Comparison of Track XI fluorometric immunoassay with Bio-EnzaBead enzyme-linked immunosorbent assay for detection of serum antibody to mouse hepatitis virus.

The Track XI system (Microbiological Associates, Bethesda, Md.) was compared with the Bio-EnzaBead assay (Organon Teknika, Durham, N.C.) for the detection of antibody to mouse hepatitis virus (MHV). Strain A/J mice were inoculated intranasally with MHV type 3. Sera were collected at 1, 2, 4, and 9 weeks postinoculation and tested. Individual serum samples were retested twice by each method. The results suggested that the Track XI system was more sensitive and reliable than the Bio-EnzaBead assay in detecting antibody to MHV type 3 in individual serum samples from A/J mice.     

Mercury (II) alters mitochondrial activity of monocytes at sublethal doses via oxidative stress mechanisms

The perennial controversy about the safety of mercury in dental amalgams has adversely affected the availability and the quality of dental care. Chronic Hg(II) blood concentrations above 300 nM are known to alter function of the nervous system and the kidney. However, the effects of blood concentrations of 10 to 75 nM, far more common in the general population, are not clear and mechanisms of any effects are not known. The monocyte is an important potential target of Hg(II) because of its critical role in directing inflammatory and immune responses. In the current study we tested the hypothesis that concentrations of Hg(II) of 10 to 300 nM alter monocyte activity via a redox-dependent mechanism. Mitochondrial activity was used to establish inhibitory concentrations of Hg(II) following 6 to 72 h of exposures to THP1 human monocytic cells. Then subinhibitory concentrations were applied, and total glutathione levels and reactive oxygen species (ROS) were measured. Antioxidants [N-acetyl cysteine, (NAC); Na2SeO3, (Se)] and a pro-oxidant (tert-butylhydroquinone, tBHQ) were used to support the hypothesis that Hg(II) effects were redox-mediated. After 72 h of exposure, 20 microM of Hg(II) inhibited monocytic mitochondrial activity by 50%. NAC mitigated Hg(II)-induced mitochondrial suppression only at concentrations of greater than 10 microM, but Se had few effects on Hg-induced mitochondrial responses. tBHQ significantly enhanced mitochondrial suppression at higher Hg(II) concentrations. Hg(II) concentrations of 75 and 300 nM (0.075 and 0.30 microM, respectively) significantly increased total glutathione levels, and NAC mitigated these increases. Se plus Hg(II) significantly elevated Hg-induced total cellular glutathione levels. Increased ROS levels were not detected in monocytes exposed to mercury. Hg(II) acts in monocytic cells, at least in part, through redox-mediated mechanisms at concentrations below those commonly associated with chronic mercury toxicity, but commonly occurring in the blood of some dental patients.     

Long-term protective immune response elicited by vaccination with an expression genomic library of Toxoplasma gondii

Immunization of BALB/c mice with an expression genomic library of Toxoplasma gondii induces a Th1-type immune response, with recognition of several T. gondii proteins (21 to 117 kDa) and long-term protective immunity against a lethal challenge. These results support further investigations to achieve a multicomponent anti-T. gondii DNA vaccine.     

Sputum Cytokine Levels in Patients with Pulmonary Tuberculosis as Early Markers of Mycobacterial Clearance

Sputum and serum from patients with active pulmonary tuberculosis (TB), healthy purified protein derivative-positive adults, and patients with bacterial pneumonia were collected to simultaneously assess local immunity in the lungs and peripheral blood. To determine whether cytokine profiles in sputum from TB patients and control subjects were a reflection of its cellular composition, cytospin slides were prepared in parallel and assessed for the presence of relative proportions of epithelial cells, neutrophils, macrophages, and T cells. Gamma interferon (IFN-γ) in sputum from TB patients was markedly elevated over levels for both control groups. With anti-TB therapy, IFN-γ levels in sputum from TB patients decreased rapidly and by week 4 of treatment were comparable to those in sputum from controls. Further, IFN-γ levels in sputum closely followed mycobacterial clearance. Although detected at fourfold-lower levels, IFN-γ immunoreactivities in serum followed kinetics in sputum. TNF-α, interleukin 8 (IL-8) and IL-6 also were readily detected in sputum from TB patients at baseline and responded to anti-TB therapy. In contrast to IFN-γ, however, TNF-α and IL-8 levels also were elevated in sputum from pneumonia controls. These data indicate that sputum cytokines correlate with disease activity during active TB of the lung and may serve as potential early markers for sputum conversion and response to anti-TB therapy.     

PG-M1: A New Monoclonal Antibody Directed against a Fixative-Resistant Epitope on the Macrophage-Restricted Form of the CD68 Molecule

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.     

Decreased availability of intestinal dopamine in transmural colitis may relate to inhibitory effects of interferon-gamma upon L-DOPA uptake

AIMS: The present study aimed to evaluate the relationship between intestinal inflammation, interferon-gamma (IFN-gamma) levels and intestinal levels of dopamine, its precursor l-3,4-dihydroxyphenylalanine (L-DOPA), and the activity of aromatic L-amino acid decarboxylase (AADC) activity along the digestive tract in a rat experimental model of colitis. METHODS: Colitis was induced by rectal administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS). Catechol derivatives were assayed by means of HPLC-EC. RESULTS: It is shown that dopamine and noradrenaline levels in the distal colon (inflamed mucosa), but not in the ileum (non-inflamed mucosa), of TNBS-treated rats were markedly lower than in control animals. A slight decrease in L-DOPA tissue levels, no changes in AADC activity and an increase in plasma IFN-gamma levels accompanied this decrease in dopamine levels. Exposure of Caco-2 cells, a human intestinal epithelial cell line, to human IFN-gamma resulted in a concentration-dependent and long-lasting inhibition of L-DOPA uptake, which most likely explains the decrease in dopamine levels in the inflamed mucosa. CONCLUSION: Changes in tissue levels of noradrenaline and dopamine in experimental colitis in the rat follow a similar pattern to that observed in patients with Crohn's disease and ulcerative colitis. In this model of experimental colitis, the decrease in dopamine levels is most likely explained by the inhibitory effect of IFN-gamma on L-DOPA uptake by intestinal epithelial cells.     

Regulation of glutathione by oxidative stress in bovine pulmonary artery endothelial cells

Glutathione plays important roles as an intracellular antioxidant and in the maintenance of cellular thiol-disulfide balance. In addition, glutathione may regulate cell growth signaling induced by oxidative stress. We previously reported that cellular glutathione is up-regulated by bleomycin in bovine pulmonary artery endothelial cells. The present study examined effects of hydrogen peroxide (H(2)O(2)) on cell growth and glutathione levels. Exogenous addition of H(2)O(2) induced biphasic effects on cell growth; 1 micro M was stimulatory and >10 micro M was inhibitory. However, both growth-promoting and inhibitory levels of H(2)O(2) increased cellular glutathione levels. Whereas 1 micro M H(2)O(2) moderately but significantly increased glutathione, 30 micro M caused a more substantial increase. Like bleomycin, both concentrations of H(2)O(2) activated DNA binding of antioxidant response element (ARE), a regulatory element in the promoter of the gamma-glutamylcysteine synthetase heavy chain subunit, a key regulator of glutathione synthesis. However, only high concentrations of H(2)O(2) activated p44/42 mitogen-activated protein (MAP) kinase. Thus, cellular glutathione is up-regulated by H(2)O(2), perhaps via activating ARE-binding factors in a mechanism independent of MAP kinase. H(2)O(2)-mediated increase in glutathione and activation of ARE binding may play important roles in growth and death of pulmonary artery endothelial cells.