Carlos Velazquez

Antibacterial and free‐radical scavenging activities of Sonoran propolis

Aims:  To evaluate the antibacterial and free‐radical scavenging (FRS) activities of propolis collected from three different areas of Sonoran Desert in northwestern Mexico [Pueblo de Alamos (PAP), Ures (UP) and Caborca (CP)].

Low number of peripheral blood B lymphocytes in patients with pulmonary tuberculosis.

The cellular immune response plays a critical role in the containment of persistent Mycobacterium tuberculosis infection; however, the immunological mechanisms that lead to its control are not completely identified. The goal of this study was to evaluate B (CD19+) and T (CD3+) peripheral blood lymphocyte profiles and T-cell subsets (CD4+ and CD8+) in patients with pulmonary tuberculosis (TB). Percentages (p = 0.02) and absolute numbers (p = 0.005) of B cells were significantly lower in patients with pulmonary TB than in healthy donors. In contrast, percentages (p = 0.12) and absolute numbers (p = 0.14) of T cells were similar in TB patients and healthy donors. No significant differences in percentages of CD4+ (p = 0.19) or CD8+ (p = 0.85) T cells between patients and healthy donors were observed. In summary, patients with pulmonary tuberculosis had a lower number of peripheral blood B lymphocytes than healthy controls.

Giardia lamblia infection induces different secretory and systemic antibody responses in mice.

The adult mouse model of Giardia lamblia infection serves as an excellent animal model to understand the immunological mechanisms involved in the control and clearance of Giardia infection. Little is known about the G. lamblia‐specific antigens that stimulate the humoral immune response in this model of giardiasis. We analysed the secretory and systemic antibody responses to G. lamblia during primary and secondary infection in C3H/HeJ adult mice. Faecal IgA and Serum IgG anti‐G. lamblia antibodies were observed at week 2 post‐infection. Serum IgG responses remained constant over the next several weeks, whereas faecal IgA titres continued to rise from weeks 2–6 post‐infection. Western blot analysis revealed that intestinal IgA and serum IgG antibody responses were directed toward several distinct proteins of G. lamblia. Certain proteins appeared to be recognized by both faecal IgA and serum IgG, whereas other antigens were specific for either the secretory or systemic antibody responses. G. lamblia primary and secondary infections were associated with differences in the antibody recognition pattern. The biochemical and immunological characterization of these antigens will help us to better understand the immunobiology of the G. lamblia–host interaction.

Physical properties and antibacterial activity of chitosan/acemannan mixed systems.

The aim of the present study was to investigate the mechanical and thermal properties of mixed chitosan-acemannan (CS-AC) mixed gels and the antibacterial activity of dilute mixed solutions of both polysaccharides. Physical hydrogels of chitosan comprising varying amounts of non-gelling acemannan were prepared by controlled neutralization of chitosan using ammonia. As the overall acemannan concentration in the mixed hydrogel increased while fixing that of CS, the mechanical strength decreased. These results indicate that AC perturbs the formation of elastic junctions and overall connectivity as it occurs in the isolated CS network. Heterotypic associations between CS and AC leading to the formation of more compact microdomains may be at play in reducing the density of the gel network consolidated by CS, possibly due to shorter gel junctions. Micro-DSC studies at pH 12.0 seem consistent with the suggestion that molecular heterotypic associations between CS and AC may be at play in determining the overall physical properties of the mixed gel systems. In dilute solution, CS showed antimicrobial activity against Staphylococcus aureus but not against Escherichia coli; AC did not exert antimicrobial activity against any of the two bacterial species. In blended solutions of both polysaccharides, as the amount of AC increased, the antimicrobial activity of the system against S. aureus ceased. In conclusion, this study demonstrates that it is feasible to incorporate acemannan in chitosan-acemannan gels and that although the mechanical strength decreases due to the presence of AC, the gel network persists even at high amount of AC. This study anticipates that the CS-AC mixed gels may offer promise for the future development of biomaterials such as scaffolds to be used in wound therapy.

