An abrupt and concordant initiation of apoptosis: antigen-dependent death of CD8+ CTL.

The ability of CD8+ cytotoxic T lymphocytes (CTL) to clear viral infections may be limited when high avidity CTL encounter supra-optimal antigen density on antigen-presenting cells (APC) and undergo antigen-dependent apoptosis of CTL (ADAC). Previously, we have shown ADAC in CD8+ populations to be Fas independent, TNF-alpha receptor 2 (TNFR2) mediated, caspase dependent, and accompanied by a decrease in Bcl-2. We now employ flow cytometry to follow ADAC within individual CD8+ cells to demonstrate that the intense TCR signal induced in high avidity CTL by supra-optimal antigen density results 8 - 16 h later in a caspase-independent TNFR2 down-modulation that is directly related to the stimulating APC antigen density and concludes in a rapid onset of apoptosis by 18 - 24 h. Individual CTL undergoing apoptosis exhibit a dramatic and concurrent: (1) positive staining with Annexin V and propidium iodide; (2) transformation to a smaller cell size characteristic of apoptosis; and (3) a nearly complete loss of Bcl-2, c-IAP1, and TRAF2. We conclude that the antigen-dependent apoptosis of CD8+ CTL occurs when a tandem TCR/TNFR2 signal initiates an abrupt and concordant onset of multiple apoptotic events.

Comparison of practical treatment methods to eradicate pinworm (Dentostomella translucida) infections from Mongolian gerbils (Meroines unguiculatus).

This study evaluates the efficacy of various treatment methods to eradicate Dentostomella translucida from Mongolian gerbil colonies. The following five treatment methods were instituted in naturally infected groups of 10 gerbils each: topical ivermectin misting, ivermectin-medicated drinking water, piperazine citrate-medicated drinking water, fenbendazole-medicated feed, and a combination of ivermectin-medicated drinking water and fenbendazole-medicated feed. Treatment success was assessed by using weekly fecal flotations, with necropsy examinations performed on fecal-negative gerbils (except those in the misted group) at 5 weeks after the last treatment. Topical ivermectin misting left 40% of gerbils fecal-positive. With piperazine citrate-medicated drinking water, 60% of the gerbils were fecal-positive; the remaining 40% had adult worms in their digestive tract at necropsy. Ivermectin-medicated drinking water caused 80% of the gerbils to be negative on fecal flotation. On necropsy, however, all but one of these gerbils harbored adult pinworms. Treatments with fenbendazole-supplemented feed alone or in combination with ivermectin-treated water resulted in no fecal shedding or evidence of adult pinworms on necropsy examination. Of the five treatments evaluated, only those using fenbendazole-medicated feed (150 ppm) provided a practical and reliable treatment method to eradicate pinworm infections in Mongolian gerbil colonies.

Competitive inhibition in vivo and skewing of the T cell repertoire of antigen-specific CTL priming by an anti-peptide-MHC monoclonal antibody.

We have recently described a mAb, KP15, directed against the MHC-I/peptide molecular complex consisting of H-2D(d) and a decamer peptide corresponding to residues 311-320 of the HIV IIIB envelope glycoprotein gp160. When administered at the time of primary immunization with a vaccinia virus vector encoding gp160, the mAb blocks the subsequent appearance of CD8(+) CTL with specificity for the immunodominant Ag, P18-I10, presented by H-2D(d). This inhibition is specific for this particular peptide Ag; another H-2D(d)-restricted gp160 encoded epitope from a different HIV strain is not affected, and an H-2L(d)-restricted epitope encoded by the viral vector is also not affected. Using functional assays and specific immunofluorescent staining with multivalent, labeled H-2D(d)/P18-I10 complexes (tetramers), we have enumerated the effects of blocking of priming on the subsequent appearance, avidity, and TCR Vbeta usage of Ag-specific CTL. Ab blocking skews the proportion of high avidity cells emerging from immunization. Surprisingly, Vbeta7-bearing Ag-specific TCR are predominantly inhibited, while TCR of several other families studied are not affected. The ability of a specific MHC/peptide mAb to inhibit and divert the CD8(+) T cell response holds implications for vaccine design and approaches to modulate the immune response in autoimmunity.

