Tritium forms discrimination in ryegrass under constant tritium exposure: From seed germination to seedling autotrophy.

Uncertainties remain regarding the fate of atmospheric tritium after it has been assimilated in grasslands (ryegrass) in the form of TFWT (Tissue Free Water Tritium) or OBT (Organically Bound Tritium). One such uncertainty relates to the tritium forms discrimination during transfer from TFWT to OBT resulting from photosynthesis (OBTphoto), corresponding to the OBTphoto/TFWT ratio. In this study, the OBT/TFWT ratio is determined by experiments in the laboratory using a ryegrass model and hydroponic cultures, with constant activity of tritium in the form of tritiated water (denoted as HTO) in the "water" compartment (liquid HTO) and "air" compartment (HTO vapour in the air). The OBTphoto/TFWT ratio and the exchangeable OBT fraction are measured for three parts of the plant: the leaf, seed and root. Plant growth is modelled using dehydrated biomass measurements taken over time in the laboratory and integrating physiological functions of the plant during the first ten days after germination. The results suggest that there is no measurable discrimination of tritium in the plant organic matter produced by photosynthesis.

TREM-1 modulation during early stages of dengue virus infection.

Uncontrolled and intricate production of inflammatory factors is the characteristic feature of dengue infection. The triggering receptor expressed in myeloid cells-1 (TREM-1), expressed on the surface of monocytes and neutrophils, is capable of enhancing and regulating the inflammatory response via the production of different mediators in bacterial and viral infections. Here, both the expression of TREM-1 on human monocytes and neutrophils from peripheral blood of dengue infected individuals, as well as the levels of the soluble form of TREM-1 (sTREM-1) in the sera of these patients were compared against healthy controls. A significant reduction of TREM-1 expression was observed in neutrophils during the first days of infection, followed by a gradual recovery throughout the course of infection. Also, sera from DENV-infected patients exhibited significantly higher sTREM-1 levels than healthy individuals. The difference was more pronounced during the first 5 days after the onset of symptoms. These findings highlight the dynamic process of TREM-1 expression during DENV infection. We hypothesized that increment of free sTREM-1 could be a compensatory mechanism aiming to counteract the inflammatory process elicited during DENV infection.

Frequency of Mycobacterium bovis as an etiologic agent in extrapulmonary tuberculosis in HIV-positive and -negative Mexican patients.

Mycobacterium bovis can be an important etiological agent for extrapulmonary (EP) manifestations of tuberculosis, especially in HIV-infected persons. From January 2000 to December 2003, M. bovis as a cause of EP tuberculosis was investigated at the Pneumonology Service, Hospital General de Mexico, Mexico City. Eighty HIV-positive (HIV+) patients and 83 HIV-negative (HIV-) with EP involvement (ganglionar, genitourinary, meningeal, cutaneous, peritoneal, and pericardial) were analyzed using clinical, immunological, bacteriological, histopathological, and molecular biology methods. Mycobacterium species were identified by hsp65-RFLP analysis and species of M. tuberculosis complex isolates by spoligotyping. M. bovis was present in 6 HIV- cases (7.2%; 3 with lymphadenitis and 3 genitourinary) vs 11 in HIV+ cases (13.75%; 7 with lymphadenitis, 3 genitourinary, and 1 meningeal). Favorable response to retroviral and specific M. bovis chemotherapy was observed. Spoligotyping showed a unique profile in each isolate, 16 belonging to BOV1 lineage and 1 to BOV2 lineage. M. bovis is an significant re-emerging cause of EPTB in Mexico. Consumption of unpasteurized dairy products is the most likely source of transmission. Successful treatment depends on the adequate and opportune identification of the agent responsible.

Analysis of antibody response in human dengue patients from the Mexican coast using recombinant antigens.

