ABSTRACT
At the present time, gut microbiota inspires great interest in the field of neuroscience as a function of its role in normal physiology and involvement in brain function. This aspect suggests a specific gut-brain pathway, mainly modulated by gut microbiota activity. Among the multiple actions controlled by microbiota at the brain level, neuronal plasticity and cognitive function represent two of the most interesting aspects of this cross-talk communication. We address the possible action of two-months implementation of gut Bifidobacteria using a mixture of three different strains (B-MIX) on hippocampal plasticity and related cognitive behavior in adult healthy Sprague Dawley rats. B-MIX treatment increases the hippocampal BDNF with a parallel gain in dendritic spines' density of hippocampal CA1 pyramidal neurons. Electrophysiological experiments revealed a significant increment of HFS-induced LTP formation on the CA1 hippocampal region in B-MIX treated rats. All these effects are accompanied by a better cognitive performance observed in B-MIX treated animals with no impairments in locomotion activity. Therefore, in adult rats, the treatment with different strains of bifidobacteria is able to markedly enhance neuronal plasticity and the CNS function influencing cognitive behavior, an effect that may suggest a potential therapeutic treatment in brain diseases associated with cognitive functions.
Subject(s)
Bifidobacterium/physiology , Hippocampus/microbiology , Learning/physiology , Neuronal Plasticity , Animals , Brain-Derived Neurotrophic Factor/metabolism , Dendritic Spines/microbiology , Dendritic Spines/physiology , Male , Memory/physiology , Pyramidal Cells/cytology , Pyramidal Cells/microbiology , Pyramidal Cells/physiology , Rats, Sprague-Dawley , Spatial Learning/physiologyABSTRACT
The role of specific sex-related patterns in olfactory dysfunctions of Parkinson's disease (PD) patients is unclear. The aim of this study was to assess the presence of specific sex-related patterns in olfactory dysfunctions excluding the possibility of confounding effects in patients with Parkinson's disease. One hundred and sixty-eight participants (99 PD patients and 69 controls) were enrolled and evaluated using Sniffin' Sticks Extended test (SSET). There was no significant sex difference in the control group for the SSET parameters. By contrast, in the PD group male patients scored significantly lower on odor discrimination (OD), identification (OI), and Threshold-Discrimination-Identification (TDI) score than females. On multivariable linear regression analysis, the only significant predictors of TDI score were sex and apathy. Among PD patients, men showed a significantly greater impairment compared to women in OI, OD and TDI score, but not in odor threshold (OT). These findings highlighted the possible role of sex differences in the development of associated PD non-motor symptoms.
Subject(s)
Olfaction Disorders/physiopathology , Parkinson Disease/physiopathology , Sensory Thresholds/physiology , Sex Characteristics , Aged , Female , Humans , Male , Middle Aged , Olfaction Disorders/etiology , Parkinson Disease/complicationsABSTRACT
UNLABELLED: ESSENTIALS: The differential diagnosis among thrombotic microangiopathies (TMAs) is challenging. We studied a case of TMA with neurologic symptoms, no renal impairment and normal ADAMTS-13 levels. Two novel mutations in complement factor I and thrombomodulin genes were identified. Complement-regulator genes can be involved in TMAs with normal ADAMTS-13 regardless of renal damage. BACKGROUND: Thrombotic microangiopathies (TMAs) often represent a challenge for clinicians, because clinical, laboratory, and even genetic features are not always sufficient to distinguish among different TMAs. OBJECTIVES: The aim of this study was to investigate the pathogenetic mechanisms underlying an acute case of TMA with features of both thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). PATIENTS/METHODS: We report the case of a 49-year-old woman who developed an acute TMA with neurologic involvement and no renal impairment. ADAMTS-13, von Willebrand factor, and complement-system biochemical characterization was performed on acute phase samples. Exome sequencing and direct Sanger sequencing of previously aHUS-associated genes were performed. The functional consequences of the thrombomodulin (THBD) mutation were investigated by in vitro expression studies. RESULTS: Despite a clinical diagnosis of TTP, the patient had normal ADAMTS-13 levels and increased VWF antigen levels with ultra-large von Willebrand factor multimers. C3, C4, and complement factors H and I (CFI) were normal. Molecular analysis confirmed two novel heterozygous mutations in CFI (c.805G>A, p.G269S) and THBD (c.1103C>T, p.P368L), and in vitro expression studies showed a reduction in the generation of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) caused by mutated THBD. This proinflammatory condition, associated with the p.G269S mutation in CFI, probably leads to a complement-mediated endothelial activation, with a relevant prothrombotic potential in case of transient environmental triggers. CONCLUSIONS: This study identified the first case of acute TMA without renal involvement but with neurological damage carrying two novel mutations in complement-regulator genes, highlighting the possible role of the complement system as a common pathogenetic mechanism in TMAs.
