Detection of Rickettsia felis in a New World flea species, Anomiopsyllus nudata (Siphonaptera: Ctenophthalmidae)

The flea and rodent samples studied in this project were collected from field study sites in New Mexico from winter 1998 to spring 2001. During this period, 155 small rodents (14 different species) were live-trapped and combed for the presence of fleas. A total of 253 fleas were collected, comprising 21 species. Two of the 253 fleas collected were polymerase chain reaction (PCR) positive for the Rickettsia 17-kDa protein gene. These two fleas were both Anomiopsyllus nudata Baker, each collected from an individual Neotoma albigula Hartley, on two occasions. Individual fleas positive for the Rickettsia 17-kDa protein gene were then tested with primers targeting the rickettsial genes for citrate synthase (gltA) and two major outer membrane proteins (ompA and ompB). The nucleotide sequences of the PCR products of these two fleas were identical to each other and were 100% (394/394), 100% (1150/1150), 99.8% (469/470), and 99.3% (818/824) similar to the corresponding sequences of the 17-kDa, gltA, ompA, and ompB genes of Rickettsia felis, respectively. Flea homogenates of individual PCR-positive fleas were inoculated into shell vials seeded with Vero cells, and the Gimenez stain technique was used to demonstrate the presence of Rickettsia-like organisms in detached cells found in aspirated medium 19 d after inoculation. These cells were harvested and tested by PCR, targeting portions of the 17-kDa and gltA genes, resulting in products 100% identical to R. felis. This work comprises the first report of R. felis detection in a flea species (A. nudata) endemic to the New World.     

Development and comparison of enzyme immunoassays for diagnosis of Chagas' disease using fixed forms of Trypanosoma cruzi (Epimastigotes, Amastigotes, and Trypomastigotes) and assessment of antigen stability for the three assays

Three enzyme immunoassays (EIAs) for diagnosis of Chagas' disease were developed with fixed forms of Trypanosoma cruzi using a panel of 435 sera from the following groups: Venezuelan subjects positive by immunofluorescence (n = 70), Venezuelan healthy controls (n = 85), healthy Canadians (n = 166), and subjects with other parasitic diseases (n = 114). All assays achieved 100% sensitivity and reasonable specificity for amastigotes (97.6%), epimastigotes (98.3%), and trypomastigotes (99.3%). The fixed-trypomastigote assay was stable over 4 months at 4 degrees C and room temperature. These data suggest that a fixed-trypomastigote EIA may be a suitable candidate for blood bank screening.     

Morphologic Study of the Participation of the Complement System in Hyperacute Rejection of Renal Xenotransplants

The role of polymorphonuclear leukocyte (PMN) and platelet infiltration in the hyperacute rejection of renal xenotransplants was studied. In a first group, a dog kidney was grafted to rabbit recipients with intact immune adherence and chemotaxis. A second group included recipients depleted of PMN's with nitrogen mustard, and in a third group, immune adherence and chemotaxis were modified by depleting the third component of complement by means of cobra venom factor. Serial kidney biopsies were studied with light and electron microscopic technics. A semiquantitative evaluation of PMN and platelet glomerular infiltration indicated that a reduction in the number of PMN's or platelets is associated with an increased survival time of the transplanted kidney.     

Accuracy of a commercial enzyme-linked immunosorbent assay for CagA in patients from Brazil with and without gastric carcinoma

We validated a commercial enzyme-linked immunosorbent assay for the detection of anti-CagA antibodies in Brazilian patients with Helicobacter pylori infection. The test presented high sensitivity (97.4%) and specificity (88.9%) when employed in patients without gastric carcinoma. However, in gastric carcinoma patients, the test was neither sensitive nor specific enough to detect cagA-positive H. pylori infection.     

Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration

The maintenance of a benign chronic Toxoplasma gondii infection is mainly dependent on the persistent presence of gamma interferon (IFN-gamma) in the central nervous system (CNS). However, IFN-gamma-activated microglia are paradoxically involved in parasitism control and in tissue damage during a broad range of CNS pathologies. In this way, nitric oxide (NO), the main toxic metabolite produced by IFN-gamma-activated microglia, may cause neuronal injury during T. gondii infection. Despite the potential NO toxicity, neurodegeneration is not a common finding during chronic T. gondii infection. In this work, we describe a significant down-modulation of NO production by IFN-gamma-activated microglia in the presence of conditioned medium of T. gondii-infected astrocytes (CMi). The inhibition of NO production was paralleled with recovery of neurite outgrowth when neurons were cocultured with IFN-gamma-activated microglia in the presence of CMi. Moreover, the modulation of NO secretion and the neuroprotective effect were shown to be dependent on prostaglandin E(2) (PGE(2)) production by T. gondii-infected astrocytes and autocrine secretion of interleukin-10 (IL-10) by microglia. These events were partially eliminated when infected astrocytes were treated with aspirin and cocultures were treated with anti-IL-10 neutralizing antibodies and RP-8-Br cyclic AMP (cAMP), a protein kinase A inhibitor. Further, the modulatory effects of CMi were mimicked by the presence of exogenous PGE(2) and by forskolin, an adenylate cyclase activator. Altogether, these data point to a T. gondii-triggered regulatory mechanism involving PGE(2) secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia. This may reduce host tissue inflammation, thus avoiding neuron damage during an established Th1 protective immune response.     

Toxoplasma gondii prevents neuron degeneration by interferon-gamma-activated microglia in a mechanism involving inhibition of inducible nitric oxide synthase and transforming growth factor-beta1 production by infected microglia

Interferon (IFN)-gamma, the main cytokine responsible for immunological defense against Toxoplasma gondii, is essential in all infected tissues, including the central nervous system. However, IFN-gamma-activated microglia may cause tissue injury through production of toxic metabolites such as nitric oxide (NO), a potent inducer of central nervous system pathologies related to inflammatory neuronal disturbances. Despite potential NO toxicity, neurodegeneration is not commonly found during chronic T. gondii infection. In this study, we describe decreased NO production by IFN-gamma-activated microglial cells infected by T. gondii. This effect involved strong inhibition of iNOS expression in IFN-gamma-activated, infected microglia but not in uninfected neighboring cells. The inhibition of NO production and iNOS expression were parallel with recovery of neurite outgrowth when neurons were co-cultured with T. gondii-infected, IFN-gamma-activated microglia. In the presence of transforming growth factor (TGF)-beta1-neutralizing antibodies, the beneficial effect of the parasite on neurons was abrogated, and NO production reverted to levels similar to IFN-gamma-activated uninfected co-cultures. In addition, we observed Smad-2 nuclear translocation, a hallmark of TGF-beta1 downstream signaling, in infected microglial cultures, emphasizing an autocrine effect restricted to infected cells. Together, these data may explain a neuropreservation pattern observed during immunocompetent host infection that is dependent on T. gondii-triggered TGF-beta1 secretion by infected microglia.     

Cytogenetic analysis of 100 consecutive newly diagnosed cases of acute lymphoblastic leukemia in Rio de Janeiro

We report the cytogenetic analysis of newly diagnosed Brazilian children with acute lymphocytic leukemia (ALL). We investigated 100 ALL cases from four different institutions in Rio de Janeiro. The frequency of chromosomal abnormalities was 92.3%. The karyotype profile and recurrent abnormalities found in this study do not differ essentially from those described by other groups. Although the Brazilian population is usually the product of different ethnic groups, our results show that the frequency of each recurrent abnormality is similar to that found in populations without our degree of diverse ethnic composition. Hence, our results suggest that childhood ALL in Brazil has the same biological features as that in developed countries, supporting the use of similar treatment protocols. We can therefore expect to reach the same survival rates in the coming years, depending possibly on the efficacy of the support therapy and extent of social assistance.