Chagas disease.

Chagas disease is the clinical condition triggered by infection with the protozoan Trypanosoma cruzi. The infection is transmitted by triatomine insects while blood feeding on a human host. Field studies predict that one third of an estimated 18 million T cruzi-infected humans in Latin America will die of Chagas disease. Acute infections are usually asymptomatic, but the ensuing chronic T cruzi infections have been associated with high ratios of morbidity and mortality: Chagas heart disease leads to unexpected death in 37.5% of patients, 58% develop heart failure and die and megacolon or megaoesophagus has been associated with death in 4.5%. The pathogenesis of Chagas disease appears to be related to a parasite-induced mutation of the vertebrate genome. Currently, treatment is unsatisfactory.

Evidence of rickettsial spotted fever and ehrlichial infections in a subtropical territory of Jujuy, Argentina.

Between November 1993 and March 1994, a cluster 6 pediatric patients with acute febrile illnesses associated with rashes was identified in Jujuy Province, Argentina. Immunohistochemical staining of tissues confirmed spotted fever group rickettsial infection in a patient with fatal disease, and testing of serum of a patient convalescing from the illness by using an indirect immunofluorescence assay (IFA) demonstrated antibodies reactive with spotted fever group rickettsiae. A serosurvey was conducted among 16 households in proximity to the index case. Of 105 healthy subjects evaluated by IFA, 19 (18%) demonstrated antibodies reactive with rickettsiae or ehrlichiae: 4 had antibodies reactive with Rickettsia rickettsii, 15 with Ehrlichia chaffeensis, and 1 with R. typhi. Amblyomma cajennense, a known vector of R. rickettsii in South America, was collected from pets and horses in the area. These results are the first to document rickettsial spotted fever and ehrlichial infections in Argentina.

Poorly differentiated insular thyroid carcinoma. A case report with identification of intact insulae with fine needle aspiration biopsy.

BACKGROUND: Subsequent to the publication of a report in 1984 entitled "Poorly Differentiated ("Insular") Carcinoma: A Reinterpretation of Langhans "wuchernde Struma," poorly differentiated insular thyroid carcinoma (PDITC) has become recognized as a distinct thyroid neoplasm. It is classified morphologically and biologically as an intermediate entity between well-differentiated (papillary and follicular) and undifferentiated (anaplastic) thyroid carcinomas. Only a few publications have addressed the findings with fine needle aspiration biopsy (FNAB). CASE: A 67-year-old female presented for evaluation of a massively enlarged thyroid gland. Fine needle aspiration biopsy of the thyroid with a 22-gauge needle showed many large, multilayered, round to oval nests of tumor cells, 0.2-0.4 mm in diameter. Rosettelike configurations of 8-15 cells, 0.025-0.050 mm in diameter, were also observed. Nests of neoplastic cells in the histologic sections were virtually identical to those in the fine needle aspiration biopsy specimens. When the patient developed metastatic cervical adenopathy one year later, a microfollicular pattern was seen on both the FNAB and histologic sections. CONCLUSION: When nests of tumor cells, 0.2-0.4 mm in diameter, are identified in a thyroid FNAB specimen, PDITC should be included in the differential diagnosis. A microfollicular pattern in a metastatic lymph node does not exclude the possibility that the primary tumor is a PDITC.

A novel receptor mediated ATP transport system regulated by tyrosine and serine/threonine phosphokinases in Trypanosoma cruzi trypomastigotes.

