![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Infection and Immunity, November 2003, p. 6205-6212, Vol. 71, No. 11
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.11.6205-6212.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Sezione di Microbiologia, Dipartimento di Patologia e Medicina di Laboratorio, Università degli Studi di Parma, Parma,1 Sezione di Chimica Biologica, Dipartimento di Biologia Molecolare, Università degli Studi di Siena, Siena,2 Dipartimento Malattie Infettive, Parassitarie ed Immuno-Mediate, Istituto Superiore di Sanità, Rome, Italy3
Received 23 May 2003/ Returned for modification 8 July 2003/ Accepted 28 July 2003
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To obtain standardizable reagents in sufficient amounts for therapeutic assays, KT-IdAb were produced in the monoclonal (KT-MAb) and recombinant single chain fragment variable (KT-scFv) format by hybridoma and phage display molecular technologies from animals vaccinated with mAbKT4 (18, 24). All these novel KT-mimicking antibodies displayed in vitro and in vivo microbicidal activity against KTR-possessing microbial pathogens (6, 18, 30).
In an attempt to identify biologically active fragments for possible therapeutic use, we have sequenced KT-scFv and synthesized a number of peptides, with special focus on those pertaining to CDR domains which are expected to express antigen specificity (15). Here we report on the synthesis and optimization, through alanine scanning, of a decapeptide derived from the sequence of the variable region and containing part of the CDR1 segment of the KT-scFv light chain. By the use of C. albicans as a model of a variety of KT- and KT-IdAb-susceptible microorganisms, we demonstrate the potent in vitro and in vivo activities of this novel microbicidal peptide. We also provide some circumstantial evidence that killer peptide (KP), like KT and KT-IdAb, interacts with ß-glucan constituents of the cell wall. Overall, this is to our knowledge the first engineered peptide fragment derived from a microbicidal recombinant IdAb capable of exerting a remarkable antimicrobial activity in vitro and in vivo.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Synthesis of decapeptides from the KT-scFv sequence. Studies have been carried out on the correlation between amino acid sequences and functions by synthesizing a series of decapeptides belonging to the KT-scFv sequence, with special regard to CDR regions and CDR peptides as well. A synthetic decapeptide (P6) containing the first three amino acids of the CDR-L1 region (SAS; sequence positions 24 to 26) and exerting remarkable candidacidal activity in vitro on a molar basis (see below) was selected for large-scale synthesis as soluble product. Solid-phase synthesis was carried out in a MultiSynTech Syro automatic peptide synthesizer (Witten, Germany) employing Fmoc chemistry with HOBt activation and Rink amide MBHA resin as solid support. Peptides were cleaved from the resins and deprotected by treatment with trifluoroacetic acid containing ethanedithiol, water, triisobutylsilane, and anisole (93:2.5:2:1.5:1). After precipitation by ethylic ether, the crude peptides were purified by using a Vydac C18 column (25 cm by 1 cm) and characterized by amino acid analysis and mass spectrometry.
Optimization of a killer decapeptide. The decapeptide P6 was analyzed by alanine scanning in order to identify the functional contribution of each residue. The derivative showing the highest candidacidal activity in vitro (hereafter designated KP) was reduced by COOH-terminal deletions of up to three residues (KP derivatives) to establish the ability to retain the candidacidal activity.