Subject(s)
Atypical Hemolytic Uremic Syndrome/genetics , Complement Factor I/genetics , Mutation , Purpura, Thrombotic Thrombocytopenic/genetics , Thrombomodulin/genetics , ADAMTS13 Protein/blood , Atypical Hemolytic Uremic Syndrome/blood , Atypical Hemolytic Uremic Syndrome/diagnosis , Atypical Hemolytic Uremic Syndrome/immunology , Biomarkers/blood , Carboxypeptidase B2/blood , DNA Mutational Analysis , Female , Genetic Predisposition to Disease , HEK293 Cells , Heterozygote , Humans , Middle Aged , Phenotype , Purpura, Thrombotic Thrombocytopenic/blood , Purpura, Thrombotic Thrombocytopenic/diagnosis , Purpura, Thrombotic Thrombocytopenic/immunology , Transfection , von Willebrand Factor/metabolismSubject(s)
Acute Kidney Injury/physiopathology , Kidney Failure, Chronic/surgery , Kidney Transplantation , Adult , Female , Humans , Male , Prognosis , Tissue Donors , Young AdultABSTRACT
SUMMARY: Crisponi syndrome (CS) is a rare, autosomal recessive disorder, characterized by hyperthermia, extensive muscular contractions in the face after even minimal stimuli or crying, hypertonia, opisthotonus, camptodactyly, and typical facial features. Recently, it has been demonstrated that CRLF1 (cytokine receptor-like factor 1) gene mutation is associated with CS. Here we report a case of CS with a new mutation in the CRLF1 gene associated with moderate clinical phenotype.
Subject(s)
Fever/genetics , Hand Deformities, Congenital/genetics , Mutation/genetics , Receptors, Cytokine/genetics , Trismus/congenital , Death, Sudden , Facies , Fatal Outcome , Female , Genotype , Humans , Hyperhidrosis , Infant , Muscle Contraction/genetics , Phenotype , Trismus/geneticsABSTRACT
Hemolytic uremic syndrome (HUS) is a disease of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. About 90% of cases are secondary to infections by Escherichia coli strains producing Shiga-like toxins (STEC-HUS), while 10% are associated with mutations in genes encoding proteins of complement system (aHUS). We describe two patients with a clinical history of STEC-HUS, who developed end-stage renal disease (ESRD) soon after disease onset. They received a kidney transplant but lost the graft for HUS recurrence, a complication more commonly observed in aHUS. Before planning a second renal transplantation, the two patients underwent genetic screening for aHUS-associated mutations that revealed the presence of a heterozygous CFI mutation in patient #1 and a heterozygous MCP mutation in patient #2, and also in her mother who donated the kidney. This finding argues that the two cases originally diagnosed as STEC-HUS had indeed aHUS triggered by STEC infection on a genetic background of impaired complement regulation. Complement gene sequencing should be performed before kidney transplantation in patients who developed ESRD following STEC-HUS since they may be undiagnosed cases of aHUS, at risk of posttransplant recurrence. Furthermore, genetic analysis of donors is mandatory before living-related transplantation to exclude carriers of HUS-predisposing mutations.
