Fluorescent two-photon nanolithography.

Improving the excitation conditions in two-photon fluorescent lithography reduces the size of the fabricated structures to nanometre scales. We demonstrate that a precise control of the illumination profile and the scanning speed of the laser beam is enough to decrease the photo-polymerization volume of resins by orders of magnitude. This work also shows experimental evidence of surface effects that yield a different polymerization intensity threshold compared with bulk. Such phenomenon enables to perform a non-linear optical nanolithography in a simple way, allowing fast-prototyping procedures. We present a detailed study of the polymer growth process using fluorescence and atomic force microscopy.

Detection of coproantibodies and faecal immune complexes in human trichinellosis.

Trichinella spiralis is the nematode causative agent of trichinellosis, an intestinal and tissular parasitosis. Even though an early diagnosis during the intestinal phase is essential to limit the infection in humans, to date, there are no available tests to achieve this goal. Based on the immune response generated by the host's intestinal mucosa, the aim of this work was to develop ELISAs to assess the presence of coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes in stool samples of 18 individuals belonging to different outbreaks that have arisen in Argentina. By the methodologies developed in this work it was found that anti-muscle larva excretory-secretory products (ML-ESP) CAbs were detected in 89% of the samples analysed regardless of the time p.i. Anti-ML-ESP IgA, IgG, IgE and IgM were detected in 56%, 56%, 28% and 22% of the individuals respectively. Those samples negative for anti-ML-ESP total immunoglobulins proved positive for anti-adult worm-ESP CAbs. No CAgs were detected in any of the samples. The results obtained in this work indicate that the intestinal immune response in human trichinellosis is featured by all the isotypes of specific immunoglobulins. Furthermore, the detection of antibodies in stool samples, in either the free or complexed form, could be applied to confirm early human trichinellosis.

Early pulmonary response in rats infected with Trichinella spiralis.

The migratory stage of Trichinella spiralis, the newborn larva, travels along the pulmonary microvascular system on its way to the striated muscle cells. In the present study, an important inflammatory reaction was observed on days 5 and 14 post-infection (p.i.) in the lungs of infected rats. This inflammation was characterized by a Th2 cell phenotype of hyperplastic bronchus-associated lymphoid tissue and by goblet cell hyperplasia. Among the inflammatory cells were eosinophils and mast cells scattered over the pulmonary parenchyma. On day 5 p.i. the number of IgE(+), CD4(+) and CD5(+) cells in the bronchus-associated lymphoid tissue were increased and IgE-secreting lung cells were also detected. At the end of the migratory phase of the infection (day 14 p.i.), only IgE(+) cells were detected in high numbers and in the bronchoalveolar lavage fluid, an increment in the total IgE levels as well as the presence of IgE and IgA anti-larvae surface were also detected. In cytotoxicity assays, cells from the bronchoalveolar lavage had considerable biological activity since they were able to kill the larvae even in the absence of specific antibodies. These results show that the lung is an organ involved in the immune response developed early during a T. spiralis infection and suggest its importance in the protection of the host.

In vitro and in vivo effects of progesterone on Trichinella spiralis newborn larvae.

We have previously demonstrated that during pregnancy there exists an increased parasiticide activity against Trichinella spiralis newborn larvae (NBL) in infected rats. In this work we analysed the contribution of peritoneal cells from noninfected pregnant rats to the mortality of the NBL in cytotoxicity assays, and evaluated the role of progesterone in this effector mechanism. Our findings suggest that progesterone can induce activation of effector peritoneal cells to destroy the NBL in a rapid and antibody-independent manner. The administration of progesterone to ovariectomized rats also led to a significant decrease in the parasite load of the animals, thus demonstrating that progesterone induces the increase of the parasiticide activity of the leukocytes involved in the mechanisms of NBL death.

