Cardiac responses to ß-adrenoceptor stimulation is partly dependent on mitochondrial calcium uniporter activity.

BACKGROUND AND PURPOSE: Despite the importance of mitochondrial Ca(2+) to metabolic regulation and cell physiology, little is known about the mechanisms that regulate Ca(2+) entry into the mitochondria. Accordingly, we established a system to determine the role of the mitochondrial Ca(2+) uniporter in an isolated heart model, at baseline and during increased workload following ß-adrenoceptor stimulation. EXPERIMENTAL APPROACH: Cardiac contractility, oxygen consumption and intracellular Ca(2+) transients were measured in ex vivo perfused murine hearts. Ru360 and spermine were used to modify mitochondrial Ca(2+) uniporter activity. Changes in mitochondrial Ca(2+) content and energetic phosphate metabolite levels were determined. KEY RESULTS: The addition of Ru360 , a selective inhibitor of the mitochondrial Ca(2+) uniporter, induced progressively and sustained negative inotropic effects that were dose-dependent with an EC50 of 7 µM. Treatment with spermine, a uniporter agonist, showed a positive inotropic effect that was blocked by Ru360 . Inotropic stimulation with isoprenaline elevated oxygen consumption (2.7-fold), Ca(2+) -dependent activation of pyruvate dehydrogenase (5-fold) and mitochondrial Ca(2+) content (2.5-fold). However, in Ru360 -treated hearts, this parameter was attenuated. In addition, ß-adrenoceptor stimulation in the presence of Ru360 did not affect intracellular Ca(2+) handling, PKA or Ca(2+) /calmodulin-dependent PK signalling. CONCLUSIONS AND IMPLICATIONS: Inhibition of the mitochondrial Ca(2+) uniporter decreases ß-adrenoceptor response, uncoupling between workload and production of energetic metabolites. Our results support the hypothesis that the coupling of workload and energy supply is partly dependent on mitochondrial Ca(2+) uniporter activity.

The potential role of lung epithelial cells and beta-defensins in experimental latent tuberculosis.

Mycobacterium tuberculosis is a facultative intracellular pathogen capable of producing both progressive disease and latent infection. Latent infection is clinically asymptomatic and is manifested only by a positive tuberculin test or a chest radiograph that shows scars or calcified nodules indicative of resolved primary tuberculosis infection. In this study, we used a well-characterized model of latent tuberculosis infection in B6D2F1 mice to compare the production of beta-defensin-3 by infected bronchial epithelial cells and macrophages. We demonstrated by immunolectronmicroscopy that M. tuberculosis can actually infect epithelial cells and induce significant higher production of beta-defensin-3 associated to mycobacteria than infected macrophages. These results demonstrate that lung epithelium harbour mycobacteria during experimental chronic infection; being a possible reservoir of latent mycobacteria in vivo, beta-defensins might participate in bacilli killing or dormancy induction.

Enhanced wear resistance of ball-and-socket joints of dental implants by means of titanium gaseous nitriding.

The aim of this research is the surface hardening of the ball-and-socket joint of dental implants by means of heat treatments in order to obtain titanium nitrides. These nitrides minimize the wear of the titanium used in prosthesis. In this paper the optimum heat treatment, the hardness and the wear resistance are described in relation to the ball-and-socket joint without heat treatment.

Low frequency of BCL-2/J(H) translocation in peripheral blood lymphocytes of healthy Japanese individuals.

The incidence of follicular lymphoma differs significantly between white and Japanese individuals. Translocation between the BCL-2 and immunoglobulin heavy chain genes is detected in 85% to 90% of all follicular lymphomas in whites. Recently, BCL-2/J(H) translocation was detected in peripheral blood lymphocytes from more than 50% of healthy white individuals. To clarify the reason for the difference in incidence of follicular lymphoma between whites and Japanese, the frequency of BCL-2/J(H) translocation in peripheral blood lymphocytes of healthy Japanese individuals was compared with that of German individuals. The prevalence of BCL-2/J(H) translocation in Japanese adults appeared to be significantly lower than that in German adults. The present data suggest that the low frequency of BCL-2/J(H) translocation in the Japanese general population may be one of the major reasons for the difference in incidence of follicular lymphoma between whites and Japanese.

Pulmonary mononuclear cell responses to antigens of Mycobacterium tuberculosis in healthy household contacts of patients with active tuberculosis and healthy controls from the community.

