134 research outputs found
T Cell Proliferation and Apoptosis in HIV-1-Infected Lymphoid Tissue: Impact of Highly Active Antiretroviral Therapy
No full textT cell turnover was studied in situ in tonsillar lymphoid tissue (LT) from HIV-1-infected individuals during 48 weeks of highly active antiretroviral therapy (HAART) and compared to that of HIV-1-negative controls. Prior to therapy, CD4 cell proliferation (%CD4+ Ki67+) and apoptosis (%CD4+ TUNEL+) were increased in HIV-1-infected LT and both parameters correlated with tonsillar viral load. CD8 cell proliferation (%CD8+ Ki67+) was increased 4- to 10-fold, mainly in the germinal centers. Apoptotic CD8+ T cell levels (%CD8+ TUNEL+) were raised preferentially in the tonsillar T cell zone. The frequency of CD8+ Ki67+ and CD8+ TUNEL+ T cells correlated with tonsillar viral load and with the fraction of CD8+ T cells expressing activation markers. During HAART, CD4 cell turnover normalized while CD8 cell turnover was dramatically reduced. However, low level viral replication concomitant with slightly elevated levels of CD8 cell turnover indicated a persistent cellular immune response in LT. In conclusion, enhanced T cell turnover may reflect effector cells related to HIV-1 infection.Anne Ma Dyrhol-Riise, Maria Ohlsson, Kathrine Skarstein, Svein J. T. Nygaard, Jan Olofsson, Roland Jonsson and Birgitt Åsjöhttp://www.elsevier.com/wps/find/journaldescription.cws_home/622806/description#descriptio
Cytokine Patterns in Tuberculosis Infection; IL-1ra, IL-2 and IP-10 Differentiate Borderline QuantiFERON-TB Samples from Uninfected Controls
No full textBackground: Interferon gamma release assays (IGRAs) do not discriminate between active tuberculosis (TB) and latent TB infection (LTBI), which limit their use in TB endemic areas. Subjects with QuantiFERON-TB (QFT) results around the diagnostic cut-off more likely show inconsistent results on serial testing which makes the interpretation of the assay difficult. We have studied potential biomarkers in patients with various stages of TB infection and with borderline QFT tests compared to those with higher values. Methods: 27 soluble biomarkers were analysed in QFT supernatants from patients with active TB (n = 18), individuals with LTBI (n = 48) and from QFT negative controls (n = 16) by the Multiplex bead assay. The LTBI group was classified into two groups according to QFT IFN-γ levels; QFT borderline (0.35–0.70 IU/mL, n = 11) or QFT high (>0.70 IU/mL, n = 36). Results: The levels of IL-1ra, IL-2, IL-13, IL-15, IFN-γ, IP-10 and MCP-1 in background corrected TB antigen stimulated supernatants (TBAg-Nil) significantly distinguished both active TB and LTBI QFT high groups from the QFT negative controls (p≤0.004). In addition, IL-1ra, IL-2 and IP-10 significantly differentiated the QFT borderline group from the controls (p≤0.001). Still, in the QFT borderline group the IL-1ra and IP-10 levels were not significant different from neither the QFT high nor the active TB group, whereas the IL-2 levels were lower (p≤0.003). The level of IP-10 showed the best separation between the QFT borderline group and the QFT negative controls (AUC 0.92) and offered 100% sensitivity for active TB. Conclusion: IL-1ra, IL-2 and IP-10 differentiate QFT borderline samples from uninfected controls and the majority of QFT borderline subjects were classified as LTBI by these markers. Still, inconsistency was seen, and further studies are needed to examine the performance of alternative markers before concluded if they could be used as diagnostics tools
Screening for latent tuberculosis in Norwegian health care workers: high frequency of discordant tuberculin skin test positive and interferon-gamma release assay negative results
No full textBackground: Tuberculosis (TB) presents globally a significant health problem and health care workers (HCW) are at increased risk of contracting TB infection. There is no diagnostic gold standard for latent TB infection (LTBI), but both blood based interferon-gamma release assays (IGRA) and the tuberculin skin test (TST) are used. According to the national guidelines, HCW who have been exposed for TB should be screened and offered preventive anti-TB chemotherapy, but the role of IGRA in HCW screening is still unclear. Methods: A total of 387 HCW working in clinical and laboratory departments in three major hospitals in the Western region of Norway with possible exposure to TB were included in a cross-sectional study. The HCW were asked for risk factors for TB and tested with TST and the QuantiFERON®TB Gold In-Tube