Subject(s)
Complement Factor I/genetics , Escherichia coli Infections/complications , Hemolytic-Uremic Syndrome/complications , Kidney Failure, Chronic/etiology , Membrane Cofactor Protein/genetics , Mutation/genetics , Adult , Case-Control Studies , DNA Primers/chemistry , DNA Primers/genetics , Escherichia coli Infections/genetics , Escherichia coli Infections/microbiology , Female , Genetic Testing , Graft Rejection/diagnosis , Graft Rejection/etiology , Hemolytic-Uremic Syndrome/genetics , Hemolytic-Uremic Syndrome/microbiology , Heterozygote , Humans , Kidney Failure, Chronic/pathology , Kidney Failure, Chronic/therapy , Kidney Transplantation , Male , Middle Aged , Pedigree , Prognosis , Recurrence , Risk Factors , Shiga-Toxigenic Escherichia coli , Thrombocytopenia/complications , Thrombocytopenia/genetics , Thrombocytopenia/microbiology , Young AdultABSTRACT
We report our experience in long-term treatment of relapsing remitting multiple sclerosis patients with natalizumab (N). In November 2009 we evaluated 141 patients (126 AIFA criterion A, 15 AIFA criterion B). We paid particular attention to the treatment period and the patients follow-up; during the whole therapeutic program, they undergone to clinical and radiological evaluation for every 3 months. The compliance was optimal and we found no significant side effects. 26 patients completed the 24 monthly doses. After 24 months 51% of patients were free from disease activity. We found a reduction in relapses and EDSS, moreover the clinical improvement was also confirmed by radiological examinations. Our results show that the best therapeutic results are achieved by early initiation of treatment.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Multiple Sclerosis/therapy , Adult , Cohort Studies , Disease Management , Female , Follow-Up Studies , Humans , Italy/epidemiology , Magnetic Resonance Imaging/trends , Male , Multiple Sclerosis/epidemiology , Multiple Sclerosis/immunology , Natalizumab , Survival Analysis , Time FactorsABSTRACT
Genetic evidence indicates a central role of cerebral accumulation of beta-amyloid (Abeta) in the pathogenesis of Alzheimer's disease (AD). Beside presenilin 1 and 2, three other recently discovered proteins (Aph 1, PEN 2 and nicastrin) are associated with gamma-secretase activity, the enzymatic complex generating Abeta. Alterations in genes encoding these proteins were candidates for a role in AD. The PEN 2 gene was examined for unknown mutations and polymorphisms in sporadic and familial Alzheimer patients. Samples from age-matched controls (n=253), sporadic AD (SAD, n=256) and familial AD (FAD, n=140) were screened with DHPLC methodology followed by sequencing. Scanning the gene identified for the first time a missense mutation (D90N) in a patient with FAD. Three intronic polymorphisms were also identified, one of which had a higher presence of the mutated allele in AD subjects carrying the allele epsilon4 of apolipoprotein E than controls. The pathogenic role of the PEN-2 D90N mutation in AD is not clear, but the findings might lead to new studies on its functional and genetic role.
Subject(s)
Alzheimer Disease/genetics , Genetic Predisposition to Disease , Membrane Proteins/genetics , Mutation , Aged , Amyloid Precursor Protein Secretases , Base Sequence , Blotting, Western , Chromatography, High Pressure Liquid , Female , Humans , Male , Pedigree , Polymorphism, Genetic , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A series of 6-thioxopyrimidines (5, 6), their 6-oxo- analogs (11-14), and pyrimidine-2,4-diones (20-26), were synthesized and evaluated for their antitumoral activity against 60 tumoral cell lines. The activity of propenethioamide (3, 4) and propeneamide (7-10 and 15-19) intermediates is also reported. Among the tested compounds the thioxopyrimidine 5c, bearing an N'-benzyl group, showed the best cytostatic activity. Furthermore, high selectivity and cytotoxic activity on the HOP-92 cell line of non-small cell lung cancer was exhibited by 3-amino-2-[(methylamino)thioxamethyl]-3-pyrrolidino-2-propenenitrile (3a).
