RESUMO
The factors affecting the in vitro lysis of PARA-7 tumor cells mediated by antibody and complement were examined with the use of the 51Cr-release assay. The addition of immune syngeneic hamster antisera (HA) to 51Cr-labeled PARA-7 target cells 30 minutes before the addition of guinea pig complement (GPC) failed to produce significant levels of lysis. Similarly, the addition of rabbit anti-PARA-7 serum (RAS) 30 minutes prior to the addition of GPC resulted in low levels of lysis. Longer incubations in the presence of RAS, but not RAS and GPC, increased levels of lysis. Extended incubation in the presence of RAS increased the amount of antibody bound to the target cells. This increase did not appear to be due to the expression of additional antigen, because the increase occurred at 4 degrees C and was not sensitive to inhibitors of protein synthesis. The inability to obtain increased levels of lysis by extended incubation in the presence of both RAS and GPC appeared to be due to the inhibitory effects of GPC on the interaction of antibody and antigen. Similar inhibitory effects could be produced by inactivated GPC and various other substances. When extended incubations in the presence of antiserum were used for the reexamination of the cytolytic activity of syngeneic immune HA, significant lysis was detectable.
Assuntos
Anticorpos Antineoplásicos , Proteínas do Sistema Complemento , Testes Imunológicos de Citotoxicidade , Neoplasias Experimentais/imunologia , Antígenos de Neoplasias , Linhagem Celular , Fatores de TempoRESUMO
Spleen cells from syngeneic tumor-bearing mice were examined for direct cell-mediated cytotoxicity (DCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC). In the DCMC assay specific cytotoxicity against the homologous tumor cell was assessed. In the LDCC assay cytotoxicity was nonspecifically assessed against EL-4 cells in the presence of concanavalin A or phytohemagglutinin. Most tumor lines tested (19/22) produced no cytotoxic reactivity in either the DCMC or LDCC assays. In the case of the remaining tumor lines (EL-4, BW5147-3, and P815 Y-3), significant LDCC, but not DCMC, was detected, which indicated that although cytotoxic effector cells had been activated, the reactivity was not directed toward the homologous tumor cell or could not be expressed in the DCMC assay. The EL-4 and BW5147-3 cell lines proved to be sporadic in terms of their ability to induce LDCC, whereas the P815 Y-3 cell line produced consistent LDCC. Reactivity induced by P815 Y-3 cells appeared to be due to the constitutive production and release of a soluble component which could activate cytotoxic T-cells in vivo.
Assuntos
Concanavalina A , Citotoxicidade Imunológica , Neoplasias Experimentais/imunologia , Fito-Hemaglutininas , Animais , Linhagem Celular , Feminino , Leucemia Experimental/imunologia , Ativação Linfocitária , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Transplante de Neoplasias , Especificidade da Espécie , Transplante IsogênicoRESUMO
The association of glutathione (GSH) with putative external chemoreceptors elicits feeding behavior in Hydra. In the present study, solubilized membrane proteins were chromatographed on an affinity column of immobilized GSH in order to isolate GSH-binding proteins that may represent the Hydra GSH chemoreceptor. The most abundant of the affinity-purified proteins was a triplet of peptides ranging in molecular weight from 24.5-26 kDa. Antiserum generated against the 24.5-26 kDa triplet peptides inhibited GSH-stimulated feeding behavior by 47%, implicating a role for one or more of these peptides in Hydra chemoreception.
Assuntos
Proteínas de Transporte/isolamento & purificação , Células Quimiorreceptoras/metabolismo , Glutationa/metabolismo , Hydra/química , Animais , Membrana Celular/química , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Comportamento Alimentar/efeitos dos fármacos , Glutationa/farmacologia , ImmunoblottingRESUMO
Lymphoid preparations from nonimmune rainbow and brook trout were found to lyse murine tumor cells (EL-4 & P815Y) in vitro in an 18 hr 51Cr-release assay conducted at 16-18 degrees C. Lysis was proportional to the effector: target cell ratio, required direct cell to cell contact, and was not depleted by the removal of nylon wool adherent cells. Lymphoid populations from peripheral blood, the thymus, and the anterior kidney, but not the spleen, were active in the cytotoxicity assay. Individual fish varied considerably in their ability to lyse one or both target cells. These data and the results of unlabelled target cell inhibition studies suggest that the reaction is selective if not specific. The addition of PHA to the reaction mixture resulted in markedly enhanced cytotoxic reactivity. In the presence of PHA lysis was readily detectable at 4 hr. The data demonstrate that nonimmune Salmonids possess a cytolytic effector cell population which has considerable cytotoxic potential and may represent a heterogeneous "natural killer cell" population.
