Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent.

The choice of host cell receptor and the mechanism of binding (opsonic versus nonopsonic) may influence the intracellular fate of Mycobacterium tuberculosis. We have identified two substrains of M. tuberculosis H37Rv, designated H37Rv-CC and -HH, that differed in their modes of binding to complement receptor type 3 (CR3) expressed in transfected Chinese hamster ovary (CHO-Mac-1) cells: H37Rv-CC bound nonopsonically, whereas H37Rv-HH bound only after opsonization in fresh serum. H37Rv-CC also bound nonopsonically to untransfected CHO cells, whereas H37Rv-HH binding was enhanced by serum and was mediated by the 1D1 antigen, a bacterial adhesin previously identified as a polar phosphatidylinositol mannoside. H37Rv-CC and -HH had identical IS6110 DNA fingerprint patterns. Of five M. tuberculosis clinical isolates examined, four displayed the same binding phenotype as H37Rv-CC, as did the Erdman strain, whereas one isolate, as well as Mycobacterium smegmatis, behaved like H37Rv-HH. Nonopsonic binding of H37Rv-CC to CHO cell-expressed CR3 was apparently to the beta-glucan lectin site, as it was cation independent and inhibited by laminarin (seaweed beta-glucan) and N-acetylglucosamine; laminarin also inhibited the binding of H37Rv-CC to monocyte-derived macrophages. Further, binding of H37Rv-CC to CHO-Mac-1 cells was inhibited by prior agitation of bacteria with glass beads (which strips outer capsular polysaccharides) and by preincubation with amyloglucosidase, as well as by the presence of capsular D-glucan and D-mannan from M. tuberculosis Erdman, but not by Erdman D-arabino-D-mannan, yeast mannan, or capsular components from H37Rv-HH. Analysis of capsular carbohydrates revealed that H37Rv-CC expressed 5-fold more glucose and 2.5-fold more arabinose and mannose than H37Rv-HH. Flow cytometric detection of surface epitopes indicated that H37Rv-CC displayed twofold less surface-exposed phosphatidylinositol mannoside and bound complement C3 less efficiently than H37Rv-HH; these differences were eliminated after treatment of H37Rv-CC with glass beads. Thus, outer capsular polysaccharides mediate the binding of H37Rv-CC to CR3, likely to the beta-glucan site. Moreover, there are strain-dependent differences in the thickness or composition of capsular polysaccharides that determine the mode of binding of M. tuberculosis to mammalian cells.     

Characterization of human folylpolyglutamate synthetase expressed in Chinese hamster ovary cells

Chinese hamster AUX B1 cells lack the enzyme folylpolyglutamate synthetase (FPGS) responsible for adding polyglutamates to folie acid. The human gene for FPGS was introduced into AUX B1 cells by cotransfection with human genomic DNA and either pSV2neo or pSV2gpt plasmid DNA. Cells were coselected for FPGS expression by growth in the absence of glycine, adenosine, and thymidine, and for drug resistance by growth in geneticin or mycophenolic acid. The presence of human enzyme in transfected cells was identified by relative enzyme activity determinations with ATP or dATP as substrates. The human HeLa cell enzyme displayed a 50% higher activity with dATP, and the hamster enzyme showed a 50% higher activity with ATP. Hamster and human enzymes also differed in the chain length of polyglutamates on cellular folate. Only monoglutamates were detected in AUX B1 cells, while wild-type hamster cells had predominantly hexa- and heptaglutamates, and human HeLa cells had predominantly hepta- and octaglutamates. Transformants with FPGS activity that showed a human enzyme preference for dATP also had folate polyglutamate chain lengths characteristic of the human enzyme.     

Proton conductance of human transient receptor potential-vanilloid type-1 expressed in oocytes of Xenopus laevis and in Chinese hamster ovary cells

Transient receptor potential-vanilloid type-1 (TRPV1) is a ligand-gated cation channel with preference for divalent cations, especially Ca(2+) (sequence of conductances: Ca(2+)>Mg(2+)>Na(+) approximately/= K(+) approximately/= Cs(+)). In the present study, the two-electrode voltage-clamp technique was used on oocytes of Xenopus laevis expressing TRPV1 to evaluate whether human TRPV1 also conducts protons. In medium devoid of K(+), Na(+), Mg(2+), and Ca(2+), capsaicin 1 microM induced a significant inward current (62% of the current in physiological medium). The effects of capsaicin were abolished in the presence of capsazepine 3 microM. The capsaicin-induced currents in medium devoid of Na(+), K(+), Mg(2+), and Ca(2+) were dependent on pH, causing larger inward currents and less negative reversal potentials at low pH and vice versa. The same current was also demonstrated in Chinese hamster ovary cells expressing human TRPV1. We conclude that TRPV1 conducts protons, in addition to Na(+), K(+), Mg(2+), and Ca(2+). The proton conductance may help to initiate action potentials and to translocate H(+) dependent on TRPV1 activation and membrane potential.     