Aspectos clinicos, serologicos y parasitologicos de un brote de triquinelosis humana en villa mercedes, san luis, argentina las fases aguda y cronica de la infeccion / Clinical, serological and parasitological aspects of an outbreak of human trichinellosis in Villa Mercedes, San Luis, Argentina: the acute and chronic phases of the infection

Se describen las características clínicas y serológicas de individuos cursando las fases aguda (n: 54) y crónica (n: 32) de triquinelosis, de un brote epidémico ocurrido en una zona endémica de Argentina, originado por el consumo de productos de cerdo de origen comercial. Se evaluaron los datos epidemiológicos, signos y síntomas de la parasitosis y estudios de laboratorio. Se realizaron estudios parasitológicos en pacientes y en chacinados. Los resultados mostraron que: a) la carga parasitaria de los productos de cerdo fue de 200.0±18.3 larvas/g; b) se demostró la presencia de larvas musculares en 10 de 11 pacientes estudiados; c) durante la fase aguda se observó fiebre (94%), eosinofilia (90%), mialgias (85%), cefalea (81%), edema facial (54%), diarrea (52%) y anticuerpos anti-Trichinella (64%); d) el 15% de los pacientes debió ser hospitalizado presentando el 7% de ellos complicaciones; e) durante la fase crónica se observó mialgia (72%), alteraciones visuales (22%), desórdenes gastrointestinales (31%) y la persistencia de anticuerpos (77%). Este estudio evidencia el impacto socioeconómico de la triquinelosis debido a las características clínicas de la fase aguda y a la presencia de síntomas en la fase crónica. .

Aspectos clinicos, serologicos y parasitologicos de un brote de triquinelosis humana en villa mercedes, san luis, argentina las fases aguda y cronica de la infeccion / Clinical, serological and parasitological aspects of an outbreak of human trichinellosis in Villa Mercedes, San Luis, Argentina: the acute and chronic phases of the infection

Se describen las características clínicas y serológicas de individuos cursando las fases aguda (n: 54) y crónica (n: 32) de triquinelosis, de un brote epidémico ocurrido en una zona endémica de Argentina, originado por el consumo de productos de cerdo de origen comercial. Se evaluaron los datos epidemiológicos, signos y síntomas de la parasitosis y estudios de laboratorio. Se realizaron estudios parasitológicos en pacientes y en chacinados. Los resultados mostraron que: a) la carga parasitaria de los productos de cerdo fue de 200.0±18.3 larvas/g; b) se demostró la presencia de larvas musculares en 10 de 11 pacientes estudiados; c) durante la fase aguda se observó fiebre (94%), eosinofilia (90%), mialgias (85%), cefalea (81%), edema facial (54%), diarrea (52%) y anticuerpos anti-Trichinella (64%); d) el 15% de los pacientes debió ser hospitalizado presentando el 7% de ellos complicaciones; e) durante la fase crónica se observó mialgia (72%), alteraciones visuales (22%), desórdenes gastrointestinales (31%) y la persistencia de anticuerpos (77%). Este estudio evidencia el impacto socioeconómico de la triquinelosis debido a las características clínicas de la fase aguda y a la presencia de síntomas en la fase crónica. . (AU)

Human serum antibodies against shared antigens of different stages of Trichinella spiralis: relevance of glycan and protein epitopes.

Taking into account that antibodies against the surface antigens of newborn larvae (anti-NBL Abs) present in sera from individuals with chronic trichinellosis recognize antigenic determinants of the excretory-secretory muscle larva products (ML-ESP), and that these products are mainly glycoproteins, the aim of this work was to assess the frequency of anti-NBL Abs in sera from individuals with acute and chronic trichinellosis, to analyse the relevance of glycan and protein epitopes of the ML-ESP in the cross-reactivity phenomenon, and its correlation with the host's serum response towards these products. Anti-NBL surface Abs were determined in sera by indirect immunofluorescence. The degree of recognition by serum and purified anti-NBL Abs was evaluated comparatively before and after chemical deglycosylation of ML-ESP by immunoelectrotransfer blot assay. Results showed that 64% of the sera from individuals with acute trichinellosis and 35% of those belonging to the chronic phase had anti-NBL Abs, and also that the protein epitopes are the major ones responsible for the cross-reactivity phenomenon involving the ML-ESP and the NBL surface during both phases of the infection, while glycan epitopes are immunodominant in the stimulation of the host's immune system. A modulatory phenomenon in the immune response generated towards Trichinella spiralis NBL driven by the ML-ESP is postulated.

