Biomedical subjects

J M Alunda

Publications and source records attributed to J M Alunda.

At least 19 recordsLinked to original sources

PubMed2006

Real-time reverse transcription-PCR quantification of cytokine mRNA expression in golden Syrian hamster infected with Leishmania infantum and treated with a new amphotericin B formulation.

A real-time quantitative reverse transcription-PCR assay was developed for the quantification of cytokine mRNA expression in the golden Syrian hamster Mesocricetus auratus infected with Leishmania infantum and treated with amphotericin B (AMB) formulated in microspheres made of human serum albumin (HSA). Treatment was administered intravenously on days 69, 71, and 73 postinfection (p.i.) with 10(7) metacyclic promastigotes, at doses of 2 and 40 mg/kg of AMB. High infection levels were recorded for untreated animals by day 76 p.i., with parasite loads always about 2 log10 per gram higher in the liver than in the spleen. Treatment was highly effective with both doses, but at 40 mg/kg, almost complete parasite elimination was achieved. mRNA expression of gamma interferon (IFN-gamma) and, to a lesser extent, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) in spleen cells was up-regulated in most animals of the untreated group. The mRNA expression of interleukin-4 was strongly down-regulated in untreated as well as treated infected animals. Treatment with the lower dose of AMB-HSA down-regulated the mRNA expression of IFN-gamma and TNF-alpha, with no effect on the deactivating cytokine TGF-beta. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN-gamma and TNF-alpha and strong suppression of TGF-beta. Treatment of noninfected animals did not alter the cytokine expression pattern with regard to untreated controls. Our results suggest that treatment of L. infantum-infected Syrian hamsters with highly effective nontoxic doses of AMB-HSA causes deactivation of the anti-inflammatory cytokine TGF-beta, which in turn results in up-regulation of the Th1 cytokines IFN-gamma and TNF-alpha.

Amphotericin B↗

PubMed2005

Influence of the vehicle on the properties and efficacy of microparticles containing amphotericin B.

New microparticles containing amphotericin B (AMB) have been developed and manufactured by spray drying. To this end albumin, polylactic-co-glycolic acids (PLGA) and poly(sebacic anhydride) have been employed as drug carriers. The selection of the solvent used to disperse the drug and the vehicle before spray drying was critical on production yields and physical properties of the microparticles. Once particle size, morphology and dispersability in some aqueous media were shown to be acceptable for an intravenous administration, in vivo efficacy was evaluated and compared with the reference medicine Fungizone. Microparticles prepared with albumin, albumin heated at a high temperature, some kinds of PLGA or polyanhydride, as well as Fungizone, were tested in an experimental hamster model of infection with Leishmania infantum, by evaluating the evolution of parasitic burdens in spleen, liver and antibody responses. After the injection of three doses corresponding to 2 mg of AMB per kilogram each, diverse reactions were reported depending on the vehicle. The best dispersability, reduction of parasites and antibody response were achieved when the treatment was performed with AMB in albumin microspheres.

Albumins↗

PubMed2004

Amphotericin B molecular organization as an essential factor to improve activity/toxicity ratio in the treatment of visceral leishmaniasis.

An in vivo study has been performed in order to determine the influence of amphotericin B (AMB) molecular organization on the toxicity and activity of this drug in the treatment of experimental visceral leishmaniasis. Three formulations with similar composition but different drug molecular self-association in aqueous media were prepared. Acute toxicity was evaluated by injecting them in healthy hamsters. Sub-acute toxicity and efficacy were studied administering them to animals previously infected with Leishmania infantum. The preparation with drug molecules completely dissolved into monomers (formulation "C") and produced the highest acute toxicity. The formulation whose AMB molecules were disposed as non-water-soluble multi-aggregates (formulation "B") proved to provide the lowest acute toxicity. This formula also showed an improved activity, mainly in the liver, if compared with the third tested formulation containing AMB molecules disposed as smaller dimerical "water-soluble" aggregates (formulation "A"). As a conclusion, molecular aggregation in biological media should be an important factor to consider when researching or optimizing medicines containing AMB. The liberation of molecules as large dispersed non-water-soluble multi-aggregates seems to improve the narrow therapeutic margin attached to the use of this drug.

Amphotericin B↗

PubMed2004

Anti-leishmanial activity of a new formulation of amphotericin B.