Characterization of a human monoclonal antibody against Shiga toxin 2 expressed in Chinese hamster ovary cells

Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 microg/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.     

Plasminogen activator inhibitor type 2 cDNA transfected into Chinese hamster ovary cells is stably expressed but not secreted

Immunological screening with a monoclonal antibody probe developed against the plasminogen activator inhibitor type 2 (PAI-2) detected 6 positive clones from approximately 1.7 × 10⁵ recombinant phages in a λgt₁₁ expression library containing cDNA inserts prepared from human placental mRNA. Hybridisation experiments at high stringency indicated that the 6 clones were related. One positive clone was found to produce a fusion protein of Mr 170 000 that was recognised by both monoclonal and polyclonal antibodies against PAI-2. In order to obtain a full length cDNA clone for PAI-2, an additional cDNA library was screened. From this screening, we obtained a cDNA clone that encoded all but a portion of the 5′-untranslated region of the PAI-2 mRNA. Primer extension experiments determined that the 5'-untranslated region was 74 nucleotides in length. The PAI-2 mRNA has an open reading frame of 1245 nucleotides and encodes a 46,6 kDa protein. Analysis of the predicted amino acid sequence revealed that like ovalbumin, PAI-2 has an internal non-cleaved signal peptide. The PAI-2 coding sequence is followed by a 3'-untranslated region of 581 nucleotides. In order to study secretion of PAI-2, a plasmid construct containing the PAI-2 cDNA preceded by the SV40 early promotor was transfected into Chinese hamster ovary cells. The PAI-2 cDNA was efficiently expressed in these cells but unexpectedly the protein was not secreted into the culture medium. The absence of PAI-2 secretion in Chinese hamster ovary cells may be due to the lack of a ‘tissue specific secretory mechanism’ that is present in tissues which normally express PAI-2     

Valinomycin-induced apoptosis in Chinese hamster ovary cells

Accumulating evidence endorses that excessive K(+) efflux is an ionic mechanism underlying apoptosis both in neuronal and non-neuronal cells. K(+) channels play important roles in mediating the pro-apoptotic K(+) efflux. Chinese hamster ovary (CHO) cells have been widely used for gene transfection experiments. These cells lack detectable endogenous voltage-gated K(+) channels. We were interested in knowing whether the absence of endogenous K(+) channels would render wild-type CHO cells more resistant to apoptotic death. We also wished to determine if direct stimulation of K(+) efflux would trigger apoptosis in these cells. Exposing CHO cells to hypoxia (1% O(2)) or to a typical apoptotic insult of serum deprivation for up to 24h did not affect cell survival. On the other hand, the K(+) ionophore valinomycin caused substantial cell death within 12h of its application. Valinomycin-treated CHO cells underwent several apoptotic events, including phosphatidylserine (PS) membrane translocation, caspase-3 activation, and mitochondrial membrane depolarization during the first few hours of exposure. Reducing K(+) efflux by elevating extracellular K(+) concentrations noticeably attenuated valinomycin-induced cell death. This study reinforces a K(+) efflux-mediated apoptotic mechanism in CHO cells and may help to explain the unique feature of their higher tolerance to apoptosis.     

Laminin on Toxoplasma gondii mediates parasite binding to the beta 1 integrin receptor alpha 6 beta 1 on human foreskin fibroblasts and Chinese hamster ovary cells.

We investigated the role of parasite-bound laminin and the host cell beta 1 integrin receptors for this extracellular matrix protein in Toxoplasma gondii binding to fibroblasts. Laminin but not fibronectin was detected on extracellular tachyzoites by immunofluorescence and immunoblotting. Binding of parasites to CHO cells was inhibited by polyclonal antibodies to laminin and by a monoclonal antibody directed against the globular carboxyl-terminal portion of the long arm of laminin (at or near the suggested ligand-binding sites for alpha 3 beta 1 and alpha 6 beta 1), but not by a monoclonal antibody directed against the lateral short arms of laminin near the cross region of the molecule. Antibodies to the alpha 6 but not the alpha 2, alpha 3, or alpha 5 chains of the beta 1 family of integrins blocked parasite attachment to human foreskin fibroblasts and CHO cells. Attachment of T. gondii to cells via laminin on the parasite surface and laminin receptors on the mammalian cell is consistent with the capacity of the parasite to invade almost all nucleated cells.     