Assuntos
Citotoxicidade Imunológica , Linfócitos/imunologia , Salmonidae/imunologia , Truta/imunologia , Animais , Adesão Celular , Técnicas In Vitro , Células Matadoras Naturais/imunologia , Lectinas/imunologia , Leucemia Experimental/imunologia , Sarcoma de Mastócitos/imunologia , CamundongosRESUMO
Salmonella typhimurium SR-11 is extremely virulent at a dose as low as 10(5) colony forming units (cfu) when administered perorally to BALB/c mice. Utilizing mini-transposon mutagenesis, a mutant of S. typhimurium SR-11 was isolated that was unable to utilize oleate and citrate as carbon sources. This mutant, designated S. typhimurium SR-11 Fad- (Fatty acid), was found to utilize sugars under cya/crp control as sole carbon sources, suggesting that the mutation is not in either of these genes. In addition, SR-11 Fad- utilized pyruvate and succinate, but was unable to utilize either acetate or isocitrate as sole carbon source. In contrast to SR-11, SR-11 Fad- was found to be avirulent, i.e. BALB/c mice were completely healthy after oral infection with 10(9) S. typhimurium SR-11 Fad- cells. Moreover, 21 days after SR-11 Fad- infection, BALB/c mice were found to be protected against an oral challenge with 10(9) cells of S. typhimurium SR-11.
Assuntos
Vacinas Bacterianas/imunologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Animais , Ácido Cítrico/metabolismo , Elementos de DNA Transponíveis/genética , Ácidos Graxos/metabolismo , Fezes/microbiologia , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Insercional , Salmonelose Animal/prevenção & controle , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Baço/microbiologia , Vacinação , VirulênciaRESUMO
Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18Col-, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice alone but is eliminated when fed together with E. coli F-18. Recently we randomly cloned E. coli F-18 DNA into E. coli F-18Col- and let the mouse intestine select the best colonizer. In this way, we isolated a 6.5-kb E. coli F-18 DNA sequence that simultaneously stimulated synthesis of type 1 fimbriae and enhanced E. coli F-18Col- colonizing ability. In the present investigation we show that the gene responsible for stimulation of type 1 fimbriae synthesis appears to be leuX, which encodes a tRNA specific for the rare leucine codon UUG. Moreover, it appears that expression of leuX may be regulated by two proteins (22 kDa and 26 kDa) encoded by genes immediately adjacent to leuX.
Assuntos
Escherichia coli/genética , Fímbrias Bacterianas/metabolismo , RNA de Transferência/genética , Aglutinação , Aderência Bacteriana , Códon , Colicinas/genética , Escherichia coli/patogenicidade , Manose/metabolismo , Mapeamento por Restrição , Virulência/genéticaAssuntos
Ciclofosfamida/uso terapêutico , Resistência a Medicamentos , Neoplasias Experimentais/tratamento farmacológico , Adenoviridae , Animais , Biópsia , Células Cultivadas , Cricetinae , Ciclofosfamida/administração & dosagem , Rejeição de Enxerto , Vírus Auxiliares , Transplante de Neoplasias , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/imunologia , Transplante Homólogo , Infecções Tumorais por VírusRESUMO
Mice were challenged with high (10(8)) or low (10(4)) numbers of allogenic tumor cells and assessed for cellular immunity. The responses obtained indicated that high dose challenge produced both delayed-type hypersensitivity (DTH) and cell-mediated cytotoxic reactivity (DCMC), while low dose challenge produced DTH, an apparent suppressive effect, and little or no DCMC. Pretreatment with 100 mg/kg of cyclophosphamide (CTX) 3 d before antigen failed to alter this pattern, but treatment 3 d after antigen administration abrogated both DTH and DCMC. Animals given a combined modulating protocol consisting of an initial low dose challenge followed on day 3 by CTX treatment and day 6 by a high dose challenge developed DCMC in the presence of a greatly reduced or absent DTH response. These results demonstrate the differential effects of allogeneic challenge dose on the development of cellular immunity; the differential effects of CTX treatment given prior to or following alloimmunization, and demonstrate how these effects can be combined to modulate the immune response by selectively activating subpopulations of T-lymphocytes.