Antimutagenicity and Antiproliferative Studies of Lipidic Extracts from White Shrimp (Litopenaeus vannamei)

An organic extract from fresh shrimp (Litopenaeus vannamei) was studied for antimutagenic and antiproliferative properties using Salmonella typhimurium tester strains TA98 and TA100 with metabolic activation (S9) and a cancer cell line (B-cell lymphoma), respectively. Shrimp extract was sequentially fractionated by thin layer chromatography (TLC) and each fraction was tested for antimutagenic and antiproliferative activities. Crude organic extracts obtained from shrimp reduced the number of revertants caused by aflatoxina B1, showing a dose-response type of relationship. Sequential TLC fractionation of the active extracts produced several antimutagenic and/or antiproliferative fractions. These results suggested that the lipid fraction of the tested species contained compounds with chemoprotective properties that reduce the mutagenicity of AFB1 and proliferation of a cancer cell line.

Independent Selection by I-Ak Molecules of Two Epitopes Found in Tandem in an Extended Polypeptide Antigen

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31–47 and 48–63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48–63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31–47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31–47 or 48–62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.

Host defences against Giardia lamblia.

Giardia spp. is a protozoan parasite that inhabits the upper small intestine of mammals and other species and is the aetiological agent of giardiasis. It has been demonstrated that nitric oxide, mast cells and dendritic cells are the first line of defence against Giardia. IL‐6 and IL‐17 play an important role during infection. Several cytokines possess overlapping functions in regulating innate and adaptive immune responses. IgA and CD4+ T cells are fundamental to the process of Giardia clearance. It has been suggested that CD4+ T cells play a double role during the anti‐Giardia immune response. First, they activate and stimulate the differentiation of B cells to generate Giardia‐specific antibodies. Second, they act through a B‐cell‐independent mechanism that is probably mediated by Th17 cells. Several Giardia proteins that stimulate humoral and cellular immune responses have been described. Variant surface proteins, α‐1 giardin, and cyst wall protein 2 can induce host protective responses to future Giardia challenges. The characterization and evaluation of the protective potential of the immunogenic proteins that are associated with Giardia will offer new insights into host–parasite interactions and may aid in the development of an effective vaccine against the parasite.

Apoptotic induction by pinobanksin and some of its ester derivatives from Sonoran propolis in a B-cell lymphoma cell line

Propolis is a resinous substance produced by honeybees (Apis mellifera) from the selective collection of exudates and bud secretions from several plants. In previous works, we reported the antiproliferative activity of Sonoran propolis (SP) on cancer cells; in addition we suggested the induction of apoptosis after treatment with SP due to the presence of morphological changes and a characteristic DNA fragmentation pattern. Herein, in this study we demonstrated that the antiproliferative effect of SP is induced through apoptosis in a B-cell lymphoma cancer cell line, M12.C3.F6, by an annexin V-FITC/Propidium iodide double labeling. This apoptotic effect of SP resulted to be mediated by modulations in the loss of mitochondrial membrane potential (ΔΨm) and through activation of caspases signaling pathway (3, 8 and 9). Afterward, in order to characterize the chemical constituents of SP that induce apoptosis in cancer cells, an HPLC-PDA-ESI-MS/MS method followed by a preparative isolation procedure and NMR spectroscopy analysis have been used. Eighteen flavonoids, commonly described in propolis from temperate regions, were characterized. Chrysin, pinocembrin, pinobanksin and its ester derivatives are the main constituents of SP and some of them have never been reported in SP. In addition, two esters of pinobanksin (8 and 13) are described by first time in propolis samples in general. The antiproliferative activity on M12.C3.F6 cells through apoptosis induction was exhibited by pinobanksin (4), pinobanksin-3-O-propanoate (14), pinobanksin-3-O-butyrate (16), pinobanksin-3-O-pentanoate (17), and the already reported galangin (11), chrysin (9) and CAPE. To our knowledge this is the first report of bioactivity of pinobanksin and some of its ester derivatives as apoptosis inducers. Further studies are needed to advance in the understanding of the molecular basis of apoptosis induction by SP and its constituents, as well as the structure-activity relationship of them.