二氢叶酸还原酶缺陷型中华仓鼠卵巢细胞偏爱密码子的研究

目的研究二氢叶酸还原酶缺陷型中华仓鼠卵巢细胞（CHO dhfr-）的偏爱密码子。方法建立CHO dhfr-细胞高丰度mRNA的cDNA文库，对该文库进行签定以获得合格的编码蛋白质的cDNA序列，统计密码子使用频率并与CUTG数据库中的中华仓鼠比较，同时对所获得的cDNA序列进行对应统计分析。结果获得了50个合格的CHO dhfr-细胞高丰度蛋白的cDNA序列，与中华仓鼠密码子使用频率比较，除了Pro，Arg外，其余氨基酸的最高使用频率的密码子在CHO dhfr-细胞和中华仓鼠中都相同。同时根据对应分析确定了能解释最多变量（14．7％）的第一主成分，确定了22个同义密码子为CHO dhfr-细胞偏爱密码子。结论CHO dhfr-细胞存在自身偏爱的密码子，提示密码子的偏爱性是不同哺乳动物细胞功能差异的因素之一，也为外源基因在CHO dhfr-细胞中通过优化密码子而获得高表达提供了新思路。     

Induction of a type 1 immune response to a recombinant antigen from Mycobacterium tuberculosis expressed in Mycobacterium vaccae.

A 19-kDa lipoprotein from Mycobacterium tuberculosis was expressed as a recombinant antigen in the nonpathogenic mycobacterial host strain M. vaccae. Immunization of mice with the recombinant M. vaccae resulted in induction of a strong type 1 immune response to the 19-kDa antigen, characterized by immunoglobulin G2a (IgG2a) antibodies and gamma interferon (IFN-gamma) production by splenocytes. Immunization with the same antigen in incomplete Freund's adjuvant induced a strong IgG1 response with only low levels of IFN-gamma. Subsequent intravenous and aerosol challenges of immunized mice with virulent M. tuberculosis demonstrated no evidence of protection associated with the response to the 19-kDa antigen; in fact, the presence of the recombinant 19-kDa antigen abrogated the limited protection conferred by M. vaccae (vector control). The recombinant M. vaccae system is a convenient approach to induction of type 1 responses to M. tuberculosis antigens. However, the unexpected reduction in protective efficacy of M. vaccae expressing the 19-kDa antigen highlights the complexity of testing recombinant subunit vaccines and the need for a better understanding of the immune mechanisms required for effective vaccination against tuberculosis.     

Cytogenetic response to coffee in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes

We have investigated the genotoxic effects of three differentbrands and three types of coffee (freshly brewed regular, instantregular and freshly brewed decaffeinated) in two mammalian systems:the Chinese hamster ovary (CHO) AUXB1 cell line and human peripherallymphocytes. Sisterchromatid exchanges (SCEs) and endoreduplicatedcells (ERCs) were used as the endpoints. Coffee was preparedaccording to the manufacturer's suggestions, and after cooling,administered to cultured cells at dilutions ranging up to 11%that of full-strength coffee. Each brand and type of coffeeinduced significant levels of SCEs and ERCs in AUXB1 cells.SCEs, but not ERCs, were induced in human peripheral lymphocytes.Bisulfite, which complexes with carbonyls and reduces theirgenotoxicity, significantly diminished the number of SCEs andERCs found after administration of coffee. Catalase and peroxidase,enzymes that destroy hydrogen peroxide activity, had no significanteffect upon the SCE and ERC frequencies in AUXB1 cultures treatedwith freshly brewed regular coffee. These experiments indicatethat different brands and types of coffee have sufficient genotoxicactivity to increase SCEs and ERCs at levels only a fractionof those normally consumed. 1,2-Dicarbonyls alone and peroxidesalone do not appear to be responsible for the majority of SCEsand ERCs that were observed to be induced by dilute coffee.     

Increased adhesion of Chinese hamster ovary cells to substratum by cholera enterotoxin.