test (QFT). A logistic regression model analyzed the associations between risk factors for TB and positive QFT or TST. Results: A total of 13 (3.4%) demonstrated a persistent positive QFT, whereas 214 (55.3%) had a positive TST (≥ 6 mm) and 53 (13.7%) a TST ≥15 mm. Only ten (4.7%) of the HCW with a positive TST were QFT positive. Origin from a TB-endemic country was the only risk factor associated with a positive QFT (OR 14.13, 95% CI 1.37 - 145.38, p = 0.026), whereas there was no significant association between risk factors for TB and TST ≥ 15 mm. The five HCW with an initial positive QFT that retested negative all had low interferon-gamma (IFN-γ) responses below 0.70 IU/ml when first tested. Conclusions: We demonstrate a low prevalence of LTBI in HCW working in hospitals with TB patients in our region. The “IGRA-only” seems like a desirable screening strategy despite its limitations in serial testing, due to the high numbers of discordant TST positive/IGRA negative results in HCW, probably caused by BCG vaccination or boosting due to repetitive TST testing. Thus, guidelines for TB screening in HCW should be updated in order to secure accurate diagnosis of LTBI and offer proper treatment and follow-up
Internettbasert læringsressurs i infeksjonsmedisin og mikrobiologi
No full textVårt prosjekt er et internettbasert læringsprogram i infeksjonsmedisino og mikrobiologi for medisinstudenter og andre som er interessert i disse fagområdene. Det skal fungere som et supplement til forelesninger, smågruppeundervisning og lærebøker i infeksjonsmedisin og mikrobiologi. Undervisningen i infeksjonsmedisin og mikrobiologi er spredt over ulike semestre, og vi ønsket derfor å opprette en ressurs der studentene kan få en oversikt over disse fagfeltene. Læringsmålene i disse to fagfeltene har vært vårt utgangspunkt for valget av de ulike emnene i E-læringssiden. Vi har brukt ulike kilder og markert dem med fotnoter fortløpende. Målet vårt er at studentene skal gis den mest kunnskapsbaserte informasjonen, men medisin er i kontinuerlig utvikling og innholdet i E-læringssiden vil dermed kunne utløpe. Innholdet kan enkelt redigeres og forandres, og revisjon vil være nødvendig i takt med endring i kunnskapsbasert medisin. Avslutningsvis har vi en samling av pasientkasuser for å gjøre læringsprogrammet interaktivt og for å understreke de viktigste temaene. Veilederne våre på denne oppgaven har vært Anne Ma Dyrhol Riise MD, førstemanuensis ved Institutt for klinisk medisin, UIO og Halvor Rollag, Professor ved mikrobiologisk avdeling, UIO. Vi ønsker å takke dem for god veiledning
IP-10 measured by Dry Plasma Spots as biomarker for therapy responses in Mycobacterium Tuberculosis infection
No full textTuberculosis (TB) has huge impact on human morbidity and mortality and biomarkers to support rapid TB diagnosis and ensure treatment initiation and cure are needed, especially in regions with high prevalence of multi-drug resistant TB. Soluble interferon gamma inducible protein 10 (IP-10) analyzed from dry plasma spots (DPS) has potential as an immunodiagnostic marker in TB infection. We analyzed IP-10 levels in plasma directly and extracted from DPS in parallel by ELISA from 34 clinically well characterized patients with TB disease before and throughout 24 weeks of effective anti-TB chemotherapy. We detected a significant decline of IP-10 levels in both plasma and DPS already after two weeks of therapy with good correlation between the tests. This was observed both in pulmonary and extrapulmonary TB. In conclusion, plasma IP-10 may serve as an early biomarker for anti-TB chemotherapy responses and the IP-10 DPS method has potential to be developed into a point-of care test for use in resource-limited settings. Further studies must be performed to validate the use of IP-10 DPS in TB high endemic countries
T Cell Responses and Regulation and the Impact of In Vitro IL-10 and TGF-beta Modulation During Treatment of Active Tuberculosis