Protective immunity against Mycobacterium tuberculosis requires CD4+ lymphocyte-mediated immune responses and IFN-gamma activity. As the primary portal of entry of M. tuberculosis is the lung, pulmonary immune responses against multiple M. tuberculosis Ags were compared between both M. tuberculosis-exposed tuberculin skin test-positive healthy household contacts (HHC) of patients with active sputum smear and culture-positive tuberculosis and tuberculin skin test-positive healthy control individuals from the community (CC). Frequencies of M. tuberculosis Ag-specific IFN-gamma-producing cells, IFN-gamma concentrations in culture supernatants, and DNA synthesis in bronchoalveolar cells (BAC) and PBMC were studied in HHC (n = 10) and CC (n = 15). Using enzyme-linked immunospot assay we found higher frequencies of IFN-gamma-producing cells with specificity to M. tuberculosis-secreted Ag 85 (Ag 85) in BAC from HHC than in BAC from CC (p < 0.022) and relative to autologous PBMC, indicating compartmentalization of Ag 85-specific cells to the lungs. Further, IFN-gamma-producing cells with specificity to components A and B of Ag 85 were specifically compartmentalized to the lungs in HHC (p < 0. 05). IFN-gamma concentrations in culture supernatants of BAC and Ag-specific DNA synthesis were low and comparable in the two subject groups. Increased immune responses to Ag 85 at the site of repeated exposure to M. tuberculosis (the lung) may represent an important component of protective immunity against M. tuberculosis. Correlates of protective immunity against M. tuberculosis are required for assessment of the efficiency of anti-tuberculous vaccines.

Expression of transforming growth factor-beta but not tumor necrosis factor-alpha, interferon-gamma, and interleukin-4 in granulomatous lung lesions in tuberculosis.

The expression of transforming growth factor (TGF-beta 1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were assessed in lung tissues from patients with tuberculosis. Vimentin, a constitutively expressed cellular protein, was present in 12 of 19 tissue sections indicating adequate preservation of tissue proteins in these cases. Immunohistochemical studies for cytokines were done in the vimentin positive sections only. TGF-beta 1 was localized to mononuclear phagocytes of tuberculous lung lesions in 4 of 12 tuberculosis patients. TNF-alpha, IFN-gamma, and IL-4 were absent in sections from all tuberculosis patients. The failure to detect the latter cytokines may indicate that these molecules may not be expressed at the site of disease, or are not a feature of the late stages of tuberculous granulomas. TGF beta-1, although not universally expressed, may be involved in the development and/or consequences of tuberculous granuloma formation. These data substantiate further the role of TGF-beta 1 in the immunopathology of tuberculosis.

Application of stable isotope ratio analysis to the characterization of the geographical origin of olive oils.

To gain information about the geographical origin of oil samples, measurements of delta(13)C and delta(18)O of the whole oil and some of its fractions have been performed on samples coming from fruits of Olea europaea L. produced in Greece, Morocco, Spain, Italy, Tunisia, and Turkey. The results obtained by applying statistical procedures have given pieces of evidence that oil samples have shown the trend to cluster according to the different climatic areas of growing environment of fruits. Some confusion has been observed for samples coming from neighboring countries having similar climates.

Latent infection and reactivation of human herpesvirus 6 in two novel myeloid cell lines.

It has been reported that reactivation of human herpesvirus-6 (HHV-6) causes a failure of hematopoiesis. To clarify the mechanisms of bone marrow suppression induced by HHV-6 infection, it is necessary to establish an in vitro model of HHV-6 infection in hematopoietic progenitor cells. We have established two novel Philadelphia chromosome-positive myeloid cell lines, SAS413 and SAS527, which possess different hematologic characteristics and show distinct susceptibility to infection by HHV-6, from a patient with blast crisis of chronic myelogenous leukemia (CML). HHV-6 subgroup A (HHV-6A) showed marked replication in SAS413, forming syncytia and inducing cell lysis in short-term culture. On the other hand, HHV-6A-inoculated SAS527 continued to proliferate without cell lysis and only a few cells showed HHV-6 antigen expression. In contrast to HHV-6A infection, inoculation with HHV-6 subgroup B (HHV-6B) did not induce any cytopathic effect (CPE) or viral antigen expression in either of the cell lines. Although HHV-6B replication was undetectable, the presence of the HHV-6 genome in both cell lines was shown by polymerase chain reaction (PCR) during culture for more than 10 months, suggesting that HHV-6B latently infected SAS413 and SAS527. Phorbol ester treatment of SAS527 latently infected with HHV-6B resulted in reactivation of HHV-6, as shown by the appearance of a CPE, positive reactivity for the HHV-6 antigen, and isolation of infectious HHV-6. These novel cell lines should be useful for studying the mechanisms of HHV-6-induced hematopoietic failure and HHV-6 latency and reactivation, as well as differentiation, of the myeloid cell lineage.

