Biochemical characterization of new strains of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis> and<Emphasis Type="Italic"> T. rangeli</Emphasis> isolates from Peru and Mexico

Seven trypanosome stocks isolated have been characterized by lectin agglutination, isoenzyme analysis, and the end products excreted. The stocks were isolated from different geographic areas—one from Mexico (TM5), and six from Peru, four of these isolated from different species of triatoma (TP504, TP702, TP704 and TP706), the other two isolated from the salivary glands of Rhodnius ecuadorensis (TRa605 and TRa606). Additionally, one strain of Trypanosoma cruzi isolated from a human case (strain TC-Maracay) and one strain of T. rangeli (TRa, Cajamarca-Peru strain), characterized and maintained in our laboratory, were used as reference strains. According to statistical study, the stocks were grouped into three clusters: (1) cluster I included the reference strain of T. cruzi (TC-Maracay); (2) cluster II was subdivided into two groups—subcluster IIA for the Mexican isolate (TM5) and subcluster IIB for the Peruvian ones, isolated from the salivary glands of Rhodnius ecuadorensis (TRa 605 and TRa 606) and the reference strain T. rangeli (TRa); these two new isolates were classified as T. rangeli; and (3) cluster III for the rest of the Peruvian isolates, which should be considered at least as a different strain from the T. cruzi strain Maracay. We show that the identification of T. cruzi and T. rangeli in mixed infections is readily achieved by biochemical methods. These findings identified three clusters of Mexican and Peruvian stocks that correlate with geographic origin, although assignment to a T. cruzi linage was not possible.     

Currant allergy and the Rosaceae-grass pollen allergy syndrome: a case report.

BACKGROUND: Despite the increasing use of currants in culinary recipes, currant allergy has rarely been reported. OBJECTIVES: To study a case of currant allergy and to explore cross-reactivity between grass pollen and Rosaceae family fruit allergens. METHODS: Skin prick tests to pollen and skin prick-to-prick tests with currants and peach were performed. Specific IgE levels were determined using the CAP method. We prepared a protein extract of 0.1 mg/mL in phosphate-buffered saline using red currant in the presence of protease inhibitors. Immunoblot inhibition studies were performed to explore cross-reactivity between grass pollen and currant allergens. RESULTS: Skin prick test results were positive to Dactylis, arizonic, and olive pollens. Results of skin prick-to-prick tests with fresh red and black currants were negative and positive, respectively, to peach. The specific IgE level was 5.7 KU/L to red currant and 2.92 KU/L to peach (CAP). Western blot analysis with red currant extract revealed specific IgE protein bands of 37 and 26 kDa. Preincubation of sera with extracts from red currant and peach inhibited both IgE bands, and preincubation with Dactylis pollen inhibited the 37-kDa band only. CONCLUSIONS: We report a case of allergy to grass pollen with an oral allergy syndrome involving several fruits from 2 different families of the Rosidae subclass confirmed by in vitro tests. Inhibition studies demonstrated cross-reactivity between different fruits (currant and raspberry) from the Rosidae subclass and were incomplete with grass pollen allergens.     

A double mutant [N543H+2393del9] allele in the LDL receptor gene in familial hypercholesterolemia: effect on plasma cholesterol levels and cardiovascular disease

Familial hypercholesterolemia is a genetic disorder caused by mutations in the LDL receptor gene. During a survey of mutations of LDL receptor gene in Spanish FH patients we found two mutations in the same allele: a missense N543H mutation in exon 11 and a 9bp inframe deletion (2393del9) located in exon 17. This double mutant allele was founded in 10 out of 458 unrelated patients: one homozygous FH [N543H+2393del9] + [N543H+2393del9], one compound heterozygote [N543H+2393del9] + [W-18X+E256K] and 8 heterozygotes. Flow cytometric analysis showed a defective LDL binding (20% of normal value) and internalization (23%) in lymphocytes from the homozygous patient; furthermore, studies of mitogen-stimulated lymphocytes demonstrated that the ability of LDL to support cell proliferation was impaired. Unexpectedly, not all carriers of the double mutant allele develop hypercholesterolemia and, furthermore, cholesterol-lowering treatment of the homozygous patient resulted in a 58% LDL cholesterol reduction. In conclusion, the phenotypic expression in the homozygous and heterozygous patients presented here, as well as the LDL-receptor residual activity, allowed the classification of this mutation as mild extending the group of mild mutations found at homozygosity.     