In vitro evaluation of killer activity of synthetic peptides. The candidacidal activity in vitro was determined essentially as previously reported (18, 24). In brief, approximately 150 viable KT-susceptible germinating C. albicans UP10 cells, suspended in 10 µl of H2O, were added to 90 µl of H2O containing synthetic decapeptides or peptides representing CDRs to obtain a final concentration of 100 µg/ml. The same concentration of an irrelevant peptide (TSTTSLELD) was used as a control. P6 and the products obtained by alanine scanning were further tested at the concentrations of 100, 25, and 6.25 µg/ml in comparison with a scramble decapeptide properly synthesized as P6 altered sequence as a control. After incubation for 6 h at 37°C with the respective reagents, the fungal cells were dispensed and streaked on the surface of Sabouraud dextrose agar plates which were then incubated at 30°C, and CFU were enumerated after 48 h. Each experiment was performed in triplicate. KP was also tested in vitro in comparison with its own scramble decapeptide (SP) at scalar dilutions (100 to 0.8 µg/ml) in order to establish the minimal fungicidal concentration corresponding to 100% killing of C. albicans cells. The above-mentioned CFU assay was also carried out in the presence of scalar concentrations (800 to 12.5 µg/ml) of pustulan (ß1,6-glucan; Calbiochem, La Jolla, Calif.; approximate molecular weight [MW] of 20,000) and laminarin (ß1,3-glucan; Sigma Chemical Co., St. Louis, Mo; approximate MW of 5,800; see also reference 21). Both polysaccharides were coincubated with KP or SP (both 25 µg/ml) for 6 h at 37°C. KP and equivalent molar concentrations of its derivatives were also tested at scalar dilutions to establish the peptide concentration (in moles per liter) corresponding to the 50% inhibitory concentration (IC50). Assays were performed in triplicate, and the IC50 of each peptide was calculated by nonlinear regression analysis of curves obtained by plotting the number of CFU versus log peptide concentration using GraphPad Prism 3.02 software.
In vivo evaluation of KP therapeutic activity. (i) Vaginal infection model. A previously established experimental model of vaginal infection by C. albicans in oophorectomized, estrogen-treated rats was used (9). Estrogen-conditioned rats (five animals/group) were inoculated intravaginally with 107 cells of fluconazole-sensitive (SA-40) or fluconazole-resistant (AIDS 68) C. albicans strains and intravaginally administered different doses of KP at 1, 24, and 48 h postchallenge. Both yeast strains were originally isolated from a human vaginal infection and maintained in stock collections in the Department of Bacteriology and Medical Mycology of the Istituto Superiore di Sanità, Rome, Italy. The vaginal C. albicans burden was quantitated by CFU enumeration from the vaginal fluid taken each day by using a special calibrated loop. Vaginal smears were also stained by the periodic acid-Schiff-van Gieson method. The outcome of treatment was assessed in terms of microscopic reduction of the hyphal growth in the vagina as well as by acceleration of fungal CFU clearance over a period of 28 days. Negative controls (untreated rats and rats treated with SP) were included. As a positive control, rats received 50 or 100 µg of fluconazole (Pfizer)/ml in phosphate-buffered saline (0.1 ml) at 1, 24, and 48 h after the yeast challenge. The person dealing with animal treatments and vaginal smears was blinded with regard to the nature of the material being injected.
(ii) Systemic infection model. Groups of eight BALB/c or severe combined immunodeficient (SCID) female mice, both obtained from Charles River (Calco, Italy), weighing 18 to 21 g, were challenged with five 50% lethal doses (LD50's; corresponding to 107 cells of C. albicans strain SA-40) by the intravenous route. Fifty micrograms of KP was therefore administered intraperitoneally for 3 days starting on day 0, i.e., 1 h after the fungal challenge, and at 24 and 48 h thereafter. Untreated animals or animals treated with SP (same dosage and treatment schedule as those treated with KP) were the controls. The person who injected the animals was blinded with regard to the nature of the material being injected. The animals were then followed for mortality and internal organ invasion for 60 days.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
The IC50 values of KP derivatives shortened by progressive COOH-terminal amino acid deletion were also calculated. There was a decay of candidacidal activity of almost 3 orders of magnitude with the deletion of the COOH-terminus serine and of >4 orders of magnitude with the deletion of the last four COOH-terminal residues. This shows that the amino acids belonging to CDR1 were indeed critical for KP activity.