Enhanced responses to Mycobacterium tuberculosis antigens by human alveolar lymphocytes during active pulmonary tuberculosis.

Responses to mycobacterial and nonmycobacterial antigens were examined in bronchoalveolar cells (BAC) and peripheral blood mononuclear cells (PBMC) from patients with active pulmonary tuberculosis (n=16) and healthy subjects (n=23). DNA synthesis in BAC (but not PBMC) from tuberculosis patients was significantly increased in response to the mycobacterial antigens purified protein derivative (PPD), antigen 85, and mannose-capped lipoarabinomannan but not to nonmycobacterial antigens. The response to PPD was also increased in enriched alveolar lymphocytes from tuberculosis patients (P<.05). The frequency of interferon-gamma but not interleukin-4- or -10-producing cells by ELISAspot was increased in PPD-stimulated BAC from patients with tuberculosis (P<.05). Accessory function of alveolar macrophages for T lymphocyte responses was similar and suppressive activity was variably decreased in tuberculosis patients. Thus, there is compartmentalization of mycobacterial antigen-specific lymphocytes to the lungs during active tuberculosis that on challenge produce a Th1-type cytokine host response.

Chemokines induced by infection of mononuclear phagocytes with mycobacteria and present in lung alveoli during active pulmonary tuberculosis.

The capacity of Mycobacterium tuberculosis (MTB) to induce production of chemokines with known chemotactic activity for monocytes and lymphocytes, the cellular building blocks of granulomas, was investigated. These chemokines included regulated upon activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha). MTB stimulated production of MCP-1 and MIP-1alpha by blood monocytes (MN) and alveolar macrophages (AM). MTB infection of MN and AM stimulated release but not production of RANTES. AM produced or released significantly higher levels than MN of RANTES (by 2.1-fold), MCP-1 (by 6.9-fold), and MIP-1alpha (by 5. 5-fold) (P < 0.05 for each). This study also confirmed that MTB-infected AM produce the chemokine interleukin (IL)-8. MTB infection of AM resulted in increased steady-state expression of messenger RNA (mRNA) for MCP-1 and MIP-1alpha and minimal increased expression of RANTES mRNA. Both an avirulent (H37Ra) and a virulent (H37Rv) strain of MTB and purified protein derivative of H37Rv but not latex beads induced production of chemokines. Supernatants of MTB-infected cells demonstrated chemotactic activity for both monocytes and lymphocytes partially inhibitable by neutralizing antibodies against the chemokines studied. Bronchoalveolar lavage fluid from patients with active pulmonary tuberculosis as compared with healthy control subjects contained increased levels of RANTES (by 8-fold), MCP-1 (by 2.7-fold), and IL-8 (by 8.9-fold) (P < 0.05), but not MIP-1alpha, as compared with healthy control subjects. Thus, multiple chemokines may be involved in recruitment of cells for granuloma formation in tuberculosis.

Involvement of the Fas/Fas ligand pathway in activation-induced cell death of mycobacteria-reactive human gamma delta T cells: a mechanism for the loss of gamma delta T cells in patients with pulmonary tuberculosis.

Although the identity of T cells involved in the protection against Mycobacterium tuberculosis (Mtb) in humans remain unknown, patients with pulmonary tuberculosis (TB) have reduced numbers of Mtb-reactive, V gamma 9+/V delta 2+ T cells in their blood and lungs. Here we have determined whether this gamma deltaT loss is a consequence of Mtb Ag-mediated activation-induced cell death (AICD). Using a DNA polymerase-mediated dUTP nick translation labeling assay, 5% or less of freshly isolated CD4+ alpha beta or gamma delta T cells from normal healthy individuals and TB patients were apoptotic. However, during culture Mtb Ags induced apoptosis in a large proportion of V gamma 9+V delta 2+ peripheral blood T cells from healthy subjects (30-45%) and TB patients (55-68%); this was increased further in the presence of IL-2. By contrast, anti-CD3 did not induce any significant level of apoptosis in gamma delta T cells from healthy subjects or TB patients. Mtb Ag stimulation rapidly induced Fas and Fas ligand (FasL) expression by gamma delta T cells, and in the presence of metalloproteinase-inhibitors >70% of gamma delta T cells were FasL+. Blockade of Fas-FasL interactions reduced the level of Mtb-mediated gamma delta T cell apoptosis by 75 to 80%. Collectively, these findings demonstrate that Mtb-reactive gamma delta T cells are more susceptible to AICD and that the Fas-FasL pathways of apoptosis is involved. AICD of gamma delta T cells, therefore, provides an explanation for the loss of Mtb-reactive T cells during mycobacterial infection.