Assuntos
Ciclofosfamida/farmacologia , Citotoxicidade Imunológica , Hipersensibilidade Tardia/imunologia , Animais , Relação Dose-Resposta Imunológica , Imunidade Celular , Sarcoma de Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Overall, the risk assessment data have shown not only that recombinant DNA research using E. coli strains is safe but also that E. coli strains, in general, including K-12 strains, can colonize the mammalian colon in individuals undergoing antibiotic treatment. However, very little is known about how E. coli colonizes the mammalian colon and it is possible that different strains use different adhesins and different colonic receptors in the process. Through the approach described here it has been possible to begin to identify both E. coli adhesins and colonic receptors which may play an important role in the colonization process. Hopefully, continued research into the molecular basis of E. coli colonic colonization will lead to the development of healthy E. coli strains for recombinant DNA research which are unable to colonize the human colon under any circumstance.
Assuntos
Colo/microbiologia , Contenção de Riscos Biológicos , Escherichia coli/crescimento & desenvolvimento , Adesividade , Animais , Fímbrias Bacterianas/fisiologia , Humanos , Lipopolissacarídeos/fisiologia , CamundongosRESUMO
Escherichia coli F-18 Col- was previously shown to be a poor colonizer of the streptomycin-treated mouse large intestine, relative to its parent, E. coli F-18. Prior to attempting to clone genes responsible for the colonization phenotype of E. coli F-18 into E. coli F-18 Col-, a suitable cloning vector had to be found. In this investigation, we report that the commonly used cloning vectors pBR322, pHC79, and pBR329 all segregate from E. coli F-18 Col- both when grown in L broth under conditions of nonselection (i.e., in vitro) and when fed to streptomycin-treated mice (i.e., in vivo). Insertion of the cer region (which promotes resolution of replicating plasmids into monomeric forms) into pHC79 stabilized this plasmid in E. coli F-18 Col- in vitro and in vivo. In contrast, two independent cer insertions into pBR329 did not stabilize the plasmid completely in E. coli F-18 Col- in vitro, and feeding the strain to streptomycin-treated mice resulted in rapid segregation of the plasmids in vivo. Also, stability of all three plasmids in E. coli F-18 Col- in vitro was achieved by insertion of the parB region of plasmid R1, which encodes a cell-killing protein, Hok, that is active only postsegregationally. However, as with cer, complete in vitro and in vivo stabilization was achieved only in parB constructs of pBR322 and pHC79.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Vetores Genéticos , Intestino Grosso/microbiologia , Plasmídeos , Animais , Cosmídeos , Escherichia coli/patogenicidade , Fezes/microbiologia , Camundongos , Estreptomicina/farmacologiaRESUMO
Escherichia coli F-18, isolated from the feces of a healthy human in 1977, is an excellent colonizer of the streptomycin-treated mouse large intestine and displays normal motility and chemotaxis ability. A chemotaxis-defective derivative of E. coli F-18, E, coli F-18 CheA-, and a nonflagellated derivative, E. coli F-18 Fla-, were constructed. These strains were found to colonize the streptomycin-treated mouse large intestine as well as E. coli F-18 when mice were fed both E. coli F-18 and either the CheA- or Fla- derivative at high levels (10(10) CFU of each strain per mouse) or low levels (10(4) CFU of each strain per mouse). Furthermore, E. coli F-18 lost motility and chemotaxis ability when grown in colonic or cecal mucus in vitro despite retaining the ability to synthesize flagella. Thus, it appears that neither motility nor chemotaxis plays a role in the ability of E. coli F-18 to colonize because this strain becomes functionally nonmotile upon growth in the streptomycin-treated mouse large intestine.
Assuntos
Movimento Celular , Quimiotaxia , Escherichia coli/fisiologia , Intestino Grosso/microbiologia , Estreptomicina/farmacologia , Animais , Contagem de Colônia Microbiana , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura , Fezes/microbiologia , Motilidade Gastrointestinal , Humanos , Intestino Grosso/efeitos dos fármacos , Masculino , Camundongos , Camundongos EndogâmicosRESUMO
The heat shock response of Borrelia burgdorferi B31 cells was characterized with regard to the heat shock proteins (Hsps) produced. Five to seven Hsps were detected by sodium dodecyl sulfate-gel electrophoresis and fluorography of proteins from cells labeled with [35S]methionine after shifts from 33 degrees C to 37 or 40 degrees C or from 20 degrees C to 33, 37, or 40 degrees C. Analysis of [35S]methionine-labeled Hsps by two-dimensional electrophoresis and autoradiography revealed 12 Hsps. Western immunoblot analysis with antisera to highly conserved Escherichia coli and Mycobacterium tuberculosis Hsps revealed a single 72-kilodalton (kDa) protein band that reacted with antibodies to E. coli DnaK and with antibodies to the M. tuberculosis 71-kDa Hsp homolog of E. coli DnaK. Two proteins with apparent molecular masses of 66 and 60 kDa reacted with antibodies against the M. tuberculosis 65-kDa Hsp homolog of E. coli GroEL. Human immune sera collected from patients with Lyme disease reacted with both the 66-kDa Hsp and the 60-kDa Hsp but failed to react with the 72-kDa Hsp. These data are discussed with regard to the possibility that host recognition of highly conserved epitopes of GroEL homologs of B. burgdorferi may result in autoimmune reactions causing arthritis and other pathologies.