Trypanosoma cruzi has a plasma membrane ATP transport system that may consist of an exterior receptor domain (ATP-R) and an interior domain regulated by tyrosine and serine/threonine kinases. The addition of exogenous ATP to freely swimming trypomastigotes resulted in a receptor-mediated inward movement of the nucleotide, and the system obeyed mass action law (Km, 9.42 microM and Vmax, 77.7 nmol x min(-1) x 4 x 10(6) trypomastigotes(-1)). Preloaded [3H]ADPi was not exchanged for ATP following the addition of increasing concentrations of exogenous ATPo to swimming trypomastigotes. Trypomastigote [ATP]o <--> ATP-R --> [ATP]i transport was [ATP]o-dependent and saturable at 100 microM. [ATP]o <--> ATP-R --> [ATP]i transport was abrogated by the tyrosine kinase inhibitors, genistein and lavendustin A. [ATP]o <--> ATP-R --> [ATP]i transport was also inhibited by the serine/threonine kinase inhibitor, staurosporin. Suramin, the antagonist of P2x and P2y purinergic receptors and the inhibitor of tyrosine kinase growth factor receptors, was also a very effective competitive inhibitor of the trypomastigote ATP transport system. The action of exogenous [gamma32P]ATPo resulted in the initial and simultaneous phosphorylation of a 63-kDa polypeptide (p63) and of a 92.4-kDa polypeptide (p92.4), which was followed by the abrupt phosphorylation of many other substrate proteins. The trypomastigote p63/p92.4 polypeptides may represent substrate proteins of a putative ATP-R-related tyrosine phosphokinase, and ATP receptors may transmit their signals by phosphorylation of specific substrate proteins.

Attenuation of parasite cAMP levels in T. cruzi-host cell membrane interactions in vitro.

Previous investigations have shown that the adhesion of T. cruzi plasma membrane vesicles (PMV) to monolayers of host cell myoblasts and to immobilized heart muscle sarcolemma membranes (PAM) on polyacrylamide beads is mediated by the interaction of T. cruzi attachment sites with the muscarinic cholinergic and beta-adrenergic receptors of the host cell membrane. It has also been shown that this interaction is blunted by the specific antagonists of the mammalian receptors atropine and propranol, respectively. In the studies reported here, PAM also rapidly attached to swimming T. cruzi trypomastigotes in a complex, concentration-dependent fashion and binding isotherms showed that the equilibrium between free and bound PAM is rapidly reached within 2 minutes of incubation in physiologically balanced salt solutions. In this time frame, trypomastigote cAMP levels are significantly reduced from steady state values within 30 seconds of the addition of PAM in a buffer system containing a diesterase inhibitor. Maximal attenuation of cAMP levels was measured between 1 and 2 minutes of the addition of PAM to T. cruzi trypomastigotes. The degree of cAMP level attenuation was reduced by blocking PAM attachment with either atropine or propranol. On the basis of these results we propose that a likely pathway for the negative parasite signal generated upon adhesion of host muscle cell membranes to the surface of the flagellates is from the parasite's surface attachment sites directly to a Pertussis toxin sensitive inhibitory protein Gi, thereby blunting adenyl cyclase activity and cAMP formation.

Characterization of the tobacco glycoprotein surface binding property of heart and skeletal muscle cells. I. Modulation of the heart cell membrane TGP interaction by anti-TGP IgG.

Monolayers of L6 rat skeletal myoblast cells formed surface binding isotherms with the purified tobacco leaf glycoprotein TGP1 and the enriched cigarette tar glycoprotein TGP2. Scatchard analysis showed that the binding in the range of the limited concentrations tested was to a single class molecule and the calculated affinity constant (Kd) for TGP1 and TGP2 showed similar values (9.78 x 10(-13) M and 3.09 x 10(-13) M, respectively). The bound TGPs were almost totally displaced by excess nonradiolabeled molecules. The calculated Bmax of the L6 myoblast monolayer was 2.93 fmol for TGP1 and 0.217 fmol for TGP2 per 32.2 mm2. Guinea pig heart sarcolemma binding isotherms were also formed with radiolabeled TGP1 and TGP2. The interaction of tobacco leaf TGP1 with the heart cell membranes was irreversible because only 15-20% of the bound TGP1 was displaced by 100-fold, non-labeled molecules but the interaction of tar TGP2 with heart sarcolemma was reversible and probably saturable. The heart sarcolemma TGP2 affinity constant (Kd) was 5.88 x 10(-7) M and the Bmax, 2.45 x 10(-8) M per 12.5 micrograms sarcolemma. Pretreatment of heart sarcolemma with increasing concentrations of leaf TGP1 did not displace tar TGP2 binding but its absorption on the membrane resulted in increased TGP2 sarcolemma attachment by a complex and unexplained mechanism. Increasing concentrations of the sera of 10 of 15 guinea pigs (67%) that received mainstream emissions of tobacco smoke from a University of Kentucky cigarette smoking machine for 152 days, displaced cigarette tar TGP2 heart cell sarcolemma attachment and this inhibition was significantly different from that produced by the sera of sham smoked and of non-exposed animals (Mann-Whitney test, p = 0.0082). Staphylococcus protein A inhibited the displacement of TGP2 produced by the sera of cigarette smoke exposed guinea pigs and this observation indicated that this action was mediated by IgG molecules. The specific immunoprecipitation of a radiolabeled surface epitope of the L6 myoblast monolayers pretreated with TGP1 or TGP2 by immune IgG against TGP2 and by the IgG of an antiserum against standard TGP showed that the tobacco glycoproteins attached to a unit polypeptide of the plasma membrane of the muscle cells of approximately 76 kDa. These data support the notion that TGP molecules in cigarette smoke are absorbed systemically on smoking and may have a direct toxic effect when they attach to the surface TGP binding proteins of heart and skeletal muscle cells.