The combination of zidovudine and interferon alpha-2B in the treatment of adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is frequently a very aggressive malignancy with a poor survival despite aggressive multiagent chemotherapy. The combination of the antiretroviral drug zidovudine (AZT) and interferon alpha (IFNalpha) has been reported to induce remissions in patients with ATL. The purpose of this study was to evaluate the clinical response and toxicity following administration of a combination of IFNalpha-2b and AZT in patients with human T-cell lymphotropic virus type I (HTLV-I)-associated ATL. Eighteen patients with ATL (chronic. crisis, acute or lymphoma type) were treated with the combination of AZT (50 - 200 mg orally 5 times a day) and IFNalpha-2b (2.5 - 10 million units subcutaneously daily). Three patients had objective responses lasting more than one month. One patient had a clinical complete remission, lasting 21.6 months and two patients had partial remissions lasting 3.7 and 26.5 months. Six patients were not considered evaluable for response due to short and/or interrupted periods of treatment. Seventeen patients have died with a median survival time after initiation of therapy of 6 months. Neutropenia and thrombocytopenia were the dose limiting toxicities. In conclusion, the response rate in this study was lower than noted in the two previous published series. This may be due to the amount and type of prior treatment our patients had received.

Two intermediate-avidity cytotoxic T lymphocyte clones with a disparity between functional avidity and MHC tetramer staining.

The efficacy of cytotoxic T lymphocytes (CTL) has been shown to be highly dependent upon their functional avidity (the sensitivity of their cellular response to MHC-peptide complexes). To examine this relationship, we employed target cell lysis as a quantitative measure and established a set of four CTL clones that exhibited a range of functional avidities spanning more than three orders of magnitude. Within this set, clones displayed a linear correlation between functional avidity and the TCR down-regulation that occurred in response to increasing antigen density. Staining intensity of MHC-peptide tetramer, however, correlated only with the very highest and very lowest avidity clones; the two intermediate-avidity clones showed an inverse relationship between tetramer staining and functional avidity. Compensation for differences in surface levels of TCR improved the correlation, but failed to fully account for this discrepancy. Comparison of TCR signals generated by stimulation of CTL with substrate-bound soluble MHC-peptide or antigen-presenting cells suggested that internal TCR signaling efficiency accounts for at least a portion of the observed functional avidity and suggests the need for caution in directly relating tetramer staining to avidity.

High-avidity CTL exploit two complementary mechanisms to provide better protection against viral infection than low-avidity CTL.

Previously, we observed that high-avidity CTL are much more effective in vivo than low-avidity CTL in elimination of infected cells, but the mechanisms behind their superior activity remained unclear. In this study, we identify two complementary mechanisms: 1) high-avidity CTL lyse infected cells earlier in the course of a viral infection by recognizing lower Ag densities than those distinguished by low-avidity CTL and 2) they initiate lysis of target cells more rapidly at any given Ag density. Alternative mechanisms were excluded, including: 1) the possibility that low-avidity CTL might control virus given more time (virus levels remained as high at 6 days following transfer as at 3 days) and 2) that differences in efficacy might be correlated with homing ability. Furthermore, adoptive transfer of high- and low-avidity CTL into SCID mice demonstrated that transfer of a 10-fold greater amount of low-avidity CTL could only partially compensate for their decreased ability to eliminate infected cells. Thus, we conclude that high-avidity CTL exploit two complementary mechanisms that combine to prevent the spread of virus within the animal: earlier recognition of infected cells when little viral protein has been made and more rapid lysis of infected cells.

Bioassays of APONIN-3 and -4 with rabbit erythrocytes.

Rabbit erythrocytes in methanolic phosphate medium were used to bioassay the activity of authentic samples of methyl stearate and methyl palmitate (in 10% methanol:90% water, v/v), which had been identified as apparent oceanic naturally occurring cytolins (APONIN-3 and -4) produced by Nannochloris oculata. The two natural products are notable for cytolytic activity toward the unarmoured dinoflagellate, Gymnodinium breve Davis, an organism responsible for red tides consisting of harmful algal blooms in the Gulf of Mexico and along the eastern coast of the United States. Bioassays were done with heparinized rabbit blood. The absorbance at 540 nm was observed for 15 min in comparison with a sample treated with haemolysing agent. The results indicated that at reasonable concentrations of 1-10 ppm, neither was a haemolysin, although such concentrations caused cytolysis of G. breve cultures.

GDNF is trophic for mouse motoneurons that express a mutant superoxide dismutase (SOD-1) gene.

