human tumor cell lines and primary human tumor cells established from biopsies were readily transfected in vitro resulting in the expression of IL-2. Following in vitro transfection, IL-2 expression continued up to several weeks post-transfection in primary tumor cells. In preclinical efficacy studies in a murine model of renal cell carcinoma (RCC), the direct intratumoral administration of an IL-2 plasmid DNA/DMRIE/DOPE complex resulted in the generation of tumor specific lymphocytes and complete tumor regression in the majority of the mice. In preclinical animal safety studies, repeated administration of Leuvectin was safe and well-tolerated. Following these promising preclinical results, Leuvectin has entered clinical trials and two pilot phase I/II trials are described.

A cancer treatment is described in which i.m. injection of plasmid DNA (pDNA) encoding murine interferon alpha (mIFN-alpha) leads to potent antitumor effects on primary and metastatic tumors in mice. Mice bearing s.c. B16F10 melanoma, Cloudman melanoma, or glioma 261 tumors were injected i.m. with mIFN-alpha pDNA. In all three tumor models, a significant reduction in tumor volume and enhancement of survival was found after IFN pDNA therapy. The mIFN-alpha pDNA could be injected as infrequently as once every other week and still produce a significant antitumor effect, and, in a metastatic tumor model, the therapy markedly reduced the number of lung tumor metastases. Depletion of immune cell subsets indicated that CD8(+) T cells were required for the antitumor response. These studies demonstrate that primary and metastatic tumors can be treated systemically by i.m. injection of a plasmid encoding a cytokine gene.

Intratumoral (i.t.) injection of a plasmid DNA vector encoding the murine interleukin-2 (IL-2) gene was used to treat established renal cell carcinoma (Renca) tumors in BALB/c mice. Tumor regression was observed in 60-90% of mice that were injected i.t. for 4 days with IL-2 plasmid DNA complexed with the cationic lipid DMRIE/DOPE ((+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propa naminium bromide/dioleoylphosphatidylethanolamine). The mice remained tumor-free until the conclusion of the study, which was 4 months after tumor challenge. In a rechallenge experiment, mice that were rendered tumor-free for 6 months by IL-2 plasmid DNA treatment rejected a subsequent challenge of Renca cells but could not reject a challenge with the unrelated, syngeneic CT-26 tumor. Spleen cells from cured mice contained Renca-specific cytotoxic T lymphocytes, and adoptive transfer of mixed lymphocyte cultures into naive mice at 2 days after challenge with Renca cells prevented tumor growth. In vivo depletion of T-cell subsets at the time of i.t. injection with IL-2 plasmid DNA demonstrated that CD8+ T cells, but not CD4+ T cells, were the primary effectors of the antitumor response.

This research provides the first evidence for specific receptor binding of polyhedron-derived baculovirus (PDV) to host cells and to lepidopteran brush border membrane vesicles (BBMV) and demonstration of entry via a nonendocytotic pathway involving direct membrane fusion. The technique of fluorescence-activated cell sorting analysis was used to investigate the specificity of binding between the PDV phenotype of Lymantria dispar nuclear polyhedrosis virus (LdNPV) and host membranes. Fluorescein isothiocyanate-labeled PDV was found to bind in a saturable manner to the gypsy moth cell line IPLB-LdEIta and to L. dispar BBMV. The IPLB-LdEIta cell line was found to possess approximately 10(6) PDV-specific receptor sites per cell. Excess levels of unlabeled PDV were highly efficient in competing with fluorescein isothiocyanate-labeled PDV for limited receptor sites, further supporting the specificity of the interaction. Major reductions in virus binding (as high as 70%) after protease treatment of cells indicated that a protein receptor is involved. A fluorescence dequenching assay of membrane fusion with octadecyl rhodamine B (R18)-labeled PDV was used to identify PDV fusion to host cells and BBMV. Direct membrane fusion of PDV occurred at 27 degrees C to both target membranes as well as at 4 degrees C at approximately 55% of the levels achieved at 27 degrees C. Viral fusion to BBMV occurred throughout the pH range of 4 to 11, with dramatically increased fusion levels (threefold) under the alkaline conditions normal for lepidopteran larval midguts. Treatment of cells with chloroquine, a lysosomotropic agent, did not significantly affect PDV fusion to cells or infectivity in tissue culture assays.

Polymerase chain reaction (PCR) technology was employed to detect baculovirus DNA sequences from viral occlusion bodies (OB) contaminating the surface of gypsy moth eggs. The level of sensitivity of the technique was as low as 5 viral genome copies and DNA from 1 OB equivalent. Thirty minutes of shortwave UV irradiation of eggs contaminated with 8.4 x 10(4) OBs prevented amplification of viral DNA sequences from OBs on the egg surface. These methods are important for providing a better understanding of gypsy moth virus epizootiology as well as for the examination of insect eggs for the persistence of baculovirus gene sequences inside the egg or on the egg surface. In addition, these methods can be easily modified for monitoring the persistence of genetically engineered baculoviruses in insect populations as well as the fate of genes that these viruses might carry.

The present paper has discussed available test systems for determination of the response of G-6-PD-deficient human erythrocytes to environmental agents. The limitations and advantages of each model have been examined, and the results of research using each model have been presented. The future development of suitable animal models or in vitro test systems may rely on advances in fields such as genetics and biochemistry. Genetic engineering may allow researchers to develop cells with a genetic deficiency of G-6-PD. These deficient cells could then be used to simulate human G-6-PD-deficient erythrocyte responses to various agents. Advances in biochemistry, in areas such as metabolism and enzymology, may also have an impact on future test systems. Due to the fact that present model systems are limited and their predictions often unreliable, the establishment of safe environmental health standards will depend upon advances in modern science and the converging of developments from various disciplines.