A new, simple assay method using Chinese hamster ovary cells was devised for cholera enterotoxin. The effect of the cholera toxin was measured by an increase in cell adhesion to the substratum. The increase was dependent on the concentration of the toxin used and was effective for a longer period of time than that of adhesion increased by dibutyryl cyclic adenosine monophosphate and theophyline. There was a delay of about 60 min before the increase in adhesion, using the toxin, appeared, whereas the increase caused by dibutyryl cyclic adenosine monophosphate appeared almost at once.     

Legionella pneumophila inhibits protein synthesis in Chinese hamster ovary cells.

Legionella pneumophila is a gram-negative facultative intracellular parasite that causes Legionnaires disease. To explore the interactions between L. pneumophila and host cells, we have developed a continuous cell line model of infection. We show that about 80% of Chinese hamster ovary (CHO) cells were associated with L. pneumophila after incubation for 3 h at a multiplicity of infection of 20 bacteria per cell. Within 3 to 4 h of incubation with L. pneumophila, protein synthesis of CHO cells was markedly inhibited, as shown by the reduction of incorporation of radiolabeled amino acids into proteins. L. pneumophila did not inhibit transport of amino acids or cause degradation of newly synthesized proteins in CHO cells. Cytochalasin D blocked internalization of L. pneumophila by CHO cells, yet CHO cell protein synthesis was inhibited. These results indicated that L. pneumophila could inhibit host protein synthesis from the cell exterior. L. pneumophila that had been killed with antibiotics prior to incubation with CHO cells still inhibited protein synthesis, indicating that the inhibition of CHO cell protein synthesis occurred in the absence of de novo protein synthesis by L. pneumophila.     

人γδT细胞TCR分子与结核杆菌多肽抗原特异性结合的证据

目的探讨γδT细胞对结核杆菌抗原(Mtb—Ag)识别的分子机制。方法采集人外周血(PB)，分别经抗TCRγδ-PE和FITC标记的Mtb—Ag及其纯化的不同组分，结核杆菌高分子量蛋白组分(Mtb—HW-Ag)、低分子量多肽组分(Mtb—LW-Ag)或Mtb-Ag峰3单一主肽抗原(Mtb-Ag-Pk3)进行双标记染色，或使用过量Mtb—AS先与细胞预处理，然后再做上述染色，用流式细胞仪检测γδT细胞上结合的抗TCRγδ-PE荧光强度变化。将人外周血PBMC经Mtb—Ag刺激24h后，使用抗TCRγδ-PE和Mtb—Ag-FITC染色，用激光共聚焦显微镜观察γδT细胞表面TCRγδ分子的聚集。结果经流式细胞仪分析，Mtb—Ag及其纯化的Mtb—LW-Ag、Mtb-Ag-Pk3均可与γδT细胞发生特异性直接结合，而且Mtb—Ag可有效阻断抗TCRγδ单抗与γδT细胞的结合。而Mtb—HW-Ag与γδT细胞并无特异性结合效应。经激光共聚焦显微镜观察，Mtb—Ag可显著激活γδT细胞而诱导TCRγδ分子发生功能性聚集化。结论γδT细胞通过其表面的TCRγδ分子可直接结合Mtb—Ag，而Mtb-Ag-Pk3多肽可能是Mtb—Ag发挥其结合效应的有效成分。Mtb—Ag可激活γδT细胞并诱导TCRγδ分子聚集，而可能参与功能性脂筏(raft)的形成。     

Binding of the 68-kilodalton protein of Mycobacterium avium to alpha(v)beta3 on human monocyte-derived macrophages enhances complement receptor type 3 expression.

