Quantitative nucleic acid sequence-based assay as a new molecular tool for detection and quantification of Leishmania parasites in skin biopsy samples

Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.     

Serological differentiation of experimentally induced Candida dubliniensis and Candida albicans infections

Using a rabbit model of systemic infection, we show that it is possible to differentiate infections caused by Candida dubliniensis and other Candida species by detecting the antibody response mounted by the infected animals. These results confirm our previous observation in a patient with C. dubliniensis candidemia and suggest that detection of C. dubliniensis-specific antibodies is useful in the diagnosis of invasive candidiasis caused by this yeast.     

Changes in the infectivity, pyruvate kinase activity, acid phosphatase activity and P-glycoprotein expression in glibenclamide-resistant Leishmania mexicana

We previously demonstrated susceptibility of Leishmania sp. to glibenclamide, a K⁺-ATP transport blocker which interacts with members of the superfamily of adenosine 5′ triphosphate-binding cassette transporters. In order to characterize the molecular differences between a sensitive Leishmania strain, NR(Gs), and an experimentally selected glibenclamide-resistant strain, NR(Gr), specific biochemical and functional parameters have been evaluated both in the wild type and in the resistant strain. Most noteworthy, NR(Gr) exhibit an increased expression of P-glycoprotein and a decreased activity of functional key enzymes such as acid phosphatase, a prominent virulent factor of the parasite, and pyruvate kinase, a key control enzyme for both carbohydrate and protein metabolism. The specific biochemical, metabolic and functional changes observed in the resistant strain correlated with a reduced infectivity of stationary phase NR(Gr) in J774 macrophages and suggested a mechanism to overcome the effect of glibenclamide. Received: 21 January 2000 / Accepted: 1 March 2000     

Development of the human elbow joint

Many studies have been published on the development of the human elbow joint, but authors disagree on its morphogenetic timetable. Most discrepancies center on the cavitation of the elbow joint (including the humeroradial, humeroulnar, and superior radioulnar joints), and the organization of the tunnel of the ulnar nerve. We summarize our observations on the development of the elbow joint in 49 serially sectioned human embryonic (n = 28) and fetal (n = 21) upper limbs. During week 12, ossification begins in the epiphyses of the elements comprising the elbow joint. At the end of the embryonic period, the shallow groove between the posterior aspect of the medial epicondyle and the olecranon process, begins to be visible. The elbow joint cavity appears in O'Rahilly stage 21 (51 days) at the level of the humeroulnar and humeroradial interzones. Formation of the cavity begins at the medialmost portion of the humeroradial interzone and the lateralmost portion of the humeroulnar interzone. The annular ligament begins to develop in O'Rahilly stage 21 (51 days), and the superior radioulnar joint cavity appears between this ligament and the lateral aspect of the head of the radius during O'Rahilly stage 23 (56 days). We established the morphogenetic timetable of the human elbow joint.     

Hydrocephalus induced by immunological blockage of the subcommissural organ–Reissner's fiber (RF) complex by maternal transfer of anti-RF antibodies

Stenosis of the cerebral aqueduct seems to be a key event for the development of congenital hydrocephalus. The causes of such a stenosis are not well known. Overholser et al. in 1954 (Anat Rec 120:917-933) proposed the hypothesis that a dysfunction of the subcommissural organ (SCO) leads to aqueductal stenosis and congenital hydrocephalus. The SCO is a brain gland, located at the entrance of the cerebral aqueduct, that secretes glycoproteins into the cerebrospinal fluid that, upon release, assemble into a fibrous structure known as Reissner's fiber (RF). By the permanent addition of new molecules to its rostral end, RF grows and extends along the aqueduct, fourth ventricle, and central canal of the spinal cord. The immunological blockage of the SCO-RF complex has been used to test Overholser's hypothesis. The following was the sequence of events occurring in pregnant rats that had been immunized with RF glycoproteins: the mother produced anti-RF antibodies and transferred them to the fetus through the placenta and to the pup through the milk, and the antibodies reached the brain of the fetus and pup and blocked the SCO-RF complex. This resulted in a permanent absence of RF that was followed by stenosis of the cerebral aqueduct, and then by the appearance of hydrocephalus. The latter was patent until the end of the 6-month observation period. The chronic hydrocephalic state appeared, in turn, to induce new alterations of the SCO. It is concluded that a selective immunological knock out of the SCO-RF complex leads to hydrocephalus.     

Increased myeloid colony growth in vitro of precultured progenitor cells

Human peripheral blood myeloid progenitor cells (GM-CFU) form markedly more colonies in soft agar following a short-term preculture in vitro. This enhancement is independent of monocytes and T lymphocytes and reflects an alteration of progenitor cells themselves. The preculture increases the number of cells being in the S phase of cell cycle and renders them relatively independent of exogenous CSF.     

Human placentae membrane receptor for IgG—i. Studies on properties and solubilization of the receptor

Characterization of the human placental membrane receptor for human ¹²⁵I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10⁷ mole^?1, and 2.0 ± 0.16 × 10¹⁵ IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 mM), and periodate (4 mM) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 mM or 20 mM) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of ¹²⁵I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.     

Topographic studies on medial reticular nucleus.