Identification of T‐cell stimulating antigens from Giardia lamblia by using Giardia‐specific T‐cell hybridomas

T‐cell immune response plays an important role in controlling Giardia lamblia infections. Little is known about the G. lamblia‐specific antigens that stimulate a cell‐mediated immune response. The aim of the present study was to identify T‐cell stimulating G. lamblia antigens. For this purpose, we generated a group of Giardia‐specific T‐cell hybridomas (2F9, 4D5, 6D10, 8B9, 9B10, 10F7 and 10G5). Hybridomas were screened for reactivity with G. lamblia protein extract by the CTLL bioassay. These T‐cell hybridomas did not exhibit any significant activation either in the absence of G. lamblia protein extract or in the presence of irrelevant antigen (hen white egg lysozyme). To further characterize the T‐cell hybridomas generated, we selected three hybridomas (10G5, 4D5 and 9B10). Giardia lamblia proteins of 90–110, 65–77 and 40–64 kDa showed T‐cell stimulating activity for the hybridomas 10G5, 4D5 and 9B10, respectively, in a concentration‐dependent manner. Protein extract obtained from different G. lamblia strains (GS/M‐83‐H7, WB C6 and a clinical isolate (YJJ)) stimulated all T‐cell hybridomas, indicating that T‐cell‐stimulating antigens are expressed among different G. lamblia strains. In conclusion, we identified T‐cell stimulating G. lamblia antigens by using Giardia‐specific T‐cell hybridomas. To our knowledge, these hybridomas are the first‐described T‐cell hybridomas specific for G. lamblia.

Sonoran propolis and some of its chemical constituents inhibit in vitro growth of Giardia lamblia trophozoites.

Propolis is a cereus resin with a complex chemical composition that possesses a wide range of biological activities. The aim of this study was to evaluate the in vitro anti-Giardia lamblia activity of Sonoran propolis collected from three different areas of Sonoran Desert in northwestern Mexico (Caborca, Pueblo de Alamos, and Ures) and some of its chemical constituents. Additionally, we also analyzed the seasonal effect on the anti-G. lamblia activity of propolis. G. lamblia trophozoite cultures were treated with different concentrations of Sonoran propolis or chemical compounds during 48 h cell proliferation and cell viability were determined. Ures propolis showed the highest inhibitory activity against G. lamblia (IC50 63.8 ± 7.1 µg/mL) in a dose-dependent manner (Ures > Pueblo de Alamos > Caborca). Season had a significant effect on the in vitro anti-G. lamblia activity of Ures propolis. Summer propolis showed the highest inhibitory effect on the G. lamblia trophozoite growth (IC50 23.8 ± 2.3 µg/mL), followed by propolis collected during winter (IC50 59.2 ± 34.7 µg/mL), spring (IC50 102.5 ± 15.3 µg/mL), and autumn (IC50 125.0 ± 3.1 µg/mL). Caffeic acid phenethyl ester, an Ures propolis exclusive constituent, had the highest growth-inhibitory activity towards G. lamblia [IC50 63.1 ± 0.9 µg/mL (222.1 ± 3.2 µM)]. To our knowledge, this is the first study showing that caffeic acid phenethyl ester possesses antiparasitic activity against G. lamblia. Naringenin [IC50 125.7 ± 20.7 µg/mL (461.8 ± 76.3 µM)], hesperetin [IC50 149.6 ± 24.8 µg/mL (494.9 ± 82.2 µM)], and pinocembrin [IC50 174.4 ± 26.0 µg/mL (680.6 ± 101.7 µM)] showed weak anti-G. lamblia activity. On the other hand, chrysin and rutin did not show significant antiparasitic activity. In conclusion, our results suggest that Sonoran propolis and some of its chemical constituents had inhibitory effects on the in vitro growth of G. lamblia trophozoites.