The "ABC" of Virus-Specific T Cell Immunity in Solid Organ Transplantation.

Transplant patients are at increased risk of viral complications due to impaired control of viral replication, resulting from HLA mismatching between graft and host and the immunosuppression needed to avert alloimmune reactions. In the past decade, quantitative viral load measurements have become widely available to identify patients at risk and to inform treatment decisions with respect to immunosuppressive drugs and antiviral therapies. Because viral loads are viewed as the result of viral replication and virus-specific immune control, virus-specific T cell monitoring has been explored to optimize management of adenovirus, BK polyomavirus and cytomegalovirus ("ABC") in transplant patients. Although most studies are descriptive using different technologies, the overall results show that the quantity and quality of virus-specific T cells inversely correlate with viral replication, whereby strong cellular immune responses are associated with containment of viral replication. The key obstacles to the introduction of assays for virus-specific T cells into clinical practice is the definition of reliable cutoffs for clinical decision making, the poor negative predictive value of some assays, and the absence of interventional trials justifying changes of antiviral treatment or immunosuppression. More clinical research is needed using optimized assays and targets before standardization and commutability can be envisaged as achieved for viral load testing.

Alloreactive T Cells to Identify Risk HLA Alleles for Retransplantation After Acute Accelerated Steroid-Resistant Rejection.

The risk of rejection by cellular alloreactivity to the transplant donor is not routinely assessed. Here we analyzed alloreactive T cells in kidney transplant recipients and report how their detection may have helped to prevent rejection of a second kidney graft in a patient with a history of acute accelerated steroid-resistant nonhumoral rejection. Alloreactive CD4 and CD8 T cells were quantified using a flow-cytometric mixed lymphocyte reaction assay based on interferon-γ induction. A group of 16 nonrejecting transplant recipients did not show any alloreactive T-cell immunity to their respective donors, whereas alloreactivity to third-party controls was detectable. In the patient with rejection, HLA-specific antibodies were not detectable before and shortly after rejection, but after transplantation the patient showed exceptionally high frequencies of alloreactive T cells against 2 of 11 HLA-typed controls (0.604% and 0.791% alloreactive CD4 T cells and 0.792% and 0.978% alloreactive CD8 T cells) who shared HLA alleles (HLA-A*24, -B*44, -C*02, -DQB1*5) with the kidney donor. These HLA alleles were subsequently excluded for allocation of a second graft. No alloreactive T cells were observed toward the second kidney donor, and this transplantation was performed successfully. Thus, shared HLA alleles between the donor and third-party controls may suggest that alloreactive T cells had contributed to rejection of the first graft. The rejecting patient highlights that determination of cellular alloreactivity before transplantation may be applied to identify unacceptable mismatches and to reduce the risk for acute cellular rejection episodes.

Superior Sensitivity of Ex Vivo IFN-γ Release Assays as Compared to Skin Testing in Immunocompromised Patients.

Comparative assessment of the tuberculin skin testing (TST) and commercial IFN-γ release-assays (IGRAs) is hampered by the use of different antigens (tuberculin PPD in TST vs. ESAT-6/CFP-10 in IGRAs). Thus, PPD was used as a common stimulus to compare performance of the TST and three IGRAs in 72 controls, 101 hemodialysis patients and 100 renal transplant recipients. Results of the TST were compared with PPD-induced IFN-γ induction in vitro detected by ELISPOT, ELISA or a flow-cytometric FACS assay. Percentages of positive tests were significantly lower in TST (9.2%) compared to ELISA (55.3%), ELISPOT (45.3%) and FACS (44.9%, p < 0.0001). Agreement between TST and IGRAs was highest for controls (κ = 0.19-0.32) and poor in immunocompromised patients (κ = 0 for transplant patients, κ = 0.06-0.13 for hemodialysis patients). Discrepant results were largely TST negative and IGRA positive. Among IGRAs, agreement was highest between ELISPOT and FACS (κ = 0.61). Unlike TST, all IGRAs were associated with variables of mycobacterial exposure. Among IGRAs, the FACS assay was least affected by the level of immunosuppression. In conclusion, both the percentage of positive results and between-test-agreement were higher with IGRAs as compared to TST. This indicates superiority of IGRAs in detecting a PPD-specific immune response which may also apply for immunity toward Mycobacterium tuberculosis-specific antigens.

