A novel experimental immunomodulatory therapy against Nocardia brasiliensis in a BALB/c murine model.

BACKGROUND: Mycetoma is recognized as a neglected tropical disease and there are still therapeutic challenges, especially in cases recalcitrant to standard therapy or with high risk of dissemination. Subcultures have been used previously to decrease the virulence of human pathogens. Previous reports have demonstrated that after carrying out 200 subcultures of Nocardia brasiliensis, a decrease in virulence was observed. AIM: To evaluate the effect of attenuated N. brasiliensis strains on the development of lesions in an established mycetoma infection. METHODS: Female 8-12-week-old BALB/c mice were injected with N. brasiliensis suspension to establish a mycetoma. Sixty mice were selected and divided into three groups: two of these groups were inoculated in the dorsum with N. brasiliensis subcultured 200 and 400 times, respectively, while the third group served as control. The thickness of each lesion was measured with calipers every week for 12 weeks. RESULTS: After 12 weeks, we observed that inoculation of 1 × 105 colony-forming units of attenuated N. brasiliensis strains was able to modify the natural history of the infection, with a decrease in the size of the lesions, particularly with P400, compared with the control group (P < 0.01). CONCLUSION: In this experimental evaluation of an immunomodulatory therapy with attenuated N. brasiliensis strains in a murine model, there was a greater stability in the size of the lesion over time in BALB/c mice inoculated with the P400 strain. This treatment could open the possibility of using the attenuated strain as immunomodulatory therapy in patients recalcitrant to standard therapy, with high risk of dissemination or who develop drug-related adverse effects.

"CLUPS": A New Culture Medium for the Axenic Growth of Entamoeba histolytica.

Amebiasis remains a major health problem in Mexico. Therefore, the search for better culture media and low-cost diagnostic and therapeutic tools is fundamental. We present a new culture medium for Entamoeba histolytica which allows the microbe to preserve its virulence factors and ability to induce hepatic abscesses in animal models. The novel CLUPS medium is an improved version of the PEHPS medium, previously designed in our laboratory. The main difference is the substitution of raw beef liver in PEHPS by raw beef lung in the CLUPS medium. To compare the performance of three-culture media (traditional TYI-S-33, PEHPS, and CLUPS), E. histolytica trophozoites were cultured in quintuplicate, followed by the evaluation of phospholipase activity and the induction of liver abscesses in golden hamsters. E. histolytica trophozoites grew significantly better in CLUPS medium than in TYI-S-33. Likewise, CLUPS-cultured trophozoites produced significantly more phospholipases than TYI-S-33-cultured trophozoites. Finally, trophozoites grown in any of the three tested media had similar potential to induce liver abscesses.

Snapshot of Quantiferon TB gold testing in Northern Mexico.

Most people infected with Mycobacterium tuberculosis have an asymptomatic condition named latent tuberculosis. These people do not have bacilli in the corporal secretions and are hard to diagnose by conventional laboratory tests. Diagnosis of latent tuberculosis infection (LTBI) in México is based on the tuberculin skin test (TST). This test has disadvantages, principally because the vaccine containing the Bacille Calmette-Guérin (BCG) is applied to 99% of this population and causes false positive TST outcomes. Recently, interferon-gamma release assays (IGRA) have been demonstrated to be a good test to detect latent tuberculosis with equal or better sensitivity to TST and without interference from BCG. However, in México the IGRA are an uncommon test due to the higher cost compared to TST. The main objective of this work was demonstrate the potential utility of the Quantiferon TB(®) gold in tube (QTB(®)-GIT) test to detect latent TB in a population from northern México. Samples from 106 subjects with close contact, or without contact, with actively infected TB patients were tested to detect LTBI. Our results show a significant difference between individuals in close contact with active TB patients (39.7%) compared to those without contact (3.2%), p < 0.01. The concordance between TST and QTB(®)-GIT was poor (κ = 0.31). Our preliminary results show that the QTB(®)-GIT has better capacity than TST to detect latent tuberculosis infection.

Association between human papilloma virus-type infections with micronuclei frequencies.

To determine the association between Human papillomavirus (HPV)-type infections with the frequency of Micronucleus (MN), a hospital-based, unmatched case-control study was carried out. We evaluated and compared the average number of MN/1,000 cells among three groups of Mexican females. Twenty one women ranging in age from 31-56 years and divided into three groups were studied. Group I comprised seven control women without cervical lesions and with HPV-negative, Group II was composed of seven women with Squamous intraepithelial lesions (SIL) infected with low-risk HPV low-risk, and Group III was made up of seven women with SIL infected with high-risk HPV infection. Analysis of variance (ANOVA) test revealed differences among Groups I (5.14+/-3.02), II (13.43+/-3.41), and III (25.43+/-3.41) (F=67.46; P=0.0001). We demonstrated an association between HPV type infection and higher MN frequencies. However, a larger controlled study with sufficient follow-up will be required to further evaluate the usefulness of this test in the clinical management of women with HPV infection.