Inhibition of KP activity by glucan. Since (i) KT and its mimicking KT-IdAb interact with cell surface ß-glucan (4, 12, 13), (ii) KP contained part of the sequence of a CDR domain potentially involved in the binding, and (iii) as suggested above, this sequence was critical for KP activity, we examined whether KP activity was affected by soluble glucan molecules. In addition, we tested for any competition by KP on an IdAb (KT-MAb) binding to C. albicans germ tubes, the growth forms of the fungus mostly expressing KT- and KT-IdAb receptor components.
We observed that the candidacidal activity of KP was strongly and dose dependently inhibited by laminarin, a soluble ß1-3 glucan, but not by pustulan, a soluble ß1-6 glucan (Table 1). In addition KP, but not SP, completely inhibited the binding of the KT-MAb to germinating cells of C. albicans, as seen by immunofluorescence assay (data not shown; see also reference 24). Altogether, these data suggest that the candidacidal decapeptide did effectively compete for the site of binding of KT-IdAb on the fungal cell wall, presumably constituted by, or containing, ß1-3 glucan as receptor component.
|
(i) Vaginal infection in oophorectomized, estrogen-treated rats. In a preliminary, dose-finding experiment, rats were treated intravaginally with 10, 25, 50, and 100 µg of KP, 1, 24, and 48 h after the fungus challenge, and monitored for C. albicans clearance from the vagina as described in Materials and Methods. A dose-response therapeutic effect, but with 50 and 100 µg of the compound being similarly efficacious, was observed. Thus, in all subsequent experiments, 50 µg of KP was used in the established three-dose administration schedule.
Figure 2 shows the results of a typical experiment (out of three performed with comparable results) where the animals were intravaginally infected with C. albicans cells and then each treated with 50 µg of KP. SP and fluconazole (an efficacious, widely used anticandidal drug) served as negative and positive controls, respectively. KP was seen to accelerate the early rate of clearance (1 to 5 days) of the fungus from rat vagina as significantly as fluconazole. KP also provided a substantial resolution of the infection (less than 103 CFU/ml of vaginal fluid) within 3 weeks of challenge, when the untreated controls still had from 2 x 104 to 4 x 104 Candida CFU/ml of vaginal fluid. No acceleration of the fungal clearance and no effect on resolution of infection was provided by SP administration. Overall, the therapeutic benefit of KP was substantially comparable with that of fluconazole (Fig. 2). Noteworthy, KP proved to have a therapeutic effect also in rat vaginal infection caused by a C. albicans strain (AIDS 68) which was resistant to treatment with up to 100 µg of fluconazole (Table 2).
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this line, we have originally exploited the KT phenomenon and the Id network to produce KT-IdAb, in the polyclonal, monoclonal, and recombinant format, capable of mimicking in vitro and in vivo the potent wide-spectrum, microbicidal activity of the KT from P. anomala (19). These antibodies were effective in vitro against a panel of relevant opportunistic and frank pathogens such as C. albicans, P. carinii, and M. tuberculosis, including multidrug-resistant strains and, more recently, penicillin-, methicillin-, and vancomycin-resistant cocci (7). They were also effective in several experimental models of infection (5, 18, 23). In particular, KT-scFv exerted a therapeutic effect against C. albicans rat vaginal infection when intravaginally administered as such or vectored by the human commensal Streptococcus gordonii colonizing the vaginal mucosa (3, 18).
Nonetheless, the intrinsic limitations of the reported approach in terms of potential for treatment of human infections are easily recognized. A practical problem related to therapeutic treatment with KT-scFv is the requirement for continuous administration of a potentially expensive and labile product. The mucosal delivery of the candidacidal antibody fragment permanently expressed by a colonizing human commensal bacterium would appear to be safe and cost-effective, but the means for large-scale production and standardization and the regulatory pathway for the acceptance of new transgenic commensals would need to be defined. In addition, this last approach could not be considered for the treatment of systemic infections.