No full textMycobacterium tuberculosis (Mtb) is particularly challenging for the immune system being an intracellular pathogen, and a variety of T cell subpopulations are activated by the host defence mechanism. In this study, we investigated T cell responses and regulation in active TB patients with drug-sensitive Mtb (N = 18) during 24 weeks of efficient anti-TB therapy. T cell activation, differentiation, regulatory T cell (Treg) subsets, Mtb-induced T cell proliferation and in vitro IL10 and TGF-beta modulation were analysed by flow cytometry at baseline and after 8 and 24 weeks of therapy, while soluble cytokines in culture supernatants were analysed by a 9-plex Luminex assay. Successful treatment resulted in significantly reduced co-expression of HLA-DR/CD38 and PD-1/CD38 on both CD4(+) and CD8(+) T cells, while the fraction of CD4+ CD25 high CD127 low Tregs (P = 0.017) and CD4(+) CD25(high) CD127(low) CD147(+) Tregs (P = 0.029) showed significant transient increase at week 8. In vitro blockade of IL-10/TGF-beta upon Mtb antigen stimulation significantly lowered the fraction of ESAT-6-specific CD4(+) CD25(high) CD127(low) Tregs at baseline (P = 0.047), while T cell proliferation and cytokine production were unaffected. Phenotypical and Mtb-specific T cell signatures may serve as markers of effective therapy, while the IL-10/ TGF-beta pathway could be a target for early inhibition to facilitate Mtb clearance. However, larger clinical studies are needed for verification before concluding
Regulation of Gag- and Env-Specific CD8+ T Cell Responses in ART-Naïve HIV-Infected Patients: Potential Implications for Individualized Immunotherapy.
No full textStrategies to develop a functional cure for HIV infection will likely require boosting of effector T cell responses to eliminate reactivated, latently infected cells. We have recently explored an assay for assessing antigen-specific regulation of T cell proliferation, which was related to clinical progression in untreated patients and to vaccine efficacy in two trials of therapeutic Gag-based vaccines. We here expand the same assay to further investigate regulation mediated by various inhibitory pathways. Peripheral blood mononuclear cells from 26 asymptomatic HIV-infected, antiretroviral therapy-naïve patients were stimulated with Gag and Env overlapping peptide panels for 5 days. Monoclonal antibodies (mAbs) blocking inhibitory mediators interleukin (IL) 10, transforming growth factor (TGF) β, programmed death ligand (PD-L) 1 and herpes virus entry mediator (HVEM) were added to parallel cultures. Functional T cell regulation (FTR) was defined as the difference in proliferation between stimulated cultures with and without blocking mAbs. FTR was detected in 54% of patients. Blockade of IL-10/PD-L1 and IL10/TGF-β detected all cases with Gag- and Env-associated FTR, respectively. In accordance with previous findings, isolated Env FTR was associated with higher plasma HIV RNA and lower CD4 counts, while patients with both Gag and Env FTR also had higher Gag- and Env-specific proliferative CD8+ T cell responses. There was no association between FTR and frequencies of activated regulatory T cells. In conclusion, we observed substantial heterogeneity in FTR between patients, inhibitory pathways and HIV antigens. FTR may help to individualize immunomodulation and warrants further assessment in clinical immunotherapy trials
HIV patients with latent tuberculosis living in a low-endemic country do not develop active disease during a 2 year follow-up; a Norwegian prospective multicenter study
No full textBackground
Interferon-γ release assays (IGRA) serve as immunodiagnostics of tuberculosis (TB) infection to identify individuals with latent TB infection (LTBI) eligible for preventive anti-TB therapy. In this longitudinal study of HIV-infected LTBI patients we have observed for possible progression to active TB as well as evaluated repeated IGRA testing in a TB low-endemic setting.
Methods
QuantiFERON TB-Gold In-tube® assay (QFT), TB-SPOT.TB® (TSPOT) and tuberculin skin test (TST) were performed on 298 HIV-patients recruited from seven out-patient clinics in Norway. Patients with active TB, LTBI and negative IGRA were followed with repeat QFTs and clinical evaluation over a period of 24 months.
Results
Seven HIV-patients (median CD4 count 270; IQR 50–340) were diagnosed with active TB at inclusion, all IGRA positive. Sixty-four (21%) HIV-patients (median CD4 count 471; IQR 342–638) were diagnosed with LTBI and of these 39 (61%) received TB preventive treatment. Neither treated nor untreated HIV-infected LTBI patients developed active TB during the 24 months. At baseline, the median interferon-γ (INF-γ) level measured by QFT was 3.48 IU/ml (IQR 0.94 – 8.91 IU/ml) for treated LTBI compared to 1.13 IU/ml (IQR 0.47 – 4.25 IU/ml) for untreated LTBI patients (p = 0.029). The QFT reversion rates were 75% for active TB, 23% for treated LTBI and 44% for untreated LTBI, whereas the conversion rate for the non-TB group was 7% despite no new TB exposure. There was no significant difference in the trend of INF-γ levels over time between treated and untreated LTBI patients.