Subject(s)
Antineoplastic Agents/chemical synthesis , Neoplasms/drug therapy , Pyrimidines/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Chemistry, Pharmaceutical , Humans , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
A detailed analysis of the endogenous sterols present in the clinically relevant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14alpha demethylase inhibitor ketoconazole and two inhibitors of delta24(25)-sterol methyl transferase, 20 piperidin-2-yl-5alpha-pregnan-3beta-20-R-diol (22,26-azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated and purified from their host cells and neutral lipids were extracted, separated and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-cholesta-7-en-3beta-ol (ergosta-7-en-3beta-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3beta-ol; these cells also contained cholesterol (up to 80% by weight of total sterols), probably derived from host cells. Amastigotes that proliferated in the presence of 10 nM ketoconazole (minimal inhibitory concentration, MIC) for 24 h had a sharply reduced content of endogenous 4-desmethyl sterols with a concomitant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrolanosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100-300 nM) accumulated large amounts of C27 sterols whose structure suggested, in the case of 22,26-azasterol, that delta14 sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorporation of [2-(14)C]-acetate into the parasite's endogenous C4-desmethyl sterols with an IC50 of 50 nM, indistinguishable from the value reported previously for the extracellular epimastigote form. Taken together, the results showed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both delta5 and delta22 reductases. They also suggest that the 100-fold higher potency of antifungal azoles as antiproliferative agents against amastigotes, when compared with epimastigotes, is most probably due to a smaller pool of endogenous sterols in the intracellular parasites.
Subject(s)
Sterols/biosynthesis , Trypanosoma cruzi/metabolism , Animals , Chlorocebus aethiops , Cholestanol/analogs & derivatives , Cholestanol/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Ketoconazole/pharmacology , Lanosterol/analogs & derivatives , Lanosterol/pharmacology , Methyltransferases/antagonists & inhibitors , Oxidoreductases/antagonists & inhibitors , Sterol 14-Demethylase , Trypanosoma cruzi/chemistry , Trypanosoma cruzi/drug effects , Vero CellsABSTRACT
We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against Trypanosoma (Schizotrypanum) cruzi, the etiological agent of Chagas' disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30- to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite's endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3 beta-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite's sterol C-14 alpha demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37 degrees C, the MIC of SCH 56592 was 0.3 nM, again 33- to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas' disease, SCH 56592 given at > or = 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCR-based tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas' disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against T. cruzi and may be useful in the treatment of human Chagas' disease.
Subject(s)
Antiparasitic Agents/pharmacology , Chagas Disease/drug therapy , Triazoles/pharmacology , Trypanosoma cruzi/drug effects , Animals , Dose-Response Relationship, Drug , Female , Mice , Triazoles/therapeutic use , Trypanocidal AgentsABSTRACT
Chagas' disease, a protozoan infection by the kinetoplastid Trypanosoma cruzi, constitutes a major public health problem in Latin America. With the use of mouse models of both short- and long-term forms of the disease, the efficacy of D0870, a bis-triazole derivative, was tested. D0870 was able to prevent death and induced parasitological cure in 70 to 90 percent of animals, in both the short- and long-term disease. In contrast, currently used drugs such as nifurtimox or ketoconazole prolonged survival but did not induce significant curing effects. D0870 may be useful in the treatment of human long-term Chagas' disease, a condition that is currently incurable.
Subject(s)
Chagas Disease/drug therapy , Triazoles/therapeutic use , Trypanocidal Agents/therapeutic use , Animals , Base Sequence , Chagas Disease/parasitology , Drug Administration Schedule , Ketoconazole/therapeutic use , Molecular Sequence Data , Nifurtimox/therapeutic use , Sterols/biosynthesis , Time Factors , Triazoles/administration & dosage , Triazoles/pharmacology , Trypanocidal Agents/administration & dosage , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/metabolismABSTRACT