Comparative analysis of assays for detection of cell-mediated immunity toward cytomegalovirus and M. tuberculosis in samples from deceased organ donors.

Cell-mediated immunity assays could be valuable for risk assessment of organ donors, but no data exist on their feasibility in deceased donors. In this study, 105 deceased donors (52.3 ± 16.9 years) were screened at the time of organ procurement. Pathogen-specific stimulation was performed using a cytomegalovirus (CMV) lysate, tuberculin (purified protein derivative [PPD]) and soluble Mycobacterium tuberculosis-specific ESAT-6/CFP-10 proteins in combination with an in-house fluorescence-activated cell sorting (FACS) assay or commercial assay formats (QuantiFERON-CMV/TB for ELISA, T-SPOT.TB for ELISPOT). CMV-IgG antibody titers were determined as gold standard for CMV infection; 51.4% of samples were CMV seropositive. Indeterminate results were observed in 47.6% of ELISA, 12.5% of FACS and 0% of ELISPOT assays. Agreement with serology was highest for FACS (95.6%, κ = 0.91), followed by ELISPOT (84.0%, κ = 0.68) and ELISA (80.0%, κ = 0.60). Agreement between ELISA and serology increased if the CMV lysate was used as stimulus (96.7%, κ = 0.92). Among the T cell assays, agreement between ELISPOT and FACS was highest (κ = 0.70). PPD-positive results among valid samples differed between assays (26.5% for ELISA, 23.1% for FACS and 50.5% for ELISPOT); 2.0% were QuantiFERON-TB positive, 3.3% were ESAT-6/CFP-10-positive in FACS and 13.4% were positive in the T-SPOT.TB assay. In conclusion, cellular immunity may be analyzed from samples of deceased donors, although the assays differ in the rate of positivity and indeterminate results.

BK polyomavirus-specific cellular immune responses are age-dependent and strongly correlate with phases of virus replication.

BK polyomavirus (BKPyV) infection is widespread and typically asymptomatic during childhood, but may cause nephropathy in kidney transplant recipients. However, there is only limited knowledge on BKPyV-specific immunity in children and adults, and its role in BKPyV-replication and disease posttransplant. We therefore characterized BKPyV-specific immunity from 122 immunocompetent individuals (1-84 years), 38 adult kidney recipients with (n = 14) and without BKPyV-associated complications (n = 24), and 25 hemodialysis (HD) patients. Blood samples were stimulated with overlapping peptides of BKPyV large-T antigen and VP1 followed by flow-cytometric analysis of activated CD4 T cells expressing interferon-γ, IL-2 and tumor necrosis factor-α. Antibody-levels were determined using enzyme-linked immunosorbent assay. Both BKPyV-IgG levels and BKPyV-specific CD4 T cell frequencies were age-dependent (p = 0.0059) with maximum levels between 20 and 30 years (0.042%, interquartile range 0.05%). Transplant recipients showed a significantly higher BKPyV-specific T cell prevalence (57.9%) compared to age-matched controls (21.7%) or HD patients (28%, p = 0.017). Clinically relevant BKPyV-replication was associated with elevated frequencies of BKPyV-specific T cells (p = 0.0002), but decreased percentage of cells expressing multiple cytokines (p = 0.009). In conclusion, BKPyV-specific cellular immunity reflects phases of active BKPyV-replication either after primary infection in childhood or during reactivation after transplantation. Combined analysis of BKPyV-specific T cell functionality and viral loads may improve individual risk assessment.

Confirmation of Bacterial Leaf Streak Caused by Xanthomonas oryzae pv. oryzicola on Rice in Madagascar.