BACKGROUND AND METHODS: An in vitro system of motoneurons was established from mice carrying a transgene for a human superoxide dismutase-1 (SOD-1) with a gly93ala mutation that has been linked to familial amyotrophic lateral sclerosis (FALS). These cultures were characterized and used to compare the effects of glial cell line-derived neurotrophic factor (GDNF) on motoneurons expressing the mutant gene with those on normal motoneurons. RESULTS: Recombinant human GDNF (100 ng/ml) significantly promoted the survival of a subpopulation of choline acetyltransferase (ChAT)-immunoreactive motoneurons that were also immunoreactive for the homeoprotein islet-1 in cultures from both wild type and mutant SOD-1 mice. However, GDNF did not increase the total number of ChAT-immunoreactive neurons in cultures from either wild type or transgenic mice. A distinct subpopulation of islet-1-immunoreactive motoneurons characterized by a soma 3 1/2 times larger and a ten-fold increase in neurite length was observed exclusively in GDNF-treated cultures. In cultures from mutant SOD-1 mice, there were 3 1/2 times as many motoneurons of this subpopulation as in wild type cultures at 6 days in vitro. In addition, this subpopulation of neurons survived for 10 days in vitro, the longest time point studied, in culture from mutant SOD-1 mice, but not in cultures from wild type mice. This subpopulation was also present at 6 days in vitro in cultures from mutant SOD-1 mice that received GDNF at 3 days in vitro instead of at the time of plating, suggesting that GDNF promotes the differentiation of these neurons. CONCLUSION: Our observations suggest that the expression of a mutant SOD-1 gene, as occurs in familial ALS, does not compromise the trophic effects of GDNF on motoneuron survival, but may affect the development of motoneurons.

Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections.

We used several approaches to develop enhanced vaccines for chronic viral infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). 1) Selected epitopes were used to avoid potentially harmful immune responses. 2) Linkage between helper and cytotoxic T-lymphocyte (CTL) epitopes was found to be important. 3) We developed an "epitope enhancement" approach modifying the sequences of epitopes to make more potent vaccines, including examples for HIV and HCV epitopes presented by murine class II and human class I major histocompatibility complex (MHC) molecules. 4) CTL avidity was found to be important for clearing viral infections in vivo, and the mechanism was examined. High-avidity CTLs, however, were found to undergo apoptosis when confronted with high-density antigen, through a mechanism involving tumor necrosis factor (TNF), TNF-RII, and a permissive state induced through the T-cell receptor. 5) We employed cytokines in the adjuvant to steer immune responses toward desired phenotypes, and showed synergy between cytokines. 6) Intrarectal immunization with peptide vaccine induced mucosal and systemic CTL. Local mucosal CTL were found to be critical for resistance to mucosal viral transmission and this resistance was enhanced with mucosally delivered interleukin-12. 7) We used an asymmetry in induction of mucosal and systemic immune responses to circumvent pre-existing vaccinia immunity for use of recombinant vaccinia vaccines.

Gene transfer into the mammalian inner ear using HSV-1 and vaccinia virus vectors.

The introduction of foreign genes into cells has become an effective means of achieving intracellular expression of foreign proteins, both for therapeutic purposes and for experimental manipulation. Gene delivery to the nervous system has been extensively studied, primarily using viral vectors. However, to date less work has focused on gene delivery to the inner ear, and existing studies have primarily used adenovirus and adeno-associated virus. Using two recombinant viral vectors, herpes simplex type 1 (HSV-1), and vaccinia virus, bearing the Escherichia coli lacZ gene, we tested gene delivery to the guinea pig cochlea in vivo with beta-galactosidase staining as an assay. The HSV-1 and vaccinia virus vectors were both found to infect and elicit transgene expression successfully in many cells in the guinea pig cochlea, including cells in the organ of Corti. These data demonstrate the feasibility of gene delivery to the inner ear using these two viral vectors. Such techniques may facilitate study of the auditory systems, and might be used to develop gene therapy strategies for some forms of hearing loss.

Interaction of murine MHC class I molecules with tapasin and TAP enhances peptide loading and involves the heavy chain alpha3 domain.

