Immunomodulatory Peptide from Cystatin, a Natural Cysteine Protease Inhibitor, against Leishmaniasis as a Model Macrophage Disease▿
Identifieur interne : 000194 ( Pmc/Corpus ); précédent : 000193; suivant : 000195Immunomodulatory Peptide from Cystatin, a Natural Cysteine Protease Inhibitor, against Leishmaniasis as a Model Macrophage Disease▿
Auteurs : Snigdha Mukherjee ; Anindita Ukil ; Pijush K. DasSource :
- Antimicrobial Agents and Chemotherapy [ 0066-4804 ] ; 2007.
Abstract
Cystatin, a natural cysteine protease inhibitor, has strong antileishmanial activity, which is due to its potential to induce nitric oxide (NO) generation from macrophages. Cysteine protease-inhibitory activity and NO-up-regulatory activity correspond to different regions, as revealed by the dissection of cystatin cDNA into nonoverlapping fragments. By using synthetic overlapping peptides, the NO-up-regulatory activity was found to be confined to a 10-mer sequence. In addition to having reasonable inhibitory effects on amastigote multiplication within macrophages (50% inhibitory concentration, 5.2 μg/ml), 97 and 93% suppression, respectively, of liver and spleen parasite burdens was achieved with the 10-mer peptide at a dose of 0.5 mg/kg of body weight/day, given consecutively for 4 days along with a suboptimal dose of gamma interferon in a 45-day mouse model of visceral leishmaniasis. Peptide treatment modulated the levels of cytokine secretion by infected splenocytes, with increased levels of interleukin-12 and tumor necrosis factor alpha and increased inducible NO synthase production, and also resulted in resistance to reinfection. The generation of a natural peptide from cystatin with robust immunomodulatory potential may therefore provide a promising therapeutic agent for macrophage-associated diseases.
Url:
DOI: 10.1128/AAC.01555-06
PubMed: 17339373
PubMed Central: 1855531
Links to Exploration step
PMC:1855531Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Immunomodulatory Peptide from Cystatin, a Natural Cysteine Protease Inhibitor, against Leishmaniasis as a Model Macrophage Disease<xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Mukherjee, Snigdha" sort="Mukherjee, Snigdha" uniqKey="Mukherjee S" first="Snigdha" last="Mukherjee">Snigdha Mukherjee</name><affiliation><nlm:aff id="aff0">Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India</nlm:aff></affiliation></author><author><name sortKey="Ukil, Anindita" sort="Ukil, Anindita" uniqKey="Ukil A" first="Anindita" last="Ukil">Anindita Ukil</name><affiliation><nlm:aff id="aff0">Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India</nlm:aff></affiliation></author><author><name sortKey="Das, Pijush K" sort="Das, Pijush K" uniqKey="Das P" first="Pijush K." last="Das">Pijush K. Das</name><affiliation><nlm:aff id="aff0">Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">17339373</idno><idno type="pmc">1855531</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855531</idno><idno type="RBID">PMC:1855531</idno><idno type="doi">10.1128/AAC.01555-06</idno><date when="2007">2007</date><idno type="wicri:Area/Pmc/Corpus">000194</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000194</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Immunomodulatory Peptide from Cystatin, a Natural Cysteine Protease Inhibitor, against Leishmaniasis as a Model Macrophage Disease<xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Mukherjee, Snigdha" sort="Mukherjee, Snigdha" uniqKey="Mukherjee S" first="Snigdha" last="Mukherjee">Snigdha Mukherjee</name><affiliation><nlm:aff id="aff0">Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India</nlm:aff></affiliation></author><author><name sortKey="Ukil, Anindita" sort="Ukil, Anindita" uniqKey="Ukil A" first="Anindita" last="Ukil">Anindita Ukil</name><affiliation><nlm:aff id="aff0">Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India</nlm:aff></affiliation></author><author><name sortKey="Das, Pijush K" sort="Das, Pijush K" uniqKey="Das P" first="Pijush K." last="Das">Pijush K. Das</name><affiliation><nlm:aff id="aff0">Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India</nlm:aff></affiliation></author></analytic><series><title level="j">Antimicrobial Agents and Chemotherapy</title><idno type="ISSN">0066-4804</idno><idno type="eISSN">1098-6596</idno><imprint><date when="2007">2007</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Cystatin, a natural cysteine protease inhibitor, has strong antileishmanial activity, which is