This study was undertaken to evaluate the feasibility of using recombinant dengue proteins to discriminate between acute dengue infections versus uninfected dengue samples. Dengue virus proteins E, NS1, NS3, and NS4B were cloned as fusion proteins and expressed in Escherichia coli. Recombinant products were tested in 100 serum samples obtained from acute dengue fever cases collected from 3 states of Mexico where dengue is endemic. Sera from 75 healthy individuals living in nonendemic areas for dengue were used as a control group. In sera from the dengue patients group, antibody responses to E protein were demonstrated in 91% of cases and NS1 protein was recognized to various extents (99%) within the first 7 days of infection. The antibody responses to NS3 and NS4B were frequently of low magnitude. Consistent negative antibody responses to all proteins were found in sera from the control group. These data suggest that the glutathione-S-transferase (GST)-dengue fusion proteins may be feasible antigens for a sensitive and specific serological assay.

Peripheral blood CD161+ T cells from asthmatic patients are activated during asthma attack and predominantly produce IFN-gamma.

In humans, T cells expressing the CD161 molecule NKR-P1A constitute around 20% of the circulating CD3(+) cells and are potentially immunoregulatory in several diseases. Their role in asthma is not well known, but they could participate in asthma attacks. To determinate whether activation of CD161(+) T cells and their cytokine production correlate with clinical status of asthma, we analysed blood samples from asthma attack patients (AAP) and stable asthma patients (SAP) in comparison with healthy non-atopic controls (HC). There was a significant higher baseline expression of CD69 on T cells from AAP and the difference was more notorious on CD161(+) T cells; upregulation of CD69 was observed on both CD161(-) and CD161(+) T cells driven by Dermatophagoides pteronyssinus crude extract, whereas polyclonal stimulation with phorbol 12-myristate 13-acetate plus ionomycin predominantly induced IFN-gamma but no IL-4, IL-5 and IL-13 by CD161(+) T cells in all groups; upon polyclonal stimulation, there were more CD161(+) T cells producing IFN-gamma and less CD161(-) T cells producing this cytokine, contrasting with the opposite results observed in SAP and HC groups. Our results indicate that, during asthma attack, CD161(+) T cells are activated and are able to produce predominantly IFN-gamma but no Th2 cytokines. We hypothesize that during an asthma attack, IFN-gamma produced by CD161(+) T cells could help to reestablish the Th1/Th2 equilibrium. These observations may contribute to the understanding of the immune mechanisms involved in asthma attacks.

Prevalence of Escherichia coli and Salmonella spp. in street-vended food of open markets (tianguis) and general hygienic and trading practices in Mexico City.

Street-vendors in Mexico City provide ready-to-eat food to a high proportion of the inhabitants. Nevertheless, their microbiological status, general hygienic and trading practices are not well known. During spring and summer 2000, five tianguis (open markets) were visited and 48 vendors in 48 stalls interviewed. A total of 103 taco dressings were sampled for E. coli and Salmonella spp.: 44 (43%) contained E. coli and 5 (5%) Salmonella (2 S. Enteritidis phage type 8, 1 S. Agona, 2 S. B group). Both E. coli and salmonellas were isolated from three samples. Of Salmonella-positive stalls 80% (4/5) had three or more food-vendors and 80% of vendors were males, compared with 37.3% (16/43) and 46.4% (20/43) in the Salmonella-negative stalls respectively. Food-vendors kept water in buckets (reusing it all day), lacked toilet facilities, and prepared taco dressings the day before which remained at the tianguis without protection for 7.8 h on average. Consumption of street-vended food by local and tourist populations poses a health risk.

Evaluation of the polymerase chain reaction in the diagnosis of miliary tuberculosis in bone marrow smear.

