Metadata-driven comparative analysis tool for sequences (meta-CATS): an automated process for identifying significant sequence variations that correlate with virus attributes.

The Virus Pathogen Resource (ViPR; www.viprbrc.org) and Influenza Research Database (IRD; www.fludb.org) have developed a metadata-driven Comparative Analysis Tool for Sequences (meta-CATS), which performs statistical comparative analyses of nucleotide and amino acid sequence data to identify correlations between sequence variations and virus attributes (metadata). Meta-CATS guides users through: selecting a set of nucleotide or protein sequences; dividing them into multiple groups based on any associated metadata attribute (e.g. isolation location, host species); performing a statistical test at each aligned position; and identifying all residues that significantly differ between the groups. As proofs of concept, we have used meta-CATS to identify sequence biomarkers associated with dengue viruses isolated from different hemispheres, and to identify variations in the NS1 protein that are unique to each of the 4 dengue serotypes. Meta-CATS is made freely available to virology researchers to identify genotype-phenotype correlations for development of improved vaccines, diagnostics, and therapeutics.

[Treatment of the acute lung abscess without sequestration].

The study aimed to assess the efficacy of the adopted principles of the treatment of lung abscess without sequestration on the example of 2397 patients. Treatment led to the complete recovery in 1731 (72,2%) patients. The 614 (25,6%) patients showed the chronization of the process and 52 (2,2%) died. The surgical treatment of lung abscess without sequestration (performed in cases of uneffective conservative treatment) led to the complete recovery on 45.7% more often and the chronization of the process was registered on 26.8% more seldom, whereas the lethality rate was higher than by conservative treatment on 8%.

Effects of pentobarbital on upper airway patency during sleep.

We hypothesised that pentobarbital would improve upper airway mechanics based on an increase in latency to arousal and amplitude of the phasic genioglossus electromyogram (EMG), and a decrease in the active upper airway critical closing pressure (P(crit)). 12 healthy subjects received pentobarbital (100 mg) or placebo in a double-blind, crossover protocol. During wakefulness, we measured the genioglossus reflex response to negative pressure pulses. During sleep, carbon dioxide was insufflated into the inspired air. Airway pressure was then decreased in a stepwise fashion until arousal from sleep. With basal breathing during sleep: flow rate was lower in volunteers given pentobarbital; end-tidal CO(2) concentration and upper airway resistance were greater; and P(crit) was unaffected (pentobarbital mean ± SD -11.7 ± 4.5 versus placebo -10.25 ± 3.6 cmH(2)O; p = 0.11). Pentobarbital increased the time to arousal (297 ± 63s versus 232 ± 67 s; p<0.05), at which time phasic genioglossus EMG was higher (6.2 ± 4.8% maximal versus 3.1 ± 3%; p<0.05) as were CO(2) levels. The increase in genioglossus EMG after CO(2) administration was greater after pentobarbital versus placebo. Pentobarbital did not affect the genioglossus negative-pressure reflex. Pentobarbital increases the time to arousal and stimulates genioglossus muscle activity, but it also increases upper airway resistance during sleep.

Neostigmine but not sugammadex impairs upper airway dilator muscle activity and breathing.

BACKGROUND: Cholinesterase inhibitor-based reversal agents, given in the absence of neuromuscular block, evoke a partial upper airway obstruction by decreasing skeletal upper airway muscle function. Sugammadex reverses neuromuscular block by encapsulating rocuronium. However, its effects on upper airway integrity and breathing are unknown. METHODS: Fifty-one adult male rats were anaesthetized with isoflurane, tracheostomized, and a femoral artery and vein were cannulated. First, we compared the efficacy of sugammadex 15 mg kg(-1) and neostigmine 0.06 mg kg(-1) to reverse respiratory effects of rocuronium-induced partial paralysis [train-of-four ratio (T4/T1)=0.5]. Subsequently, we compared the safety of sugammadex and neostigmine given after recovery of the T4/T1 to 1, by measuring phasic genioglossus activity and breathing. RESULTS: During partial paralysis (T4/T1=0.5), time to recovery of minute volume to baseline values was 10.9 (2), 75.8 (18), and 153 (54) s with sugammadex, neostigmine, and placebo, respectively (sugammadex was significantly faster than neostigmine and placebo, P<0.05). Recovery of T4/T1 was also faster for sugammadex than neostigmine and placebo. Neostigmine administration after complete recovery of T4/T1 decreased upper airway dilator muscle activity to 64 (30)% of baseline and decreased tidal volume (P<0.05 for both variables), whereas sugammadex had no effect on either variable. CONCLUSIONS: In contrast to neostigmine, which significantly impairs upper airway dilator muscle activity when given after recovery from neuromuscular block, a reversal dose of sugammadex given under the same conditions does not affect genioglossus muscle activity and normal breathing. Human studies will be required to evaluate the clinical relevance of our findings.