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola is an important disease of rice. BLS is prevalent in Asia and West Africa, where it was first reported in Nigeria and Senegal in the early 1980s (4). Recently, molecular analysis of strains from Mali (2) and Burkina Faso (5) further confirmed the presence of BLS in West Africa. In Madagascar, BLS symptoms were first reported in the 1980s by Buddenhagen but the causal agent was not unequivocally determined (1). To confirm Buddenhagen's observations using modern molecular typing tools, we surveyed several rice fields in the Antananarivo and Antsirabe districts in March 2013. BLS symptoms were observed on cultivated Oryza sativa grown under both upland and lowland conditions, with a proportion of diseased individuals varying from 30% up to 80%. Symptomatic leaves presenting water-soaked lesions that developed into translucent, yellow streaks with visible exudates at the surface were sampled. One to four centimeter long pieces of diseased leaves were ground using the Qiagen TissueLyser system at 30 rps for 30 s (Qiagen, Courtaboeuf, France). The ground tissue was then macerated in 1 ml of sterile water for 1 h at 4°C. Non-diluted and 10-fold diluted tissue macerates were plated on semi-selective PSA medium (peptone 10 g/liter, sucrose 10 g/liter, glutamic acid 1 g/liter, bacto agar 16 g/liter, actidione 50 mg/liter, cephalexin 40 mg/liter, and kasugamycin 20 mg/liter) and incubated for 3 to 7 days at 28°C. Single, yellow, Xanthomonas-like colonies were isolated on non-selective PSA medium. Diagnostic multiplex PCR was performed on single colonies for pathovar identification (3). Five strains that produced three diagnostic bands corresponding to the X. oryzae pv. oryzicola pattern were further analyzed for pathogenicity on 3-week-old O. sativa cv. Nipponbare plants. Bacteria grown on PSA plates and adjusted to 1 × 108 CFU/ml were infiltrated into rice leaves with a needleless 1-ml syringe (2 × 3 infiltrations per plant and strain). Seven days after incubation in the greenhouse (27 ± 1°C with a 12-h photoperiod), inoculated leaves showed water-soaked lesions that produced yellow exudates corresponding to those initially observed in rice fields and observed for leaves challenged with the X. oryzae pv. oryzicola reference strain BLS256. Symptomatic leaf tissues were ground and plated on non-selective PSA medium, resulting in colonies with typical Xanthomonas morphology that were confirmed as X. oryzae pv. oryzicola by multiplex PCR typing (3), thus fulfilling Koch's postulates. Finally, the five strains were subjected to gyrB sequencing upon PCR amplification using the universal primers XgyrB1F (5'-ACGAGTACAACCCGGACAA-3') and XgyrB1R (5'-CCCATCARGGTGCTGAAGAT-3'). The 743-bp partial gyrB sequences were 100% identical to the gyrB sequence of strain BLS256. As expected, the gyrB sequence of strains KACC10331, MAFF311018, and PXO99A of the X. oryzae pv. oryzae pathovar respectively showed nine, 16, and 10 mismatches in comparison to the Malagasy strains, thus further supporting that they belong to the pathovar oryzicola. References: (1) I. W. Buddenhagen. Int. Rice Comm. Newsl. 34:74, 1985. (2) C. Gonzalez et al. Mol. Plant Microbe Interact. 20:534, 2007. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) D. O. Niño-Liu et al. Mol. Plant Pathol. 7:303, 2006. (5) I. Wonni et al. Plant Dis. 95:72, 2011.

PD-1 analysis on CD28(-) CD27(-) CD4 T cells allows stimulation-independent assessment of CMV viremic episodes in transplant recipients.