Chemokine receptors that mediate B cell homing to secondary lymphoid tissues are highly expressed in B cell chronic lymphocytic leukemia and non-Hodgkin lymphomas with widespread nodular dissemination

B cell neoplasms present heterogeneous patterns of lymphoid organ involvement, which may be a result of the differential expression of chemokine receptors. We found that chemokine receptor (CCR)7, CXC chemokine receptor (CXCR)4, or CXCR5, the main chemokine receptors that mediate B cell entry into secondary lymphoid tissues and their homing to T cell and B cell zones therein, were highly expressed in B malignancies with widespread involvement of lymph nodes. Conversely, those pathologies with little or no nodular dissemination showed no expression to very low levels of CCR7 and CXCR5 and low to moderate levels of CXCR4. These findings provide evidence for the role of CCR7, CXCR4, and CXCR5 in determining the pattern of lymphoid organ involvement of B tumors. Functional studies were performed on B malignancies expressing different levels of CCR7, CXCR5, and CXCR4. Multiple myeloma (MM) cells did not express CCR7 nor CXCR5 and did not migrate in response to their ligands; a moderate expression of CXCR4 on MM cells was accompanied by a migratory response to its ligand, CXCL12. By contrast, cells from B cell chronic lymphocytic leukemia (B-CLL) expressed the highest levels of these chemokine receptors and efficiently migrated in response to all ligands of CCR7, CXCR4, and CXCR5. In addition, the migration index of B-CLL cells in response to both of the CCR7 ligands correlated with the presence of clinical lymphadenopathy, thus indicating that the high expression of functional chemokine receptors justifies the widespread character of B-CLL, representing a clinical target for the control of tumor cell dissemination.     

Microplate technique to determine hemolytic activity for routine typing of Listeria strains.

Because the hemolysis produced by Listeria monocytogenes and Listeria seeligeri on blood agar is frequently difficult to interpret, we developed a microplate technique for the routine determination of hemolytic activity with erythrocyte suspensions. This microtechnique is a simple and reliable test for distinguishing clearly between hemolytic and nonhemolytic strains and could be used instead of the CAMP (Christie-Atkins-Munch-Petersen) test with Staphylococcus aureus in the routine typing of Listeria strains. Furthermore, our results suggest that the quantitation of the hemolytic activity of the Listeria strains, along with the D-xylose, L-rhamnose, and alpha-methyl-D-mannoside acidification tests, allows the differentiation of L. monocytogenes, L. seeligeri, and Listeria ivanovii. We also observed that the treatment of erythrocytes with crude exosubstances of rhodococcus equi, Pseudomonas fluorescens, Acinetobacter calcoaceticus, and S. aureus enhanced the hemolytic activity of all Listeria strains with this characteristic.     

Microhardness of superficial and deep sound human dentin

Our purpose in this study was to determine the microhardness of superficial and deep dentin by means of two indentation methods (Knoop and Vickers) under two different applied loads. Twelve dentin discs approximately 2-mm thick were obtained from both superficial and deep dentin by transversally sectioning the crowns of sound, extracted human third molars with a diamond blade under water irrigation. Dentin surfaces were sequentially polished, and indentations (n = 20 per surface) were performed with either Vickers indentor at loads of 300 and 500 g, respectively, or Knoop indentor at loads of 50 and 100 g, respectively. Average Vickers hardness number (VHN) and Knoop hardness number (KHN) were calculated and treated with two-way analysis of variance (ANOVA) and Student's t test. Microhardness of dentin was not influenced by the different loads applied for both indentation methods. Knoop hardness was significantly higher for superficial than for deep dentin (p < 0.05). Conversely, Vickers hardness was not significantly different for both substrates (p > 0.05). Differences in dentin hardness as a function of depth exist, but they might not be relevant, and no alteration of the distribution of stresses along the adhesive interface is expected.     

The Immune Reactivity Role of HCV-Induced Liver Infiltrating Lymphocytes in Hepatocellular Damage

Liver infiltrating lymphocytes (LIL) were isolated from HCV-positive (+) and HCV-negative (–) end-stage livers. Phenotypic analysis and functional studies using proliferative and lymphocytotoxic assays were performed with the isolated LIL. Two CD3+ lymphocyte populations were found in LIL using FITC anti-CD3 monoclonal antibodies (mAb). One was a bright fluorescence intensity population (as in PBL), and the other dim. We calculated the number of FITC-anti-CD3 mAbs bound per lymphocyte on PBL and LIL and found 80,040 ± 4628 and 39,615 ± 3932, respectively. Therefore, HCV+ and HCV– patient PBL contained approximately twice the number of CD3 molecules per cell than patient CD3+ LIL. LIL also contained approximately a threefold higher concentration of TCR+, CD4–CD8–, and CD56,16 (NK) cells than the patient PBL. Thus, a major subset of LIL is phenotypically similar to mouse NK1.1+ intermediate T cells. LIL freshly isolated from HCV+ livers exhibited weak CTL activity against EBV- or Con A-transformed lymphoblast targets infected with vaccinia–HCV recombinant virus (rHCV) or primary hepatocyte cultured cells. However, after in vitro coculture of LIL with rHCV, these cells developed a strong cytotoxicity for the above targets. In contrast, LIL from HCV– livers were not cytotoxic against the same targets. Histochemical studies (in situ) demonstrated that these hepatocytes express CD95, and stains demonstrated apoptosis. The HCV+ hepatocytes also express class I MHC molecules and ICAM-1. The addition of mAb specific for these adhesion molecules inhibited CML activity. Short-term cultured hepatocytes (targets) from HCV+ and HCV– patients produced low levels of cytokines IL-1, IL-2, IL-6, TNF, and IFN- but a high level of IL-8. It is speculated that LIL expressing reduced numbers of CD3 molecules may even function as immune regulators as proposed for intermediate T cells in mice.     