The effectiveness of albumin microspheres loaded with amphotericin B was tested in an in vivo model of visceral leishmaniasis using the golden hamster. Free and encapsulated amphotericin B was tested at the dose of 1 mg/kg given by the intracardiac route on days 25, 26 and 27 post-infection (p.i.) to treat animals previously infected with 10(7) stationary promastigotes by the intracardiac route. Encapsulated amphotericin was highly effective against infection causing a reduction of 88.8% and 87.2% in the early stage of infection (day 32 p.i.) and of 66.7% and 54% in a later stage of infection (day 135 p.i.) in liver and spleen parasite load respectively, compared with untreated animals, whereas free amphotericin was inactive. Lymphocyte proliferation was restored together with an increase in CD4(+) subsets in animals treated with encapsulated amphotericin B, but not in those treated with the non-encapsulated compound. Antibody responses did not increase after treatment with encapsulated amphotericin B with antibody levels remaining at base levels for most animals in contrast to those of untreated or treated with free amphotericin, where in most animals the antibody levels sharply increased. This new formulation could be a more economical alternative to liposomes for the treatment of visceral leishmaniasis with amphotericin B.

Amphotericin B↗

PubMed2004

Treatment of experimental visceral leishmaniasis with amphotericin B in stable albumin microspheres.

Hydrophilic albumin microspheres are proposed as a new delivery system for amphotericin B (AMB; AMB microspheres). The acute toxicity of AMB microspheres was lower than that of the AMB-deoxycholate (AMB-Doc) reference formulation in hamsters. Lethal doses in healthy and infected animals were improved at least eight times. Intravenous bolus administration of doses of AMB microspheres up to 40 mg/kg of body weight did not produce acute symptoms of toxicity. The efficacy of this new formulation was tested against Leishmania infantum-infected hamsters at doses of 2, 10, 20, and 40 mg/kg. With the 2-mg/kg dose, the activity of AMB, as assessed through the parasite load reductions in the liver and spleen and the evolution of antibody levels, was also improved (P < 0.05) by use of the AMB microsphere system. At the higher doses of 10, 20, and 40 mg/kg, reductions in parasite levels of more than 99% were achieved in the liver and spleen after the administration of AMB microspheres. A pharmacokinetic study was performed to study the serum, liver, and spleen AMB concentrations after administration of AMB microspheres and the reference formulation. Interestingly, a significant accumulation of AMB in the spleen and liver was observed after AMB microsphere administration. Our results suggest that this new formulation is a promising alternative to the conventional AMB-Doc formulation for the treatment of visceral leishmaniasis.

Amphotericin B↗

PubMed2003

Leishmania-specific lymphoproliferative responses and IgG1/IgG2 immunodetection patterns by Western blot in asymptomatic, symptomatic and treated dogs.

Peripheral cell responses against Leishmania infantum and serology by IFAT and WB were determined in 87 untreated dogs from an endemic area (Madrid, Spain) and in 15 treated dogs (antimonials, allopurinol). All untreated symptomatic dogs (nine) did not show any lymphoproliferative response, whereas 21 out of 78 untreated asymptomatic dogs had a positive cellular response. Serum IgG(2) from dogs with clinical signs of patent leishmaniosis reacted with a variety of peptides (26, 29, 34-35.4, 42, 45, 50-57 and 67 kDa), but IgG(1) response was mainly directed against a 67-kDa peptide. Successfully treated dogs displayed a low immunoreactivity of both IgG(1) and IgG(2), particularly against 67 kDa, thus indicating the potential prognostic value of this region. Positive cellular response of dogs treated with good clinical progress was only observed up to 5-12 months post treatment. Untreated asymptomatic dogs with positive cell response showed a clear recognition by IgG(2) of approximately 67 and 45 kDa antigens, whereas IgG(1) did not recognise any antigen.

Animals↗

PubMed2003

Immunization against ovine haemonchosis with three low molecular weight somatic antigens of adult Haemonchus contortus.

Individual bands (15) from electroblotted soluble extracts of adult Haemonchus contortus were excised and three peptides of molecular weight ca. 56 (F4), 39 (F8) and 18.5 kDa (F14) used to vaccinate 4-4.5-months-old lambs against the nematode. Immunizing doses from each peptide were administered in 1 ml Freund complete adjuvant (first 50 microg injection) and 1 ml Freund incomplete adjuvant (second and third 50 microg injections) to six lambs. Two weeks after last immunization, animals were challenged with 300 L-3/kg live weight (LW). Lambs were slaughtered 34 days after challenge. Immunization induced a strong antibody response estimated by enzyme-linked immunosorbent assay, whereas no peripheral lymphoproliferative response was observed. Lambs in the F8-vaccinated group showed on average delayed pre-patent period, lower faecal egg counts and reduction of abomasal worm burdens, although the differences were not statistically significant.

Animals↗

PubMed2001

Seasonal changes in prevalence and intensity of Hypoderma actaeon in Cervus elaphus from central Spain.

Hypodermosis in red deer, Cervus elaphus Linnaeus (Artilodactyla: Cervidae), caused by the warble fly, Hypoderma actaeon Latreille (Diptera: Oestridae), was studied at Quintos de Mora (Toledo, Spain) over a one-year period between October 1994 and September 1995. One hundred and twenty-five red deer were examined for the presence of warble fly larvae. The prevalence of warbles was 44.8% and the intensity of infection was 38.29 (SD +/- 61.32) warbles/deer infected. Clear seasonal variations were observed in prevalence and intensity, with the highest values in the autumn and winter. Statistically significant differences were observed in prevalence and intensity among age groups. In contrast, no differences were found in prevalence and intensity between males and females.

Age Factors↗

PubMed2001

Onchocercosis in red deer (Cervus elaphus) from Spain.

Onchocercosis, caused by Onchocerca flexuosa, was observed in red deer (Cervus elaphus) from Spain for the first time. Adult specimens of P. flexuosa were found in nodules in subcutaneous tissues in 42 of 125 (33%) red deer between October 1994 and September 1995; intensity of infection +/- SD was 3.93 +/- 5.26 nodules per infected host. A clear seasonal pattern in the distribution of nodules was observed, with higher values of prevalence and intensity in fall and winter in contrast to spring and summer. Significant differences were found among age groups in prevalence, but not in the mean intensity. No differences in infection were apparent between male and female adult red deer.

Age Factors↗

PubMed2000

Vaccination of Manchego lambs against Haemonchus contortus with a somatic fraction (p26/23) of adult parasites.

A low molecular weight fraction from adult Haemonchus contortus containing two peptides (p26/23) was used to vaccinate Manchego female lambs between 3.5 and 5 months of age. Immunizing injections were given three times on days 0, 14 and 28 of the experiment. On day 43, lambs were challenged with 400 third stage larvae/kg live weight. Vaccination induced a lengthening of prepatent periods, significant reduction (> 60%) in mean faecal egg counts and smaller variations in packed cell volume values. At necropsy, average worm burden in the vaccinated lambs was significantly lower (61.6%) than that found in unvaccinated challenged animals. A clear correlation was found between protection and serum antibody response in immunized lambs.

Animals↗

PubMed2000

Serodiagnosis of haemonchosis with a somatic antigen (Hc26) in several breeds of sheep.

Sera from 53 sheep belonging to Castellano, Churro, Manchego, and Merino breeds were analyzed to test the diagnostic value of a 26-kD antigen from adult Haemonchus contortus at prepatency and early and late patency of experimental haemonchosis. Animals that received zero, 1, or 2 infections with the parasite were tested. In addition, sera from 20 experimentally infected and 10 noninfected Texel sheep were used to test the antigen. Sera from 37 infected animals at prepatency as well as at patency in primary and secondary infection were found positive with the 26-kD antigen. However, sera from 10 animals with the lowest worm burdens (second infection) did not recognize the antigen during early patency (day 28 postinfection). IgG1 was the only isotype implicated in antigen recognition because IgG2, IgA, and IgM, in the same sera, showed no reactivity with the peptide. Antigen specificity was confirmed because hyperimmune sera against infective larvae and adult stages of the most common gastrointestinal nematodes found in natural infections in sheep (Trichostrongylus colubriformis and Teladorsagia circumcincta) did not recognize this peptide. The antigen was recognized only by anti-adult H. contortus hyperimmune sera and appeared to be absent in the L3 parasite stage. In addition, the partial N-terminal amino acid sequence of the diagnostic peptide is reported.

Animals↗

PubMed2000

Pharyngeal bot flies in Cervus elaphus in central Spain: prevalence and population dynamics.

The prevalence and intensity of infestations by bot flies Pharyngomyia picta and Cephenemyia auribarbis in red deer (Cervus elaphus) from Quintos de Mora (Toledo, Spain) were determined over a 1-yr period. Bots were present all year. No clear correlations were found between age or sex of the host and parasitization levels (prevalence and intensity). Considerable variation was found in prevalence and intensity, with larger values from December to March. Cephenemyia auribarbis was restricted from November to March, with maximum numbers of L-3 in February. Pharyngomyia picta showed a more complex profile with 2 peaks (March and August), indicating 2 generations per year.