OBJECTIVE: Miliary tuberculosis (MTB) is difficult to diagnose. When prompt diagnosis is necessary, the polymerase chain reaction (PCR) to detect mycobacterial DNA may be valuable. SETTING: Tuberculosis clinic in an academic tertiary-level hospital in Mexico. DESIGN: Bone marrow (BM) aspiration samples from 30 consecutive clinically suspected MTB patients and 58 non-tuberculosis hematologic patients were evaluated by in-house PCR using a fragment of the insertion sequence IS6110; results were compared with those obtained by acid-fast-stained smears, culture in Löwenstein-Jensen medium, histology, and serology. RESULTS: Tuberculosis diagnosis was confirmed in all MTB suspects, 28 by microscopy and culture in pulmonary or extra-pulmonary samples other than BM, and two by clinical and radiologic improvement after antituberculosis treatment. In fresh BM specimens, in-house PCR was positive in 21/30 (70%) suspects, contrasting with only one positive (3.3%) in staining and culture, and four with compatible histologic findings (13.3%). BM samples from the control group showed negative results in bacteriologic and histologic studies, except in nine who had positive PCR results. These nine control cases had malignant processes. CONCLUSION: PCR in aspirates of BM is a useful diagnostic assay in cases of MTB, mainly when bacteriological results are negative.

Tuberculosis is still a major cause of cervical lymphadenopathies in adults from developing countries.

To establish the frequency of infectious aetiology in Mexican adult patients with cervical lymphadenopathies (CLAs), 87 consecutive patients with enlarged cervical lymphatic nodes, HIV negative and without anti-tuberculous treatment, were selected from a tertiary-level speciality concentration hospital. Histopathological studies, investigation of acid-fast bacilli, cultures in Löwenstein Jensen and Mycobacterium growth indicator tube (MGIT) media, and in-house polymerase chain reaction (PCR) with IS6110-based primers for Mycobacterium tuberculosis complex were performed in resected lymphatic nodes. Non-infectious aetiology corresponded to 45 cases (52 %). Tuberculosis was suspected in 42 cases (48%) by histology and confirmed positive results were obtained by staining in 8 (19%), by culture in 23 (55%), and by PCR in 34 (81 %) patients. All were confirmed after therapeutic success. In addition to the epidemiological transition process occurring in Mexico, tuberculosis remains an important cause of CLA. Histopathology with confirmatory studies including PCR can detect tuberculous aetiology.

Influx and accumulation of Cs(+) by the akt1 mutant of Arabidopsis thaliana (L.) Heynh. lacking a dominant K(+) transport system.

An extensive literature reports that Cs(+), an environmental contaminant, enters plant cells through K(+) transport systems. Several recently identified plant K(+) transport systems are permeable to Cs(+). Permeation models indicate that most Cs(+) uptake into plant roots under typical soil ionic conditions will be mediated by voltage-insensitive cation (VIC) channels in the plasma membrane and not by the inward rectifying K(+) (KIR) channels implicated in plant K nutrition. Cation fluxes through KIR channels are blocked by Cs(+). This paper tests directly the hypothesis that the dominant KIR channel in plant roots (AKT1) does not contribute significantly to Cs(+) uptake by comparing Cs(+) uptake into wild-type and the akt1 knockout mutant of Arabidopsis thaliana (L.) Heynh. Wild-type and akt1 plants were grown to comparable size and K(+) content on agar containing 10 mM K(+). Both Cs(+) influx to roots of intact plants and Cs(+) accumulation in roots and shoots were identical in wild-type and akt1 plants. These data indicate that AKT1 is unlikely to contribute significantly to Cs(+) uptake by wild-type Arabidopsis from 'single-salt' solutions. The influx of Cs(+) to roots of intact wild-type and akt1 plants was inhibited by 1 mM Ba(2+), Ca(2+) and La(3+), but not by 10 microM Br-cAMP. This pharmacology resembles that of VIC channels and is consistent with the hypothesis that VIC channels mediate most Cs(+) influx under 'single-salt' conditions.

Underestimation of Mycobacterium tuberculosis infection in HIV-infected subjects using reactivity to tuberculin and anergy panel.