Plasma membrane ATP receptors in Trypanosoma cruzi trypomastigotes.

Trypomastigotes of Trypanosoma cruzi were impermeable to exogenous radiolabeled ATP for up to 2 h at 4 degrees C. Radioligand binding assays in the cold showed that trypomastigotes had two populations of saturable ATP receptors (ATP-Rs) and the radioligand interaction was reversed by nonlabeled ATP in concentration-dependent assays. The Kds of high- and low-affinity ATP-Rs were 7.27 x 10(-8) and 4.32 x 10(6)M, respectively. The BmaxS for ATP-R1 and ATP-R2 were 2.05 x 10(-14) and 2.50 x 10(-12) mol/4 x 10(-6) flagellates, respectively, or 3100 ATP-R1 copies per trypomastigote and 376,000 ATP-R2 copies per trypomastigote. ATP,2-methyl-thio-ATP,ITP, and ADP displaced ATP-Rs (IC50s: 2.59 x 10(-6) to 7.84 x 10(-6)M/4 x 10(6) trypomastigotes). UTP, CTP, ADP beta S, Cibacron blue, and azido-ATP were 10-100 times less effective. Atractyloside, adenosine, suramin, cAMP, Basilen blue, and GTP failed to displace T. cruzi ATP-Rs. Infective vertebrate stage trypomastigote ATP-Rs were localized to a family of 63 kDa surface glycopolypeptides and ATP-Rs appeared to be stage-regulated because the noninfective insect epimastigote forms had ATP-Rs with KdS that are not capable of metabolic interactions with host-cell micromolar ATP levels. Trypomastigote ATP-Rs may play an important role in the induction of the process of T. cruzi intracellular parasitosis.

Fine-needle aspiration of breast biopsy specimens: correlation of histologic and cytologic findings.

The accuracy of fine-needle aspiration (FNA) cytologic diagnosis of nonpalpable breast lesions and the prevalence of neoplasm occurring in areas unrelated to the radiologic abnormality were studied. Template-guided FNA cytologic examination was performed in 101 surgically excised breast specimens. The exact area of the mammographic abnormality was aspirated with radiographic control. Despite accurate placement of the needle for aspiration, seven of 101 aspirates (7%) yielded insufficient cytologic material. Ninety-four of the 101 aspirates (93%) were adequate for diagnosis. The cytologic diagnosis was benign in 58 (62%), atypical in seven (7%), suspicious for malignancy in four (4%), and malignant in 25 (27%). All cases diagnosed as suspicious or malignant and five of 58 cases diagnosed as benign at cytologic examination proved to be malignant at histologic examination. In three of these five the malignancy was in the area of the radiologic abnormality; in two it was not. FNA cytologic examination can be helpful in evaluating nonpalpable breast lesions, but it is not as accurate as histologic examination of surgically excised lesions.

Trypanosoma cruzi attachment to lymphocyte muscarinic cholinergic and beta adrenergic receptors modulates intracellular signal transduction.