Attachment to and uptake by host cells are important early events in the pathogenesis of intracellular organisms such as Mycobacterium avium. Monocyte-derived macrophages (MDM) are known to express multiple surface receptors that play a role in binding to and uptake of M. avium. These include complement receptor type 3 (CR3), fibronectin receptor, mannose receptor, and transferrin receptor. In addition to these, we have previously reported that the integrin receptor alpha(v)beta3 also plays a role in binding to M. avium in a nonopsonic environment. Further, we have shown that a 68-kDa surface protein of M. avium binds to human monocytes and plays a role in attachment of M. avium to MDM. The present study provides direct evidence that this protein mediates attachment of M. avium to MDM by binding to alpha(v)beta3. Using the technique of cell surface enzyme-linked immunosorbent assay, we have shown that the M. avium 68-kDa protein inhibits the binding of monoclonal antibodies (MAb) against alpha(v)beta3 to MDM compared to control proteins such as ovalbumin and laminin (P < 0.05). Dual-labeling studies were performed to demonstrate that after phagocytosis, alpha(v)beta3 is present along with M. avium in phagosomes of M. avium-infected MDM. In addition, we have demonstrated that this interaction between alpha(v)beta3 and the M. avium 68-kDa protein resulted in enhancement of CR3 expression, which is known to play a role in complement-mediated uptake of M. avium. Attachment of MDM to wells coated with the M. avium 68-kDa protein resulted in a twofold increase in CR3 expression compared to attachment of MDM to wells coated with ovalbumin. This enhancement was completely inhibited by pretreatment of MDM with MAb against alpha(v)beta3. In summary, M. avium binds to MDM via alpha(v)beta3 with the help of the M. avium 68-kDa protein, and this ligation enhanced the expression of CR3 on MDM. Since CR3 has been known to play a role in M. avium uptake, enhanced expression of this receptor mediated by M. avium-alpha(v)beta3 interaction indicates a complex mechanism of communication among different receptors that participate in M. avium attachment and uptake. These findings add to current understanding of the roles played by multiple receptor-ligand systems in uptake and pathogenesis of intracellular pathogens such as M. avium.     

Synthesis of prostaglandins in cholera toxin-treated Chinese hamster ovary cells

The prostaglandin (PG) and adenosine 3',5'-cyclic monophosphate (cAMP) responses of Chinese hamster ovary (CHO) cells were measured after cholera toxin (CT) exposure to evaluate dose and kinetic relationships. Release of prostaglandin E2 (PGE2) and the accumulation of cAMP were dependent on the dose of CT, with an effective dose of approximately 10-100 ng/ml within 4 h; the PGE2 response was about four- to six-fold more than that of PGE1. CHO cells exposed to CT also released increased amounts of thromboxane B2 (TxB2), PGF2 gamma, and 6-keto PGF1 gamma (a non-enzymatic degradation product of prostacyclin). Kinetic analysis of CT-treated cells revealed that small peaks of cAMP accumulation and of PGE1 and PGE2 release were detected at approximately 30 min, but larger, progressive PG and cAMP responses were measured 2-4 h later. Exposure of the cells to relatively high doses of membrane-permeable derivatives of cAMP (1 mM) and forskolin (10 microM) caused PGE2 release. Concomitantly, exogenous PGE2 (100 microM) increased intracellular levels of cAMP. We have considered the interrelationship of the cyclo-oxygenase and the cyclic nucleotide pathways relative to the molecular mechanism of CT.     

Vitronectin mediates internalization of Neisseria gonorrhoeae by Chinese hamster ovary cells.

Gonococci producing a distinct opacity protein (OpaA in strain MS11) adhere to and are efficiently internalized by cultured epithelial cells such as the Chang conjunctiva cell line. Both adherence and uptake require interactions between OpaA and heparan sulfate proteoglycans on the mammalian cell surface. Chinese hamster ovary (CHO) cells also support adherence of gonococci through interactions of OpaA with cell surface heparan sulfate proteoglycans. However, despite this similarity in the requirements for adherence, CHO cells are not capable of internalizing gonococci. In this report, we characterized this apparent deficiency and identified a factor in fetal calf serum (FCS) which is capable of mediating uptake of gonococci by CHO cells. In the absence of FCS, OpaA+ gonococci adhered to but were not internalized by CHO cells, whereas in the presence of up to 15% FCS, the bacteria were efficiently internalized by the cells. Preincubation of bacteria, but not cells, with FCS also stimulated internalization, suggesting that a factor present in FCS was binding to the surface of gonococci and subsequently stimulating entry. Using a combination of chromatographic purification procedures, we identified the adhesive glycoprotein vitronectin as the serum factor which mediates the internalization of gonococci by CHO cells. Vitronectin-depleted serum did not support gonococcal entry, and this deficiency was restored by the addition of purified vitronectin. Further experiments using a set of gonococcal recombinants, each expressing a single member of the family of Opa outer membrane proteins, demonstrated that vitronectin bound to the surface of OpaA-producing gonococci only and that the vitronectin-mediated uptake by the CHO cells was limited to this bacterial phenotype. To our knowledge, our data are the first example that vitronectin can serve as a molecule that drives bacterial entry into epithelial cells.