BACKGROUND: This study aimed to evaluate purified protein derivative (PPD) reactivity and its interrelationship with anergy panel and CD4+ lymphocytes in HIV-infected subjects as compared to PPD reactivity in HIV-uninfected individuals in a tuberculosis endemic and high Bacillus Calmette-Guérin (BCG) coverage environment. METHODS: Clients of four Mexico City HIV detection centres were screened for HIV-1 antibodies (ELISA or haemagglutination, Western Blot); reactivity to PPD (Mantoux PPD, 5TU RT-23), Candida (1:1000, 0.1 ml), and tetanus toxoid (10Lf, 0.1 ml); and CD4+ T cells. Active tuberculosis was excluded. Informed consent was obtained. RESULTS: From 5130 clients 1168 subjects were enrolled; of these 801 (68.6%) were HIV positive. Reactivity to PPD among HIV-positive subjects was found in 174 (22%), 261 (32.6%), and 296 (37%), at PPD cutoff levels of > or =10 mm, > or =5 mm, and > or =2 mm as compared to 224 (61%) of 367 HIV-negative individuals' reactors to PPD (> or =10 mm) (P < 0.001). After exclusion of anergic individuals using two cutoff levels for cutaneous allergens (< or =2 mm and < or =5 mm), PPD reactivity between HIV-infected and uninfected individuals continued to be significantly different. Only HIV-infected individuals with CD4+ T cells > or =500 cells/mm3 had similar reactivity to PPD as HIV-uninfected individuals. Variables associated with PPD reactivity were CD4+ T cell counts, BCG scar, HIV infection and age. CONCLUSIONS: PPD reactivity was useful to diagnose tuberculosis infection only among HIV-infected individuals with CD4+ counts > or =500 cells/mm3. Among individuals with lower counts, lowering cutoff levels or using anergy panel did not permit comparable reactivity as that observed among HIV-uninfected individuals.

What are the effects of nitrogen deficiency on growth components of lettuce?

Relationships between nitrogen (N) content and growth are routinely measured in plants. This study determined the effects of N on the separate morphological and physiological components of plant growth, to assess how N-limited growth is effected through these components. Lettuce (Lactuca sativa) plants were grown hydroponically under contrasting N-supply regimes, with the external N supply either maintained continuously throughout the period of study, or withdrawn for up to 14 d. Richards' growth functions, selected using an objective curve-fitting technique, accounted for 99.0 and 99.1% of the variation in plant dry weight for control and N-limited plants respectively. Sublinear relationships occurred between N and relative growth rates under restricted N-supply conditions, consistent with previous observations. There were effects of treatment on morphological and physiological components of growth. Leaf weight ratio increased over time in control plants and decreased in N- limited plants. Shoot:root ratio followed a similar pattern. On a whole-plant basis, assimilation of carbon decreased in N-limited plants, a response paralleled by differences in stomatal conductance between treatments. Changes in C assimilation, expressed as a function of stomatal conductance to water vapour, suggest that the effects of N limitation on growth did not result directly from a lack of photosynthetic enzymes. Relationships between plant N content and components of growth will depend on the availability of different N pools for remobilization and use within the plant.

Testing a process-based model of tree seedling growth by manipulating.

A model was developed that simulated photosynthesis, growth and allocation in tree seedlings. The model was parameterized with data from experiments on seedlings of sycamore (Acer pseudoplatanus L.), Sitka spruce (Picea sitchensis (Bong) Carr.) and young birch trees (Betula pendula Roth.). In these experiments, CO2 concentration ([CO2]) and nutrient addition rate were varied. Parameters quantifying nutrient uptake, translocation and starch synthesis were fitted, based on data from control treatments. Elevated [CO2] and low-nutrient treatments were then used to test the predicted response of growth and allocation against observations. The model accurately predicted total seedling growth in the elevated [CO2] treatments. A response of growth to elevated [CO2] was seen in the birch and sycamore experiments, but not in the Sitka spruce, because of photosynthetic down-regulation. Predictions of allocation were reasonably accurate in the birch and Sitka spruce experiments, but were notably poorer in the sycamore experiments, possibly because of differences in sink strength between root and shoot. In the birch and sycamore experiments, little change in allocation with elevated [CO2] was observed or predicted. This was ascribed to the relative values of K(Tc) and K(Tn), the translocation coefficients that determine the sensitivity of allocation to carbon and nitrogen uptake rates, respectively. Growth and allocation in the low-nutrient treatments were poorly predicted by the model. In Sitka spruce, it was suspected that the photosynthetic parameters measured in August 1994 had been higher earlier in the season, before nutrients became depleted. In sycamore, the discrepancies were thought to relate to differences in sink strength between root and shoot that could not be described by the model.

