Recombinant Fasciola hepatica Fatty Acid Binding Protein as a Novel Anti-Inflammatory Biotherapeutic Drug in an Acute Gram-Negative Nonhuman Primate Sepsis Model.

Due to their phylogenetic proximity to humans, nonhuman primates (NHPs) are considered an adequate choice for a basic and preclinical model of sepsis. Gram-negative bacteria are the primary causative of sepsis. During infection, bacteria continuously release the potent toxin lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to death. Our previous research has demonstrated in vitro and in vivo using a mouse model of septic shock that Fh15, a recombinant variant of the Fasciola hepatica fatty acid binding protein, acts as an antagonist of Toll-like receptor 4 (TLR4) suppressing the LPS-induced proinflammatory cytokine storm. The present communication is a proof-of concept study aimed to demonstrate that a low-dose of Fh15 suppresses the cytokine storm and other inflammatory markers during the early phase of sepsis induced in rhesus macaques by intravenous (i.v.) infusion with lethal doses of live Escherichia coli. Fh15 was administered as an isotonic infusion 30 min prior to the bacterial infusion. Among the novel findings reported in this communication, Fh15 (i) significantly prevented bacteremia, suppressed LPS levels in plasma, and the production of C-reactive protein and procalcitonin, which are key signatures of inflammation and bacterial infection, respectively; (ii) reduced the production of proinflammatory cytokines; and (iii) increased innate immune cell populations in blood, which suggests a role in promoting a prolonged steady state in rhesus macaques even in the presence of inflammatory stimuli. This report is the first to demonstrate that a F. hepatica-derived molecule possesses potential as an anti-inflammatory drug against sepsis in an NHP model. IMPORTANCE Sepsis caused by Gram-negative bacteria affects 1.7 million adults annually in the United States and is one of the most important causes of death at intensive care units. Although the effective use of antibiotics has resulted in improved prognosis of sepsis, the pathological and deathly effects have been attributed to the persistent inflammatory cascade. There is a present need to develop anti-inflammatory agents that can suppress or neutralize the inflammatory responses and prevent the lethal consequences of sepsis. We demonstrated here that a small molecule of 14.5 kDa can suppress the bacteremia, endotoxemia, and many other inflammatory markers in an acute Gram-negative sepsis rhesus macaque model. These results reinforce the notion that Fh15 constitutes an excellent candidate for drug development against sepsis.

Meningoencefalitis por Cryptococcus neoformans en adolescente con desnutrición / Cryptococcus neoformans meningoencephalitis in a malnourished teenager

La criptococosis es una micosis causada por dos hongos levaduriformes encapsulados del género Cryptoccoccus, ingresa al organismo por vía inhalatoria con diseminación al sistema nervioso central, su prevalencia es mayor en inmunodeprimidos por VIH SIDA. Presentamos el caso de un paciente masculino de 13 años de edad, VIH negativo, de la etnia Lenca, procedente de zona rural de Honduras, con historia de contacto prolongado con heces de paloma (Columba livia), quien se presentó con síndrome de hipertensión endocraneana. Tinta china de líquido cefalorraquídeo reportó levaduras encapsuladas compatibles con Cryptococcus neoformans spp confirmado por cultivo. Su único antecedente de inmunosupresión fue la desnutrición. Tuvo evolución favorable con la terapia combinada anfotericina b y fluconazol

Cryptococcosis is a fungal infection caused by two encapsulated yeasts of the genus Cryptoccoccus. The microorganism enters the body through inhalation and it disseminates to the central nervous system. Its prevalence is higher in immunocompromised patients, especially those with HIV-AIDS. We report the case of a 13-year old male patient, negative for HIV infection, who belongs to the Lenca ethnic group, from a rural area in Honduras and a positive history of contact with pigeon feces (Columba livia). The patient presented with intracranial hypertension syndrome. An India-ink examination of his cerebrospinal fluid revealed encapsulated yeasts compatible with Cryptococcus neoformans spp., confirmed by culture. The presentation of this case is important because of being a pediatric HIV negative patient, whose only previous immunosuppression was malnutrition. Additionally, he had a history of contact with pigeon feces, resulting in risk factors for developing the disease. We should also take into account the imp ortance of the epidemiological correlation in order to achieve an early diagnosis and an adequate response to therapy. In this case, the combined therapy with fluconazole and amphotericin, led to a favorable outcome

Reproductive outcomes of Alpine goats primed with progesterone and treated with human chorionic gonadotropin during the anestrus-to-estrus transition season.