We have studied the antiproliferative effects of two sterol analogs previously reported as potent inhibitors of delta 24(25) sterol methyl transferase (E.C. 2.1.1.43) of yeasts and fungi on epimastigotes and amastigotes on Trypanosoma (Schizotrypanum) cruzi, the causative agents of Chagas disease, as well as its chemotherapeutic effects in a murine model of the disease. On the epimastigote form proliferating in liver infusion tryptose medium at 28 degrees C 22,26-azasterol (AZA), a cholestanol analog with a 6-membered aza ring as a side chain produced a dose-dependent reduction of the growth rate up to 3 microM, but at 10 microM complete growth arest and cell lysis took place after 120-144 h. For 24(R,S),25-epiminolanosterol (EIL), complete growth arrest and lysis took place with 6 microM. In both cases the antiproliferative effects were potentiated by the simultaneous incubation of the epimastigotes with inhibitors of sterol C-14 alpha-demethylase such as ketoconazole or SDZ 89,485, as indicated by concave isobolograms and fractional inhibitory concentrations ranging from 0.11 to 0.46. Analysis of the sterol composition in control and treated cells by thin-layer and capillary gas-liquid chromatography coupled to mass spectrometry showed that growth inhibition correlated with the complete disappearance of the native endogenous sterols of the parasite (ergosterol and 24-ethyl analogs) and the accumulation of 24-desalkyl sterols. Against the clinically relevant amastigote form proliferating inside cultured Vero cells at 37 degrees C, AZA eradicated the parasite of 100 nM, while the corresponding concentration for EIL was 300 nM. Synergic effects of both inhibitors when combined with ketoconazole against this form of the parasite was demonstrated using a three-dimensional analytic method which allowed the identification of optimal drug concentrations. Finally, it was found that daily oral administration of AZA at 50 mg/kg/day for a total of 43 doses to mice infected with a lethal inoculum of T. cruzi allowed survival of all treated animals 25 days after infection, while all control (untreated) animals were dead at this point of time. Increased survival correlated with a 90% reduction in parasitemia in the treated animals. The antiparasitic effects of the azasterol were potentiated in combined treatments with ketoconazole. This is the first report of a successful application of a sterol methyl transferase inhibitor as a chemotherapeutic agent in a protozoal infection.
Subject(s)
Chagas Disease/drug therapy , Enzyme Inhibitors/pharmacology , Methyltransferases/antagonists & inhibitors , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/drug effects , Animals , Chagas Disease/enzymology , Chagas Disease/metabolism , Cholestanol/analogs & derivatives , Cholestanol/pharmacology , Drug Synergism , Enzyme Inhibitors/therapeutic use , Female , In Vitro Techniques , Ketoconazole/pharmacology , Lanosterol/analogs & derivatives , Lanosterol/pharmacology , Lipids/chemistry , Mice , Trypanocidal Agents/therapeutic use , Trypanosoma cruzi/chemistry , Trypanosoma cruzi/enzymologyABSTRACT
OBJETIVOS: Nos propusimos comprobar si los factores hormonales tienen influencia en la actividad y evolución del Lupus Eritematoso Sistémico (LES), determinando los niveles séricos de estradiol, testosterona, FSH y de la concentración de receptores estrogénicos en células monoclonales de sangre periférico(CMSP). METODOLOGIA: Se seleccionaron 22 pacientes con diagnóstico de LES basados en los criterios de la ARA 1982, del sexo femenino culminando sólo 10 de ellos, la investigación. Se realizó registro clínico, inmunoserológico, niveles séricos de estradiol, testosterona, FSH y concentración de receptores estrogénicos en CMSP tanto para los pacientes como para los controles. El análisis estadístico se realizó mediante la "t" de Student y el índice de correlación lineal de Pearson. RESULTADOS: En el 80 por ciento de los pacientes se observó Anyti-Adn elevados. Factores del Complemento disminuidos en el 90 por ciento y complejos inmunes circulantes elevados en la totalidad de los casos. La creatinina sérica fue normal contrastando con el hallazgo de nefritis lúpica en el 100 por ciento. Los estrógenos séricos elevados significativamente (p<0,01), testosterona disminuida (p<0,01), FSH sin diferencia estadística en ambos grupos (p<0,5). No hubo correlación entre el estradiol, testosterona en relación a la concentración de receptores estrogénicos en CMSP e igualmente no se constató correlación entre la concentración de receptores estrogénicos y los índices de actividad de la enfermedad. CONCLUSION: La determinación de los factores del coplemento viene a ser índices precisos de actividad, superiores a los anticuerpos Anti-Adn bicatenario. En todo paciente lúpico, existe nefropatía. Proponemos que las pacientes con LES, tienen a poseer estradiol y por otra parte, la testosterona disminuida en relación a la población normal. En cuanto a la concentración de receptores de estrógeno en CMSP, no se encontró diferencia entre los grupos. No pareciera relacionarse los niveles elevados de estradiol con la actividad de la enfermedad, ni existir correlación entre la actividad estrogénica y la lúpica.