Gnathostomosis, an emerging foodborne zoonotic disease in Acapulco, Mexico.

Between 1993 and 1997, 98 gnathostomosis cases were clinically identified in Acapulco, Mexico. Intermittent cutaneous migratory swellings were the commonest manifestation. Larvae were identified in 26 cases, while in 72, final diagnosis was made on the basis of epidemiologic data, food habits, and positive enzyme-linked immunosorbent assay and Western blot results.

Antibody response to Klebsiella pneumoniae 60 kDa protein in familial and sporadic ankylosing spondylitis: role of HLA-B27 and characterization as a GroEL-like protein.

OBJECTIVE: To study the antibody response of HLA-B27+ patients with ankylosing spondylitis (AS) and their first degree relatives to the 60 kDa protein of Klebsiella pneumoniae and to characterize this protein. METHODS: Sera from 84 individuals were analyzed by ELISA to determine the titer of antibodies against the 60 kDa protein of K. pneumoniae. Subjects were divided into 3 categories: Group 1: 44 HLA-B27+ AS related individuals (35 patients, 9 healthy controls); Group 2: 28 healthy B27- AS related individuals; and Group 3: 12 healthy B27- non-AS related subjects. The 60 kDa protein of K. pneumoniae was induced at 45 degrees C and purified by electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was characterized as a GroEL-like heat shock protein (HSP). The recognition of GroEL-like protein was confirmed by immunoblot of 2 dimension electrophoresis. The response to GroEL-like protein from other bacteria and the response to lipopolysaccharide (LPS) was also analyzed by immunoblot. RESULTS: HLA-B27+ individuals (Group 1), independent of their disease status, showed a significant higher response to the 60 kDa protein of K. pneumoniae than HLA-B27- subjects from Groups 2 and 3 (p < 0.0001). This protein was characterized as a HSP of the GroEL family and designated HSP60Kp. The GroEL of other enterobacteria as well as that of Mycobacterium leprae were recognized by HLA-B27+ individuals by immunoblot, whereas HLA-B27- individuals did not. LPS was not recognized by HLA-B27 positive or negative subjects. CONCLUSION: These findings suggest a relationship between HLA-B27 and the response to a GroEL-like protein that could have implications in AS.

[Importance of molecular methodology in diagnosis]. / La trascendencia de la metodología molecular en el diagnóstico.

The National Institute for Epidemiological Diagnosis and Reference (INDRE) partially supports epidemiological surveillance programs through the identification of most infectious agents prevalent in the country. The success of a program for the control or eradication of a particular infectious disease mainly depends on the opportune and accurate identification of the corresponding etiologic agent. For laboratory diagnosis at INDRE, both conventional methodology using direct or microscopic examinations of specimens or growth in culture media followed by physiological or immunological characterization of the isolate, as well as new techniques based in biochemical, immunochemical and molecular biology procedures are carried out. Antigens can be detected in clinical samples by ELISAs with polyclonal or monoclonal antibodies. Specific nucleic acids can be extracted, identified and typed with techniques like electrophoresis, hybridization with genomic probes, polymerase chain reaction or fragment restriction length polymorphism. Recombinant molecules or highly purified antigens are being obtained and used for the determination of antibodies, mainly with indirect ELISA, IgM capture-ELISA and Western Blot. The better performance, specificity and sensitivity of these laboratory procedures, provide faster results, with equal or greater accuracy than traditional ones, at lower cost.

