Enhancement of diagnostic efficiency by a gamma interferon release assay for pulmonary tuberculosis

This study was designed to examine the use of the QuantiFERON-TB Gold assay as an aid in the diagnosis of active pulmonary tuberculosis (TB) in Brazilian patients. Using the receiver operating characteristic curve, the cutoff was adjusted to >= 0.20 IU/ml. The sensitivity increased to 86%, with 100% specificity. All TB patients with negative sputum smear microscopy and negative culture results were positive using this test.

Monitoring of vaccine-specific gamma interferon induction in genital mucosa of mice by real-time reverse transcription-PCR.

Monitoring of T-cell responses in genital mucosa has remained a major challenge because of the absence of lymphoid aggregates and the low abundance of T cells. Here we have adapted to genital tissue a sensitive real-time reverse transcription-PCR (TaqMan) method to measure induction of gamma interferon (IFN-gamma) mRNA transcription after 3 h of antigen-specific activation of CD8 T cells. For this purpose, we vaccinated C57BL/6 mice subcutaneously with human papillomavirus type 16 L1 virus-like particles and monitored the induction of CD8 T cells specific to the L1(165-173) H-2D(b)-restricted epitope. Comparison of the responses induced in peripheral blood mononuclear cells and lymph nodes (LN) by L1-specific IFN-gamma enzyme-linked immunospot assay and TaqMan determination of the relative increase in L1-specific IFN-gamma mRNA induction normalized to the content of CD8b mRNA showed a significant correlation, despite the difference in the readouts. Most of the cervicovaginal tissues could be analyzed by the TaqMan method if normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA was used and a significant L1-specific IFN-gamma induction was found in one-third of the immunized mice. This local response did not correlate with the immune responses measured in the periphery, with the exception of the sacral LN, an LN draining the genital mucosa, where a significant correlation was found. Our data show that the TaqMan method is sensitive enough to detect antigen-specific CD8 T-cell responses in the genital mucosa of individual mice, and this may contribute to elaborate effective vaccines against genital pathogens.

Immune response to mouse mammary tumor virus in mice lacking the alpha/beta interferon or the gamma interferon receptor.

Mouse mammary tumor virus (MMTV) is a retrovirus which induces a strong immune response and a dramatic increase in the number of infected cells through the expression of a superantigen (SAg). Many cytokines are likely to be involved in the interaction between MMTV and the immune system. In particular, alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) exert many antiviral and immunomodulatory activities and play a critical role in other viral infections. In this study, we have investigated the importance of interferons during MMTV infection by using mice with a disrupted IFN-alpha/beta or IFN-gamma receptor gene. We found that the SAg response to MMTV was not modified in IFN-alpha/betaR(0/0) and IFN-gammaR(0/0) mice. This was true both for the early expansion of B and T cells induced by the SAg and for the deletion of SAg-reactive cells at later stages of the infection. In addition, no increase in the amount of proviral DNA was detected in tissues of IFN-alpha/betaR(0/0) and IFN-gammaR(0/0) mice, suggesting that interferons are not essential antiviral defense mechanisms during MMTV infection. In contrast, IFN-gammaR(0/0) mice had increased amounts of IL-4 mRNA and an altered usage of immunoglobulin isotypes with a reduced frequency of IgG2a- and IgG3-producing cells. This was associated with lower titers of virus-specific antibodies in serum early after infection, although efficient titers were reached later.

Parameter estimation and use of gamma interferon assay for the diagnosis of bovine tuberculosis in Brazil

This study aimed to evaluate the interference of tuberculin test on the gamma-interferon (INFg) assay, to estimate the sensitivity and specificity of the INFg assay in Brazilian conditions, and to simulate multiple testing using the comparative tuberculin test and the INFg assay. Three hundred-fifty cattle from two TB-free and two TB-infected herds were submitted to the comparative tuberculin test and the INFg assay. The comparative tuberculin test was performed using avian and bovine PPD. The INFg assay was performed by the BovigamTM kit (CSL Veterinary, Australia), according to the manufacturer's specifications. Sensitivity and specificity of the INFg assay were assessed by a Bayesian latent class model. These diagnostic parameters were also estimate for multiple testing. The results of INFg assay on D0 and D3 after the comparative tuberculin test were compared by the McNemar's test and kappa statistics. Results of mean optical density from INFg assay on both days were similar. Sensitivity and specificity of the INFg assay showed results varying (95% confidence intervals) from 72 to 100% and 74 to 100% respectively. Sensitivity of parallel testing was over 97.5%, while specificity of serial testing was over 99.7%. The INFg assay proved to be a very useful diagnostic method.