Plasma membrane vesicles of Trypanosoma cruzi (PMVs) formed saturation binding isotherms with naive murine T lymphocytes. Parasite membrane attachment to the muscarinic cholinergic receptors of Lyt 2.2+T cells (suppressor cells) resulted in the synthesis of cGMP, attenuation of cAMP levels and in the secretion of prostaglandin E2, an immunoregulator effector substance. These T suppressor cell signals were blunted by atropine and by monospecific antibody against T. cruzi surface epitopes. The interaction of T. cruzi PMVs with the beta adrenergic receptors of Lyt L3T4+T cells (helper cells) resulted in the synthesis of cAMP and in the attenuation of cGMP levels. T helper cells did not secrete prostaglandin E2 when T. cruzi PMVs were added to this system. These T helper cell signals were blunted by propranolol and by monospecific antibody against T. cruzi surface epitopes. The interaction of T. cruzi with T lymphocytes may result, therefore, in the down-regulation of the immune response induced by prostaglandin E2 T suppressor cell secretion and by cAMP inhibition of proliferation of T helper cells.

Association of elevated anti-sarcolemma, anti-idiotype antibody levels with the clinical and pathologic expression of chronic Chagas myocarditis.

Antibody F(ab')2 fragments derived from the sera of four patients with histology-proven chronic Chagas myocarditis [cF(ab')2]-complexed antibody F(ab')2 fragments of children with acute Trypanosoma cruzi infection [aF(ab')2] in significantly higher molar ratios than those measured with F(ab')2 antibody fragments of normal subjects [nF(ab')2] from nonendemic areas (p less than 0.05). Anti-idiotype [cF(ab')2 X aF(ab')2] immune-complex formation was significantly blunted by preabsorption of cF(ab')2 with porcine heart atria sarcolemma (PAMs) immobilized on sepharose beads (inhibition, mean, 78.1 +/- 2.4%, n = 4). cF(ab')2 X nF(ab')2] immune-complex formation was also inhibited by pretreatment of cF(ab')2 with PAMs (inhibition, mean, 48.7 +/- 7.5%, n = 4). The sera of three groups of subjects from a geographic zone endemic for T. cruzi infection in the northeast of Brazil were assayed for free and immune-complexed IgG anti-acute T. cruzi infection F(ab')2. The indexed levels of free IgG anti-idiotype antibody activity and levels of IgG anti-idiotype immune complexes (IC') were markedly elevated in hospitalized patients with severe, decompensated chronic Chagas myocarditis (n = 23), and their IC' indexes were significantly higher than those measured in asymptomatic seropositive subjects from a nearby endemic village of the northeast of Brazil (Moniz Ferreira, n = 92, p less than 0.001) and in healthy seronegative villagers (n = 84, p less than 0.001). There exists a strong correlation between elevated IgG anti-sarcolemma, anti-idiotype activity levels and the clinical and pathologic expression of chronic Chagas myocarditis.

Modulation of Trypanosoma cruzi adhesion to host muscle cell membranes by ligands of muscarinic cholinergic and beta adrenergic receptors.

Plasma membrane vesicles (PMVs) of Trypanosoma cruzi adhered to L6 myoblast host cells as a function of time and concentration in saturation phenomena in a similar fashion to that reported in a previous publication. The initial adhesion rate (A0) of T. cruzi PMVs to L6 myoblasts in tissue culture was inhibited by acetylcholine (10(-5) M), isoproterenol (10(-5) M) and norepinephrine (10(-8) M) (range 29.1-50.3% of control). Atropine, the antagonist of muscarinic cholinergic receptors (10(-5) M), and propranolol or pindolol, the antagonists of beta-adrenergic receptors (10(-5) M), were also equal inhibitors of the T. cruzi PMV to L6 myoblast adhesion rate (range 26.1 to 55.5% of control). The alpha-adrenergic receptor ligands yohimbine and phentolamine (10(-5) M) showed no A0 inhibitory activity in similar assays. The interaction of T. cruzi PMVs with type I host muscle sarcolemma receptors was clearly defined in assays which used porcine heart atrial membranes (PAMs) immobilized on cationic polyacrylamide beads. In this parasite membrane-host cell membrane assay system, 10(-10) M atropine and 5 x 10(-9) M propranolol produced a shift of an S-shaped T. cruzi PMV to PAM saturation isotherm to the right, suggesting that a negative cooperative interaction was produced between the intramembrane ligand binding site and another, surface heterotropic T. cruzi PMV adhesion site. Atropine and propranolol were equieffective inhibitors of the T. cruzi striated muscle sarcolemma recognition process, raising the possibility that T. cruzi attachment molecules annexed pairs of muscarinic cholinergic and beta-adrenergic receptors to effect adhesion of the two membrane surfaces.