Expression of the inhibitory receptor programmed death 1 (PD-1) on cytomegalovirus (CMV)-specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28(-) CD27(-) double negativity is known as a typical phenotype of CMV-specific CD4 T cells. Therefore, the co-expression of inhibitory receptors on CD28(-) CD27(-) CD4 T cells was assessed as a rapid, stimulation-independent parameter for monitoring CMV complications after transplantation. Ninety-three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross-sectional and longitudinal manner. CMV-specific CD4 T cell levels quantified after stimulation were compared to levels of CD28(-) CD27(-) CD4 T cells. PD-1 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression on CD28(-) CD27(-) CD4 T cells were related to viremia. A percentage of ≥0.44% CD28(-) CD27(-) CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV-specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD-1 expression levels on CD28(-) CD27(-) CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA-4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD-1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD-1 expression on CD28(-) CD27(-) CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation-independent parameter to guide duration of antiviral therapy.

Blockade of programmed death receptor-1 signaling restores expression of mostly proinflammatory cytokines in anergic cytomegalovirus-specific T cells.

BACKGROUND: Programmed death receptor-1 (PD-1) compromises cytomegalovirus (CMV)-specific T-cell responses and has been linked to CMV viremia after transplantation. An impaired functional and proliferative capacity of PD-1-positive CMV-specific T cells may be reversed by the antibody-mediated blockade of PD-1 signaling. However, knowledge is limited on changes in "cytokinome" expression profiles associated with reversal of functional exhaustion. METHODS: The "cytokinome" was analyzed by 27-plex Luminex technology comparing renal transplant recipients with low (n = 5) and high (n = 5) PD-1 expression on CMV-specific T cells. The effect of blocking PD-1 by PD-ligand (PD-L) antibodies on restoration of cytokine expression was examined. RESULTS: CMV-specific cytokine release and proliferation was lower in patients with high PD-1 expression on CMV-specific T cells. Antibody-mediated blockade of PD-L in CMV-stimulated samples restored expression levels of interleukin (IL)-1ß, IL-2, IL-6, IL-9, IL-10, granulocyte colony-stimulating factor, interferon-γ, macrophage inflammatory protein-1α, and tumor necrosis factor-α. By contrast, no profound effect was observed for controls or patients with low PD-1 expression, or in staphylococcal enterotoxin B-stimulated cells. CONCLUSION: Taken together, this pilot study provides evidence that a high PD-1 expression on CMV-specific T cells actively impairs proliferation and "cytokinome" responses in an antigen-specific manner. Importantly, blockade of PD-L restores CMV-specific T-cell proliferation and expression of a panel of different proinflammatory and/or type 1 cytokines, suggesting a common but as yet unknown regulatory principle. We conclude that PD-1 exhaustion is reversible and potentially amenable to therapeutic ex vivo and possibly in vivo manipulation. However, detailed knowledge of the differential effects on the "cytokinome" will be necessary to increase the safety and the efficacy of such manipulations.

Diagnosis and management of tuberculosis in transplant donors: a donor-derived infections consensus conference report.

Mycobacterium tuberculosis is a ubiquitous organism that infects one-third of the world's population. In previous decades, access to organ transplantation was restricted to academic medical centers in more developed, low tuberculosis (TB) incidence countries. Globalization, changing immigration patterns, and the expansion of sophisticated medical procedures to medium and high TB incidence countries have made tuberculosis an increasingly important posttransplant infectious disease. Tuberculosis is now one of the most common bacterial causes of solid-organ transplant donor-derived infection reported in transplant recipients in the United States. Recognition of latent or undiagnosed active TB in the potential organ donor is critical to prevent emergence of disease in the recipient posttransplant. Donor-derived tuberculosis after transplantation is associated with significant morbidity and mortality, which can best be prevented through careful screening and targeted treatment. To address this growing challenge and provide recommendations, an expert international working group was assembled including specialists in transplant infectious diseases, transplant surgery, organ procurement and TB epidemiology, diagnostics and management. This working group reviewed the currently available data to formulate consensus recommendations for screening and management of TB in organ donors.

TBNET - Collaborative research on tuberculosis in Europe.