In human cells the association of MHC class I molecules with TAP is thought to be mediated by a third protein termed tapasin. We now show that tapasin is present in murine TAP-class I complexes as well. Furthermore, we demonstrate that a mutant H-2Dd molecule that does not interact with TAP due to a Glu to Lys mutation at residue 222 of the H chain (Dd(E222K)) also fails to bind to tapasin. This finding supports the view that tapasin bridges the association between class I and TAP and implicates residue 222 as a site of contact with tapasin. The inability of Dd(E222K) to interact with tapasin and TAP results in impaired peptide loading within the endoplasmic reticulum. However, significant acquisition of peptides can still be detected as assessed by the decay kinetics of cell surface Dd(E222K) molecules and by the finding that prolonged viral infection accumulates sufficient target structures to stimulate T cells at 50% the level observed with wild-type Dd. Thus, although interaction with tapasin and TAP enhances peptide loading, it is not essential. Finally, a cohort of Dd(E222K) molecules decays more rapidly on the cell surface compared with wild-type Dd molecules but much more slowly than peptide-deficient molecules. This suggests that some of the peptides obtained in the absence of an interaction with tapasin and TAP are suboptimal, suggesting a peptide-editing function for tapasin/TAP in addition to their role in enhancing peptide loading.

Supraoptimal peptide-major histocompatibility complex causes a decrease in bc1-2 levels and allows tumor necrosis factor alpha receptor II-mediated apoptosis of cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) are primary mediators of viral clearance, but high viral burden can result in deletion of antigen-specific CTLs. We previously reported a potential mechanism for this deletion: tumor necrosis factor (TNF)-alpha-mediated apoptosis resulting from stimulation with supraoptimal peptide-major histocompatibility complex. Here, we show that although death is mediated by TNF-alpha and its receptor (TNF-RII), surprisingly neither the antigen dose dependence of TNF-alpha production nor that of TNF-RII expression can account for the dose dependence of apoptosis. Rather, a previously unrecognized effect of supraoptimal antigen in markedly decreasing levels of the antiapoptotic protein Bc1-2 was discovered and is likely to account for the gain in susceptibility or competence to sustain the death signal through TNF-RII. This decrease requires a signal through the TCR, not just through TNF-RII. Although death mediated by TNF-RII is not as widely studied as that mediated by TNF-RI, we show here that it is also dependent on proteolytic cleavage by caspases and triggered by a brief initial encounter with antigen. These results suggest that determinant density can regulate the immune response by altering the sensitivity of CTLs to the apoptotic effects of TNF-alpha by decreasing Bc1-2 levels.

Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge.

Mucosal tissues are major sites of HIV entry and initial infection. Thus, the induction of a mucosal cytotoxic T lymphocyte (CTL) response is an important feature for an effective HIV vaccine. However, little is known about approaches to induce such a protective CTL response in the mucosa. Here for the first time we show that intrarectal immunization with a synthetic, multideterminant HIV peptide plus cholera toxin adjuvant induced long-lasting, antigen-specific CTL memory in both the inductive (Peyer's patch) and effector (lamina propria) mucosal sites, as well as in systemic sites (spleen), whereas systemic immunization induced specific CTL only in the spleen. Cholera toxin adjuvant, while enhancing the response, was not essential. The CTL recognized target cells either pulsed with HIV peptide or expressing endogenous whole envelope glycoprotein of Mr 160,000 (gp160). Exploring the requirements for CTL induction, we show that mucosal CTL responses are both interleukin 12 and interferon-gamma dependent by using antibody-treated and knock-out mice. Finally, to determine whether a mucosal response is actually protective against local mucosal challenge with virus, we show that intrarectal immunization with the synthetic HIV peptide vaccine protected mice against infection via mucosal challenge with a recombinant vaccinia virus expressing HIV-1IIIB gp160. These studies provide an approach to development of an HIV vaccine that induces CTL immunity in the mucosal and systemic immune systems and protects against mucosal infection with a virus expressing HIV-1 gp160.

CCG repeats in cDNAs from human brain.

Expansion mutations of trinucleotide repeats and other units of unstable DNA have been proposed to account for at least some of the genetic susceptibility to a number of neuropsychiatric disorders, including bipolar affective disorder, schizophrenia, autism, and panic disorder. To generate additional candidate genes for these and other disorders, cDNA libraries from human brain were probed at high stringency for clones containing CCG, CGC, GCC, CGG, GCG, and GGC repeats (referred to collectively as CCG repeats). Some 18 cDNAs containing previously unpublished or uncharacterized repeats were characterized for chromosomal locus, repeat length polymorphism, and similarity to genes of known function. The cDNAs were also compared with the 37 human genes with eight or more consecutive CCG triplets in GenBank. The repeats were mapped to a number of loci, including 1p34, 2p11.2, 2q30-32, 3p21, 3p22, 4q35, 6q22, 7qter, 13p13, 17q24, 18p11, 19p13.3, 20q12, 20q13.3, and 22q12. Length polymorphism was detected in 50% of the repeats. The newly cloned cDNAs include a complete transcript of human neurexin-1B, a portion of BCNG-1 (a newly described brain-specific ion channel), a previously unreported polymorphic repeat located in the 5' UTR region of the guanine nucleotide-binding protein (G-protein) beta2 subunit, and a human version of the mouse proline-rich protein 7. This list of cDNAs should expedite the search for expansion mutations associated with diseases of the central nervous system.