Several distinct classes of neurons have been identified in the medial reticular nucleus of the medulla and pons and in proximity thereto. Neurons projecting down the spinal cord comprised the principal class with two subclasses according as the neurons did or did not receive monosynaptic inputs from the fastigial nuclei of the cerebellum. Two other classes were recognized accordings as they projected to the cerebellum or rostrally to the mesencephalon. Topographic planar maps giving the location of these neurons have been constructed by exploring the nucleus with series of microelectrode tracks in parasagittal or in transverse planes. The different classes of neurons were not arranged in large discrete nuclei. In part they appeared to be randomly distributed, but many colonies of one or another class of neurons could be recognized with 3-11 neurons in zones with dimensions of a millimeter or so. Because of the limitations of sampling by microelectrode tracks at spacings of 0.5 mm, single colonies might have an actual population of 100 or more. Many of the class of neurons projecting to the cerebellum were in the region of the perihypoglossal nucleus. However, almost as many were located deep in the medial reticular nucleus. None was found at the pontine level. Reticulospinal neurons with fast axonal conduction velocities tended to be located dorsally to those with slow velocities. Correlation with the findings of Ito et al. leads to the conjecture that the neurons with fast axons are excitatory, while those with slow axons are primary inhibitory neurons. There is a brief reference to the problems raised by the admixture of the various neuronal classes, there being discrete colonies immersed in a scattered arrangement of all classes.     

Differences in hormonal and inflammatory parameters in male Lewis and Long Evans rats with adjuvant arthritis

The purpose of the present study was (i) to compare secretory responses of prolactin and corticosterone to the acute stress of immobilization in male rats of the Lewis and Long Evans strains and (ii) to compare secretion of the two hormones in rats with fully developed adjuvant arthritis (AA) and their relationship with the intensity of the inflammatory reaction. A short immobilization of 5 min induced equal elevations of both hormones in both strains, but 20-min immobilization produced significantly stronger responses in Long Evans rats than in Lewis rats. AA inhibited prolactin secretion equally in both strains (from 11.6 +/- 1.3 ng/ml to 4.2 +/- 0.6 ng/ml in Lewis rats, p < 0.01, and from 3.7 +/- 0.6 to 2.12 +/- 0.1 ng/ml in Long Evans rats, p < 0.05), but caused a conspiciously larger elevation of corticosterone in the Long Evans than in the Lewis animals (11.5 +/- 1.2 microg/dl in Long Evans rats versus 5.1 +/- 0.5 microg/dl in Lewis rats, p < 0.01) while basal levels were similar. The larger corticosterone response in the Long Evans rats was associated with a stronger inflammatory reaction assessed by hind paw swelling (2.3 +/- 0.1 ml for Long Evans rats versus 1.8 +/- 0.08 ml for Lewis rats, p < 0.01) and plasma levels of nitric oxide (47.5 +/- 5.7 microM for Long Evans rats versus 28.7 +/- 2.5 microM for Lewis rats, p < 0.01) than in the Lewis males with lower corticosterone levels. In conclusion, there are significant, obviously genetically based, differences in the corticosterone responses to both immobilization and AA between the two strains, with the Long Evans rats reacting more strongly than the Lewis rats. The lack of the expected inverse relationship between corticosterone levels and the intensity of the inflammation indicates that the activity of corticosterone is not its primary determinant and that other important factors are involved.     

Breakpoints for predicting Pseudomonas aeruginosa susceptibility to inhaled tobramycin in cystic fibrosis patients: use of high-range Etest strips

Inhaled administration of tobramycin assures high concentrations in cystic fibrotic lungs, improving the therapeutic ratio over that of parenteral tobramycin levels, particularly against Pseudomonas aeruginosa. Conventional Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) breakpoints only consider parenteral levels and do not take into account these high antimicrobial concentrations. The Spanish Antibiogram Committee (The MENSURA Group) has tentatively defined specific breakpoint values for inhaled tobramycin when testing P. aeruginosa isolates from cystic fibrosis (CF) patients (susceptible, < or =64 microg/ml; resistant, > or =128 microg/ml). The antimicrobial susceptibilities of 206 prospectively collected CF P. aeruginosa isolates were determined by the reference agar dilution method. For tobramycin, the performance of high range tobramycin Etest strips (AB Biodisk, Solna, Sweden) and conventional tobramycin disks were assessed with the same collection. Applying MENSURA proposed breakpoints, 95.1% of the strains were categorized as susceptible to tobramycin, either using agar dilution or Etest high-range strips (99% categorical agreement between both methods). With CLSI breakpoints, susceptibility rates decreased to 79.1 and 81.1% for agar dilution and Etest strips, respectively (83.5% categorical agreement). Minor, major, and very major errors for Etest strips (CLSI criteria) were 13.6, 1.2, and 14.8%, respectively. Upon applying the new proposed criteria for inhaled tobramycin, only one major and one very major error were observed with Etest strips. Whenever inhaled tobramycin is considered for therapy, we suggest that P. aeruginosa strains from CF patients categorized as intermediate or resistant to tobramycin according to the CLSI criteria should be retested with high-range Etest strips and recategorized using MENSURA interpretive criteria. CLSI breakpoints should still be followed when intravenous tobramycin is used in CF patients, particularly during the course of exacerbations.