In recognition of these constraints, and because of our interest in dissecting the biological activity of KT-scFv, we addressed the biological activity of peptides synthesized and optimized from the KT-scFv sequence, with special regard to the CDR domains which express the antibody-specific binding to the susceptible cells. For this, we were also encouraged by the recent identification of KTR as a ß-glucan component of the cell wall (12). Importantly, glucan and glucan-like molecules are largely present in the microbial world, where they exert critical structural and/or virulence properties. Thus, targeting these components with antibodies or antibody derivatives could provide novel and general therapeutic approaches.
To prove the above concept, we have selected as a model organism C. albicans, a KT-, KT-MAb-, and KT-scFv-susceptible opportunistic pathogen of ever-increasing medical importance. This fungus is one of the most frequently involved microbial pathogens in the etiology of mucosal infections, being the principal agent of vulvo-vaginal (acute, chronic, and recurrent) infection and oral pathology in human immunodeficiency virus-infected subjects (17). In addition, it causes serious, highly lethal systemic infections in immunocompromised or otherwise debilitated hosts against which the current therapeutic choices are limited, also due to increasing antimycotic resistance problems (1, 10). Nonetheless, other KT-sensitive microbial pathogens are, in principle, to be considered in this approach since they have also been demonstrated to be susceptible to KT-MAb and KT-scFv (5-7).
Through KT-scFv sequence determination, a synthetic decapeptide (P6) inclusive of three CDR1 residues of the VL chain was selected for large-scale synthesis as soluble product because of its strong candidacidal activity in vitro in comparison with other decapeptides pertaining to CDRs as well as CDR-constituting peptides.
P6 was analyzed by alanine scanning in an attempt to potentiate its activity, thus obtaining a potent in vitro killer decapeptide denominated KP. Importantly, the candidacidal activity of KP required the presence of the three CDR1 amino acids SAS and was clearly inhibited, in a dose-dependent fashion, by laminarin, a ß1-3 glucan preparation. This latter effect was highly specific, since it was not reproduced by pustulan, a ß1-6 glucan preparation, thus suggesting that the peptide preferentially interacts with a receptorial component containing ß1-3 glucan. This would also suggest that germinating C. albicans cells, which in contrast to nongerminating yeast cells are highly susceptible to KP, express ß1-3 glucan on their surface. Indeed, ß1-6 glucan is rather expressed by nongerminating yeast cells (32). Germination of yeast cells is mandatory for tissue invasion by C. albicans, and ß-glucan synthesis is absolutely required for this event to occur. In fact inhibitors of ß1-3 glucan synthase are candidacidal (11). Whichever the precise ß-glucan configuration recognized by KP, our data suggest that, similarly to KT and KT-IdAb, the decapeptide also interacts with ß-glucans of KTR, as strengthened by the observation that KP, like KT, was able to inhibit the binding of a KT-MAb to KTR of C. albicans cells in a direct immunofluorescence assay (data not shown; see also references 24 and 26). Taken together, our findings constitute circumstantial evidence that the candidacidal activity of KP is, by still-unknown mechanisms, mediated in part or as a whole by KP interaction with ß-glucan.
The true reason why P6 and consequently KP, rather than the whole light chain CDR1 or other CDRs, proved to be a particularly strong KP is not known. The expectation that the anticandidal activity, if present, would have been expressed at the highest level by one or more CDRs was not supported by the experimental data. For instance, the VH CDR3 (which was the most active, on a molar basis, among all CDRs) was slightly less active than P6, and its activity was substantially unaffected by alanine replacement which, in contrast, increased P6 activity by more than 2 logs. Even though KP cannot be considered a structural mimotope of KT, it acted as a functional mimotope and was adopted for therapeutic purposes with reference to its enhanced activity in vitro. The functional mimicry, based on a critical interaction of KP with ß-glucan, i.e., the KTR, deserves further investigations aimed at unravelling the mechanism of action of KP on sensitive microbial cells. Central to these ongoing investigations are those studies concerning the nature of the molecular interaction between KP and its glucan receptor, which is expected to be a rather widespread molecule in consideration of the fact that KP, exactly as its source antibody molecules KT-MAb and Sc-Fv (5, 7) are, is active in vitro against several fungi and some bacteria, for instance, M. tuberculosis (unpublished data).