Networking is a key feature of scientific success. The Tuberculosis Network European Trialsgroup (TBNET) was founded in 2006 as a non-profit, non-governmental peer-initiated scientific organization to collaboratively address research priorities in the area of tuberculosis in Europe. Today, TBNET is the largest tuberculosis research organization in Europe with nearly 500 members from 22 EU countries and 49 countries worldwide (www.tb-net.org). Apart from small multicenter basic research studies, a particular strength of TBNET is the performance of large collaborative projects, pan-European multicenter studies and database projects. In recent years, research from TBNET has substantially contributed to the understanding of the management, risk and prognosis of patients with multidrug (MDR) and extensively drug-resistant (XDR) tuberculosis and led to a better understanding of the clinical value of novel tests for the identification of adults and children with tuberculosis and latent infection with Mycobacterium tuberculosis. In 2009, two branches of TBNET were founded to specifically address tuberculosis in the pediatric population (ptbnet) and non-tuberculous mycobacterial diseases (NTM-NET). In addition to the research activities, TBNET is developing expert consensus documents for clinical management and provides training and capacity building especially for members from Eastern European countries, where tuberculosis is still a prevalent health problem.

Interferon-γ release assays for the diagnosis of active tuberculosis: a systematic review and meta-analysis.

Interferon-γ release assays (IGRAs) are now established for the immunodiagnosis of latent infection with Mycobacterium tuberculosis in many countries. However, the role of IGRAs for the diagnosis of active tuberculosis (TB) remains unclear. Following preferred reporting items for systematic reviews and meta-analyses (PRISMA) and quality assessment of diagnostic accuracy studies (QUADAS) guidelines, we searched PubMed, EMBASE and Cochrane databases to identify studies published in January 2001-November 2009 that evaluated the evidence of using QuantiFERON-TB® Gold in-tube (QFT-G-IT) and T-SPOT.TB® directly on blood or extrasanguinous specimens for the diagnosis of active TB. The literature search yielded 844 studies and 27 met the inclusion criteria. In blood and extrasanguinous fluids, the pooled sensitivity for the diagnosis of active TB was 80% (95% CI 75-84%) and 48% (95% CI 39-58%) for QFT-G-IT, and 81% (95% CI 78-84%) and 88% (confirmed and unconfirmed cases) (95% CI 82-92%) for T-SPOT.TB®, respectively. In blood and extrasanguinous fluids, the pooled specificity was 79% (95% CI 75-82%) and 82% (95% CI 70-91%) for QFT-G-IT, and 59% (95% CI 56-62%) and 82% (95% CI 78-86%) for T-SPOT.TB®, respectively. Although the diagnostic sensitivities of both IGRAs were higher than that of tuberculin skin tests, it was still not high enough to use as a rule out test for TB. Positive evidence for the use of IGRAs in compartments other than blood will require more independent and carefully designed prospective studies.

Challenges and perspectives for improved management of HIV/Mycobacterium tuberculosis co-infection.

HIV and Mycobacterium tuberculosis (MTB) are two widespread and highly successful microbes whose synergy in pathogenesis has created a significant threat for human health globally. In acknowledgement of this fact, the European Union (EU) has funded a multinational support action, the European Network for global cooperation in the field of AIDS and TB (EUCO-Net), that brings together experts from Europe and those regions that bear the highest burden of HIV/MTB co-infection. Here, we summarise the main outcome of the EUCO-Net project derived from an expert group meeting that took place in Stellenbosch (South Africa) (AIDS/TB Workshop on Research Challenges and Opportunities for Future Collaboration) and the subsequent discussions, and propose priority areas for research and concerted actions that will have impact on future EU calls.

The risk of tuberculosis related to tumour necrosis factor antagonist therapies: a TBNET consensus statement.

