Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells

Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4(+) and CD8(+) T cells secreting INFgamma, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFgamma-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4(+) and CD8(+) T cell subsets, IFNgamma-secreting RV-specific CD8(+) but not CD4(+) T cells were detected in recently infected children. Using the same approach, both CD4(+) and CD8(+) RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFgamma-secreting CD4(+) T cells from adult volunteers preferentially express the intestinal homing receptor alpha4beta7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFgamma-secreting CD4(+) T cells preferentially express L-selectin but not alpha4beta7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFgamma is independent of the expression of these homing receptors.     

Aging and the human vestibular nuclei: morphometric analysis

The data concerning the effects of age on the brainstem are scarce and few works are devoted to the human vestibular nuclear complex. The study of the effects of aging in the vestibular nuclei could have clinical interest due to the high prevalence of balance control and gait problems in the elderly. We have used in this work eight human brainstems of different ages sectioned and stained by the formaldehyde-thionin technique. The neuron's profiles were drawn with a camera lucida and Abercrombie's method was used to estimate the total number of neurons. The test of Kolmogorov-Smirnov with the correction of Lilliefors was used to evaluate the fit of our data to a normal distribution and a regression analysis was done to determine if the variation of our data with age was statistically significant. Aging does not affect the volume or length of the vestibular nuclear complex. Our results clearly show that neuronal loss occurs with aging in the descending (DVN), medial (MVN), and lateral (LVN) vestibular nuclei, but not in the superior (SVN). There are changes in the proportions of neurons of different sizes but they are not statistically significant. The neuronal loss could be related with the problems that elderly people have to compensate unilateral vestibular lesions and the alterations of the vestibulospinal reflexes. The preservation of SVN neurons can explain why vestibulo-ocular reflexes are compensated after unilateral vestibular injuries.     

Morphological features in human cortical brain microvessels after head injury: a three-dimensional and immunocytochemical study

We studied the morphology of cortical microvessels in the brains of 10 patients who had died after receiving a traumatic head injury (THI). Scanning electron microscopy (SEM) of vascular corrosion casts, confocal microscopy of histological sections after immunocytochemistry, and detection of apoptosis by terminal dUTP nick end labeling (TUNEL) were used. Microvascular casts showed an angioarchitectonic distribution that was defined as normal according to results obtained in a previous, nontraumatic series of subjects. However, when we compared them with previous works, the cast surface of some of the microvessels showed three types of morphological alterations: longitudinal folds, sunken surfaces with craters, and a significant flattening with reduction of lumen. The vessels that were primarily affected were the arterioles and capillaries of the middle and deep cortical vascular zones. Immunostaining with the monoclonal antibody MAS-336 against endothelial cells also showed the presence of longitudinal folds with a thinning of the vascular lumen, cytoplasmic round bodies, and a thickening of the endothelial cell membrane. The TUNEL technique revealed a positive staining of some endothelial cells. The structural alterations we observed indicate that microvessels undergo endothelial cell damage after THI. We suggest that this kind of lesion and the secondary functional injury to the blood-brain barrier (BBB) could play an important role in the development of the secondary lesions that these patients show in the subacute phase.     

Molecular epidemiology of hepatitis B virus in Afro-Venezuelan populations

Summary.  Hepatitis B virus (HBV) infection among Venezuelan populations of African origin was analyzed. These populations exhibited lower HBV prevalence than the one found in the African continent. Sequence analysis of 6 isolates showed that 3 belonged to genotype F, while the 3 others were HBV genotype A. HBV genotype A was more common in the Afro-Venezuelan groups than in the general Venezuelan population. This might reflect the introduction of genotype A during the slavery period. The absence of the African genotype E among these isolates supports the hypothesis of a recent origin for this HBV genotype. HBV genotype F has already been introduced to these relatively isolated communities. Received February 18, 2002; accepted March 8, 2002 Published online July 22, 2002     

Non-pathogenic Entamoeba histolytica: functional and biochemical characterization of a monoxenic strain