Enrichment of right-side-out Trypanosoma cruzi plasma membrane vesicles.

A simple method to prepare a high yield of Trypanosoma cruzi plasma membrane vesicles (PMV) from epimastigotes and metacyclic trypomastigotes is described. The method may be applicable to other protozoa. Solid-phase immunoassay to bind surface T. cruzi epitopes showed that this preparation was enriched with 80-82% PMV and that most of these were right-side out (81-92%). The method was based on the extraction of extrinsic proteins and subpellicular tubules with mild high and low ionic strength buffers without detergents (pH 7.4) and on the differential centrifugation of PMV based on their specific density (1.049 g/ml, 4 degrees C). Transmission electron microscopy of PMV pellets showed a heterogeneous population of vesicles without other significant cytoskeletal contaminants. T. cruzi PMV were also enriched with an ouabain- and oligomycin-insensitive magnesium-ATPase and contained an adenylyl cyclase, preserved for at least 3 months at -70 degrees C in storage buffer. Measurements of the [14C]-dextran and the 3H2O space indicated that T. cruzi PMV were not sealed, explaining why Lubrol PX and NaF failed to stimulate the adenylyl cyclase activity further and why T. cruzi PMV were unable to concentrate 86Rb in flow dialysis assays. No detectable DNA and RNA was found. The preparation was not capable of removing 51Cr or [3H]glucosamine from live L6 myoblast surfaces in physiologic conditions and acid phosphatase was extracted by this method. The contaminating fraction (18-20% by immunoassay) consisted of endoplasmic reticulum membranes with NADH oxidase activity and of kinetoplast membranes with cytochrome c oxidase and oligomycin sensitive magnesium-ATPase activity. The biologically active T. cruzi PMV retained the ability of living forms to trigger the alternate pathway of complement by releasing the Bb activation fragment from human Factor B.

Complementary surface epitopes, myotropic adhesion and active grip in Trypanosoma cruzi-host cell recognition.

Plasma membranes with complementary surface epitopes and with essentially the same orientation as tissue infective metacyclic trypomastigotes and epimastigotes of Trypanosoma cruzi adhere to L6 myoblast host cells preferentially to smooth muscle and epithelial cells as a function of time, surface area and concentration in saturation phenomena at 4 and 37 degrees C. The initial adhesion rates are partially calcium ion dependent and, at saturation, they are also dependent on high energy phosphorylated intermediates, exerting an active grip on adherent parasite membranes. These phenomena are consistent with the existence of parasite attachment molecules on the external surface of the plasma membrane and complementary host cell receptor structures with the capacity to bind them.

Pathoimmune polymyositis induced in C3H/HeJ mice by Trypanosoma cruzi infection.

The pathogenic mechanisms related to the development of idiopathic inflammatory skeletal muscle disease are unknown. The myotropic protozoa Trypanosoma cruzi and Toxoplasma gondii are implicated in the induction of myositis in experimental animals (1) and humans (2). In the experiments reported here, a model of pathoimmune myositis is described in C3H/HeJ T. cruzi-infecteéd mice. The results showed a significant occurrence of acquired, T. cruzi antigen-dependent spleen T cell cytotoxicity to syngeneic skeletal muscle myoblasts of C3H mice which developed an apparently sterile lymphoid polymyositis. Further experiments with polymyositic C3H mice suggest that spleen B cells do not secrete antibody capable of inducing complement-mediated skeletal muscle myoblast lysis. However, the T. cruzi sensitized splenic lymphocytes did produce a lymphokine which was capable of inducing lysis of syngeneic myoblasts by chromium-51 release assay.

Primary muscle disease: definition of a 25-kDa polypeptide myopathic specific chagas antigen.