Cytokine profiles for peripheral blood lymphocytes from patients with active pulmonary tuberculosis and healthy household contacts in response to the 30-kilodalton antigen of Mycobacterium tuberculosis.

Patients with active tuberculosis (TB) have a stronger humoral but a poorer cellular immune response to the secreted 30-kDa antigen (Ag) of Mycobacterium tuberculosis than do healthy household contacts (HHC), who presumably are more protected against disease. The basis for this observation was studied by examining the Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-gamma])- and Th2 (IL-10 and IL-4)-type cytokines produced in response to the 30-kDa Ag by peripheral blood mononuclear cells (PBMC) from patients with active pulmonary TB (n = 7) and from HHC who were tuberculin (purified protein derivative) skin test positive (n = 12). Thirty-kilodalton-Ag-stimulated PBMC from TB patients produced significantly lower levels of IFN-gamma (none detectable) than did those from HHC (212 +/- 73 pg/ml, mean +/- standard error) (P < 0.001). Likewise, 30-kDa-Ag-stimulated PBMC from TB patients failed to express IFN-gamma mRNA by reverse transcription-PCR, whereas cells from HHC expressed the IFN-gamma gene. In contrast, 30-kDa-Ag-stimulated PBMC from TB patients produced significantly higher levels of IL-10 (403 +/- 80 pg/ml) than did those from HHC (187 +/- 66 pg/ml) (P < 0.013), although cells from both groups expressed the IL-10 gene. IL-2 and IL-4 were not consistently produced, and their genes were not expressed by 30-kDa-Ag-stimulated cells from either TB patients or HHC. After treatment with antituberculous drugs, lymphocytes from four of the seven TB patients proliferated and three of them expressed IFN-gamma mRNA in response to the 30-kDa Ag and produced decreased levels of IL-10.

Determination of the (13)C Content of Glycerol Samples of Different Origin.

The average carbon isotope value (delta(13)C) of 63 samples of glycerol from over 30 different sources has been determined. The results indicate that it is possible to distinguish the glycerol obtained from the glycerides produced in plants following C-3 and C-4 carbon fixation pathways. The samples obtained from animal sources seem to reflect the composition of the material consumed, as well as that produced by sugar fermentation.

Human herpesvirus 7 infection of lymphoid and myeloid cell lines transduced with an adenovirus vector containing the CD4 gene.

It has been reported recently that CD4 is a major component of the receptor for human herpesvirus 7 (HHV-7), which has been newly identified as a T-lymphotropic virus. To investigate further the role of CD4 in HHV-7 infection, we examined the susceptibility to HHV-7 infection of various CD4-negative or weakly positive cell lines into which the cDNA for CD4 was transferred using an adenovirus vector (Adex1CACD4). Of 13 cell lines transduced with Adex1CACD4, including T-lymphoid, B-lymphoid, monocytoid, and myeloid cell lines, one T-lymphoid cell line, one monocytoid cell line, and two cell lines established from the blast crisis of chronic myelogenous leukemia showed high susceptibility to HHV-7 infection. Taken together with the results of previous studies, these data suggest strongly that CD4 is a major component of the binding receptor for HHV-7. This study also shows that HHV-7 may be able to infect CD4-positive hematopoietic precursor cells as well as T lymphocytes.

Seasonal variations in the frontal organ of the frog: structural evidence and physiological correlates.

Histological study of the frontal organ in the frog, Rana esculenta, was performed during spring, summer, autumn and winter. In semithin sections stained with toluidine blue, cells containing a vacuole were clearly detected during spring, and considerably increased during summer. Such cellular elements were absent in the frontal organ during autumn and winter. This morphological evidence of seasonal variation was supported by extracellular recording in the frontal organ in different seasons. Spontaneous firing rate was found to increase from the spring to the summer, and to decrease from the autumn to the winter. Altogether, these data indicate that the frontal organ may represent an autonomic component of the pineal complex with a secretory function producing neurohormonal messages involved in the annual mechanism of the reproduction.

Mycobacterium tuberculosis (MTB)-stimulated production of nitric oxide by human alveolar macrophages and relationship of nitric oxide production to growth inhibition of MTB.