due to its potential to induce nitric oxide (NO) generation from macrophages. Cysteine protease-inhibitory activity and NO-up-regulatory activity correspond to different regions, as revealed by the dissection of cystatin cDNA into nonoverlapping fragments. By using synthetic overlapping peptides, the NO-up-regulatory activity was found to be confined to a 10-mer sequence. In addition to having reasonable inhibitory effects on amastigote multiplication within macrophages (50% inhibitory concentration, 5.2 μg/ml), 97 and 93% suppression, respectively, of liver and spleen parasite burdens was achieved with the 10-mer peptide at a dose of 0.5 mg/kg of body weight/day, given consecutively for 4 days along with a suboptimal dose of gamma interferon in a 45-day mouse model of visceral leishmaniasis. Peptide treatment modulated the levels of cytokine secretion by infected splenocytes, with increased levels of interleukin-12 and tumor necrosis factor alpha and increased inducible NO synthase production, and also resulted in resistance to reinfection. The generation of a natural peptide from cystatin with robust immunomodulatory potential may therefore provide a promising therapeutic agent for macrophage-associated diseases.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Antimicrob Agents Chemother</journal-id><journal-id journal-id-type="publisher-id">aac</journal-id><journal-title>Antimicrobial Agents and Chemotherapy</journal-title><issn pub-type="ppub">0066-4804</issn><issn pub-type="epub">1098-6596</issn><publisher><publisher-name>American Society for Microbiology (ASM)</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">17339373</article-id><article-id pub-id-type="pmc">1855531</article-id><article-id pub-id-type="publisher-id">1555-06</article-id><article-id pub-id-type="doi">10.1128/AAC.01555-06</article-id><article-categories><subj-group subj-group-type="heading"><subject>Mechanisms of Action: Physiological Effects</subject></subj-group></article-categories><title-group><article-title>Immunomodulatory Peptide from Cystatin, a Natural Cysteine Protease Inhibitor, against Leishmaniasis as a Model Macrophage Disease<xref ref-type="fn" rid="fn1">▿</xref> </article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Mukherjee</surname><given-names>Snigdha</given-names></name><xref ref-type="aff" rid="aff0"></xref></contrib><contrib contrib-type="author"><name><surname>Ukil</surname><given-names>Anindita</given-names></name><xref ref-type="aff" rid="aff0"></xref></contrib><contrib contrib-type="author"><name><surname>Das</surname><given-names>Pijush K.</given-names></name><xref ref-type="aff" rid="aff0"></xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff0">Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India</aff><author-notes><fn id="cor1"><label>*</label><p>Corresponding author. Mailing address: Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700032, India. Phone: 91-33-24140921. Fax: 91-33-473-5197, ext. 0284. E-mail: <email>pijushkdas@vsnl.com</email></p></fn></author-notes><pub-date pub-type="ppub"><month>5</month><year>2007</year></pub-date><pub-date pub-type="epub"><day>5</day><month>3</month><year>2007</year></pub-date><volume>51</volume><issue>5</issue><fpage>1700</fpage><lpage>1707</lpage><history><date date-type="received"><day>15</day><month>12</month><year>2006</year></date><date date-type="rev-recd"><day>3</day><month>2</month><year>2007</year></date><date date-type="accepted"><day>22</day><month>2</month><year>2007</year></date></history><permissions><copyright-statement>Copyright © 2007, American Society for Microbiology</copyright-statement></permissions><self-uri xlink:title="pdf" xlink:href="zac00507001700.pdf"></self-uri><abstract><p>Cystatin, a natural cysteine protease inhibitor, has strong antileishmanial activity, which is due to its potential to induce nitric oxide (NO) generation from macrophages. Cysteine protease-inhibitory activity and NO-up-regulatory activity correspond to different regions, as revealed by the dissection of cystatin cDNA into nonoverlapping fragments. By using synthetic overlapping peptides, the NO-up-regulatory activity was found to be confined to a 10-mer sequence. In addition to having reasonable inhibitory effects on amastigote multiplication within macrophages (50% inhibitory concentration, 5.2 μg/ml), 97 and 93% suppression, respectively, of liver and spleen parasite burdens was achieved with the 10-mer peptide at a dose of 0.5 mg/kg of body weight/day, given consecutively for 4 days along with a suboptimal dose of gamma interferon in a 45-day mouse model of visceral leishmaniasis. Peptide treatment modulated the levels of cytokine secretion by infected splenocytes, with increased levels of interleukin-12 and tumor necrosis factor alpha and increased inducible NO synthase production, and also resulted in resistance to reinfection. The generation of a natural peptide from cystatin with robust immunomodulatory potential may therefore provide a promising therapeutic agent for macrophage-associated diseases.