The consolidated experimental model of rat vaginal candidiasis (9) as well as a routinely adopted and well-established systemic mouse model of Candida infection (20) were used to test the therapeutic activity of KP in vivo. The results proved that KP was capable of significantly accelerating the clearance from the vagina a high fungus burden, similarly to a therapeutic course of fluconazole, a highly active, universally used anticandidal drug. KP greatly accelerated the fungus elimination from rat vagina in the first 2 to 3 days of infection, a critical period when the infecting yeast-form cells of the fungus undergo massive germination to hyphal elements and secrete critical virulence enzymes, such as the aspartyl proteinases (9). Importantly, KP did also prove to eradicate the infection caused by a fluconazole-resistant strain of C. albicans, a finding of special interest in view of the increased clinical concern about fluconazole resistance in this and allied fungi (1, 10).
A potent therapeutic effect of KP in the mouse model of systemic candidiasis was also observed. Although various natural or artificial peptides have been used to this aim, we are not aware of any of them being capable of curing a systemic experimental infection resulting in death in 2 to 3 days. Noteworthy, this curative effect was similarly exerted in normal immunocompetent mice as well as in SCID mice, demonstrating that the therapeutic benefit did not require the participation of host adaptive immunity. By its presumptive killing activity in vivo, the peptide could reduce the burden of the fungus to levels affordable by the host phagocytes, being the professional phagocytes normally active, if not more active, against the fungus in SCID mice. Neutrophils could be particularly involved, since they have a recognized critical role in the fight against systemic Candida infection (10). However, it is also possible that the KP activity does not require any help by the host because of its rapid and direct cytocidal effects, as also suggested by the results of treatment with KT-MAb in an aspergillosis model in neutropenic mice (5).
Finally, a major interest of our approach is the potential antimicrobial spectrum of KP, which may include most microorganisms possessing ß-glucan in their cell wall, inclusive of yeasts, most filamentous fungi, and some bacteria (5, 7, 16, 21). The observation that an engineered antibody fragment exerts an antimicrobial activity in vitro and in vivo comparable to that displayed by the original KT-scFv could open a new scenario in the biochemistry of antimicrobial peptides for the prevention and therapy of the numerous superficial and systemic diseases caused by the numerous KT-susceptible microorganisms.
|
ACKNOWLEDGMENTS |
|---|
We express our gratitude to Andrea Bernini and Simona Vaccari, who helped in sequence analysis, as well as to A. Botzios and A. M. Marella for their help in the preparation of the manuscript.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
| 1. | Alexander, B. D., and J. R. Perfect. 1997. Antifungal resistance trends toward the year 2000. Implications for therapy and new approaches. Drugs 54:657-678.[Medline] |
| 2. | Aliouat, E. M., J. C. Cailliez, N. Sèguy, E. Dei-Cas, L. Polonelli, M. Gerloni, S. Conti, and D. Camus. 1993. Inhibitory effect of a yeast killer toxin to the in vitro Pneumocystis carinii attachment. Serodiagn. Immunother. Infect. Dis. 5:102-106.[CrossRef] |
