Jimena Cortes

Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to U937 macrophages and A549 epithelial cells.

Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complexs strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54‐kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surface protein. Binding assays led to recognizing high activity binding peptides (HABP); five HABPs specifically bound to U937 cells, and six specifically bound to A549 cells. HABP circular dichroism suggested that they had an α‐helical structure. HABP–target cell interaction was determined to be specific and saturable; some of them also displayed greater affinity for A549 cells than U937 cells. The critical amino acids directly involved in their interaction with U937 cells were also determined. Two probable receptor molecules were found on U937 cells and five on A549 for the two HABPs analyzed. These observations have important biological significance for studying bacillus–target cell interactions and implications for developing strategies for controlling this disease.

Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells

Synthetic 20-mer long non-overlapped peptides, from STEVOR protein, were tested in RBC binding assays for identifying STEVOR protein regions having high RBC binding activity and evaluating whether these regions inhibit Plasmodium falciparum in vitro invasion. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay with high activity binding peptides (HABPs). HABP binding assays using RBCs previously treated with enzymes were carried out to study the nature of the receptor. The molecular weight of RBC surface proteins interacting with HABPs was determined by cross-linking assays and SDS-PAGE analysis. RBC binding assays revealed that peptides 30561 (41MKSRRLAEIQLPKCPHYNND60), 30562 (61PELKKIIDKLNEERIKKYIE80) and 30567 (161ASCCKVHDNYLDNLKKGCFG180) bound saturably and with high binding activity, presenting nanomolar affinity constants. HABP binding activity to RBCs previously treated with neuraminidase and trypsin decreased, suggesting that these peptides bound to RBC surface proteins and that such binding could be sialic acid dependent. Cross-linking and SDS-PAGE assays showed that the three HABPs specifically bound to 30 and 40 kDa molecular weight RBC membrane proteins. Peptides 30561, 30562 and 30567 inhibited P. falciparum in vitro invasion of red blood cells in a concentration-dependent way. Goat sera having STEVOR protein polymeric peptides antibodies inhibit parasite in vitro invasion depending on concentration. Three peptides localized in STEVOR N-terminal and central regions had high, saturable, binding activity to 30 and 40 kDa RBC membrane proteins. These peptides inhibited the parasites in vitro invasion, suggesting that STEVOR protein regions are involved in P. falciparum invasion processes during intra-erythrocyte stage.

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines.

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT‐PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25‐kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20‐amino‐acid‐long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross‐linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.

Human papillomavirus type 16 and 18 L1 protein peptide binding to VERO and HeLa cells inhibits their VLPs binding.

Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being associated with the development of anogenital cancer. The L1 Major Capsid Protein (L1) represents about 90% of total HPV protein and is involved in virus‐host cell interaction, but little is known about this binding process. L1 sequences from HPV types 16 and 18 were synthesized in 56 20‐mer peptides, covering the entire protein, HPLC‐purified, 125I‐radiolabeled and tested in VERO and HeLa cell‐binding assays to identify those peptides with high specific binding activity. Peptides 18283 (residues 54–77) and 18294 (274–308) from HPV16 L1, as well as 18312 (59–78) and 18322 (259–278) from HPV18 L1, presented high specific target cell binding activity. Peptide 18283 and 18294 affinity constants were 300 and 600 nM, respectively. Enzyme cell treatment before binding assay indicated that VERO and HeLa cell peptide receptor is a surface‐exposed protein. There was a 60% reduction in peptide 18283 binding to heparin lyase‐treated cells. Cross‐linking assays showed that these proteins molecular weights were around 69 and 54 kDa. Peptides 18283 and 18294 specifically inhibited HPV‐16 VLP binding to HeLa cells. According to the L1‐ and VLP‐reported structure, both peptides are close on the VLP‐surface, belonging to the outer surface broad pockets suggested as being potential receptor sites. Furthermore, it has been reported that a conserved motif from peptide 18294 is the target for neutralizing antibodies. These results suggest that such binding sequences are used by the virus as cell‐binding regions.

P. falciparum pro-histoaspartic protease (proHAP) protein peptides bind specifically to erythrocytes and inhibit the invasion process in vitro

Abstract Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasites invasion of erythrocytes; however, this proteins role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101LKNYIKESVKLFNKGLTKKS120) and 30610 (121YLGSEFDNVELKDLANVLSF140). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26- and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro.

Open-label randomised phase III trial of vinflunine versus an alkylating agent in patients with heavily pretreated metastatic breast cancer

Background There is no standard treatment after progression on second-line chemotherapy for metastatic breast cancer (MBC). We compared vinflunine with physicians choice of alkylating agent (AA) for patients with heavily pretreated MBC. Patients and methods In this open-label phase III trial, patients with MBC were included if they had received at least two prior chemotherapy regimens for MBC and had received anthracycline, taxane, antimetabolite and vinca alkaloid therapy. Patients were no longer candidates for these chemotherapies because of resistance and/or intolerance. Patients were randomised to either vinflunine 280 mg/m2 intravenously every 3 weeks (q3w) or AA monotherapy q3w. Stratification factors were performance status, number of prior chemotherapy lines for MBC, disease measurability and study site. The primary end point was overall survival (OS). Results A total of 594 patients were randomised (298 to vinflunine, 296 to AA). There was no difference between treatment arms in OS (hazard ratio 1.04, P = 0.67; median 9.1 months for vinflunine versus 9.3 months for AA), progression-free survival (hazard ratio 0.94, P = 0.49; median 2.5 versus 1.9 months, respectively) or overall response rate (6% versus 4%, respectively). However, the disease control rate was significantly higher with vinflunine than AA (44% versus 35%, respectively; P = 0.04). The most common adverse events (any grade) were haematological and gastrointestinal disorders and asthenia in both arms. The most common grade 3/4 adverse events were neutropenia (19% versus 11% with vinflunine versus AA, respectively) and asthenia (10% versus 4%). Conclusions Vinflunine 280 mg/m2 q3w did not improve OS compared with the physicians choice of AA as third- or later-line therapy for MBC. Vinflunine demonstrated an acceptable safety profile, suggesting that vinflunine 320 mg/m2 merits evaluation. ClinicalTrials.gov NCT01091168.

Erratum to “Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes”: [Peptides 24 (2003) 647–657]

Table 2LSA-1 protein high binding peptides and sera analogues invasion and development inhibition assays% Invasion inhibition % Development inhibition100 M 200 M 100 M 200 MPeptide20630 10 ± 434± 20± 30± 320633 48 ± 188± 115± 485± 16762 13 ± 2 100 ± 20± 557± 2Chloroquine 100 ± 2 100 ± 2 100 ± 2 100 ± 2Sera1:1011 70 ± 512 8.8 ± 7Preimmune 9.5 ± 5Chloroquine 100 ± 4Peptides belonging to LSA-1 (20630 and 20633) inhibited in vitro merozoite invasion of erythrocytes. Peptide 20633 has the highest degree of inhibitionparasite invasion and development. Peptide 6762 was used as positive control. As shown, of the two sera assayed, one monkey 11 sera, inhibited invasionby70%, whilst the other two behaved as did the control.