</p></abstract></article-meta></front><floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label><caption><p>Antileishmanial activity of cystatin through the generation of NO. (A) NO production by peritoneal macrophages (10<sup>6</sup>/ml) incubated for 48 h in culture medium with cystatin (Cys; 5 × 10<sup>−7</sup> M), <italic>L. donovani</italic> (<italic>L.d.</italic>; macrophage/parasite ratio, 1:10), IFN-γ (100 U/ml), IFN-γ plus <italic>L. donovani</italic>, IFN-γ plus cystatin, and IFN-γ plus cystatin plus <italic>L. donovani</italic>. (B) Generation of NO in peritoneal macrophages isolated from mice which received i.v. injections of either cystatin (5 mg/kg/day) or IFN-γ (10<sup>4</sup> U/mouse) or both for 4 consecutive days. Macrophages were isolated 10 h after the last injection. Data represent the means ± SD of results from three experiments. AMT (5 mg/kg/day) was used along with cystatin and IFN-γ in a separate experiment. (C) In vivo antileishmanial activity of cystatin and cystatin plus IFN-γ. Mice were challenged with 10<sup>7</sup> promastigotes, and after 10 days of infection, they were treated with various i.v. doses of cystatin with or without IFN-γ (10<sup>4</sup> U/mouse) daily for 4 consecutive days. Spleen parasite burdens were determined 45 days after infection and are expressed as the mean log<sub>10</sub> number of LDU ± SD for six animals. In another set of experiments, AMT (5 mg/kg/day) was used along with cystatin and IFN-γ. The log<sub>10</sub> number of LDU in the infected control was 2.8 ± 0.08. (D) In vitro antileishmanial activity. Macrophages were infected with <italic>L. donovani</italic> promastigotes, excess parasites were washed off, and cells were treated with graded concentrations of cystatin with or without IFN-γ (100 U/ml) for 48 h at 37°C. AMT (10 μM) was given along with cystatin plus IFN-γ in a separate set of experiments. The parasites inside each macrophage were counted. The infected controls had 7.22 ± 0.65 amastigotes/macrophage. The nature of iNOS was determined by RT-PCR analysis of the mRNA transcript (E) and by Western blot analysis of the protein levels (F) corresponding to various treatment regimens. Band intensities were analyzed by densitometry (E<sub>1</sub> and F<sub>1</sub>).</p></caption><graphic xlink:href="zac0050764840001"></graphic></fig><fig position="float" id="f2"><label>FIG. 2.</label><caption><p>Up-regulation of nitrite release by members of the cystatin superfamily complexed with papain. Papain was inactivated by reduction and alkylation. Inactivation was indicated by the complete loss of enzyme activity. The carboxymethylated papain (10<sup>−5</sup> M) was then mixed at 37°C for 45 min with 10<sup>−6</sup> M chicken cystatin, human stefin B, or T-kininogen. The free form or saturated forms were then introduced into the culture medium for 48 h before the nitrite measurement. Data represent the means ± SD of results from three experiments.</p></caption><graphic xlink:href="zac0050764840002"></graphic></fig><fig position="float" id="f3"><label>FIG. 3.</label><caption><p>Identification of the NO-stimulatory domain of cystatin. (A) Three nonoverlapping fragments representing the N-terminal (Cst I, aa 1 to 28), the intermediate (Cst II, aa 29 to 72), and the C-terminal (Cst III, aa 79 to 116) domains were cloned into a prokaryotic expression vector (pGEX-5X-2) and expressed as GST fusion proteins. Lanes 2, 4, and 6 show the Coomassie-stained SDS-PAGE gel of GST fusion proteins (∼30 kDa) with Cst I, Cst II, and Cst III, respectively, whereas lanes 1, 3, and 5 show the induced bacterial lysate. (B) Corresponding Western blot analysis using polyclonal anti-cystatin antibody. The purified recombinants as GST fusion proteins (lane 1, recombinant GST; lane 2, GST-Cst I; lane 3, GST-Cst II; and lane 4, GST-Cst III) were separated on a 10% SDS-PAGE gel (C) and immunoblotted with polyclonal anti-cystatin