Immunoparasitological parameters of the intestinal phase of trichinellosis in rats.

Enzyme-linked immunosorbent assays (ELISAs) were developed in order to detect coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes (FIC) in rats experimentally infected with Trichinella spiralis. The usefulness of these assays was compared to that of a conventional ELISA for detection of serum antibodies (Abs) to muscle larvae excretory-secretory products (ML-ESP). The ELISA for CAgs was the first parameter to give a positive result but the detection was limited only to day 2 p.i. CAbs against ML-ESP and adult worm excretory-secretory products (AW-ESP) was first positive on day 4 p.i. Anti-ML-ESP remained positive until day 12 p.i. while CAbs against AW-ESP remained positive throughout the study period. Specific IgE and IgA were found. FIC were detected between days 2 and 8 p.i. Serum Abs began to appear on day 10 p.i. Therefore, the ELISA for CAbs was a suitable assay for the detection of the enteral and early phases of the infection.

Diagnosis of human trichinellosis: pitfalls in the use of a unique immunoserological technique.

Serum samples belonging to three outbreaks in Argentina (47 patients) taken at different times post-ingestion were analysed employing IIF and ELISA simultaneously. Results show that: a) the number of patients diagnosed by a unique technique, especially by ELISA (31 patients), was lower than the one obtained by the simultaneous use of both assays (38 patients); b) four patients out of the seven diagnosed by a unique technique were negative by the other assay over the period of time evaluated. Therefore, it can be concluded that the use of a sole immunoserological technique can not only lead to the delay in the detection but also to the misdiagnosis of this parasitic infection.

Immunoelectrotransfer blot assay in acute and chronic human trichinellosis.

An immunoelectrotransfer blot assay (IETB) using excretory secretory products of muscle larvae of Trichinella spiralis (ML-ESP) and the avidin biotin system was developed in order to characterize reactivity against ML-ESP in sera from patients with acute and chronic trichinellosis. A complete pattern of up to 13 bands was developed by sera from individuals with trichinellosis where doublets, triplets, or single bands were shown to have molecular weights of roughly 66, 55, 45, 36, 29, 24, and 14 kDa. The bands at approximately 55, 36, 29, and 14 kDa proved specific for T. spiralis. The band at approximately 55 kDa was present in all trichinellosis sera, whereas the approximately 14-kDa band was present in only a small percentage of sera. The development of approximately 36- and 29-kDa bands suggests a modulation of the reactivity against ML-ESP over time. IETB proved more sensitive for the population of chronic trichinellosis under study than a conventional diagnostic enzyme-linked immunosorbent assay, allowing negative or borderline serum samples to be determined. Thus, this technique, when applied for human trichinellosis surveillance, should provide a useful tool in endemic areas.

Cytotoxicity-blocking antibodies in human chronic trichinellosis.

Antisurface newborn larva (NBL) antibodies (Abs) were found in sera from individuals chronically infected with Trichinella spiralis. These Abs were incapable of inducing NBL death by activation of normal human leukocytes of peripheral blood as determined by in vitro assays of antibody-dependent cell cytotoxicity (ADCC). Besides, such sera blocked the cytotoxic reaction mediated by Abs produced a few weeks after infection. The blocking activity could not be attributed to any particular isotype by the indirect immunofluorescence technique. Purified antisurface NBL Abs obtained from sera from chronically infected patients recognized antigens of muscle-larva excretory-secretory products (ML-ESP) in an enzyme-linked immunosorbent assay (ELISA) and an immunoelectrotransfer blot assay. Likewise, as did chronic sera, a monoclonal Ab raised against ML-ESP blocked NBL death in ADCC assays. These results suggest that during the course of an infection by T. spiralis, Abs related to ML-ESP provide an immunoevasive mechanism for avoidance by NBL of an important anti-NBL host effector mechanism.