Subject(s)
Adolescent , Adult , Middle Aged , Humans , Female , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/diagnosis , Receptors, Estrogen/analysisABSTRACT
In the present study, we investigate the immunoprotective properties of trypomastigote excretory-secretory Ag (ESA) in experimental models. In the case of BALB/c mice, the immunization with ESA resulted in the reduction of parasitemia during acute infection and a significant level of protection in terms of mortality with more than 60% survival, whereas none of the mice in the control groups survived after 39 days postinfection. The same experiments performed in Fischer rats showed a high degree of protection against acute lethal infection with 100% survival, whereas 20 to 40% of rats in the control groups survived the acute phase of T. cruzi infection. Mouse and rat immune sera presented trypanolytic activity against Trypanosoma cruzi infective forms, and recognized two major parasite components of 85 and 24 kDa. The analysis of specific isotype profiles showed a predominance of IgG1, IgG2a, and IgG2b antibody responses. Rat antisera to ESA were then used to screen a trypomastigote cDNA library. Several clones were identified, all of which encoded for the 24-kDa protein. Using a mAb (Tcr7) produced against the native protein, the 31-kDa recombinant fusion protein was purified by affinity chromatography. The antisera to the recombinant protein used in IFA and immunoelectron microscopy showed that the localization of the 24-kDa protein differs among T. cruzi developmental stages. Protection experiments were performed in BALB/c mice using two synthetic peptides (20-40 and 109-124) derived from the primary sequence of the 24-kDa polypeptide. The results obtained clearly indicated that the peptide 109 to 124 containing a putative T cell epitope represents the most protective epitope, which induced 30 to 50% of protection against mortality during acute infection, whereas the percent survival in the control groups (OVA and 20-40 OVA peptide-immunized mice) was around 16%. Moreover, analysis of T cell proliferation in response to OVA-coupled peptides clearly indicated that only the 109 to 124 peptide had the capacity to induce the proliferation of T cells from peptide-immunized mice. Interestingly, only the 109 to 124-coupled peptide induced the proliferation of T cells from T. cruzi-infected mice.
Subject(s)
Antigens, Protozoan/immunology , Epitopes/analysis , Peptide Fragments/immunology , T-Lymphocytes/immunology , Trypanosoma cruzi/immunology , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Immune Sera/immunology , Immunization , Immunoglobulin Isotypes/blood , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Rabbits , Rats , Recombinant Proteins/immunologyABSTRACT
Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a potent antiplatelet compound derived from garlic, inhibits the proliferation of both epimastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas' disease. The growth of the epimastigote form was immediately arrested by 80 microM ajoene, while 100 microM induced cell lysis in 24 hr. In the amastigote form proliferating inside VERO cells, 40 microM ajoene was sufficient to eradicate the parasite from the host cells in 96 hr. Growth inhibition of the epimastigotes was accompanied by a gross alteration of the phospholipid composition of the treated cells in which phosphatidylcholine (PC), the major phospholipid class present in control cells, dropped to the least abundant phospholipid in cells treated with 60 microM ajoene for 96 hr, while its immediate precursor, phosphatidylethanolamine (PE), became the predominant species; this was correlated with a marked drop in the incorporation of [14C-U]acetate in PC and a corresponding increase in PE. Concomitant with the change in the phospholipid headgroup composition of the cells, the fatty acids esterified to this lipid fraction underwent a dramatic alteration due to the increase in the content of saturated fatty acids and a marked reduction in the content of linoleic (18:2) acid, which is the predominant fatty acid in control cells. We also found that ajoene inhibited the de novo synthesis of neutral lipids and, in particular, of sterols in the epimastigotes, but the resultant changes in the sterol composition were not sufficient to explain the antiproliferative effects of the drug. Electron-microscopy showed a concentration-dependent alteration of intracellular membranous structures, particularly the mitochondrion and endoplasmatic reticulum. The results suggest that one important factor associated with the antiproliferative effects of ajoene against T. cruzi is its specific alteration of the phospholipid composition of these cells.
Subject(s)
Disulfides/pharmacology , Garlic , Phosphatidylcholines/biosynthesis , Plant Extracts/pharmacology , Plants, Medicinal , Platelet Aggregation Inhibitors/pharmacology , Trypanosoma cruzi/drug effects , Animals , Cell Division/drug effects , Disulfides/isolation & purification , Fatty Acids/analysis , Garlic/chemistry , Intracellular Membranes/drug effects , Phosphatidylethanolamines/biosynthesis , Phospholipids/biosynthesis , Phospholipids/chemistry , Plant Extracts/isolation & purification , Sulfoxides , Trypanosoma cruzi/metabolism , Trypanosoma cruzi/ultrastructure , Vero CellsABSTRACT
We have studied the antiproliferative effects of mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, on the protozoan parasite Trypanosoma (Schizotrypanum) cruzi and its ability to potentiate the action of specific ergosterol biosynthesis inhibitors, such as ketoconazole and terbinafine, both in vitro and in vivo. Against the epimastigote form in vitro, mevinolin produced a dose-dependent reduction of the growth rate up to 25 microM, but at 50 and 75 microM, complete growth arrest and cell lysis took place after 144 and 96 h, respectively. A systematic study of the effects of mevinolin combined with ketoconazole and terbinafine, which act at different points in the ergosterol biosynthesis pathway, on the proliferation of epimastigotes indicated a synergic action, as shown by concave isobolograms and fractional inhibitory concentration indexes ranging from 0.17 to 0.54. Analysis of the sterol composition and de novo sterol synthesis in control and treated cells by thin-layer and gas-liquid chromatographies showed that the antiproliferative effects of the drug alone and in combination were correlated with the depletion of the endogenous ergosterol pool and particularly with a critical (exogenous) cholesterol/endogenous 4-desmethyl sterol ratio in the cells. When we studied the effects of mevinolin on the amastigote form proliferating inside Vero cells in vitro, only very modest effects on the parasites were observed up to 0.75 microM; above this concentration, significant deleterious effects on the host cells were found. However, when the same concentration of the drug was combined with ketoconazole, it was able to reduce by a factor of 10 the concentration of the azole required to eradicate the parasite (from 10 to 1 nM), again indicating a synergic action. On the other hand, a combination of mevinolin and terbinafine had only additive effects on amastigotes, but a ternary combination of mevinolin, ketoconazole, and terbinafine was again clearly synergistic. In vivo studies with a murine model of Chagas' disease showed that mevinolin can also potentiate the therapeutic effects of ketoconazole in this system; combined treatment with the two drugs at doses that alone offered only limited protection against the parasite was able to essentially eliminate circulating parasites and produce complete protection against death. These results confirm the synergic action against the proliferative stages of T. cruzi both in vitro and in vivo and in vivo of combined ergosterol biosynthesis inhibitors that act at different points in the pathway and suggest that mevinolin combined with azoles, such as ketoconazole, can be used in the treatment of human Chagas' disease.
Subject(s)
Chagas Disease/drug therapy , Growth Inhibitors/pharmacology , Ketoconazole/pharmacology , Lovastatin/pharmacology , Naphthalenes/pharmacology , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/drug effects , Animals , Cell Division/drug effects , Chagas Disease/parasitology , Drug Synergism , Female , Ketoconazole/administration & dosage , Ketoconazole/therapeutic use , Lovastatin/administration & dosage , Lovastatin/therapeutic use , Mice , Naphthalenes/administration & dosage , Naphthalenes/therapeutic use , Terbinafine , Trypanocidal Agents/administration & dosage , Trypanocidal Agents/therapeutic use , Trypanosoma cruzi/growth & development , Vero CellsABSTRACT
The in vitro antiproliferative effects of ICI 195,739, a recently developed bis-triazole derivative (T. Boyle, D. J. Gilman, M. B. Gravestock, and J. M. Wardleworth, Ann. N.Y. Acad. Sci. 544:86-100, 1988; J. F. Ryley, S. McGregor, and R. G. Wilson, Ann. N.Y. Acad. Sci. 544:310-328, 1988), on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi and some aspects of its mechanism of action are described. Despite previous claims that triazole compounds act on susceptible organisms by essentially the same mechanism demonstrated for the imidazole compounds, i.e., by interfering with the synthesis of ergosterol at the level of the cytochrome P-450-dependent C-14 demethylation of lanosterol, our results indicate