| 3. | Beninati, C. M., R. Oggioni, M. Boccanera, M. R. Spinosa, T. Maggi, S. Conti, W. Magliani, F. De Bernardis, G. Teti, A. Cassone, G. Pozzi, and L. Polonelli. 2000. Therapy of mucosal candidiasis by expression of an antiidiotype in human commensal bacteria. Nat. Biotechnol. 18:1060-1064.[CrossRef][Medline] |
| 4. | Cassone, A., S. Conti, F. De Bernardis, and L. Polonelli. 1997. Antibodies, killer toxins and antifungal immunoprotection: a lesson from nature? Immunol. Today 18:164-169.[CrossRef][Medline] |
| 5. | Cenci, E., A. Mencacci, A. Spreca, C. Montagnoli, A. Bacci, K. Perruccio, A. Velardi, W. Magliani, S. Conti, L. Polonelli, and L. Romani. 2002. Killer antiidiotypes protect from early invasive aspergillosis in a murine model of allogeneic T-cell-depleted bone marrow transplantation. Infect. Immun. 70:2375-2382. |
| 6. | Conti, S., F. Fanti, W. Magliani, M. Gerloni, D. Bertoletti, S. Salati, A. Cassone, and L. Polonelli. 1998. Mycobactericidal activity of human natural, monoclonal, and recombinant yeast killer toxin-like antibodies. J. Infect. Dis. 177:807-811.[Medline] |
| 7. | Conti, S., W. Magliani, S. Arseni, E. Dieci, R. Frazzi, A. Salati, P. E. Varaldo, and L. Polonelli. 2000. In vitro activity of monoclonal and recombinant yeast killer toxin-like antibodies against antibiotic-resistant gram-positive cocci. Mol. Med. 7:613-619. |
| 8. | Conti, S., C. Cantelli, M. Gerloni, P. Fisicaro, W. Magliani, D. Bertoletti, P. Mozzoni, D. Sullivan, D. Coleman, and L. Polonelli. 1996. Killer factor interference in mixed opportunistic yeast cultures. Mycopathologia 135:1-8.[CrossRef][Medline] |
| 9. | De Bernardis, F., R. Lorenzini, and A. Cassone. 1999. Rat model of Candida vaginal infection, p. 735-740. In O. Zak and M. A. Sande (ed.), Handbook of animal models of infection. Academic Press, New York, N.Y. |
| 10. | Edwards, J. E. 1991. Invasive Candida infections: evolution of a fungal pathogen. N. Engl. J. Med. 324:1060-1062.[Medline] |
| 11. | Green, L. J., P. Marder, L. L. Mann, L. C. Chio, and W. L. Current. 1999. LY 303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability. Antimicrob. Agents Chemother. 43:830-835. |
| 12. | Guyard, C., E. Dehecq, J. P. Tissier, L. Polonelli, E. Dei-Cas, J. C. Cailliez, and F. D. Menozzi. 2002. Involvement of beta-glucan in the wide spectrum antimicrobial activity of Williopsis saturnus var. mrakii MUCL 41968 killer toxin. Mol. Med. 8:686-694.[Medline] |
| 13. | Guyards, C., N. Sèguy, J. C. Cailliez, H. Drobecq, L. Polonelli, E. Dei-Cas, A. Mercenier, and F. D. Menozzi. 2002. Characterization of a Williopsis saturnus var. mrakii high molecular weight secreted killer toxin with broad-spectrum antimicrobial activity. J. Antimicrob. Chemother. 49:961-971. |
| 14. | Jerne, N. K. 1974. Towards a network theory of the immune system. Ann. Immunol. (Inst. Pasteur) 125C:373-389. |
| 15. | Kabat, E. A. 1988. Antibody complementarity and antibody structure. J. Immunol. 141(Suppl. 7):S25-S36.[Medline] |
| 16. | Klarzynski, O., B. Plesse, J. M. Joubert, J. C. Yvin, M. Kopp, B. Kloareg, and B. Fritig. 2000. Linear ß-1,3 glucans are elicitors of defense responses in tobacco. Plant Physiol. 124:1027-1037. |
| 17. | Klein, R. S., C. A. Harris, C. Butkus Small, B. Moll, M. Lesser, and G. H. Friendland. 1984. Oral candidiasis in high risk patients as the initial manifestation of the acquired immunodeficiency syndrome. N. Engl. J. Med. 311:354-358.[Abstract] |