Chromosomal abnormalities and polymorphisms in Mexican infertile men.

A cross-sectional study was conducted to estimate the prevalence of chromosome abnormalities and normal variable chromosome features (polymorphisms) in infertile men from northeastern Mexico. Karyotyping was carried out in 326 men with diagnosis of infertility. The sperm counts showed 204 patients with oligozoospermia, 87 with azoospermia and 35 normozoospermia. Five patients with oligozoospemia and two with azoospermia presented chromosome abnormalities. Nonzoospermic men did not show chromosomal abnormalities. Polymorphisms of heterochromatin and satellite length showed a significant increased in oligozoospermic and azoospermic men with respect to normozoospermic men, respectively. This study reports the prevalence of chromosome abnormalities, polymorphisms of heterochromatin length, and polymorphisms in satellites in Mexican infertile men. The prevalence in this study was similar to other studies in world literature.

Mg2+, Mn2+, and Co2+ stimulate Entamoeba histolytica to produce chitin-like material.

The mechanism of Entamoeba histolytica cyst cell wall synthesis is not well understood. Previous research has shown that cyst-like structures formed in the presence of chitin synthase cofactors (Mg2+, Mn2+, and Co2+) resist 1% sodium dodecyl sulfate lysis (RCLS), whereas those formed in the absence of cofactors (CLS) do not, and trophozoites are immediately destroyed. This suggests that E. histolytica is able to synthesize chitin, initiating a differentiation process under axenic conditions. To test this hypothesis, polysaccharide hydrolysates from E. histolytica trophozoites, CLS, or RCLS were analyzed with high-performance liquid chromatography. The major components found in all 3 preparations were N-acetylglucosamine (NAG) and glucose (GLC), with RCLS possessing 129 and 180 times more NAG and 2.4 and 2.0 more GLC than trophozoites and CLS, respectively. After 36 hr of incubation with chitinase (16 U/ml) in a hypotonic medium (50 mOsm/kg), 68% of RCLS was lysed, and 100% lost affinity for calcofluor white M2R. The RCLS polysaccharides bound wheat germ agglutinin and appeared as long and thin or short and thick fibers. Accordingly, Mg2+, Mn2+, and Co2+ stimulated E. histolytica to synthesize a chitin-like material.

Auxotrophy to lipoproteins of Entamoeba histolytica cultivated under axenic conditions.

Entamoeba histolytica grows in media without serum but with a mixture of aminoacids, vitamins, lipoproteins, free cholesterol, phospholipids and fatty acids called PACSR. The ability of lipoproteins and free lipids to support growth of three E. histolytica strains (HK9, HMI:IMSS and HM3:IMSS) was analysed. Tubes containing 5 ml culture medium, amino acids, vitamins and either 120-1,200 microg lipoproteins/ml or 0.017-0.10 mg free lipids/ml (predissolved in absolute ethanol) were inoculated with 1x10(4) trophozoites/ml and incubated at 37 degrees C for 72 h. Amoebae died within 12 h in the presence of any free lipid combination, while those having 240-480 mg lipoproteins/ml reached densities similar to or higher than those of controls (depending on strain). The addition of ethanol (0.1%) to the media produced stable lipid solutions and did not show significant adverse effects. Accordingly, E. histolytica is auxotrophic to lipoproteins and unable to use free cholesterol, phospholipids or fatty acids.

Axenic cultivation of Trichomonas vaginalis in a serum-free medium.

Mammalian serum or bovine serum albumin are essential for Trichomonas vaginalis cultivated under axenic conditions. Unfortunately, these components inhibit several biological properties of these parasites. PACSR is a serum replacement, free of bovine serum albumin. used for axenic cultivation of Entamoeba histolytica. We show that PACSR is also useful for axenic cultivation of T. vaginalis. Tubes containing 5.5 ml PEHP, or TYI basal media, plus 8% PACSR (v/v), were inoculated with 10(3) trichomonads/ml from 3 strains (GT-3, GT-13, and GT-15) and incubated at 36.5 C for 72 hr. Depending on the strain, cultures grown in PEHP plus PACSR reached densities of 50% (GT-13), 63% (GT-3), or 82% (GT-15) as compared to controls grown in PEHPS. On the other hand, yields of GT-3, GT-13, and GT-15 maintained in TYI plus PACSR were, respectively, 53%, 57%, and 67% among those of cultures grown in TYI-S-33. In all experiments, PEHPS and TYI-S-33 contained 8% bovine serum. Yields reached in PEHPS were 2.07+/-0.275 to 4.83+/-4.41 x 10(6) trichomonads/ml, whereas in TYI-S-33, densities were 1.68+/-0.315 to 4.16+/-8.07 x 10(6) trichomonads/ml. In conclusion, PACSR added to PEHP or TYI media is useful for axenic cultivation of T. vaginalis in the absence of serum or bovine serum albumin. PACSR could be useful in performing analyses of biological properties that are inhibited by serum or any of its components.