Expression of interferon-gamma, interferon-alpha and related genes in individuals with Down syndrome and periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of interleukin-10 on the Paracoccidioides brasiliensis killing by gamma-interferon activated human neutrophils

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Study of bronchoalveolar lavage fluid in paracoccidioidomycosis: cytopathology and alveolar macrophage function in response to gamma interferon; comparison with blood monocytes

Patients with paracoccidioidomycosis (PCM) present marked involvement of the lungs during the course of the mycosis. The purpose of this work was to obtain bronchoalveolar lavage (BAL) fluid from these patients to study the cytopathology, TNF levels and the oxidative and fungicidal response of alveolar macrophages (AMs) to in vitro incubation with recombinant IFN-gamma. To compare the lung and blood compartments, these determinations were also made in plasma and blood monocytes (BMs) obtained from the same patients. The cytopathology of BAL fluid revealed a predominance of macrophages, but with the presence of neurrophil exudation, and rare lymphocytes and epithelioid and giant cells. Comparison of the oxidative status and fungicidal activity of AMs and circulating BMs demonstrated that both cell types are highly activated for these two functions when compared to control cells. However, TNF levels were higher in BAL fluid than in plasma. The possible mechanisms involved in the hyperresponsiveness of cells from PCM patients are discussed. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

IMMUNOLOGICAL AND CYTOLOGICAL INVESTIGATION OF GAMMA INTERFERON PRODUCTION IN HUMAN T-LYMPHOCYTES INDUCED BY PMA AND PHA (IMMUNOGOLD, CELL CYCLE ANALYSIS)

The present study examined cellular mechanisms involved in the production and secretion of human (gamma)IFN. The hypothesis of this investigation was that (gamma)IFN is an export glycoprotein whose synthesis in human T lymphocytes is dependent on membrane stimulation, polypeptide synthesis in the rough endoplasmic reticulum, packaging in the Golgi complex, and release from the cell by exocytosis.^ The model system for this examination utilized T lymphocytes from normal donors and patients with chronic lymphocytic leukemia (CLL) induced in vitro with the tumor promoter, phorbol 12-myristate 13-acetate (PMA) and the lectin, phytohemagglutinin (PHA) to produce (gamma)IFN. This study reconfirmed the ability of PMA and PHA to synergistically induce (gamma)IFN production in normal T lymphocytes, as measured by viral inhibition assays and radio-immunoassays for (gamma)IFN. The leukemic T cells were demonstrated to produce (gamma)IFN in response to treatment with PHA. PMA treatment also induced (gamma)IFN production in the leukemic T cells, which was much greater than that observed in similarly treated normal T cells. In these same cells, however, combined treatment of the agents was shown to be ineffective at inducing (gamma)IFN production beyond the levels stimulated by the individual agents. In addition, the present study reiterated the synergistic effect of PMA/PHA on the stimulation of growth kinetics in normal T cells. The cell cycle of the leukemic T cells was also responsive to treatment with the agents, particularly with PMA treatment. A number of morphological alterations were attributed to PMA treatment including the acquisition of an elongated configuration, nuclear folds, and large cytoplasmic vacuoles. Many of the effects were observed to be reversible with dilution of the agents, and reversion to this state occurred more rapidly in the leukemic T cells. Most importantly, utilization of a thin section immuno-colloidal gold labelling technique for electron microscopy provided, for the first time, direct evidence of the cellular mechanism of (gamma)IFN production and secretion. The results of this latter study support the idea that (gamma)IFN is produced in the rough endoplasmic reticulum, transferred to the Golgi complex for accumulation and packaging, and released from the T cells by exocytosis. ^

Successful treatment of post-influenza pseudomembranous necrotising bronchial aspergillosis with liposomal amphotericin, inhaled amphotericin B, gamma interferon and GM-CSF

A case of aspergillus tracheobronchitis following influenza A infection in an immunocompetent 35 year old woman is described that required prolonged mechanical ventilation for airways obstruction. Treatment included liposomal amphotericin, inhaled amphotericin, gamma interferon and GM-CSF. Liposomal amphotericin therapy was associated with reversible hepatosplenomegaly. Inhaled corticosteroids with continued antifungal therapy were used for the management of severe recurrent airway obstruction. After a prolonged course of treatment she survived with fixed airways obstruction unresponsive to corticosteroids.

Some biological properties of the human amniotic membrane interferon

Human amniotic interferon was investigated to define the species specificity of its antiviral action and compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cell in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.

Myosin V and iNOS expression is enhanced in J774 murine macrophages treated with IFN-gamma

Actin-based motor protein requirements and nitric oxide (NO) production are important features of macrophage activity during phagocytosis or microbicidal processes. Different classes of myosins contribute directly or indirectly to phagocytosis by providing mechanical force for phagosome closure or organelle movement. Recent data have shown the presence of myosins IC, II, V and IXb in phagosomes of bone marrow-derived murine macrophages. In our investigation we demonstrated the presence of different classes of myosins in J774 macrophages. We also analyzed the effect of gamma interferon (IFN-gamma), with or without calcium ionophore or cytochalasin B, on myosins as well as on inducible nitric oxide synthase (iNOS) expression and NO production. Myosins IC, II, Va, VI and IXb were identified in J774 macrophages. There was an increase of myosin V expression in IFN-gamma-treated cells. iNOS expression was increased by IFN-gamma treatment, while calcium ionophore and cytochalasin B had a negative influence on both myosin and iNOS expression, which was decreased. The increases in NO synthesis were reflected by increased iNOS expression. Macrophages activated by IFN-gamma released significant amounts of NO when compared to control groups. In contrast, NO production by calcium ionophore- and cytochalasin B-treated cells was similar to that of control cells. These results suggest that IFN-gamma is involved in macrophage activation by stimulating protein production to permit both phagocytosis and microbicidal activity.

Interferón gamma en el manejo de la enfermedad granulomatosa crónica en el paciente pediátrico

Antecedentes: La enfermedad granulomatosa crónica aumenta el riesgo de infecciones. El interferón gamma se ha venido utilizando como parte del tratamiento; sin embargo, su eficacia no ha sido explorada por una revisión sistemática. Objetivo: Determinar y analizar la eficacia del uso del interferón gamma en el manejo de pacientes en edad pediátrica con diagnóstico de enfermedad granulomatosa crónica. Metodología: Se realizó una revisión sistemática de la literatura. La búsqueda se hizo en cuatro bases de datos y un registro público de ensayos clínicos. Se realizó un enfoque cualitativo y cuantitativo. Para variables cualitativas se emplearon como indicadores el riesgo relativo y para las cuantitativas la diferencia del promedio. Resultados: Se incluyeron tres estudios, dos de ellos estudios no aleatorizados. Un solo ensayo clínico de buena calidad mostró evidencia sobre la utilidad del uso del interferón en la reducción de la infección severa. Conclusión: Es posible que el uso del interferón gamma se relacione con mejoría de varios desenlaces clínicos del paciente. Es necesario realizar más ensayos clínicos controlados de buena calidad que permitan explorar la eficacia del tratamiento en esta indicación. Palabras clave: Enfermedad granulomatosa crónica, interferón gamma, revisión sistemática como tópico.

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: Activation of CD8+ cells, interferon-γ recovery and reduction of lung injury

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-γ and to restrict the growth of bacilli.

Die Rolle der IFN-gamma Signalübertragung und von MyD88 in der Angiotensin-II-induzierten vaskulären Dysfunkion, Inflammation und arteriellen Hypertonie

Im Rahmen dieser Arbeit wurde die Rolle von myelomonozytären Zellen, IFN-gamma (Interferon gamma), MyD88 (myeloid differentiation factor 88) und zugrundeliegenden Signalwege in der Angiotensin II (ATII)-induzierten vaskulären Inflammation, Dysfunktion und arteriellen Hypertonie untersucht. Wie bereits veröffentlichte Vordaten aus meiner Arbeitsgruppe zeigten, schützt die Depletion von Lysozym M (LysM)+ myelomonozytären Zellen (Diphteriatoxin-vermittelt in Mäusen, die transgen für den humanen Diphtheriatoxin-Rezeptor sind, LysMiDTR Mäuse) vor der ATII-induzierten vaskulären Dysfunktion und arterieller Hypertonie, und kann durch adoptiven Zelltransfer von Wildtyp Monozyten wiederhergestellt werden. In meiner Arbeit konnte ich zeigen, dass die Rekonstitution von Monozyten-depletierten LysMiDTR Mäusen mit Wildtyp Monozyten den Phänotyp der vaskulären Dysfunktion wiederherstellen kann, die Rekonstitution mit gp91phox-/y oder Agtr1-/- Monozyten jedoch nicht. Die Hypertonus-mediierenden Effekte dieser infiltrierenden Monozyten scheinen demnach von der intakten ATII und NADPH Oxidase Signalübertragung in diesen Zellen abhängig zu sein. Vermutlich ebenfalls für die Aktivierung der Monozyten funktionell wichtig sind IFN-gamma, produziert durch NK-Zellen, und der Transkriptionsfaktor T-bet (T-box expressed in T cells), exprimiert von NK-Zellen und Monozyten. IFN-gamma-/- Mäuse waren partiell geschützt vor der ATII-induzierten vaskulären Dysfunktion und charakterisiert durch reduzierte Level an Superoxid im Gefäß im Vergleich zu ATII-infundierten Wildtyp Mäusen. IFN-gamma-/- und T-bet defiziente Tbx21-/- Mäuse zeichneten sich ferner durch eine reduzierte ATII-mediierte Rekrutierung von NK1.1+ NK-Zellen, als ein Hautproduzent von IFN-gamma, sowie CD11b+GR-1low Interleukin-12 (IL-12) kompetenten Monozyten aus. Durch Depletions- und adoptive Transferexperimente konnte ich in dieser Arbeit NK-Zellen als essentielle Mitstreiter in der vaskulären Dysfunktion identifizieren und stellte fest, dass T-bet+LysM+ myelomonozytäre Zellen für die NK-Zellrekrutierung in die Gefäßwand und lokale IFN-gamma Produktion benötigt werden. Damit wurde erstmals NK-Zellen eine essentielle Rolle in der ATII-induzierten vaskulären Dysfunktion zugeschrieben. Außerdem wurde der T-bet-IFN-gamma Signalweg und die gegenseitige Monozyten-NK-Zellaktivierung als ein potentielles therapeutisches Ziel in kardiovaskulären Erkrankungen aufgedeckt. Des Weiteren identifizierte ich in meiner Arbeit MyD88 als ein zentrales Signalmolekül in der ATII-getriebenen Inflammation und vaskulären Gefäßschädigung. MyD88 Defizienz reduzierte den ATII-induzierten Anstieg des systolischen Blutdrucks und die endotheliale und glattmuskuläre vaskuläre Dysfunktion. Zusätzlich waren die vaskuläre Superoxid-Bildung sowie die Expressionslevel der NADPH Oxidase, der wichtigsten Quelle für oxidativem Stress im Gefäß, in ATII-infundierten MyD88-/- Mäusen im Vergleich zum Wildtyp reduziert. Mit Hilfe von durchflusszytometrischen Analysen deckte ich zudem auf, dass die ATII-induzierte Einwanderung von CD45+ Leukozyten, insbesondere CD11b+Ly6G-Ly6Chigh inflammatorischen Monozyten in MyD88-/- Mäusen signifikant abgeschwächt war. Diese Resultate wurden durch immunhistochemische Untersuchung von Aortengewebe auf CD68+, F4/80+ und Nox2+ Makrophagen/Phagozyten sowie Expressionsanalysen von Inflammationsmarkern untermauert. Analysen der mRNA Expression in Aortengewebe zeigten ferner eine in Wildtyp Mäusen nach ATII Infusion tendenziell gesteigerte Expression von inflammatorischen Monozytenmakern sowie eine abnehmende Expression von reparativen Monozytenmarken, während dieser Shift zu einem proinflammatorsichen Phänotyp in MyD88-/- blockiert zu sein schien. Dies zeigt eine Rolle von MyD88 in der terminalen Differenzierung von myelomonozytären Zellen an. Um dies weitergehend zu untersuchen und aufzudecken, ob die MyD88 Effekte abhängig sind von Zellen der hämatopoetischen Linie oder Gewebszellen, wurden Knochenmarktransferexperimente durchgeführt. MyD88 Defizienz in Knochenmark-abstammende Zellen reduzierte die ATII-induzierte vaskuläre Dysfunktion und Infiltration der Gefäßwand mit CD45+ Leukozyten und inflammatorischen myelomonozytären Zellen. Die protektiven Effekte der MyD88 Defizienz in der Angiotensin II-induzierten Inflammation konnten nicht auf Signalwege über die Toll-like Rezeptoren TLR2, -7 oder -9 zurückgeführt werden, wie die Untersuchung der vaskulären Reaktivität entsprechender Knockout Mäuse zeigte. Zusammenfassend konnte ich in meiner Arbeit zeigen, dass die Infiltration der Gefäßwand mit Nox2+AT1R+T-bet+MyD88+ myelomonozytären Zellen und die Wechselwirkung und gegenseitige Aktivierung dieser Zellen mit IFN-gamma produzierenden NK-Zellen eine zentrale Bedeutung in der Pathogenese der Angiotensin II (ATII)-induzierten vaskulären Dysfunktion, Inflammation und arteriellen Hypertonie einnehmen.