The sera from patients with primary heart and skeletal muscle diseases, hospitalized patients without intrinsic muscle disease from an area endemic for Trypanosoma cruzi infections, and normal subjects (N = 693) were studied for the presence of immunoglobulin G (IgG) antisarcolemma activity using serologic methods. The prevalence of elevated serum IgG antisarcolemma activity from patients with chronic Chagas' cardiomyopathy, idiopathic cardiomyopathy, polymyositis, and Duchenne muscular dystrophy was 58.9 +/- 10.4% (N = 101) (P less than 0.001 when compared to normal subjects). Two of twelve (16.7%) patients with acute T. cruzi infection and parasitemia developed elevated antisarcolemma titers, and 9/46 (19.6%) patients with chronic T. cruzi infection without evidence of cardiomyopathy yielded high antisarcolemma titers. On the other hand, patients with chronic T. cruzi infection with advanced cardiomyopathy yielded high antisarcolemma titers in 35/74 (47.3%) (P less than 0.001 when compared to normal subjects). Radioimmunoprecipitation showed a circulating antibody to a 25-kDa T. cruzi polypeptide (P25) in 16/17 (94.1%) patients with advanced cardiomyopathy and T. cruzi infection. No such antibody was shown in 12 asymptomatic subjects with chronic T. cruzi infection.

Autoimmune myocarditis induced by Trypanosoma cruzi.

Antiheart immune reactions have been reported in patients with Chagas' disease, and we have postulated that the observed cardiac lesions are mediated by autoimmune antiheart reactions elicited by the etiologic agent Trypanosoma cruzi. In this report, BALB/c mice infected with a low inoculum of T. cruzi developed splenic lymphocyte cytotoxicity against normal syngeneic neonatal cardiac myofibers in vitro 150 days after infection, whereas splenic lymphocytes obtained from mice at 15, 45, 90, or 120 days after infection or from matched controls did not. No antiheart antibody or antibody-directed cellular cytotoxicity was observed, nor was there an increase in natural killer cell activity. Hearts from mice studied at 150 days after infection showed mononuclear cell myocarditis with myocytolysis in the absence of intracellular T. cruzi forms. Hearts from the other mice did not exhibit any histologic changes. Other reports from our laboratory have identified a cross-reacting antigen (SRA) shared by T. cruzi and striated muscle. Immunization of BALB/c mice with SRA produced immunopathogenic dynamics similar to those seen with long-term T. cruzi infection. Collectively these data indicate that the cardiac lesions seen in patients with Chagas' disease may be attributed to autoimmune reactions elicited by cross-reacting antigens of T. cruzi and striated muscle.

Reversible acquisition of a host cell surface membrane antigen by Trypanosoma cruzi.

The ability of Trypanosoma cruzi, the etiological agent of Chagas' disease to acquire host cell surface antigen was tested. Parasites emerging after intracellular replication in WOS sarcoma monolayers expressed a sarcoma-associated surface antigen. This antigen was deleted from these parasites after replication in the MNS control monolayer, which does not express WOS sarcoma-associated surface antigen, or by replication in cell-free medium. This type of reversible acquisition of host surface antigen has not been previously described in T. cruzi or other intracellular protozoan parasites.

Anti-striated muscle antibody activity produced by Trypanosoma cruzi.

We have previously shown that Trypanosoma cruzi shares antigenic determinants with preparations of the calcium-sequestering adenosine triphosphatase of sarcoplasmic reticulum. The cross-reacting antigen (SRA) is also apparently present on the sarcolemma of cardiac myofibers. Using highly specific reference antisera to either the small membranes of T. cruzi or to a tryptic fragment of striated muscle SRA, it was shown that SRA is present in the striated muscle of animals representative of the evolutionary scale ranging from nonhuman primate to fish. The small membranes of nine different T. cruzi strains isolated from widely divergent areas of the American continents also reacted with the reference antisera. This indicates that SRA is present in these T. cruzi strains and may be prevalent among all T. cruzi strains. The shared T. cruzi-striated muscle antigen, SRA, may be a heteroantigen present in all T. cruzi strains and in the striated muscle of all classes of animals. Immunization of rabbits (three of five) or chickens (five pairs of five pairs) with striated muscle membrane preparations of different classes of animals, particularly those of nonhuman primate, chicken, and turtle, gave rise to IgG anti-allogeneic striated muscle antibody activity. Immunization of rabbits (four of nine) and chickens (five pairs of six pairs) with the small membranes of different T. cruzi strains also produced IgG anti-allogeneic striated muscle. These data indicate that T. cruzi shares cross-immunogenicity with striated muscle SRA. Since SRA is apparently present on the sarcolemma of cardiac myofibers, it may be implicated in the immunopathogenesis of Chagas' disease.