Assuntos
Proteínas de Bactérias/biossíntese , Grupo Borrelia Burgdorferi/metabolismo , Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico/biossíntese , Proteínas de Bactérias/imunologia , Grupo Borrelia Burgdorferi/imunologia , Reações Cruzadas , Eletroforese em Gel Bidimensional , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/imunologia , Temperatura Alta , Humanos , Soros Imunes , Peso Molecular , Testes de PrecipitinaRESUMO
Escherichia coli F-18, a normal fecal isolate, was previously shown to be an excellent colonizer of the streptomycin-treated CD-1 mouse large intestine, whereas E. coli F-18col-, a derivative of E. coli F-18 that no longer makes the E. coli F-18 colicin, was shown to be a poor mouse colonizer. It was also shown that E. coli F-18 bound two to three times more soluble colonic mucus protein than did E. coli F-18col- and that a major receptor in CD-1 mouse colonic mucus was a 50.5-kilodalton glycoprotein. In the present investigation, an additional E. coli F-18 colonic mucus glycoprotein receptor (66 kilodaltons) and three cecal mucus glycoprotein receptors (94, 73, and 66 kilodaltons) were identified. Numerous colonic and cecal brush border protein receptors specific for E. coli F-18 were also identified. Furthermore, E. coli F-18col- was found to bind to the same mucus and brush border receptors as E. coli F-18, although to a far lesser extent. Adhesion of both E. coli F-18 and F-18col- was inhibited by D-mannose and alpha-methyl-D-mannoside, and both strains were shown to bind specifically to the mannose moiety of a mannose-bovine serum albumin glycoconjugate, although again E. coli F-18col- bound to a lesser extent. Finally, both E. coli F-18 and F-18col- were shown to be piliated. The possible role of pilus mediated adhesion in E. coli F-18 colonization of the streptomycin-treated mouse large intestine is discussed.
Assuntos
Aderência Bacteriana , Escherichia coli/genética , Mucosa Intestinal/microbiologia , Intestino Grosso/microbiologia , Receptores de Superfície Celular/fisiologia , Animais , Escherichia coli/citologia , Glicoconjugados/fisiologia , Manosídeos/metabolismo , Camundongos , Microscopia Eletrônica , Microvilosidades/fisiologia , Peso Molecular , Muco/microbiologiaRESUMO
The relative colonizing abilities of Escherichia coli F-18, isolated from the feces of a healthy human, and E. coli F-18col-, a strain derived from it which does not make the E. coli F-18 colicin, were studied. In a previous report, it was shown that when each strain was fed individually to streptomycin-treated mice, at approximately 10(10) CFU per mouse, each colonized the large intestine at between 10(7) and 10(8) CFU/g of feces indefinitely. However, when simultaneously fed to mice, although E. coli F-18 colonized at about 10(8) CFU/g of feces, E. coli F-18col- dropped to a level of 10(3) CFU/g of feces within 3 to 5 days. In the present investigation, we show that when given enough time to establish a state of colonization, E. coli F-18col- persists in feces in high numbers despite subsequent challenge by E. coli F-18. Therefore, a major defect in the ability of E. coli F-18col- to colonize in the presence of E. coli F-18 appears to be in initiating that state. In addition, when mucus was scraped from the cecal wall and, without further treatment, was inoculated with E. coli F-18 or F-18col-, both strains grew well. However, when cecal mucus was inoculated with both strains simultaneously, E. coli F-18 grew far more rapidly than E. coli F-18col-. Moreover, neither strain grew in cecal luminal contents. Together, these data suggest the possibility that both E. coli F-18 and F-18col- must grow in mucus to colonize the streptomycin-treated mouse large intestine, that E. coli F-18col- is eliminated by E. coli F-18 because it does not grow in mucus as well as E. coli F-18, and that E. coli F-18col- can resist elimination by E. coli F-18 if it is allowed enough time to establish itself within the mucus layer.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Mucosa Intestinal/microbiologia , Muco/microbiologia , Estreptomicina/farmacologia , Animais , Ceco/microbiologia , Colicinas/fisiologia , Escherichia coli/genética , Camundongos , Fatores de TempoRESUMO