Conclusion
The prevalence of LTBI is high among HIV-patients, but the risk of developing active TB seems to be low in patients with high CD4 counts in this TB low-endemic setting. In several patients, especially with baseline IFN-γ levels close to cut-offs, the QFT tests reverted to negative independent of preventive anti-TB treatment indicating possibly false positive tests. This highlights the importance of defining reliable cut-offs for immunodiagnostic tests and deferring preventive therapy in selected patients. Randomized studies with longer follow-up time are needed to identify HIV-patients that would benefit from LTBI treatment in a TB low-endemic setting
Soluble Markers of the Toll-Like Receptor 4 Pathway Differentiate between Active and Latent Tuberculosis and Are Associated with Treatment Responses
No full textBackground. Biomarkers to differentiate between active tuberculosis (TB) and latent TB infection (LTBI) and to monitor treatment responses are requested to complement TB diagnostics and control, particularly in patients with multi-drug resistant TB. We have studied soluble markers of the Toll-like-receptor 4 (TLR-4) pathway in various stages of TB disease and during anti-TB treatment. Methods. Plasma samples from patients with culture confirmed drug-sensitive TB (n = 19) were collected before and after 2, 8 and 24 weeks of efficient anti-TB treatment and in a LTBI group (n = 6). Soluble (s) CD14 and myeloid differentiation-2 (MD-2) were analyzed by the Enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide (LPS) was analyzed by the Limulus Amebocyte Lysate colorimetric assay. Nonparametric statistics were applied. Results. Plasma levels of sCD14 (p<0.001), MD-2 (p = 0.036) and LPS (p = 0.069) were elevated at baseline in patients with untreated active TB compared to the LTBI group. MD-2 concentrations decreased after 2 weeks of treatment (p = 0.011), while LPS levels decreased after 8 weeks (p = 0.005). In contrast, sCD14 levels increased after 2 weeks (p = 0.047) with a subsequent modest decrease throughout the treatment period. There was no significant difference in concentrations of any of these markers between patients with pulmonary and extrapulmonary TB or between patients with or without symptoms. Conclusion. Our data suggest that plasma levels of LPS, MD-2 and sCD14 can discriminate between active TB and LTBI. A decline in LPS and MD-2 concentrations was associated with response to anti-TB treatment. The clinical potential of these soluble TLR-4 pathway proteins needs to be further explored
In vitro analysis of antigen induced T cell-monocyte conjugates by imaging flow cytometry
No full textThere is a lack of suitable correlates of immune protection against Mycobacterium tuberculosis (Mtb) infection. T cells and monocytes play key roles in host immunity against Mtb. Thus, a method that allows assessing their interaction would contribute to the understanding of immune regulation in tuberculosis (TB). We have established imaging flow cytometer (IFC) based in vitro assay for the analysis of early events in T cell-monocyte interaction, upstream of cytokine production and T cell proliferation. This was achieved through short term stimulation of peripheral blood mononuclear cells (PBMC) from healthy Norwegian blood donors with Mycobacterium bovis Bacille Calmette-Guérin (BCG). In our assay, we examined the kinetics of BCG uptake by monocytes using fluorescently labeled BCG and T cell-monocyte interaction based on synapse formation (CD3/ TCR polarization). Our results showed that BCG stimulation induced a gradual increase in the proportion of conjugated T cells displaying NF-κB translocation to the nucleus in a time dependent manner, with the highest frequency observed at 6 h. We subsequently tested PBMC from a small cohort of active TB patients (n = 7) and observed a similar BCG induced NF-κB translocation in T cells conjugated with monocytes. The method allowed for simultaneous evaluation of T cell-monocyte conjugates and T cell activation as measured by NF-κB translocation, following short-term challenge of human PBMC with BCG. Whether this novel approach could serve as a diagnostic or prognostic marker needs to be investigated using a wide array of Mtb specific antigens in a larger cohort of patients with different TB infection status
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