This study aimed to determine the possible effects of a single injection of human chorionic gonadotropin (hCG) as a means for estrus induction in acyclic French-Alpine goats during the reproductive transition period at 25°N, 103°W. The potential effects of hCG upon ovarian function and reproductive performance of goats were also assessed. Multiparous acyclic French-Alpine goats (n = 39; 37.4 ± 8 .5 kg) were primed with 20mg progesterone (P4) 1 day prior to hCG administration. Thereafter, does were treated either with saline (hCG-0; n = 10), 50 (hCG-50; n = 9), 100 (hCG-100; n = 10), or 300 IU of hCG (hCG-300; n = 10). Ovarian structures and pregnancy were monitored by transrectal ultrasonography. In addition, after hCG application, goats were monitored twice daily (0800 and 1800 h) to detect estrus signs, with the use of aproned, sexually active bucks treated with testosterone. Goats were bred 12h after the onset of estrus. Two days after hCG administration, the number of large follicles was higher (P < 0.05) in the hCG-50 and hCG-300 groups (1.7 ± 0.1 and 1.8 ± 0.2, respectively) compared with the hCG-100 and hCG-0 groups (1.4 ± 0.2 and 1.1 ± 0.1, respectively). Although none of the hCG-0-goats depicted estrus, the estrus response from the hCG-50, hCG-100, and hCG-300 groups over the 7-d breeding period was 67%, 100%, and 90%, respectively (P > 0.05), being always accompanied by ovulation. Pregnancy rate (67, 100, and 70%), kidding rate (55%, 80%, and 70%), and litter size (1.6 ± 0.5, 1.5 ± 0.5, and 1.5 ± 0.5) for hCG-50, hCG-100, and hCG-300, respectively, did not differ among the hCG-treated does. Therefore, the combined use of P4-priming plus a 100-IU hCG injection is an effective protocol for inducing estrus in non-cycling Alpine goats during the anestrus-to-estrus transition period, which is of key importance for both goat producers and industrializers.

[The need for the use of chromogens for quantifying Leishmania promastigotes on plates from 96 wells]. / Necesidad del uso de cromógenos para cuantificar promastigotes de Leishmania en placas de 96 pozos.

The existing difficulties in the treatment of leishmaniasis justify the testing of the effect of new products on parasite forms in the search of a therapeutic alternative for the parasitosis. Given the need of establishing a method to evaluate the activity of natural synthetic products in vitro under the Cuban conditions, this paper was aimed at defining the usefulness of p-nitrophenilphosphate as a chromogenic substance for quantification of parasites in plaques from 96 wells. To standardize this colorimetric method the stages of the parasite growth curve were set. The study of linearity and selection of the sample size, which was optional for these assays, showed that it was possible to obtain maximum linear determination coefficient with 20 mm. Likewise, the variation coefficient was compared with and without the chromogen and the effect of changes in culture medium on the reading of absorbances was analyzed. The set limit of quantification proved the need of using chromogen for the purposes of this paper and the general results allow to recommend this less subjective, simpler and quicker methodology to test products of interest in this field.

[Fecal antigens of Fasciola hepatica potentially useful in the diagnosis of fascioliasis]. / Coproantígenos de Fasciola hepatica de posible utilidad en el diagnóstico de la fascioliasis.

This study describes which antigens of Fasciola hepatica are present in the feces of patients with chronic fascioliasis and in the feces of rats infected experimentally with F. hepatica metacercariae. Using a Western blot assay technique with hyperimmune serum obtained from excretory-secretory antigens of adult F. hepatica, we found in the patients' feces antigens of possible diagnostic interest, with molecular weights of 14, 19, 20, 23, 25, 32, 46, 51, and 62 kilodaltons (kDa). In addition, we showed that the peptides of 14, 20, 23, and 51 kDa are also recognized by the majority of the sera from chronic patients. We used affinity chromatography to purify the antigens present in the feces of rats that had been infected for 6 to 12 weeks, using ES78 monoclonal antibody bound to CNBr-activated Sepharose 4B. Through that approach, we identified six polypeptides, of 11, 14, 26, 32, 47, and 51 kDa; three more polypeptides, of 17, 24, and 66 kDa, could only be identified in the feces of rats that had been infected for 10 to 12 weeks. Our results suggest that these polypeptides could be antigens common to both parasitic stages. This is particularly true for the polypeptides of 14, 24, 26, and 51 kDa, because they reacted with the immune sera, the human sera, and the ES78 monoclonal antibody. These polypeptides could be important markers for acute and chronic fascioliasis.

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fa(g)) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fa(g) and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.

[Detection of antibodies against Trypanosoma cruzi in Somoto, Nicaragua, using indirect ELISA and IFI on blood samples on filter paper]. / Detección de anticuerpos contra Trypanosoma cruzi en Somoto, Nicaragua, mediante ELISA indirecto e IFI en muestras de sangre en papel de filtro.