Contributions of residue pairing to beta-sheet formation: conservation and covariation of amino acid residue pairs on antiparallel beta-strands.

In an effort to better understand beta-sheet assembly, we have investigated the evolutionary behavior of neighboring residues on adjacent antiparallel beta-strands. Residue pairs were classified according to solvent exposure as well as by whether their backbone NH and C==O groups are hydrogen bonded. The conservation and covariation of 19,241 pairs in 219 sequence alignments was analyzed. Buried pairs were found to be the most conserved, while stronger covariation was detected in the solvent-exposed pairs. However, residues on neighboring strands showed a degree of conservation and covariation similar to that of well-separated residues on the same strand, suggesting that evolutionary pressure to maintain complementarity between pairs on neighboring strands is weak. Moreover, in spite of the preference of certain amino acid pairs to occupy neighboring positions on adjacent strands, such favored pairs are neither more strongly mutually conserved nor covary more strongly than pairs of the same type in non-interacting positions. Although the beta-sheet pairs did not show outstanding evolutionary coupling, in many protein families significant conservation and covariation patterns were detected for some of the residue pairs. Overall, the weak evolutionary conservation and covariation of the beta-sheet pairs indicates that sheet structure is unlikely to be dictated by specific side-chain interactions.

Agonist peptide from a cytotoxic t-lymphocyte epitope of human carcinoembryonic antigen stimulates production of tc1-type cytokines and increases tyrosine phosphorylation more efficiently than cognate peptide.

The identification of an agonist peptide (YLSGADLNL, designated CAP1-6D) to an immunodominant cytotoxic T-lymphocyte (CTL) epitope (designated CAP1) of human carcinoembryonic antigen (CEA) has previously been reported. The agonist peptide harbors a single amino acid substitution at a non-MHC anchor residue and is proposed to exert its effects at the level of the T-cell receptor (TCR). The type and magnitude of cytokines produced by CAP1-reactive CTL upon stimulation with the agonist peptide, CAP1-6D, were compared to those obtained upon stimulation with the cognate CAP1 peptide. In addition, early events in the TCR signaling pathway were examined for differences in tyrosine phosphorylation. Upon stimulation with the agonist peptide CAP1-6D, several different CEA-specific CTL lines exhibited a marked shift in the peptide dose response, which resulted in as much as a 1,000-fold increase in the levels of GM-CSF and gamma-IFN produced as compared with the use of the CAP1 peptide. However, levels of IL-4 and IL-10, which are associated with anti-inflammatory effects, were very low or non-existent. The cytokine profile of CAP1- and CAP1-6D-specific CTL is consistent with a Tc1-type CTL. Consistent with these findings, CEA-specific CTL showed increased tyrosine phosphorylation of TCR signaling proteins ZAP-70 and TCR zeta chains in response to both peptides. However, when CAP1-6D was compared with the wild-type peptide, the increase in ZAP-70 phosphorylation was greater than the increase in zeta phosphorylation. CTL generated with the CAP1-6D agonist were shown capable of lysis of human carcinoma cells expressing native CEA. The ability to upregulate the production of GM-CSF, gamma-IFN, TNFalpha and IL-2 with the agonist peptide, as compared with CAP1, may help in initiating or sustaining anti-tumor immune responses and thus potentially prove to be useful in the treatment of CEA-positive tumors.

Strategies in the development of recombinant vaccines for colon cancer.