Structural Characteristics and Development of Ampullary Organs in Acipenser naccarii

Ampullary organs of Acipenser naccarii sturgeons were examined by optical and electronic microscopy (transmission electron microscopy and scanning electron microscopy) from hatching until 1 month later when the juvenile phase is completely established. It was observed that, when A. naccarii begins to feed actively, the ultrastructural characteristics of ampullary organs already correspond to those of adult animals. These organs may, therefore, be functional and, together with taste buds, facilitate food search after exhaustion of yolk sac food reserves. Mature ampullary organs of A. naccarii are formed by an ampulla that communicates with the exterior by means of a short channel. These ampullae correspond to the sensory portion of these receptors and are formed by two cell types: receptor cells and support cells. Receptor cells present a kinocilium on their free surface and establish ribbon synapses with axon nerve endings that arise from the underlying conjunctive tissue. Support cells enclose receptor cells, bear stereocilia and occasional cilia, and are of a secretory nature. The mucus associated with ampullary organs mainly comprises neutral mucopolysaccharides, whereas mucopolysaccharides are usually acid in other fish groups. Anat Rec, 290:1178–1189, 2007. © 2007 Wiley‐Liss, Inc.     

The Helix aspersa (brown garden snail) allergen repertoire

BACKGROUND: Ingestion of snails can induce strong asthmatic or anaphylactic responses, mainly in house-dust-mite-sensitized patients. The aim of this study was to identify the Helix aspersa (Hel a), Theba pisana (The p) and Otala lactea (Ota l) allergens and the extent of their cross-reactivity with the Dermatophagoides pteronyssinus (Der p) mite. PATIENTS AND METHODS: In 60 atopic patients, skin prick tests (SPT) to snail and D. pteronyssinus, total and specific IgE, specific IgE immunoblots, RAST and immunoblot inhibition assays were performed. RESULTS: Mean total IgE was >1,000 kU/l. Mean specific IgE (class 6 for Der p and class 2 for Hel a) SPT were positive in 44 patients for snail and in 56 for mite. Isoelectric focusing (IEF) and SDS-PAGE followed by immunoblotting of H. aspersa extract enabled the identification of 27 and 20 allergens, respectively. Myosin heavy chains from snails (molecular weight >208 kDa) disclosed two major allergens. Hel a and Der p RAST were strongly inhibited by their homologous extracts, with Hel a RAST being inhibited by the Der p extract to a much greater extent (72.6%) than the inverse (5.6%). A complete inhibition of the immunoblots by their homologous extract was obtained. However, Hel a extract did not inhibit Der p IEF separated recognition. On the other hand, mite extract extensively inhibited snail immunoblots from both IEF and SDS-PAGE separations. Immune detection on chicken, pig, rabbit, cow and horse myosins did not reveal any IgE cross recognition with snail. CONCLUSIONS: In most cases of snail allergy, mite appeared to be the sensitizing agent. Nevertheless, snails may also be able to induce sensitization by themselves. This hypothesis is supported by the finding of specific IgE to Hel a in 2 patients who did not show specific IgE to Der p, and one of them was suffering from asthma after snail ingestion.     

Patterns of reactivity to lipid transfer proteins of plant foods and Artemisia pollen: an in vivo study

BACKGROUND: Lipid transfer proteins (LTPs) are major allergens of Rosaceae fruits in the Mediterranean area. IgE-cross-reactivity has been demonstrated in vitro among LTPs from peach, apple, chestnut and Artemisia pollen. The aim of this study was to evaluate the reactivity to LTPs from peach, apple, chestnut and Artemisia pollen by means of skin prick tests (SPTs). METHODS: Forty-seven patients allergic to peach (peach group), 20 patients sensitized to Artemisia pollen with no food allergies (Artemisia group), and 12 control subjects were skin tested with fresh peach, as well as with whole extracts and purified LTPs of peach, apple, chestnut and Artemisia pollen. RESULTS: The rates of positive SPTs for peach, apple, chestnut and Artemisia LTPs were, respectively, 91, 77, 23, and 36% in the peach group, and 30, 5, 15 and 40% in the Artemisia group. No response was observed in the control subjects. SPTs with peach LTP strongly correlated with SPTs conducted with fresh peach. In the peach group, the most frequent pattern of reactivity to LTPs was the combination peach-apple (45%), followed by peach-apple-Artemisia-chestnut (21%). Significant correlations were found between peach and apple LTPs, and between Artemisia and chestnut LTPs. Positive SPTs to chestnut LTP were only observed in patients with positive SPTs to Artemisia LTP. All the patients with positive case histories to chestnut reacted to chestnut LTP. CONCLUSIONS: LTPs are plant panallergens with different patterns of cross-reactivity. They are major allergens of Rosaceae fruits and seem to be involved in allergic reactions to unrelated foodstuffs such as chestnut, probably through sensitization to the cross-reactive Artemisia LTP. Rosaceae LTPs could be useful tools for in vivo diagnosis of Rosaceae fruit allergy.