We standardized a solid-phase enzyme-linked immunosorbent assay (ELISA) in order to study the presence of Trypanosoma cruzi antibodies in asymptomatic persons who live in an area of Nicaragua endemic for Chagas' disease. The test was standardized to analyze filter-paper blood samples, which are easy to transport. In the first phase of our investigation, ELISA was used to study 18 samples of total serum and 18 eluates of blood from patients with chronic Chagas' disease; 30 samples of serum and 30 eluates of blood from healthy people, used as negative controls; and 14 samples of serum and 14 eluates of blood from patients with cutaneous or visceral leishmaniasis, which were used to study cross-reactions. Both with the total-serum and the blood-eluate samples, the ELISA test provided 100% sensitivity and 90% specificity. Cross-reactions in the patient samples were observed only with visceral leishmaniasis. The second phase of our investigation was a population study that included eight rural communities in the area of Somoto, Nicaragua. Through random sampling, filter-paper blood samples were collected from 2,434 people (1,335 men and 1,099 women) from the communities of Aguas Calientes, El Brocal, La Manzana, Las Playas, Los Canales, Santa Isabel, Santa Rosa, and Santa Teresa. Studied by ELISA and by indirect immunofluorescence (IIF), the samples included 260 found seropositive by ELISA (10.7%), of which 207 were positive according to IIF (8.5%). With both techniques, the majority of seropositives were among women, but the difference between men and women was not statistically significant. There was a high level of agreement between the results obtained with the two techniques. There was an upward trend with age, with 5.4% of those found seropositive by ELISA being persons 10 years of age or younger and 42.7% of those found seropositive being older than 50. The vast majority of the individuals analyzed were asymptomatic.

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fag) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fag and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.

[Identification, by Western blot, of Fasciola hepatica immunogens recognized by the sera of experimentally infected rats]. / Identificación, mediante western blot, de inmunógenos de Fasciola hepatica, reconocidos por los sueros de ratas infectadas experimentalmente.

This paper was aimed at identifying the main components present in the excretory-secretory antigens of adult parasite which are recognized by the sera of rats experimentally infected with metacercariae of Fasciola hepatica, by using the Western Blot technique. The cynetics of antibodies was also determined with and indirect ELISA. The results obtained allowed to find 31 components with approximate molecular weights from 11 to 136 kD. The predominant fractions were the following: 11-13 kD, 14-16 kD, 23-33 kD, 55-57 kD, 65-71 kD, and 86-136 kD. Antibodies were detected from the 2nd. week of infection in 80% of the animals and from the 3rd. week in 100% of them. There were no antibodies during the first week. The identification of these antibodies may contribute to a better understanding of the mechanisms of immunity linked with the infection by F. hepatica.

Dynamics of antigenemia and coproantigens during a human Fasciola hepatica outbreak.

In the present study the dynamics of antigenemia and coproantigens were studied in patients with Fasciola hepatica infection during an outbreak occurring in La Palma, Pinar del Río, in the West Province of Cuba. Stool and serum samples were collected from 67 patients and 40 healthy subjects. Stool samples were studied by a simple gravity sedimentation technique and an ES78 sandwich enzyme-linked immunosorbent assay (ELISA) for observation of eggs and detection of parasite coproantigens, respectively. Serum samples were also studied by the ES78 sandwich ELISA and an indirect ELISA to detect circulating antigens and antibodies, respectively. At the beginning of the study, 8 of 67 patients had patent infections and 59 had prepatent infections, which was determined by the recent consumption of lettuce contaminated with metacercariae of F. hepatica, the presence of clinical symptoms, and the absence of Fasciola eggs in their stools. Patients with prepatent infections were monitored by all techniques until patency. Circulating antigens were not detected in patients with patent infections. However, coproantigens were clearly detected in all patients with patent infections. On the other hand, 28.8% of patients with prepatent infections tested positive for circulating antigens and 81.4% tested positive for coproantigens in the first stool sample studied. Only two other coproantigen determinations were necessary to diagnose 93.2% of the patients. While circulating antigen levels diminished in all patients during the infection, coproantigen levels increased. The present study demonstrates that the ES78 sandwich ELISA is a better tool than parasitological examination for diagnosis of active early infection, since by the combination of the circulating-antigen detection assay and the coproantigen detection assay 91% of patients were able to be diagnosed at the beginning of the study. In contrast, a coprologic analysis repeated over several weeks was necessary to diagnose 100% of the patients.

Partial characterization of the epitope on excretory-secretory products of Fasciola hepatica recognized by monoclonal antibody ES78.

This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.

[The detection of antibodies, antigens and circulating immune complexes in acute and chronic fascioliasis. Preliminary results]. / Detección de anticuerpos, antígenos y complejos inmunes circulantes en las fascioliasis aguda y crónica. Resultados preliminares.