that ICI 195,739 acts on T. cruzi epimastigotes by a dual mechanism which involves blockade of ergosterol byosynthesis and a second, still-unidentified target whose alteration leads to immediate growth arrest. Although ICI 195,739 blocks ergosterol biosynthesis at the level of C-14 lanosterol demethylation, as shown by gas-liquid and thin-layer chromatography, growth arrest in ICI 195,739-treated cells is not related to the depletion of the endogenous ergosterol pool, contrary to what was previously found for ketoconazole, the reference compound among antifungal and antiprotozoal azole derivatives. Consistent with this observation is the fact that the concentration of ICI 195,739 required to inhibit de novo synthesis of ergosterol in epimastigotes by 50% is 60 nM, which is essentially identical to that previously found for ketoconazole under identical conditions, while the minimum concentration required to produce complete growth inhibition is 0.1 microM, which is 300 times lower than that of ketoconazole. With respect to the intracellular amastigote form proliferating inside vertebrate (Vero) cells, 10 nM is sufficient to eradicate the parasite completely in 96 h, with no effects on the host cells; this concentration is identical to that previously found for ketoconazole. Growth inhibition and morphological alterations induced by ketoconazole can be reserved by exogenously added ergosterol but not by cholesterol; for ICI 195, 739, neither sterol is capable of reserving the drug effects. Contrary to what was observed for ketoconazole, the in vitro antiproliferative effects of ICI 195, 739 on both forms of the parasite are not potentiated by the simultaneous presence of terbinafine, an allylamine which blocks ergosterol production by the parasite at a different level of the sterol biosynthetic pathway. These results, together with those of an accompanying study of the ultrastructural alterations induced by the drug, strongly support the notion that ICI 195, 739 acts on T. cruzi by a novel combination of biochemical and cellular effects, which could explain its extraordinary potency in vivo against the parasite.
Subject(s)
Triazoles/pharmacology , Trypanocidal Agents , Trypanosoma cruzi/drug effects , Animals , Cell Division/drug effects , Chromatography, Gas , Chromatography, Thin Layer , Ergosterol/analysis , Ergosterol/chemistry , Naphthalenes/pharmacology , Terbinafine , Trypanosoma cruzi/metabolism , Vero Cells/drug effectsABSTRACT
We have investigated the growth-inhibitory effects of two ergosterol biosynthesis inhibitors, the dioxolane imidazole ketoconazole and the allylamine SF 86-327, alone and in combination, on the proliferative stages of Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease. Proliferation of epimastigotes in liver infusion-tryptose medium at 28 degrees C was immediately arrested by any of these drugs at greater than or equal to 3 x 10(-5) M; cell lysis occurred 24 h later. Below that concentration, SF 86-327 at concentrations down to 1 x 10(-6) M stopped growth after 48 h. In contrast, ketoconazole slowed cell growth only moderately, but proliferation finally stopped and cell lysis occurred after 120 h at 3 x 10(-6) M. Synergistic effects could be observed when the two drugs were used in combination: the concentration of SF 86-327 required to reduce the cell growth to 25% of controls in 144 h was reduced 33-fold in the presence of 1 x 10(-6) M ketoconazole, which by itself reduced growth only by 30%. Amastigotes, proliferating in Vero cells at 37 degrees C, were much more susceptible to both drugs, but ketoconazole was definitely a more potent antiparasitic agent than the allylamine in this system: whereas the concentration of SF 86-327 required to reduce the number of infected cells to 50% of controls was 1 x 10(-7) M and that required to completely eradicate the parasite was 3 x 10(-6) M, for ketoconazole these concentrations were 1 x 10(-10) M and 1 x 10(-8) M, respectively. Again, strong synergistic effects were observed when the drugs were used in combination: the concentration of SF 86-327 required to reduce the number of infected cells to 50% of controls was 100-fold lower in the presence of 10(-11) M ketoconazole, which by itself had no effects on amastigote proliferation. The parasite was completely eradicated when the drugs were used in combination at concentrations as low as 10(-9) M. Synergy of the antiproliferative effects of the drugs on both froms of the parasite was further demonstrated by concave isobolograms. On the other hand, SF 86-327 at 10(-5) M had no effects on the proliferation of Vero cells, whereas ketoconazole at 10(-7) M reduced the proliferation of these cells by 50%.