Diagnosis of porcine trichinellosis: parasitological and immunoserological tests in pigs from endemic areas of Argentina.

In order to compare the reliability of serological and parasitological techniques for the diagnosis of porcine trichinellosis from endemic areas in Argentina, 116 pigs were studied: 61 animals from two separate outbreaks and 55 from a small abattoir. Direct diagnostic techniques included trichinoscopy and the artificial digestion method. Indirect diagnostic tests used in this study were the enzyme-linked immunosorbent assay (ELISA), employing the excretory-secretory products of muscle larvae (ML) as antigen, and the indirect immunofluorescence assay using as antigen ML in suspension (IIF-susp), cryostat sections of infected rat muscle or of free ML (IIF-slide). The percentage of parasitologically positive pigs was invariably lower than that of serologically positive animals (IIF-slide), even when digestion studies were carried out individually with a greater amount of muscle sample than required by current regulations. Close correlation was found between IIF using as antigen tissue sections and IIF using free ML sections, while IIF-susp proved unsuitable for diagnosis since this assay presented a high percentage of false negative results (20%). The IIF-slide technique proved positive in all parasitologically positive animals. ELISA rendered a lower percentage of positive reactions than IIF-slide, especially when worm burden was low. Since most parasitologically positive animals rendered at least two positive serological tests (two variations of IIF or IIF plus ELISA), those negative by digestion and positive by two serological methods were strongly suspected of having trichinellosis. Upon studying swine from a abattoir it was found that 9% of the pigs were positive when assayed by two serological techniques, but Trichinella spiralis infection could not be parasitologically confirmed. To sum up, serological methods may be used for screening all pigs and positive findings should be tested by the digestion method by analysing a greater quantity of pork than that required by current regulations, above all in areas with reported clinical trichinellosis in humans, to ensure that the pork is safe for human consumption.

Evaluation of an enzymatic immunohistochemical technique in human trichinellosis.

A comparative study was undertaken between an enzymatic immunohistochemical technique (EIT) developed for the diagnosis of human trichinellosis and the indirect immunofluorescence test (IIF), analysing sera from outbreaks of human trichinellosis in Argentina. The EIT was developed using a biotinylated anti-human gammaglobulin and a preformed macromolecular complex of avidin and biotinylated peroxidase. In both tests, the antigen used consisted of infected rat tissue sections containing muscle larval cysts of Trichinella spiralis. Results showed that the EIT closely correlated with IIF and also allowed diagnosis at an early stage of infection, thus helping to provide effective treatment for the disease. When the test was performed on sera from healthy individuals and those with other parasitic infections, cross-reactions were observed only with sera from patients with toxocariasis (1/8), Chagas' disease (3/17) and four out of 100 healthy individuals. No cross-reactions were observed with sera from patients with toxoplasmosis (0/7) or hydatidosis (0/8). Assay sensitivity was 100% and its specificity 93%. Since it renders no false negative results, EIT is an effective screening tool for detecting infection and should prove to be an important diagnostic technique for trichinellosis in rural areas and for epidemiological surveys.

Blocking anti-Trichinella spiralis antibodies in chronically infected rats.

Specific anti-newborn larva antibodies present in the serum of rats chronically infected with Trichinella spiralis were incapable of inducing killing of newborn larvae by activation of normal peritoneal cells. These late antibodies blocked the cytotoxic reaction induced by early antibodies produced a few weeks after infection. Passive transference of late serum to normal mice failed to induce protective immunity against infection by newborn T. spiralis larvae. When late immunoserum was fractionated by gel filtration, blocking activity was found only in the fraction containing IgG subclasses. By indirect immunofluorescence assay and cytotoxic reaction it was shown that blocking antibodies were specific for newborn larvae and could not be adsorbed with muscle larvae. It is concluded that the synthesis of anti-newborn larva antibodies is modulated in the course of a chronic infection: early antibodies developing shortly after infection are cytotoxic, whereas blocking antibodies predominate in the late population. Furthermore, the results suggest that during a chronic infection, resistance to reinfection may be modified.