24 patients with fascioliasis were studied. 19 of them were in the chronic stage and 5 in acute stage. The presence of antigens and of circulating immune complexes (CIC) was detected in 100% of the patients suffering from acute fascioliasis with less than 30 days of evolution of the clinical symptoms; whereas coproantigens were present in 100% of the chronic patients. In this group it was observed a considerable number of cases with elevated levels of antibodies. 43.7% of the cases with CIC were detected by using the precipitation technique with PEG and that of Clq deviation. A highly significant correlation was found between the eggs counting and the CIC levels by both techniques. Another important correlation was established between the eggs counting and the levels of coproantigens.

[Indirect ultra-micro-ELISA for detecting IgG antibodies in patients with fascioliasis]. / UltramicroELISA indirecto para la detección de anticuerpos IgG en pacientes con fascioliasis.

The standardization of an ultramicroELISA for the detection of IgG antibodies anti excretory-secretory antigens of Fasciola hepatica (UME-Fasciola) is described. It was studied a considerable group of sera of which 56 were from patients with fascioliasis, 168 from patients with other parasitic diseases, and 300 from sound persons that were used as negative controls. As regards the parasitology test considered as "Gold Standard", the UME-Fasciola showed a sensitivity of 100%, an specificity of 98%, and predictive values for positives and negatives of 90.3% and 100%, respectively. Cross-reaction was only observed with the sera from patients infected with Opistorchis felineus. On comparing the UME-Fasciola with the conventional ELISA, it was obtained a concordance index of 95.5%.

Detection of Fasciola hepatica antigen in cattle faeces by a monoclonal antibody-based sandwich immunoassay.

A monoclonal antibody-based sandwich immunoassay (mAb sandwich ELISA) was developed for the detection of Fasciola hepatica antigen in the faeces of cattle. The assay was applied to samples from 100 cattle infected with F hepatica, 56 animals with parasitologically proven infections of other parasites and 100 uninfected animals. F hepatica antigen was detected in all the faecal samples from animals with fasciolosis, but none of the samples from the uninfected animals or from those with other parasitic infections had significant levels of F hepatica antigens. The results indicate that the mAb sandwich ELISA is a rapid, simple and useful method for the diagnosis of active F hepatica infection in cattle.

Sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis.

A sandwich enzyme-linked immunosorbent assay has been developed for the detection of Fasciola hepatica excretory secretory (ES) antigens in stool specimens of infected humans. The assay uses antibodies against F. hepatica ES antigens. A monoclonal antibody (ES78, mouse immunoglobulin G2a) was used to capture ES antigens, and a rabbit polyclonal antibody, peroxidase conjugate, was used to identify ES antigens. Thirteen of 14 patients with parasitological evidence of fascioliasis had a detectable concentration of ES antigens (more than 15 ng/ml). None of the stool specimens from controls and from patients with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay. When the 14 patients were retested 2 months after treatment, all of the specimens from the 11 parasitologically cured patients were negative by the antigen detection assay while the specimens from the 3 patients with persisting F. hepatica eggs in their stools remained positive.

[Study of the seroprevalence of toxocariasis in an infantile population in the City of Havana]. / Estudio de seroprevalencia de toxocariasis en una población infantil de Ciudad de La Habana.

A serological study to detect antibodies to Toxocara canis in a group of 156 healthy children from City of Havana is reported for the first time in Cuba. An ELISA method was employed using excretion/secretion antigens obtained in our laboratory. Data on epidemiological factors surveyed in this group are presented. Positivity percentage was of 5.2%. Results are discussed.

[Identification and isolation of common antigens of Fasciola hepatica]. / Identificación y aislamiento de antígenos comunes de Fasciola hepatica.

Four antigenic polypeptides present in excretion-secretion antigens which were common to Fasciola hepatica somatic and tegumentary antigens, were identified by Western blotting and purified by affinity chromatography. Monoclonal antibody ES78 was used to that purpose. Molecular weights calculated for these polypeptides ranged from 37 to 13 kd; they proved to be highly reactive with sera from animals experimentally infected by Fasciola hepatica in periods as early as the second week of infection, with maximal values in weeks 6 and 10.

Detection of circulating excretory secretory antigens in human fascioliasis by sandwich enzyme-linked immunosorbent assay.

We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigen in humans with fascioliasis. The assay uses antibodies against Fasciola hepatica excretory secretory (ES) antigens. A monoclonal antibody was used to capture circulating ES antigens, and a human polyclonal antibody peroxidase conjugate was used to identify circulating ES antigens. Optimal dilutions of all reagents were determined by block titration. The antigen concentration in sera from patients was estimated by comparing the optical density at 492 nm of test sera with a standard curve. All of the serum samples from 25 patients with parasitological evidence of fascioliasis had a detectable antigen concentration (more than 10 ng/ml). None of the serum samples from 80 patients infected with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay.