Maja Nowakowski

Protection of epithelial cells against influenza A virus by a plant derived biological response modifier Ledretan-96.

A multicomponent herbal formula Ledretan‐96 was tested on an epithelial tissue culture cell line (MDCK) for its protective activity against cytopathic effects caused by influenza A virus. The whole formula and each of its 23 individual components were tested in the same system. The results indicated that the formula, when prepared according to established procedure, in the form of decoction, is active in protecting epithelial cells against damage caused by influenza A virus used at different dosages. Of the 23 components tested, only one, Terminalia chebula, showed a significant protective effect when applied to the epithelial cells individually. Controls for these studies included cell cultures exposed to individual components and the complete formula without virus; the cultures were monitored for any toxic effects by morphology and protein synthesis. The protective effects of Ledretan‐96 and Terminalia chebula could be distinguished from toxicity against the epithelial cells. In addition, the results indicated that the complete formula maintained antiviral activity at a higher therapeutic index than the Terminalia chebula extract alone.

Effect of minocycline and doxycycline on IgE responses

BACKGROUND We have recently found that the tetracycline minocycline suppresses inflammatory responses in serum immunoglobulin (Ig)E-positive asthmatic patients, and that IgE levels can decrease in these patients. The mechanism by which minocycline suppresses these responses is unknown. OBJECTIVE We have now investigated the ability of the tetracyclines, minocycline and doxycycline, to regulate IgE responses of peripheral blood mononuclear cells (PBMC) obtained from serum IgE-positive asthmatic patients. METHODS The distributions of CD3+, CD4+, CD8+, and CD19+ lymphocytes in peripheral blood of serum IgE-positive asthmatic patients and IgE-negative nonasthmatic controls, and cytokine-specific mRNA expression by their PBMC were determined by flow cytometry (reverse transcriptase-polymerase chain reaction). Serum Ig levels also were determined (nephelometry, fluoroenzymeimmunoassay, enzyme-linked immunoadsorbent assay; n = 7/group). PBMC (1.5 x 10(6)/mL) were cultured with anti-CD40 monoclonal antibody and recombinant human interleukin-4 in the presence/absence of minocycline or doxycycline (0.1 to 10 microg/mL), and IgE levels in supernatants determined on days 0, 3, and 10 (enzyme-linked immunoadsorbent assay). RESULTS Asthmatic and nonasthmatic subjects had similar numbers of blood CD4+ T cells (779/mm3 +/- 73 and 766 +/- 115, respectively) and CD19+ B-cells (239/mm3 +/- 35 and 379 +/- 95, respectively); however, CD8+ T cell numbers were decreased in asthmatic compared with nonasthmatic subjects (378/mm3 +/- 66 and 568 +/- 53, respectively; P = 0.045). High IgE levels were detected in supernatants of asthmatic PBMC on day 10 (28 ng/mL +/- 12), whereas control IgE levels did not change (<2.5 ng/mL). When either minocycline or doxycycline was included in culture, IgE production by asthmatic PBMC was strongly suppressed in dose-dependent fashion on day 10 (>80% with 10 microg/mL); control IgE did not change (<2.5 ng/mL). CONCLUSIONS The results are consistent with the idea that the therapeutic benefits obtained by asthmatic patients from minocycline may, in part, result from IgE suppression.

HIV Type 1-Specific IgE in Serum of Long-Term Surviving Children Inhibits HIV Type 1 Production in Vitro

We previously identified a group of long-term pediatric survivors who had acquired HIV-1 through maternal transmission; had not received antiretroviral therapy; are now >8 years old, in good health, and with no opportunistic infections; and have not failed to thrive, although they have greatly decreased numbers of blood CD4+ T cells (<500/mm(3)). All the children have elevated total serum IgE levels (210-2475 IU/ml) and make anti-HIV-1 IgE or IgE directed against non-HIV-1 specificities (radioimmunoassay, Western blot assay); they have no detectable antigenemia. We have now studied the ability of anti-HIV-1 IgE in serum obtained from these children to regulate (1) production of HIV-1 by interleukin 2/phytohemagglutinin (IL-2/PHA)-stimulated peripheral blood mononuclear cells (PBMCs) taken from HIV-1-seronegative donors and infected with a T cell-tropic clone of HIV-1, and (2) transmission of a primary HIV-1 strain from adult AIDS patients to uninfected IL-2/PHA-stimulated PBMCs (p24 core antigen production). High levels of HIV-1 production were observed when PBMCs were cultured for 5 days in the presence of HIV-1-seronegative donor serum that was either IgE positive or IgE negative (IgE, >100 or <100 IU/ml, respectively). HIV-1 production also was observed when PBMCs were cultured with HIV-1-infected donor serum that either contained IgE directed against non-HIV-1 specificities or was IgE negative; these levels were 40% less than those seen with sera from the HIV-1-seronegative donors. Far greater inhibition of virus production was observed if the serum in culture contained anti-HIV-1 IgE (>95%). Virus neutralization did not appear to account for the inhibition obtained with anti-HIV-1 IgE-containing serum because virus production was not suppressed in cultures to which serum was added immediately preinfection (<10%), but was strongly suppressed when serum was added 1.5 hr postinfection (>95%). The inhibition of virus production obtained with serum containing anti-HIV-1 IgE was reversed when (1) serum was depleted of IgE (immunoaffinity), but not when it was depleted of IgG (protein G-Sepharose) before inclusion in culture postinfection, (2) anti-IgE, but not anti-IgG, was included in culture, or (3) serum was heat treated before culture. The results indicate that serum from certain HIV-1-infected pediatric long-term survivors contains agents that inhibit HIV-1 production in vitro, and that these agents include anti-HIV-1 IgE. They suggest that a cytotoxic event, rather than virus neutralization, plays an important role in anti-HIV-1 IgE-mediated inhibition of virus production.

Isolation of two DNA-binding proteins from the intracellular replication complex of vaccinia virus

Abstract Two DNA-binding proteins, designated FP11 and FP14, have been purified from the viral replication complex which forms in the cytoplasm of HeLa cells during productive infection with vaccina virus. Polypeptide FP11 has an apparent molecular weight of 34,000 (by SDS-PAGE) and binds strongly to denatured DNA-cellulose columns, eluting as a narrow peak centered at 0.25 M NaCl. This polypeptide represents 30–40% of the [3H]lysine but only 17% of the [3H]methionine which can be incorporated into the complex. Polypeptide FP14 has an apparent molecular weight of 28,000 (by SDS-PAGE) and elutes from denatured DNA-cellulose as a relatively broad peak over the range 0.45–0.80 M NaCl. FP14 accounts for approximately 10% of incorporated radioactivity, regardless of whether lysine or methionine is employed as the radioactive precursor.

Characterization of a DNA-binding phosphoprotein from vaccinia virus replication complex.

Abstract The DNA-protein complex which forms in the cytoplasm of HeLa cells during the course of vaccinia virus infection contains a single DNA-binding phosphoprotein, termed FP11. This phosphoprotein has been purified to near homogeneity by chromatography on denatured DNA-cellulose. Phosphoprotein FP11 has an apparent molecular weight of 34,000, as determined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. High-voltage paper electrophoresis following acid hydrolysis reveals that the polypeptide contains phosphothreonine but not phosphoserine. The in vivo phosphorylation occurs at a minimum of four distinct phosphothreonine residues. FP11 is an acidic protein with an isoelectric point lying in the pH region 5.2–5.5

Uniform detection of HIV-1 in alveolar macrophages of pediatric but not adult AIDS patients.

Manifestations of pulmonary disease in pediatric AIDS patients differ from those in adults. To evaluate whether differences in the frequency of alveolar macrophage (AM) infection with human immunodeficiency virus type 1 (HIV‐1) could account for these clinical distinctions, we undertook a comparative analysis of HIV‐1 DNA in AMs from pediatric and adult AIDS patients by enzymatic amplification. A higher frequency of viral DNA detection in pediatric cases (100%) compared with adults (67%) was observed. The sensitivity of detection was 1 viral DNA copy per 4000 AMs; matched peripheral blood mononuclear cells from six of seven pediatric and eight of nine adult patients tested HIV‐1 DNA positive. Adult but not pediatric patients exhibited a marked alveolar lymphocytosis, 32% mean lymphocyte count compared with 7.0%, respectively. These results suggest that the burden of HIV‐1 in the lungs of pediatric AIDS patients is greater than that in adults.

Tetracycline-mediated IgE isotype-specific suppression of ongoing human and murine IgE responses in vivo and murine memory IgE responses induced in vitro

We previously reported that minocycline treatment of allergic asthmatic patients had oral steroid sparing effects and improved their clinical status and that minocycline suppressed in vitro induction of IgE responses by their PBMC. The effect of minocycline on human or animal IgE responses in vivo has not been studied. Allergic asthmatics (serum IgE: 505 +/- 535 IU ml(-1)) were given minocycline (150 mg po to 250 mg po BID) as add-on therapy to standard care for up to 10 months; control subjects (IgE: 405 +/- 472 IU ml(-1)) received standard care (n = 6 per group). Serum immunoglobulin (IgM, IgG, IgE and IgA) levels were determined monthly (Nephelometry, Unicap Total IgE Fluoroenzyme immunoassay). BALB/c mice (n = 6 per group) were injected intraperitoneally with benzylpenicilloyl(14)-Keyhole limpet hemocyanin (BPO(14)-KLH) in alum on days 0, 21 and 42, fed with minocycline or doxycycline (10-100 mg kg(-1)) on day 44 and numbers of BPO-specific IgG(1), IgE and IgA antibody-forming cell (AFC) in mesenteric LN and spleen and serum immunoglobulin levels were determined on days 46-70 (enzyme-linked immunosorbent spot assay, ELISA). The ability of minocycline or doxycycline to suppress in vitro induction of murine memory IgE responses also was investigated. Minocycline strongly suppressed serum IgE levels of allergic asthmatics (9% per month) (P = 0.012). Minocycline (and doxycycline) also strongly suppressed peak murine IgE AFC and serum IgE responses (>95, approximately 75%, respectively) and in vitro induction of memory IgE responses by murine mesenteric LN and spleen cells (>95%). Tetracycline suppression of all human and murine IgE responses was IgE isotype specific. Suppression of murine IgE responses in vivo was dose dependent and lasted 5-7 days.

Morphological and biochemical alterations of macrophages produced by a glycan psk

A glycan extracted from Coriolus versicolor (PSK, Krestin) which has antitumor and immunomodulator properties produced marked morphological and biochemical changes when added to cultures of mouse peritoneal macrophages. The cells were more spread and elongated than in control cultures, and these changes were accompanied by alterations in the rate of protein and DNA synthesis. In PSK-treated murine peritoneal macrophages the rate of protein synthesis increased above the level seen in control cultures after two days and reached a level twenty-fold higher than control on day four; this elevated rate of protein synthesis was maintained throughout the seven-day observation period. DNA synthesis was induced after four days in the presence of PSK, and reached a level ten-fold higher than control baseline on day five. This induction of DNA synthesis, however, could not be attributed to a mitogenic activity on lymphocytes. The alterations caused by PSK in macrophage metabolism may be related to the immunomodulating and antitumor activities of PSK in vivo.

Correlation of plasma complement split product levels with allergic respiratory disease activity and relation to allergen immunotherapy

BACKGROUND Allergens, including dust mite and grass pollen, and mast cell tryptase are known to generate the complement split products (CSPs) C5a and C3a, which can then trigger allergic inflammation. The relation of these anaphylatoxin levels to clinical allergic disease responses is not known. OBJECTIVE To evaluate the relationship of plasma CSP levels to allergic respiratory disease variables in an adult cohort. METHODS A cross-sectional survey was used to assess the association of plasma C5a desArg and C3a desArg levels with clinical allergic respiratory disease variables. Furthermore, a time course of the effect of routine allergen immunotherapy on plasma CSP levels and cutaneous and pulmonary responses was determined. RESULTS Adult plasma C5a desArg levels correlate with asthma severity as determined by a physician (P = .01) and by Asthma Quality of Life Questionnaire scores (P < .01). Change in plasma C5a desArg levels 1 hour after immunotherapy is associated with baseline rhinoconjunctivitis symptom severity (P = .03), change in total mean wheal diameter (P = .05), and total dust mite dosage (P = .04). Change in plasma C3a desArg levels 3 hours after immunotherapy correlates with change in total mean wheal diameter induced by dust mite (P = .01). Change in plasma CSP levels after immunotherapy did not correlate with change in spirometric outcome. CONCLUSIONS Plasma C5a desArg levels reflect allergic respiratory disease severity as assessed by physicians and correlate with Asthma Quality of Life Questionnaire scores. Changes in CSP levels after immunotherapy reflect cutaneous allergic responses, especially to dust mite allergen.

Increased Expression of CD23 (Fcε Receptor II) by Peripheral Blood Monocytes of AIDS Patients

Monocytes expressing the Fce receptor II (CD23) play important roles in inflammatory and allergic immune responses. We found that peripheral blood monocytes of AIDS patients express increased levels of CD23, compared with monocytes of healthy HIV-1-seronegative individuals (controls) (p < 0.05). We compared expression of monocyte CD23 with expression of monocyte Fcγ receptors (CD16, CD32, CD64), plasma/serum levels of IgE (also IgM, IgG, IgA), and Th1 (IFN-γ) and Th2 (IL-4, IL-10) cytokines. We found that monocyte CD23 expression directly correlated with monocyte CD16 expression (p < 0.01, R = 0.58), which was also increased in AIDS patients; there was no correlation with CD32 or CD64 or with soluble factors in plasma/serum (i.e., IgE, IL-4, IL-10, and IFN-γ). Interestingly, despite the known ability of IL-10 to downregulate monocyte CD23 expression, plasma IL-10 levels were increased in these AIDS patients compared with controls (p < 0.05). We thus evaluated the effect of AIDS and control plasma or rhIL-...