antibody (D). (E) Macrophages were incubated with graded concentrations of recombinant GST fusion products of Cst I, Cst II, and Cst III along with a suboptimal dose of IFN-γ (100 U/ml) for 48 h, and the release of NO<sub>2</sub><sup>−</sup> was quantified. Data are means ± SD of results from three experiments. The nature of iNOS expression was also determined by RT-PCR analysis of the mRNA transcript levels (F) and by Western blotting for analysis of protein levels (G) in response to 1 × 10<sup>−7</sup> M cystatin (Cys) and various recombinants as GST fusion peptides, along with 100 U of IFN-γ/ml. Band intensities were analyzed by densitometry (F<sub>1</sub> and G<sub>1</sub>). Cys II and Cys III, Cst II and Cst III.</p></caption><graphic xlink:href="zac0050764840003"></graphic></fig><fig position="float" id="f4"><label>FIG. 4.</label><caption><p>Minimal peptide sequence with NO-stimulatory potential and its antileishmanial activity. (A) Macrophages were incubated with overlapping synthetic peptides (8-mer) derived from Cst I along with IFN-γ (100 U/ml), and NO<sub>2</sub><sup>−</sup> release was quantified. (B) NO<sub>2</sub><sup>−</sup> release from macrophages in response to overlapping 10-mer peptides containing the most potent 8-mer peptide in terms of NO production was measured. (C) Dose response curves for various concentrations of compound B in combination with IFN-γ (100 U/ml) representing the release of NO<sub>2</sub><sup>−</sup> by macrophages in the presence or absence of either anti-compound B antibody or AMT (10 μM). (D) Infected macrophages were treated with graded concentrations of compound B in combination with IFN-γ (100 U/ml) in the presence or absence of either anti-compound B antibody or AMT (10 μM). Infected controls contained 7.14 ± 0.69 amastigotes/macrophages. Data are means ± SD of results from three experiments.</p></caption><graphic xlink:href="zac0050764840004"></graphic></fig><fig position="float" id="f5"><label>FIG. 5.</label><caption><p>Effect of compound B treatment on visceral infection in BALB/c mice. (A) Various doses of compound B ranging from 0.05 to 1 mg/kg/day were given i.v. along with IFN-γ (10<sup>4</sup> U/mouse) for 4 consecutive days beginning 10 days after infection. The parasite burdens in livers and spleens were then determined at 45 days after infection. Anti-peptide antibody was given along with compound B and IFN-γ in a separate set of experiments. (B) The course of visceral reinfection was studied by i.v. administration of 10<sup>7</sup> promastigotes into naïve, age-matched BALB/c mice and cured (1-mg/kg/day-compound B-treated) mice. In one group of cured mice, AMT (5 mg/kg/day) was i.v. administered 1 week after reinfection for 2 weeks. Determining liver and spleen parasite burdens, expressed as the log<sub>10</sub> numbers of LDU, allowed for the monitoring of the progression of infection in all the cases. Results are from three experiments and indicate the means ± SD for 5 to 7 mice at each time point. Ab, antibody. (C and D) Cytokine profiles of <italic>L. donovani</italic>-infected mice as analyzed by RT-PCR. The levels of expression of IL-10, IL-12, TNF-α, iNOS, and β-actin mRNA by spleen cells of infected mice treated i.v. with the peptide and/or IFN-γ in the presence or absence of anti-peptide antibody were determined and compared to those by spleen cells of mice receiving cystatin and IFN-γ treatment. RT products were visualized by ethidium bromide staining. RNA samples were obtained from five mice in each group. Results shown are representative of those for five separate samples. β-Actin expression levels were used as controls for RNA content and integrity. Band intensities were analyzed by densitometry (C<sub>1</sub> and D<sub>1</sub>).</p></caption><graphic xlink:href="zac0050764840005"></graphic></fig></floats-wrap></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Sante/explor/CovidV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000194 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000194 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Sante
|area= CovidV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:1855531
|texte= Immunomodulatory Peptide from Cystatin, a Natural Cysteine Protease Inhibitor, against Leishmaniasis as a Model Macrophage Disease▿
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:17339373" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a CovidV1
| This area was generated with Dilib version V0.6.33. |  |