On the relationship of the ATP-independent, mitochondrial associated DNA topoisomerase of Saccharomyces cerevisiae to the nuclear topoisomerase I.

A mitochondrial type I-like, ATP-independent, DNA topoisomerase, isolated from highly purified yeast mitochondria, is genetically related to nuclear topoisomerase I. We found that the mitochondrial topoisomerase activity cannot be detected in yeast mitochondrial extracts prepared from strains in which the topoisomerase I gene (TOP1) is disrupted. Thus, the topoisomerase activity associated with mitochondria is dependent upon the expression of the nuclear topoisomerase I gene.

Analysis of the effects of task preferences, task demands, and adult attention on child behavior in outpatient and classroom settings.

Two studies were conducted with children who displayed behavior problems to evaluate the effects of task preference, task demands, and adult attention on child behavior. In Study 1, we conducted brief functional analyses in an outpatient clinic to identify variables that facilitated appropriate behavior. For 8 of 10 children, distinct patterns of performance occurred; 3 children displayed improved behavior with changes in task demands, 1 child displayed improved behavior with a preferred task, and 4 children displayed improved behavior with changes in adult attention. In most cases, the children's parents carried out the assessments with adequate procedural integrity. In Study 2, we applied similar assessment methods to a classroom setting over an extended period of time. We identified independent variables controlling appropriate, on-task, and academic behavior for 2 children on two tasks, with slightly different treatment procedures across tasks for both children. In addition, the results of brief functional analyses for both children corresponded to the extended classroom assessments.

Identification of sub-populations of chick neural retinal cells by monoclonal antibodies: a fluorescence activated cell sorter screening technique.

An assay was developed which identifies monoclonal antibodies recognizing the cell surfaces of sub-populations of chick retinal cells which survive in culture. Antibodies from hybridoma culture supernatants were bound to monolayers of retina cells followed by a fluorescent secondary antibody. The quantitative fluorescence analysis ability of the fluorescence-activated cell sorter (FACS) was used to determine the fluorescence intensity associated with viable single cells and the frequency with which these cells appear in the total population. Hybridomas were generated which define both overlapping and non-overlapping retina cell populations. The cytotoxic activity of many of the monoclonal antibodies was determined on the FACS by propidium iodide exclusion, and the identity of various sub-populations was demonstrated by immunohistochemical staining of retina sections.

Monoclonal antibodies against a differentiated retinal cell population.

Two monoclonal antibodies have been isolated which bind preferentially to the plexiform layers of embryonic chick neural retina and to 50-60% of dissociated neural retinal cells in culture as determined by surface binding to cells followed by analysis in a fluorescent activated cell shorter. Each antibody appears to recognize a distinct antigenic determinant on a common cell surface antigen, a protein of approximately 230 kdalton. This antigen increases dramatically in concentration between embryonic days 7 and 11 concomitant with the elaboration of the retinal plexiform layers. The antigen appears first in the central portion of the neural retina and at later times in the periphery, an appearance consistent with the normal pattern of differentiation of the retina.

Differentiation of avian neural crest cells in vitro: absence of a developmental bias toward melanogenesis.

Neural crest cells from quail embryos grown in standard culture dishes differentiate almost entirely into melanocytes within 4 or 5 days when chick embryo extract (CEE) or occasional lots of fetal calf serum (FCS) are included in the medium. Gel fractionation showed that the pigment inducing factor(s) present in these media is of high molecular weight (greater than 400K daltons). In the absence of CEE, the neural tube can also stimulate melanocyte differentiation. Culture medium supplemented by selected lots of FCS permits crest cell proliferation but little overt differentiation after up to 2 weeks in culture if the neural tube is removed within 18 h of explantation in vitro. Subsequent addition of CEE to such cultures promotes complete melanocyte differentiation. Crest cells from White leghorn chick embryos also differentiate into melanocytes in the presence of CEE, but do not survive well in its absence. Melanocyte differentiation of crest cells from both quail and chick embryos can by suppressed by culturing under a dialysis membrane, even in the presence of the neural tube and CEE, but neuronal differentiation appears greatly enhanced.