The relative abilities of an avirulent Salmonella typhimurium strain with wild-type lipopolysaccharide (LPS) character, SL5319, and a nearly isogenic LPS-deficient mutant, SL5325, to colonize the large intestines of streptomycin-treated CD-1 mice in vivo and to penetrate colonic mucus in vitro were studied. Previously it had been shown that, when fed simultaneously to streptomycin-treated mice (approximately 10(10) CFU each), the S. typhimurium strain with wild-type LPS colonized at 10(8) CFU/g of feces indefinitely, whereas the LPS-deficient mutant dropped within 3 days to a level of only 10(4) CFU/g of feces. In the present investigation, when SL5325 was allowed to colonize for 8 days before feeding mice SL5319 or when it was fed to mice simultaneously with an Escherichia coli strain of human fecal origin (10(10) CFU each), both strains colonized indefinitely at 10(7) CFU/g of feces. Moreover, when the wild-type and LPS-deficient mutant strains were fed to mice simultaneously in low numbers (approximately 10(5) CFU each) the strains survived equally well in the large intestines for 8 days, after which the LPS-deficient mutant was eliminated (less than 10(2) CFU/g of feces), whereas the wild-type colonized at a level of 10(7) CFU/g of feces. In addition although both strains were able to adhere to mucus and epithelial cell preparations in vitro, the wild-type strain was shown to have greater motility and chemotactic activity on CD-1 mouse colonic mucus in vitro and to more rapidly penetrate and form a stable association with immobilized colonic mucosal components in vitro. Based on these data, we suggest that the ability of an S. typhimurium strain to colonize the streptomycin-treated mouse large intestine may, in part, depend on its ability to penetrate deeply into the mucus layer on the intestinal wall and subsequently, through growth, colonize the mucosa.
Assuntos
Mucosa Intestinal/microbiologia , Intestino Grosso/microbiologia , Salmonella typhimurium/crescimento & desenvolvimento , Animais , Aderência Bacteriana , Epitélio/microbiologia , Lipopolissacarídeos/deficiência , Camundongos , Muco/microbiologia , Polissacarídeos Bacterianos/deficiência , Estreptomicina/farmacologiaRESUMO
The Escherichia coli human fecal isolates F-18 and K-12 are excellent colonizers of the streptomycin-treated mouse intestine. E. coli F-18 and E. coli K-12 eda mutants (unable to utilize glucuronate, galacturonate, and gluconate) were constructed by insertional mutagenesis. Neither the E. coli F-18 eda nor the E. coli K-12 eda mutant was able to colonize the streptomycin-treated mouse intestine, whether they were fed to mice together with their respective parental strains or alone. Complementation of the eda mutants with pTC190 (containing a functional E. coli K-12 eda gene) completely restored the colonization ability of both eda mutants. Relative to their parental strains, the E. coli F-18 eda mutant and the E. coli K-12 eda mutant grew poorly in cecal mucus isolated from mice fed either normal mouse chow or a synthetic diet containing sucrose as the sole carbon source, yet the mutants and parental strains demonstrated identical growth rates in minimal medium with glucose as the carbon source. E. coli F-18 edd eda and E. coli K-12 edd eda double mutants colonized the streptomycin-treated intestine when fed to mice alone; however, when fed simultaneously with their respective parental strains, they were poor colonizers. Since the edd gene is involved only in gluconate metabolism via the Entner-Doudoroff pathway, these results implicate the utilization of gluconate and the Entner-Doudoroff pathway as important elements in E. coli colonization of the streptomycin-treated mouse large intestine.