| 18. | Magliani, W., S. Conti, F. De Bernardis, M. Gerloni, D. Bertoletti, P. Mozzoni, A. Cassone, and L. Polonelli. 1997. Therapeutic potential of antiidiotypic single chain antibodies with yeast killer toxin activity. Nat. Biotechnol. 15:155-158.[CrossRef][Medline] |
| 19. | Magliani, W., S. Conti, M. Gerloni, D. Bertolotti, and L. Polonelli. 1997. Yeast killer systems. Clin. Microbiol. Rev. 10:369-400.[Abstract] |
| 20. | Mencacci, A., A. Torosantucci, R. Spaccapelo, L. Romani, F. Bistoni, and A. Cassone. 1994. A mannoprotein constituent of Candida albicans that elicits different levels of delayed-type hypersensitivity, cytokine production, and anticandidal protection in mice. Infect. Immun. 62:5353-5360. |
| 21. | Milton, D. K., K. U. Alwis, L. Fisette, and M. Muilenberg. 2001. Enzyme-linked immunosorbent assay specific for (1 6) branched, (13)-ß-D-glucan detection in environmental samples. Appl. Environ. Microbiol. 67:5420-5424. |
| 22. | Pettoello-Mantovani, M., A. Noverino, L. Polonelli, G. Morace, S. Conti, L. di Martino, G. De Ritis, M. Iafusco, and M. Guendalini. 1995. Hansenula anomala killer toxin induces secretion, and severe acute injury in the rat intestine. Gastroenterology 109:1900-1906.[CrossRef][Medline] |
| 23. | Polonelli, L., F. De Bernardis, S. Conti, M. Boccanera, M. Gerloni, G. Morace, W. Magliani, C. Chezzi, and A. Cassone. 1994. Idiotypic intravaginal vaccination to protect against candidal vaginitis by secretory, yeast killer toxin-like antiidiotypic antibodies. J. Immunol. 152:3175-3182.[Abstract] |
| 24. | Polonelli, L., N. Sèguy, S. Conti, M. Gerloni, D. Bertoletti, C. Cantelli, W. Magliani, and J. C. Cailliez. 1997. Monoclonal yeast killer toxin-like candidacidal antiidiotypic antibodies. Clin. Diagn. Lab. Immunol. 4:142-146.[Abstract] |
| 25. | Polonelli, L., and G. Morace. 1986. Reevaluation of the yeast killer phenomenon. J. Clin. Microbiol. 24:866-869. |
| 26. | Polonelli, L., F. Fanti, S. Conti, L. Campani, M. Gerloni, M. Castagnola, G. Morace, and C. Chezzi. 1990. Detection by immunofluorescent anti-idiotypic antibodies of yeast killer toxin cell wall receptors of Candida albicans. J. Immunol. Methods 132:205-209.[CrossRef][Medline] |
| 27. | Polonelli, L., R. Lorenzini, F. De Bernardis, M. Gerloni, S. Conti, G. Morace, W. Magliani, and C. Chezzi. 1993. Idiotypic vaccination: immunoprotection mediated by antiidiotypic antibodies with antibiotic activity. Scand. J. Immunol. 37:105-110.[CrossRef][Medline] |
| 28. | Polonelli, L., and G. Morace. 1987. Production and characterization of yeast killer toxin monoclonal antibodies. J. Clin. Microbiol. 25:460-462. |
| 29. | Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. |
| 30. | Séguy, N., L. Polonelli, E. Dei-Cas, and J. C. Cailliez. 1997. Monoclonal killer toxin-like antiidiotypic antibodies to control rat pneumocystosis. J. Eukaryot. Microbiol. 44:37S. |
| 31. | Tipper, D. J., and K. A. Bostian. 1984. Double-stranded ribonucleic acid killer systems in yeasts. Microbiol. Rev. 48:125-156. |
| 32. | Torosantucci, A., P. Chiani, and A. Cassone. 2000. Differential chemokine response of human monocytes to yeast and hyphal forms of Candida albicans and its relation to the ß 1-6 glucan of the fungal cell wall. J. Leukoc. Biol. 68:923-932. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