Immune cytotoxic activity of human eosinophils against Trichinella spiralis newborn larvae.

The ability of human eosinophils to kill the newborn larvae (NBL) of Trichinella spiralis of different maturation status, in the presence of antibody, was studied. A cytotoxic in vitro test was performed using NBL less than 2 h of age (NBL2) or NBL maintained in culture at 37 degrees C for 20 h (NBL20), peripheral blood eosinophils, anti-Trichinella serum and human fresh serum as source of complement. Under these experimental conditions eosinophils from normal individuals attached to NBL2 as well as to NBL20 but only the latter were killed. On the other hand, eosinophils from volunteers with eosinophilia killed NBL regardless of larval age. Neither adherence nor significant mortality was observed in the absence of immune serum. These results indicate that NBL maturation and eosinophil activation status are crucial for antibody-dependent cellular cytotoxic reaction (ADCC).

Immune killing of newborn Trichinella larvae by human leucocytes.

The capacity of human leucocytes from normal donors to kill the newborn larvae of the nematode Trichinella spiralis in vitro, in the presence of serum from infected individuals, was studied using newborn larvae (NBL) less than 2 h of age or NBL that had been maintained in culture at 37 degrees C for 20 h. Neutrophils and monocytes attached to newborn Trichinella larvae and killed them, regardless of their age. When eosinophils were used, 20 h old NBL were killed whereas 2 h old NBL were not. Complement was essential in the cytotoxic effect of leucocytes. These results indicate that host defence against T. spiralis in humans may be a complex mechanism in which different cell types can be involved. They also show that the age of maturation of the NBL is of paramount importance in the antibody-dependent cellular cytotoxicity reaction.

[Serological, clinical, and epidemiological aspects of an outbreak of trichinosis in Azul, province of Buenos Aires]. / Aspectos serológicos, clínicos y epidemiológicos de un brote de triquinosis en Azul, Provincia de Buenos Aires.

An outbreak of human trichinellosis that took place in Southern Buenos Aires Province, an endemic area for this zoonosis, was studied. Eighteen individuals were infected by eating swine sausages lacking veterinary control. Clinical symptoms, as well as serological studies related to antibody production and cytotoxic capacity of the sera are described. Eight interdicted pigs and their products were analysed. In order to determine the transmission network of this zoonosis, wild rats were hunted in the farm where the swine were bred. The clinical symptoms of the infected individuals were: myalgia, edema, fever and cephalea. Sixty-one percent of the patients showed antibodies against muscle larvae 15-20 days after ingestion of the infected meat. Five out of eighteen patients showed anti-newborn larvae antibodies 40-60 days postinfection. The sausage burden was 5.3 muscle larvae/g. Two out of eight swine were diagnosed employing serological techniques, while parasitic techniques detected only one. The rat is an important reservoir in the domestic cycle of this zoonosis.

Aspectos serológicos, clínicos y epidemiológicos de un brote de triquinosis en Azul, Provincia de Buenos Aires. / [Serological, clinical, and epidemiological aspects of an outbreak of trichinosis in Azul, province of Buenos Aires]

An outbreak of human trichinellosis that took place in Southern Buenos Aires Province, an endemic area for this zoonosis, was studied. Eighteen individuals were infected by eating swine sausages lacking veterinary control. Clinical symptoms, as well as serological studies related to antibody production and cytotoxic capacity of the sera are described. Eight interdicted pigs and their products were analysed. In order to determine the transmission network of this zoonosis, wild rats were hunted in the farm where the swine were bred. The clinical symptoms of the infected individuals were: myalgia, edema, fever and cephalea. Sixty-one percent of the patients showed antibodies against muscle larvae 15-20 days after ingestion of the infected meat. Five out of eighteen patients showed anti-newborn larvae antibodies 40-60 days postinfection. The sausage burden was 5.3 muscle larvae/g. Two out of eight swine were diagnosed employing serological techniques, while parasitic techniques detected only one. The rat is an important reservoir in the domestic cycle of this zoonosis.