A new era involving the evaluation of recombinant vaccines for colon cancer has begun with the concurrent emergence of insights and technologies in the fields of molecular biology and immunology. These advances include (I) the identification and cloning of an array of genes associated with the neoplastic process, such as oncogenes, suppressor genes, genes encoding oncofetal antigens, and tissue lineage determinants; (2) the development of a variety of viral and bacterial vectors to deliver and present gene products; (3) the identification of numerous T-cell costimulatory molecules and the knowledge of their mode of action; (4) the cloning and analysis of the modes of action of an array of cytokines and other immunomodulatory molecules; and (5) a more sophisticated knowledge of the mode(s) of antigen presentation and T-cell activation.

Context-dependence of amino acid residue pairing in antiparallel beta-sheets.

In an effort to understand the driving forces behind antiparallel beta-sheet assembly, we have investigated the mutational tolerance of four pairs of residues in CspA, the major cold shock protein of E. coli. Two buried pairs and two exposed pairs of neighboring amino acids were separately randomized and the corresponding effects on protein stability were assessed using a protein expression screen. The thermal denaturation of a subset of the recovered proteins was measured by circular dichroism spectroscopy in order to determine the range of stabilities sampled by the expressed mutants. As anticipated, buried sites are substantially less tolerant of substitutions than exposed sites with more than half of the exposed residue combinations giving rise to stably folded proteins. The two exposed residue pairs, however, display different degrees of tolerance to substitution and accept different residue pair combinations. Except for the prohibition of proline from interior strand positions, no obvious correlations of mutant stability with any single parameter such as beta-sheet propensity or hydrophobicity can be detected. Mutant combinations recovered in both orientations (e.g. XY and YX) at a given exposed pair site often show markedly different stabilities, indicating that the local environment plays a substantial role in modulating the pairing preferences of residues in beta-sheets.

Generation of human cytolytic T lymphocyte lines directed against prostate-specific antigen (PSA) employing a PSA oligoepitope peptide.

Prostate-specific Ag (PSA), which is expressed in a majority of prostate cancers, is a potential target for specific immunotherapy. Previous studies have shown that two 10-mer PSA peptides (designated PSA-1 and PSA-3) selected to conform to human HLA class I-A2 motifs can elicit CTL responses in vitro. A longer PSA peptide (30-mer) designated PSA-OP (oligoepitope peptide), which contains both the PSA-1 and PSA-3 HLA-A2 epitopes and an additional potential CTL epitope (designated PSA-9) for the HLA-class I-A3 allele, was investigated for the ability to induce cytotoxic T cell activity. T cell lines from different HLA-A2 and HLA-A3 donors were established by in vitro stimulation with PSA-OP; the CTL lines lysed PSA-OP as well as PSA-1- or PSA-3-pulsed C1R-A2 cells, and PSA-OP and PSA-9-pulsed C1R-A3 cells, respectively. The CTL lines derived from the PSA-OP peptide also lysed PSA-positive prostate cancer cells. PSA-OP-derived T cell lines also lysed recombinant vaccinia-PSA-infected targets but not targets infected with wild-type vaccinia. PSA-OP did not bind HLA-A2 and HLA-A3 molecules. The decrease in cytotoxicity in the presence of protease inhibitors suggests that the PSA-OP is cleaved into shorter peptides, which in turn can interact with HLA-class I molecules and, as a consequence, induce CTL-mediated lysis. We have also demonstrated that it is possible to induce CTL responses in HLA-A2.1/Kb transgenic mice by immunization with PSA-OP with adjuvant. These studies thus provide evidence that oligopeptides such as PSA-OP may be useful candidates for peptide-based cancer vaccines.

Olanzapine compared with chlorpromazine in treatment-resistant schizophrenia.

OBJECTIVE: The purpose of this study was to compare the efficacy of olanzapine with that of chlorpromazine plus benztropine in patients with treatment-resistant schizophrenia. METHOD: One hundred three previously treatment-resistant patients with schizophrenia diagnosed according to the DSM-III-R criteria were given a prospective 6-week trial of 10-40 mg/day of haloperidol. Eighty-four of them failed to respond to that trial and agreed to be randomly assigned to an 8-week fixed-dose trial of either 25 mg/day of olanzapine alone or 1200 mg/day of chlorpromazine plus 4 mg/day of benztropine mesylate. RESULTS: Fifty-nine (70%) of the 84 subjects completed the trial. The primary outcome measures were Brief Psychiatric Rating Scale total score and positive symptom score, Scale for the Assessment of Negative Symptoms global score, and Clinical Global Impression score. An analysis of variance for the subjects who completed the study showed no difference in efficacy between the two drugs. Seven percent of the olanzapine-treated patients responded according to a priori criteria; no chlorpromazine-treated patients responded. The olanzapine-treated patients had fewer motor and cardiovascular side effects than the chlorpromazine-treated patients. Extrapyramidal symptoms and akathisia were similar in the two groups, although no antiparkinsonian drugs were used in the olanzapine group. CONCLUSIONS: Olanzapine and chlorpromazine showed similar efficacy, and the total amount of improvement with either drug was modest. Olanzapine-treated patients had fewer side effects than chlorpromazine-treated patients.

Cancer vaccine development.

A new era involving the evaluation of recombinant cancer vaccines has begun with the concurrent emergence of insights and technologies in the fields of molecular biology and immunology. These advances include: The identification and cloning of an array of genes associated with the neoplastic process, such as oncogenes, suppressor genes, genes encoding oncofoetal antigens and tissue-lineage determinants. The development of a variety of viral and bacterial vectors to deliver and present gene products. The identification of numerous T-cell costimulatory molecules and an understanding of their mode of action. The cloning and analysis of the modes of action of an array of cytokines and other immunomodulatory molecules. More sophisticated knowledge of the mode(s) of antigen presentation and T-cell activation. One current challenge in cancer therapy is the delineation of strategies toward the rational design and implementation of recombinant vaccines that will be of therapeutic benefit to cancer patients and/or members of groups at high risk for specific neoplasias. Numerous concepts are emerging in this regard. The study of immunologic intervention using laboratory animal models demonstrates that no one approach will prevail for all cancer types or, perhaps, for the various stages of the neoplastic process of a given tumour type. The immunological role(s) of CD8+, CD4+, natural killer and other cell types, as well as the roles of antibodies, must all be taken into consideration. This article reviews some of the strategies currently undergoing evaluation toward the development of recombinant vaccines for several carcinoma types.

Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen.

A vaccination strategy designed to enhance the immunogenicity of self-antigens that are overexpressed in tumor cells is to identify and slightly modify immunodominant epitopes that elicit T-cell responses. The resultant T cells, however, must maintain their ability to recognize the native configuration of the peptide-MHC interaction on the tumor cell target. We used a strategy to enhance the immunogenicity of a human CTL epitope directed against a human self-antigen, which involved the modification of individual amino acid residues predicted to interact with the T-cell receptor; this strategy, moreover, required no prior knowledge of these actual specific interactions. Single amino acid substitutions were introduced to the CAP1 peptide (YLSGANLNL), an immunogenic HLA-A2+-binding peptide derived from human carcinoembryonic antigen (CEA). In this study, four amino acid residues that were predicted to potentially interact with the T-cell receptor of CAP1-specific CTLs were systematically replaced. Analogues were tested for binding to HLA-A2 and for recognition by an established CTL line directed against CAP1. This line was obtained from peripheral blood mononuclear cells from an HLA-A2+ individual vaccinated with a vaccinia-CEA recombinant. An analogue peptide was identified that was capable of sensitizing CAP1-specific CTLs 10(2)-10(3) times more efficiently than the native CAP1 peptide. This enhanced recognition was shown not to be due to better binding to HLA-A2. Therefore, the analogue CAP1-6D (YLSGADLNL, Asn at position 6 replaced by Asp) meets the criteria of a CTL enhancer agonist peptide. Both the CAP1-6D and the native CAP1 peptide were compared for the ability to generate specific CTL lines in vitro from unimmunized apparently healthy HLA-A2+ donors. Whereas CAP1 failed to generate CTLs from normal peripheral blood mononuclear cells, the agonist peptide was able to generate CD8+ CTL lines that recognized both the agonist and the native CAP1 sequence. Most importantly, these CTLs were capable of lysing human tumor cells endogenously expressing CEA. The use of enhancer agonist CTL peptides may thus represent a new efficient direction for immunotherapy protocols.

In vitro generation of human cytotoxic T lymphocytes specific for peptides derived from prostate-specific antigen.

BACKGROUND: Protein antigens are presented to cytotoxic T lymphocytes as small peptides (approximately 9-10 amino acids long) bound to class I molecules of the major histocompatibility complex. The identification of tumor-associated antigens and specific peptide epitopes (i.e., antigenic determinants) may be useful in the development of anticancer vaccines. The generation of a cytotoxic T-cell response to one peptide epitope (amino acids 146-154) of human prostate-specific antigen (PSA) has been reported. PURPOSE: Our aim was to identify novel PSA peptides capable of eliciting specific cytotoxic T-cell responses. METHODS: Candidate peptides were identified on the basis of the following criteria: 1) they contained consensus amino acid motifs for binding to HLA-A2, which is the most common type of class I molecule; 2) they lacked strong homology with PSA-related kallikrein proteins; and 3) they were capable of stabilizing HLA-A2 class I molecules on the surface of human T2 (transport deletion mutant) cells, which are defective in antigen presentation. T-cell lines capable of killing (i.e., lysing) T2 target cells that had been pulsed with specific PSA peptides were generated from three different males (two disease-free individuals and one patient with prostate cancer) by incubating peripheral blood mononuclear cells with the peptides and interleukin 2. Specific cell lysis was monitored by the release of radioactivity from target cells that had been labeled with [111In]oxyquinoline. RESULTS: Two novel PSA peptides capable of eliciting cytotoxic T-cell responses were identified; these peptides were designated PSA-1 (amino acids 41-150) and PSA-3 (amino acids 154-163). Four different cytotoxic T-cell lines were generated in response to these peptides-three against PSA-3 and one against PSA-1. Specific lysis of peptide-pulsed T2 cells by the T-cell lines was blocked by the addition of a monoclonal antibody directed against class I molecules. The T-cell lines were also capable of lysing PSA-positive, HLA-A2-positive LNCaP cells (human prostate carcinoma cells). The specificity of LNCaP cell lysis was shown by the following: 1) the inability of added human K562 (chronic myelogenous leukemia) cells to inhibit it, 2) the ability of added anti-HLA-A2 antibodies to block it, and 3) the inability of the T-cell lines to induce substantial lysis of PSA-negative, HLA-A2-positive human cancer cells. IMPLICATIONS: Our studies form a rational basis for the use of PSA peptides or recombinant vectors encoding PSA in the development of anticancer vaccine immunotherapy protocols for patients with prostate cancer.

Phenotypic stability of a cytotoxic T-cell line directed against an immunodominant epitope of human carcinoembryonic antigen.

CTL lines have now been generated against defined peptides of a range of human tumor-associated antigens (TAAs). One of the potential uses of these epitope-specific CTLs is in adoptive transfer immunotherapy. This is a modality, however, that will require long-term in vitro culture of CTLs. To date, little has been reported concerning the phenotypic stability of human epitope-specific CTLs as a consequence of long-term in vitro propagation via peptide stimulation. We report here the serial phenotypic characterization of a CTL line directed against an immunodominant epitope (YLSGANLNL, designated CAP-1) of human carcinoembryonic antigen (CEA). This CTL line was derived from peripheral blood mononuclear cells of a patient with metastatic carcinoma who had been treated with a recombinant CEA-vaccinia vaccine in a Phase I trial; the CTLs were analyzed through 20 in vitro cycle passages of stimulation with CAP-1 peptide and interleukin 2 in the presence of autologous antigen-presenting cells. The CTL line was shown to be phenotypically stable in terms of high levels of cytokine (IFN-gamma, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor) production, expression of homing-adhesion molecules, ability to lyse peptide-pulsed targets, and ability to lyse human carcinoma cells endogenously expressing CEA in a MHC-restricted manner. Vbeta T-cell receptor gene usage was also analyzed. These studies thus present a rationale for the use of long-term cultured epitope-specific human CTLs, directed against a human TAA for potential adoptive transfer immunotherapy protocols.

Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4+ and CD8+ T cell responses.

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras and, thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here, we examined the capacity of a mutant K-ras 9-mer peptide to induce in vivo CD8+ cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4-12 of the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9-mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2Kd; specific binding to MHC class I may then create an immunogenic complex for the induction of anti-ras CD8+ CTL; and finally, the mutant sequence overlapped with a newly characterized anti-ras CD4+ T helper type 1 epitope, which may have implications for the coordination and activation of both anti-ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H-2Kd was demonstrated with the mutant ras4-12(V12) peptide, but not the normal ras4-12(G12) peptide, which specifically inhibited an H-2Kd-restricted, anti-nucleoprotein NP147-155 CTL response in a dose-dependent fashion. An anti-ras CD8+ T cell line was then established from immune splenocytes of BALB/c (H-2d) mice injected with ras4-12 (V12) in adjuvant, which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2Kd and strongly specific for the mutant ras peptide. Importantly, these anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene, suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12(Gly-->Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule; the ability of the mutant, but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C-terminal anchor residue; and the ras4-12(V12) peptide was immunogenic for the production of antigen-specific CD8+ CTL, which lysed in vitro a syngeneic tumor cell line harboring the mutant K-ras oncogene.

Peptide-specific activation of cytolytic CD4+ T lymphocytes against tumor cells bearing mutated epitopes of K-ras p21.

Alterations in the ras p21 protein have been associated with both rodent and human neoplasia. Thus, mutated ras p21 proteins may bear unique antigenic epitopes for immune recognition, such as by T cells, which have been implicated in host antitumor activity. Synthetic peptides that mimic segments of mutated ras p21 have been reported to be immunogenic in mice in vivo, although detailed functional analyses remains undefined. Here, in a murine model, we explored and characterized distinct effector properties of host-derived T lymphocytes reactive to mutated ras peptides, which was consistent with the CD4+ T helper type 1 (Th1) subset. BALB/c mice (H-2d) were immunized with a purified peptide, 13 amino acids in length, containing the substitution of Gly (G12) to Val (V12) at position 12, which is commonly found in human carcinomas. An alpha beta T cell receptor-positive, CD3+, CD4+, CD8- T cell line was established, which expressed peptide-specific proliferation. Cytokine assays revealed the production of interleukin-2, interferon-gamma, tumor necrosis factor and granulocyte-macrophage colony-stimulating factor. Moreover, antigen-specific cytotoxicity was demonstrable against: (1) Iad-bearing A20 tumor cells incubated with exogenously bound V12 peptide; and (2) A20 tumor cells transduced with the K-ras p21 oncogene encoding the corresponding point mutation. CD4(+)-mediated cytotoxicity was major histocompatibility complex (MHC) class II-restricted, as revealed by the absence of lysis against MHC class II- P815 targets, inhibition of A20 lysis with anti-Iad monoclonal antibodies, and induction of lysis against L cell targets transfected with E alpha A beta d. Independent isolation of a second CD4+ V12 line revealed a very similar cytolytic and MHC class II-restricted profile. Overall, these data demonstrated that peptide immunization produced a CD4+ Th1 response that specifically recognized tumor cells expressing endogenous activated K-ras epitopes, which may have implications for the development of peptide-based active immunotherapies.

Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine.

BACKGROUND: The human carcinoembryonic antigen (CEA), which is expressed in several cancer types, is a potential target for specific immunotherapy using recombinant vaccines. Previous studies have shown that when the CEA gene is placed into vaccinia virus, the recombinant vaccine (rV-CEA) can elicit T-cell responses in both rodents and non-human primates. PURPOSE: Our objective was to determine if rVCEA could elicit CEA-specific T-cell responses in humans with appropriate human leukocyte antigen (HLA) motifs. METHODS: Peripheral blood lymphocytes (PBLs) obtained from patients with metastatic carcinoma, both before and after vaccination with rV-CEA, were analyzed for T-cell response to specific 9- to 11-mer CEA peptides selected to conform to human HLA class I-A2 motifs. RESULTS: While little or no T-cell growth was seen from preimmunization PBLs of patients pulsed with CEA peptides and interleukin 2 (IL-2), T-cell lines were obtained from PBLs of patients after vaccination with one to three cycles of stimulation. Cytolytic T-cell lines from three HLA-A2 patients were established with a 9-amino acid peptide (CAP-1), and the CD8+/CD4+ double-positive T-cell line (V24T) was chosen for detailed analysis. When autologous Epstein-Barr virus (EBV)-transformed B cells were either incubated with CAP-1 peptide or transduced with the CEA gene using a retroviral vector, they were lysed by the V24T cell line, but allogeneic non-A2 EBV-transformed B cells were not. The SW403 human colon carcinoma cell line, which is CEA positive and HLA-A2 positive, was also lysed by the V24T cell line, while two non-HLA-A2 CEA-positive colon carcinoma cell lines were not. To further confirm the class I HLA-A2 restricted nature of the V24T cytotoxicity, the non-HLA-A2 SW837 CEA-expressing colon carcinoma cell line was infected with a recombinant vaccinia virus expressing the HLA class I-A2 gene, and it became susceptible to V24T lysis. Cells infected with vector alone were not lysed. CONCLUSIONS: This study demonstrates for the first time (a) the ability to generate a human cytolytic T-cell response to specific epitopes of CEA, (b) the class I HLA-A2 restricted nature of the T-cell mediated lysis, and (c) the ability of human tumor cells to endogenously process CEA to present a specific CEA peptide in the context of major histocompatibility complex for T-cell-mediated lysis. IMPLICATIONS: These findings have implications in the development of specific second-generation cancer immunotherapy protocols.

[The treatment of posttraumatic hemothorax]. / Lechenie posttravmaticheskogo gemotoraksa.

A high procoagulative activity of monocytes, a low procoagulative activity of pulmonary macrophages, and decreased proteolytic activity of leukocytes and monocytes are predominant in patients with coagulated hemothorax. The proteolytic activity of leukocytes of the pleural exudate was higher in these patients than in their blood leukocytes and lower than that of blood leukocytes from healthy donors. Differential correction of phagocytic functional activity in the pleural exudate in terms of the type of hemothorax, its stage, procoagulative and proteolytic activities of phagocytes improve the outcome of the disease.

Biologic properties of a CH2 domain-deleted recombinant immunoglobulin.

Monoclonal antibody (MAb) B72.3 reacts with TAG-72, a high-molecular-weight mucin expressed on several types of human carcinoma, and is currently being used in clinical trials for the diagnosis and therapy of human carcinoma. An expression construct containing cDNA encoding an immunoglobulin (Ig) heavy chain, with the variable region of murine MAb B72.3 and a human Ig constant region with a deletion of the CH2 domain, was generated. Immunoglobulin from the transfectoma with the highest expression of the TAG-72 immunoreactive antibody was designated MAb chimeric (c) B72.3 delta CH2. The pharmacokinetics of serum clearance of iodine-labeled MAbs cB72.3 delta CH2 and the intact cB72.3 were compared in athymic mice. By 24 hr, less than 1% of the cB72.3 delta CH2 was left in the plasma, while 36% of the cB72.3 still remained. The T1/2 alpha values of the cB72.3 delta CH2 and cB72.3 MAbs were 1.7 and 2.4 hr, respectively. The T1/2 beta values were 7.8 hr for the domain-deleted cMAb and 48.9 hr for cB72.3. Biodistribution studies in athymic mice bearing LS-174T xenografts showed a reduction in the percentage of injected dose per gram in tumor with 131I-cB72.3 delta CH2; however, the 131I-cB72.3 delta CH2 both localized to tumors faster and cleared from the blood faster than the 125I-cB72.3 MAb. Only trace amounts of the 131I-cB72.3 delta CH2 were detected in normal tissues, including kidney. The faster clearance rate, more rapid tumor targeting and lack of metabolic uptake in normal tissues demonstrated with the iodine-labeled CH2 domain-deleted cMAb may be an advantage for certain clinical protocols.