Age Factors↗

PubMed2000

Elaeophorosis in red deer from Spain.

Elaeophorosis, caused by Elaeophora elaphi, was observed in red deer (Cervus elaphus) from Toledo Province (Spain) for the first time. Adult specimens of Elaeophora elaphi were found in the hepatic vessels of nine of 151 red deer between October 1994 and September 1995; intensity of infection was two to 18 nematodes per host. Adult nematodes were only found during the period from fall through early spring. No differences were present between sex or age groups. Parasites were not found in a limited sample from fallow deer (Dama dama). Blood samples were negative for the presence of microfilariae.

Age Factors↗

PubMed1999

Serum antibody response of Castellana sheep to Haemonchus contortus infection and challenge: relationship to abomasal worm burdens.

Primary and secondary serum antibody responses to Haemonchus contortus were studied in Castellana sheep. Ten-month-old sheep were infected (200 L3/kg live weight (lw)) and challenged (400 L3/kg lw) or uninfected and equally challenged with H. contortus. Primary infections induced a partially protective response upon challenge, characterized by higher serum protein levels, longer prepatent periods, lower fecal egg counts, and significant reduction in the establishment rate of the parasite and abomasal adult and L4 worm burdens. The resistant status of the infected and challenged sheep was not clearly related either to the serum specific antibody levels (IgG: IgG1, IgG2; IgM; IgA) estimated by ELISA or to immunodetection patterns in the Western blots.

Abomasum↗

PubMed1999

Partial anaerobiosis induces infectivity of Leishmania infantum promastigotes.

Leishmania infantum stationary-phase promastigotes could acquire infectivity via preincubation in a partially anaerobic medium (95% air/5% CO2) for 16 h before the infection, whereas promastigotes were efficiently destroyed when no CO2 was present. Incubation of L. infantum promastigotes with additional glucose (20 and 50 mM) greatly increased infection parameters in the absence of CO2; this is consistent with a "reverse Pasteur effect." Results showed that culture at 33 degrees C permitted survival and amastigote multiplication (a nearly 10-fold increase in amastigotes as compared with those observed in 37 degrees C cultures). This finding was obtained with the two strains of L. infantum tested (Doba and PB75).

Anaerobiosis↗

PubMed1999

Value of Western blotting in the clinical follow-up of canine leishmaniasis.

Specific serum antibody levels in Leishmania infantum-infected dogs treated with a combination of glucantime and allopurinol were estimated by indirect immunofluorescence and Western blotting. The sensitivity of Western blot was greater than that obtained with immunofluorescence titration. In general, both diagnostic methods concurred with the post-treatment clinical status of the animals. Clinical improvement of successfully treated dogs was related to lower immunofluorescence titers and simpler and/or less reactive immunodetection patterns in Western blotting. The recognition, by infected dogs, of certain low molecular weight antigens, particularly one of approximately 26 kDa, was restricted to pretreatment samples and a single animal in relapse thus apparently constituting an active infection marker.

Allopurinol↗

PubMed1999

Short report: improved diagnosis and follow-up of canine leishmaniasis using amastigote-based indirect immunofluorescence.

A comparison of an indirect immunofluorescence test using promastigotes (IFATp) or cultured amastigotes (IFATa) in the diagnosis and follow-up of canine leishmaniasis caused by Leishmania infantum was carried out. Results obtained with both diagnostic methods were in good agreement although the IFATa titration was more sensitive than the currently used IFATp without losing specificity. The higher sensitivity of the amastigote-based IFAT resulted in an earlier diagnosis in the absence of clinical signs. Both methods showed comparable results for monitoring the clinical evolution of naturally infected and treated (meglumine antimoniate plus allopurinol) dogs.

Allopurinol↗

PubMed1998

Comparative scanning electron microscopy of third-instar Hypoderma spp. (Diptera: Oestridae).

Scanning electron microscope study of third-instar larvae of four species of Hypoderma revealed differences among species in the pattern of spination, spine morphology and morphology of the spiracular plates. These observations identify characters that enable the differentiation of Hypoderma actaeon and H. diana, parasitizing red deer (Cervus elaphus) in Europe, and provide additional characters for differentiating H. bovis and H. lineatum parasitizing cattle.

Animals↗