Assuntos
Ceco/microbiologia , Escherichia coli/patogenicidade , Gluconatos/metabolismo , Glucuronatos/metabolismo , Ácidos Hexurônicos/metabolismo , Animais , Escherichia coli/genética , Teste de Complementação Genética , Ácido Glucurônico , Mucosa Intestinal/microbiologia , Camundongos , Camundongos EndogâmicosRESUMO
The effect of a variety of metabolic inhibitors on the cell-mediated lysis of chicken erythrocytes by immune spleen cells was investigated using the Cr-release assay. The addition of cycloheximide, puromycin, emetine, pactamycin, actinomycin D or EDTA during the early stages of the reaction (0-2 h) produced partial to complete inhibition of the cytotoxic reaction, while the addition of these compounds at later time periods (2-4 h) resulted in the progressive loss of inhibitory effects. Later additions (4-6 h) of all compounds, except EDTA, resulted in a significant increase in target cell lysis. The ability of these compounds to induce increased cytotoxicity required complete inhibition of protein synthesis and the presence of reactive effector cells. It did not appear to be due to an increase in the rate of 51Cr release from previously damaged target cells, or inhibition of a target-cell repair mechanism dependent on protein synthesis. At least a portion of the increased reactivity was due to effector cell-target cell adhesions which formed after the addition of the inhibitor. The data suggests that the addition of metabolic inhibitors during the later stages of the reaction induced an increase in the efficiency or number of cytotoxic attacks.
Assuntos
Antimetabólitos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Eritrócitos/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Galinhas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Ácido Edético/farmacologia , Emetina/farmacologia , Imunidade Celular/efeitos dos fármacos , Técnicas In Vitro , Pactamicina/farmacologia , Puromicina/farmacologiaRESUMO
Escherichia coli F-17 Sr a human faecal isolate, is resistant to the T-series of bacteriophages (i.e. T2 to T7). A T2-sensitive mutant of E. coli F-17 Sr was isolated following acriflavin treatment. This mutant, E. coli F-17 Sr Ts was found to be sensitive to the entire T-series of phages. E. coli F-17 Sr and E. coli F-17 Sr Ts did not differ quantitatively in total LPS content. However, analysis of LPS revealed that a large fraction of E. coli F-17 Sr Ts was devoid of O-side-chains. This accounted for the sensitivity of this strain to bacteriophages T3, T4, and T7. In addition, E. coli F-17 Sr Ts contained only about half the amount of capsular material contained by E. coli F-17 Sr accounting for the sensitivity of the mutant to bacteriophages T2, T5, and T6. Although the two strains colonized equally well when fed individually to streptomycin-treated mice, when fed simultaneously to streptomycin-treated mice, E. coli F-17 Sr Ts colonized at a level of about 1 x 10(8) cells (g faeces)-1, whereas E. coli F-17 Sr colonized at only 1 x 10(4) cells (g faeces)-1. These studies suggest that bacterial cell surface components modulate the large intestine colonizing ability of E. coli F-17 Sr in the mouse large intestine.
Assuntos
Escherichia coli/fisiologia , Intestino Grosso/microbiologia , Absorção , Animais , Proteínas da Membrana Bacteriana Externa , Eletroforese em Gel de Poliacrilamida , Escherichia coli/análise , Escherichia coli/crescimento & desenvolvimento , Fezes/microbiologia , Humanos , Lipopolissacarídeos/análise , Masculino , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos , Mutação , Plasmídeos , Propriedades de Superfície , Fagos T/fisiologiaRESUMO
Cell-mediated cytotoxic reactivity resulting from the in vitro incubation of normal lymphocytes was assessed using nonspecific lectin-dependent cell-mediated cytotoxicity (LDCC) as a measure of overall reactivity. Spleen cells from non-immune C57BL/6 mice were incubated in vitro in RPM1-1640 supplemented with 10% fetal calf serum and 2-mercaptoethanol (2ME). Cytotoxicity was assayed against syngeneic Cr51-labeled EL-4 cells in the presence of Con A or PHA. Optimal LDCC was observed after 8 days of culture in the presence of 5 X 10(-5) M 2ME. Cytotoxicity was mediated by an activated T-lymphocyte population whose development did not appear to require macrophages. Usually LDCC in the presence of PHA was significantly greater than that obtained in the presence of Con A. The presence of 2ME during the initial phase of culture was crucial for the development of cytotoxicity, since early removal of 2ME after 1 or 3 days of culture did not alter the subsequent development of cytotoxicity, whereas delayed addition of 2ME on day 1 or 3 failed to produce cytotoxic reactivity. This rapid conversion from a 2ME sensitive state to a 2ME insensitive state may be related to a rapid loss of accessory cell viability during the early phase of culture. Together the results indicate that this system may provide a useful model for the investigation of the events leading to the development of CTL in vitro.