Anti-tumour necrosis factor (TNF) monoclonal antibodies or soluble TNF receptors have become an invaluable treatment against chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. Individuals who are treated with TNF antagonists are at an increased risk of reactivating latent infections, especially tuberculosis (TB). Following TNF antagonist therapy, the relative risk for TB is increased up to 25 times, depending on the clinical setting and the TNF antagonist used. Interferon-γ release assays or, as an alternative in individuals without a history of bacille Calmette-Guérin vaccination, tuberculin skin testing is recommended to screen all adult candidates for TNF antagonist treatment for the presence of latent infection with Mycobacterium tuberculosis. Moreover, paediatric practice suggests concomitant use of both the tuberculin skin test and an interferon-γ release assay, as there are insufficient data in children to recommend one test over the other. Consequently, targeted preventive chemotherapy is highly recommended for all individuals with persistent M. tuberculosis-specific immune responses undergoing TNF antagonist therapy as it significantly reduces the risk of progression to TB. This TBNET consensus statement summarises current knowledge and expert opinions and provides evidence-based recommendations to reduce the TB risk among candidates for TNF antagonist therapy.

LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement.

Tuberculosis control relies on the identification and preventive treatment of individuals who are latently infected with Mycobacterium tuberculosis. However, direct identification of latent tuberculosis infection is not possible. The diagnostic tests used to identify individuals latently infected with M. tuberculosis, the in vivo tuberculin skin test and the ex vivo interferon-gamma release assays (IGRAs), are designed to identify an adaptive immune response against, but not necessarily a latent infection with, M. tuberculosis. The proportion of individuals who truly remain infected with M. tuberculosis after tuberculin skin test or IGRA conversion is unknown. It is also uncertain how long adaptive immune responses towards mycobacterial antigens persist in the absence of live mycobacteria. Clinical management and public healthcare policies for preventive chemotherapy against tuberculosis could be improved, if we were to gain a better understanding on M. tuberculosis latency and reactivation. This statement by the TBNET summarises knowledge and limitations of the currently available tests used in adults and children for the diagnosis of latent tuberculosis infection. In summary, the main issue regarding testing is to restrict it to those who are known to be at higher risk of developing tuberculosis and who are willing to accept preventive chemotherapy.

Impaired detection of Mycobacterium tuberculosis immunity in patients using high levels of immunosuppressive drugs.

We have previously shown, in renal transplant recipients on maintenance immunosuppression, that a whole-blood assay was superior in detecting immunity towards purified protein derivative (PPD) compared with skin testing. As blood tests may have limitations during high-dose immunosuppression therapy, the present study was aimed at characterising the effect of high immunosuppressive drug levels on PPD-specific T-cell immunity. PPD-reactive CD4 T-cells from 13 renal transplant recipients were longitudinally quantified by the induction of cytokines using flow cytometry. To further address the effect of high and low maintenance immunosuppression, drug effects were studied in vitro and in 49 age-matched lung transplant recipients and 49 renal transplant recipients. Maintenance immunosuppression after renal transplantation did not affect PPD-specific T-cell detection (median T-cell frequencies 0.55% before and 0.46% >12 months after transplantation), whereas specific T-cell frequencies were significantly lower 3 months after transplantation (0.15%; p = 0.0002). Likewise, high-level maintenance immunosuppression after lung transplantation was associated with a significantly lower prevalence in PPD-specific T-cell reactivity compared with renal transplant recipients (16.7% versus 52.1%; p = 0.0005). In line with the observations made in vivo, calcineurin inhibitors analysed in vitro led to a dose-dependent decrease in antigen-specific T-cell reactivity. The flow cytometric assay is not adversely affected by low drug doses. In contrast, decreased levels of PPD-specific T-cells early after transplantation and low prevalence of PPD-reactivity in lung transplant recipients suggest a reduced sensitivity of in vitro testing during high-level immunosuppression.

PD-1 expression and IL-2 loss of cytomegalovirus- specific T cells correlates with viremia and reversible functional anergy.