Enzyme-linked immunosorbent assay (ELISA) with mycobacterial crude antigens for the sero-epidemiological diagnosis of active tuberculosis.

In search for reliable, nonexpensive procedures for tuberculosis diagnosis suitable for seroepidemiological studies in leprosy-endemic areas, enzyme-linked immunosorbent assays (ELISAs) with whole intact bacilli, whole lipid-free bacilli and protein-enriched soluble extracts from the H37Rv Mycobacterium tuberculosis strain were evaluated. Sera tested came from 47 active, pulmonary tuberculosis adult cases, 60 household contacts of active tuberculosis cases, 20 lepromatous leprosy adult patients, and 67 healthy adult controls obtained from low and high leprosy and tuberculosis endemicity areas. There was no influence of such endemicity levels in the number of positive results in control sera. Antibody levels obtained with each of the antigens in ELISAs were significantly different in tuberculosis patients and the control groups. Ten percent of tuberculosis contacts were positive with some of the antigens and three of them showed suggestive chest radiographs. The best combination for a high number of positive results with tuberculosis sera and low positive results with leprosy sera was the BCG soluble extract (91% and 15%, respectively). This preparation also yielded excellent sensitivity and specificity values for tuberculosis (91.5% and 92.5%, respectively). These data suggest that BCG soluble extract ELISAs could provide helpful information to estimate tuberculosis prevalence only in leprosy-free areas, under a situation of unavailability of purified antigens. In pulmonary cases, sputum microscopic examination and culture have higher sensibility than serodiagnosis; therefore, the utilization of BCG soluble extract ELISAs as a diagnostic aid in individual patients with suspected active tuberculosis only can be useful in extrapulmonary cases.

Antibody response to nitrogenase-positive and -negative Klebsiella pneumoniae strains in juvenile-onset ankylosing spondylitis patients and their first degree relatives: lack of differential recognition of the bacterial nitrogenase.

In the search for the pathogenic consequences of the molecular mimicry between the Klebsiella pneumoniae nitrogenase and the HLA-B27 antigen, sera from individuals belonging to 16 kindreds with juvenile-onset ankylosing spondylitis cases, were analyzed for antibodies against nitrogenase-positive and -negative K. pneumoniae whole bacterial extracts. An initial screening for nitrogenase producing K. pneumoniae strains was performed in 31 clinical isolates. The best nitrogenase producing strain was selected as well as a non producing one for immunoblot analysis using sera from 82 subjects, 55 HLA-B27 positive, of which 26 had some clinical manifestations. Even though electrophoretic patterns were different in both strains, there was no distinctive differential recognition of the 30-40 kDa proteins where the nitrogenase subcomponent which shares the sequence QTDRED with the HLA-B27 molecule is located. On the other hand, strong recognition of a protein of 60 kDa (p60Kp) was detected in 75% of HLA-B27 positive tested subjects independently of their clinical status. Studies on the nature of this protein and its participation in the pathogenesis of ankylosing spondylitis are now in progress.

Identification of phosphatidate nonlamellar phases on liposomes by flow cytometry.

This study is the first report that demonstrates nonlamellar arrangements, or lipidic particles, of phosphatidate inserted in the lipid bilayer of liposomes using polyclonal antibodies from mice and flow cytometry. Sera immunoreactivity was analyzed using liposomes that displayed smooth bilayers of phosphatidate particles, as shown by electron microscopy. This cytofluorimetric analysis showed that immune mice sera have a specific immunoreactivity with the phosphatide particles formed by Mn2+, which also cross-reacted with those formed by Ca2+ and with cardiolipin particles formed by Mn2+. In addition, these immune sera hardly reacted with smooth bilayered liposomes, independently of the lipid composition studied. Thus, this new methodology can be applied to demonstrate nonlamellar molecular arrangements in biological membranes.