SETTING: Although nitric oxide (NO) is a major proximate mediator of microbicidal activity in murine macrophages against intracellular pathogens including mycobacteria, its production by and effector role in human macrophages is not clear. OBJECTIVE: To determine the capacity of Mycobacterium tuberculosis (MTB) to stimulate NO in human monocytes (MN) and alveolar macrophages (AM) and to assess the relationship between NO production and intracellular growth of MTB. DESIGN: NO production (measured as nitrite) by MTB (H37Ra)-infected macrophages and intracellular growth of MTB were measured in cells from 17 healthy subjects. RESULTS: MTB (5:1, MTB:cells) stimulated little to no NO by MN, but induced NO in AM at days 4 and 7 after infection. There was, however, variability in the response by AM to MTB: among seven subjects MTB-induced NO was low (4 +/- 2 microM, mean +/- SE); six subjects were moderate (56 +/- 11); four subjects were high (502 +/- 167). NO synthase inhibitors inhibited the production of NO by AM but did not significantly affect the intracellular growth of MTB, although a trend towards increased intracellular growth was seen on day 4 of culture. Intracellular growth of MTB in AM from low NO producers was significantly higher than that in AM from moderate NO producers, P < or = 0.05. Inducible NO synthase (iNOS) mRNA by RT-PCR was constitutively expressed by both MN and AM, but was further stimulated by MTB in AM > MN; MTB-induced iNOS protein was present in both MN and AM by Western blot analysis. CONCLUSION: Thus, MTB-infected human AM are capable of producing NO and NO production correlates with intracellular growth inhibition of MTB in AM suggesting that NO may serve either directly or indirectly as a mycobactericidal mediator in human tissue macrophages.

Human herpesvirus 7 infection of CD4+(+) T cells does not require expression of the OKT4 epitope.

To evaluate the role of the OKT4 epitope in human herpesvirus 7 (HHV-7) infection, we studied the susceptibility to HHV-7 infection of CD4+ T cells isolated from two individuals with OKT4 epitope deficiency. HHV-7-infected OKT4-Leu3a+ T cells exhibited the characteristic cytopathic effect, reactivity with HHV-7-seropositive serum by immunofluorescence and down-modulation of surface CD4 in a manner similar to HHV-7-infected OKT4+Leu3a+ T cells. A semiquantitative PCR revealed that the amounts of HHV-7 replicated in OKT4+Leu3a+ T cells and OKT4-Leu3a+ T cells were not significantly different. Although it has been reported that OKT4 monoclonal antibody efficiently inhibits HHV-7 infection, the present study demonstrated that the interaction of HHV-7 with CD4+ T cells does not require participation of the epitope defined by OKT4 monoclonal antibody.

Detection of human herpesvirus 6 and human herpesvirus 7 in the submandibular gland, parotid gland, and lip salivary gland by PCR.

In order to define the major sites of persistence of human herpesvirus 6 (HHV-6) and HHV-7, PCR with DNAs from more than 100 specimens of 3 different salivary glands was performed. HHV-6 DNA was detected in 52 (88.1%) of 59 submandibular gland, 17 (63.0%) of 27 parotid gland, and 9 (52.9%) of 17 lip salivary gland specimens. On the other hand, HHV-7 DNA was detected in 59 (100%) of 59 submandibular gland, 23 (85.2%) of 27 parotid gland, and 10 (58.8%) of 17 lip salivary gland specimens. These findings demonstrate that salivary glands are a site of persistent infection of both HHV-6 and HHV-7 and that among the three types of salivary gland examined, the submandibular gland is the primary one in which these herpesviruses, especially HHV-7, persist.

T lymphocytic and immature macrophage alveolitis in active pulmonary tuberculosis.

The phenotype of bronchoalveolar cells from 11 healthy subjects and from affected and unaffected lungs of 15 patients with pulmonary tuberculosis (PTB) was determined. An immature macrophage alveolitis was found in the affected lung and the unaffected lung versus controls as determined by morphology and peroxidase activity. T lymphocytic alveolitis also was found in the affected but not the unaffected tuberculous lung compared with healthy controls. The majority of alveolar lymphocytes in unaffected and affected PTB lungs were T cells expressing the alpha beta T cell receptor. Alveolar T cells from both unaffected and affected lungs were activated, as determined by increased expression of CD69 and HLA-DR. Interleukin-2 receptor (IL-2R alpha) expression was, however, unchanged on alveolar lymphocytes from affected lung and was decreased in the unaffected lung. Thus, activated T lymphocytes and immature macrophages in the tuberculous lung are basic to the local immunopathogenesis of PTB.