Isolation of an Entamoeba histolytica intracellular alkaline phospholipase A2.

The major hemolytic activity of Entamoeba histolytica is located in a subcellular fraction called P30. Its maximal effect is observed at pH 8.0 and 1 mM Ca2+ and is due to a phospholipase A (PLA). In the present study a membrane-associated phospholipase A2 was purified from P30 to homogeneity. P30 was fractionated with ethyl ether and the insoluble fraction was extracted with 1 M KCl. The KCl-soluble material was diluted ten times with 0.1 M TRIS-HCl (pH 9.5) and passed through a chromatofocusing column with a 9-4 pH gradient. Four peaks with PLA2 activity were obtained. By affinity chromatography, peak II, the one with the highest specific activity, was resolved in three more PLA2 peaks. Peak II.2 had the highest PLA2 specific activity. When analyzed by sodium dodecyl sulfate-polyacrylamide slab-gel electrophoresis under nonreducing conditions, peak II.2 yielded a single band with an apparent molecular mass of 30 kDa. Under reducing conditions the protein dissociated into two 15-kDa monomers. The purified PLA II.2 displayed its activity at the same conditions under which the P30 hemolytic activity was maximal. The isoelectric point of PLA II.2 was 7.0. The purification procedure described above provides sufficient material for determination of the relative importance of the enzyme in the E. histolytica pathogenic mechanisms.

Identification of Entamoeba histolytica intracellular phospholipase A and lysophospholipase L1 activities.

Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with 14C or 3H, indicated the presence of phospholipase A1 and A2 as well as lysophospholipase L1 activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A2 may contribute to E. histolytica cytopathogenicity.

Entamoeba histolytica axenic growth improvement by ox bile.

Axenic Entamoeba histolytica cultures, grown in PEHPS medium, showed increasing yields and growth velocity when added with 0.03 to 0.6 g/l gallbladder ox bile, while higher doses were toxic for amebas. The highest density (505 +/- 29 x 10(3) trophozoites/ml) and shortest duplication time (Dt = 10.74 +/- 0.2 h) corresponded to cultures added with 0.24 g bile/l. Their yields were 1.74 and 3.34 times higher, respectively, than those which were reached by the non-added PEHPS controls and by growth of amebas in TYI-S-33 medium. Between 72 and 96 h of incubation a noticeable increase in trophozoite density was observed in cultures added with 0.24 g/1 bile among controls. At 72 h yields of bile-added cultures inoculated with 5, 10 and 20 x 10(3) amebas/ml increased in function of the inoculum. The improved growth of E. histolytica by adding 0.24 g/l ox bile to culture medium suggests that bile has compounds are essential for amebas.

The Entamoeba histolytica rDNA episome: nuclear localization, DNAase I sensitivity map, and specific DNA-protein interactions.

Structural and functional features of the extrachromosomal DNA element that contains the ribosomal RNA genes of Entamoeba histolytica were studied using a variety of techniques. Using in situ hybridization, the element was found to be distributed along the inner phase of the nuclear membrane in the trophozoite stage; it appears to be part of the so-called peripheral chromatin. DNAase I-sensitive regions on the episome were mapped and found to correspond to the borders of the ribosomal RNA coding region. Other DNAase I-sensitive regions were found to correspond to DNA containing a 145bp sequence that exists in the episome as tandem repeats. Electrophoretic shift assays and footprinting experiments demonstrate the existence of specific nuclear factors that bind specifically to the 145bp repeat. Preliminary analysis of the binding factors showed that a 28 kDa polypeptide is a likely candidate for a specific DNA:protein interaction involving the repetitive element. These results suggest that a protein-binding domain within the 145bp repeat may have a specific function in the episome.

A new affinity adsorbent for the purification of phospholipases A1 and A2 from animal venoms.

Dimethyl-DL-2,3-distearoyloxy-propyl-2'-hydroxy-ethylammonium++ + (Rosenthal's inhibitor) was coupled to carboxyhexyl-Sepharose 4B, through carbodiimide chemistry. Phospholipase A2 from Heloderma horridum horridum and Crotalus adamanteus bind to the immobilized ligand in the presence of Ca2+ and can be easily eluted under acidic conditions or in the presence of a chelating agent, respectively. This affinity media proved to be effective also in the purification of a Ca2(+)-independent phospholipase A1 from vespid venom.