We have cultured under monoxenic conditions and characterized an Entamoeba histolytica clone, MAV-I CINVESTAV (MAV-I), obtained from feces from an asymptomatic carrier. The clone shows the non-pathogenic E. histolytica zymodeme type I, which did not change through the process of monoxenization. Clone MAV-I was non-pathogenic in both in vivo and in vitro tests, and it did not have a functional 112-kDa adhesin. As far as we know, this is the first non-pathogenic monoxenic strain reported. Clone A (strain HM1:IMSS), a highly virulent clone with pathogenic zymodeme type II, and which has the 112-kDa adhesin, was used as a control. Protein patterns from both clones were almost identical in one-dimensional gels. In two-dimensional gels, differences in high-molecular-weight proteins were detected. Clone MAV-I adhered and phagocytosed only 12% of the red blood cells adhered and phagocytosed by clone A. MAV-I trophozoites did not destroy cell culture monolayers and did not produce hepatic abscesses in hamsters. They also showed deficiency in protease activity. The absence of virulence in clone MAV-I correlated directly with the absence of a functional 112-kDa adhesin, supporting the role that this protein plays in virulence.     

PCR assays for identification of Coccidioides posadasii based on the nucleotide sequence of the antigen 2/proline-rich antigen

A conventional nested PCR and a real-time LightCycler PCR assay for detection of Coccidioides posadasii DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family Onygenaceae remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be C. posadasii (accession number AF013256), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as C. posadasii. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens.     

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.     

Some gonad-infecting species of Philometra (Nematoda, Philometridae) from offshore fishes of South Carolina and Georgia, USA, including Philometra charlestonensis sp. nov. from the scamp Mycteroperca phenax

Three gonad-infecting species of Philometra Costa, 1845 were, for the first time, recorded from perciform fishes from estuarine and marine waters in South Carolina and Georgia, USA: Philometra charlestonensis sp. nov. from the scamp Mycteroperca phenax (Jordan et Swain) (Serranidae), P. saltatrix Ramachandran, 1973 from the bluefish Pomatomus saltatrix (Linnaeus) (Pomatomidae), and Philometra sp. from the Atlantic croaker Micropogonias undulatus (Linnaeus) (Sciaenidae). The new species is characterized mainly by males (body length 2.65–3.14 mm) with equally long, needle-like spicules (length 132–141 μm) and the gubernaculum (81–93 μm) bearing dorsal transverse lamella-like structures on its distal portion, the body length of gravid females (168–247 mm), the presence of a well-developed anterior bulbous inflation on the female oesophagus, and by the length of the first-stage larvae (544–597 μm). A key to gonad-infecting species of Philometra parasitizing marine and brackish-water fishes is provided.     

Mutations of CD40 ligand in two patients with hyper-IgM syndrome

Two patients with the X-linked form of the hyper-IgM syndrome have been studied. Both patients present: 1. Mutations in the CD40L gene (a nonsense point mutation that introduces a termination codon at the extracellular domain of the protein, and a deletion that eliminates exon 4 as consequence of an abnormal splicing). 2. Lack of CD40L expression on the lymphocyte surface after stimulation with ionomycin and PMA. 3. Altered lymphocytic proliferation in response to anti-CD3. 4. Hyper IgM, low IgG and IgA levels and neutropenia. One of the patients shows, in addition, low Natural Killer cell numbers and severe herpetic infections, which distinguishes this case from the common hyper-IgM syndrome phenotype. Finally, a hyper-IgM stable phenotype has been immortalized by Herpes virus Saimiri for the first time.     

In vivo electrical characteristics of human skin, including at biological active points

The aim is to compare the mean values of the in vivo electrical characteristics of bioiogical active points (BAPs) with those of the surrounding human skin. The impedance measurements at BAPs and on the surrounding skin are carried out in vivo on ten young, healthy people. The results of the measurements show that the BAP resistance R_P is smaller, and the capacitance C_P is higher, than the corresponding values for skin, R_S and C_S, respectively, these differences are larger at low frequencies (at f=3 Hz, R_S/R_P=3.19 and C_P/C_S=3.2). The mean values of the impedance measurements at the BAPs are different from those measured on the skin. The dependence of R_P and C_P on the pressing force, in the range of about 1–5 N, for the BAPs, has a smaller slope than that observed for the surrounding skin. An equivalent circuit for the BAPs is proposed that describes sufficiently well the experimental results obtained. These results show that the large dispersion in the observed impedance characteristics of the human body measurements in different body regions can be related to the influence of the BAPs present under the electrodes.