Cytomegalovirus (CMV) represents a major cause of infectious complications after transplantation. Recently, chronic infections with lymphocyte choriomeningitis virus (LCMV), HIV or HCV were shown to be associated with functionally exhausted T cells characterized by high expression of the programmed death (PD)-1 molecule and altered cytokine expression patterns. We therefore hypothesized that functional exhaustion of CMV-specific CD4 T cells may determine impaired CMV control in patients after renal transplantation. In viremic transplant recipients, a significantly higher proportion of CMV-specific CD4 T cells was PD-1 positive (median 40.9%, 17.0-88.7%) as compared to nonviremic transplant patients (8.8%, 0.8-80.5%), dialysis patients (8.8%, 0-36.7%) or controls (3.2%, 0.3-15.4%, p < 0.0001). In line with functional impairment, PD-1-positive T cells produced significantly less IFNgamma as compared to PD-1-negative T cells (p < 0.0001). Moreover, unlike controls or nonviremic patients, CMV-specific T cells from viremic patients showed a significant loss of IL-2 production (p < 0.0001). Interestingly, functional anergy of CMV-specific CD4 T cells was reversible in that antibody-mediated blockade of PD-1 signaling with its ligands PD-L1/-L2 led to an up to 10-fold increase in CMV-specific proliferation. In conclusion, expression of PD-1 defines a reversible defect of CMV-specific CD4 T cells that is associated with viremia, and blocking PD-1 signaling may provide a potential target for enhancing the function of exhausted T cells in chronic CMV infection.

Evaluation of latent tuberculosis infection in patients with inflammatory arthropathies before treatment with TNF-alpha blocking drugs using a novel flow-cytometric interferon-gamma release assay.

OBJECTIVE: To compare the efficacy of the conventional skin test and a novel flow cytometric whole blood assay in the diagnosis of latent tuberculosis infection (LTBI) in patients with rheumatological diseases evaluated for treatment with TNF-alpha-blocking agents. METHODS: Prospective study of 97 consecutively enrolled patients, who were assessed for the presence of LTBI through clinical history, Mendel-Mantoux skin testing and chest X-ray. In addition, T-cell reactivity towards tuberculin (PPD, purified protein derivative) and the Mycobacterium tuberculosis-specific proteins ESAT-6 and CFP-10 was determined ex vivo using a flow cytometric whole blood assay. RESULTS: After standard screening, 15% of patients receiving TNF-alpha-blocking therapy were pretreated with isoniazide (INH), another 5% of patients did not receive TNF-alpha-blocking therapy because of LTBI. PPD-reactivity in the skin was observed in 14% of patients compared with 39% with the whole blood test. Analysis of the M. tuberculosis-specific response to ESAT-6 and CFP-10 revealed positive results in 16% of patients. Using a decision tree incorporating history, chest X-ray and either skin-test or ESAT-6/CFP-10 results, 18 or 22% of the patients, respectively, were classified as latently infected with M. tuberculosis. Four patients treated with INH because of a positive skin reaction did not show reactivity to ESAT-6/CFP-10 in the whole blood assays. Another six patients not pretreated with INH because of negative skin tests would have received INH, had the results of the whole blood assay been taken into account. CONCLUSION: The Mendel-Mantoux skin test has a low sensitivity and specificity for the diagnosis of LTBI in this cohort of patients, potentially resulting in both over- and under-treatment with prophylactic INH when compared with the flow cytometric analysis of whole blood T-cell reactivity to proteins specific to M. tuberculosis. Use of T-cell based in vitro tests may help to refine diagnostic testing for LTBI.

Fitness of backcross six of hybrids between transgenic oilseed rape (Brassica napus) and wild radish (Raphanus raphanistrum).

The process of introgression between a transgenic crop modified for better agronomic characters and a wild relative could lead potentially to increased weediness and adaptation to the environment of the wild species. However, the formation of hybrid and hybrid progeny could be associated with functional imbalance and low fitness, which reduces the risk of gene escape and establishment of the wild species in the field. Our work compares the fitness components of parents and different types of backcross in the sixth generation of hybrids between transgenic oilseed rape (Brassica napus, AACC, 2n = 38) resistant to the herbicide glufosinate and wild radish (Raphanus raphanistrum, RrRr, 2n = 18). The backcross with oilseed rape cytoplasm (OBC) has a fitness value 100 times lower than that of the backcross with wild radish cytoplasm (RBC). The herbicide-resistant RBC has similar growth to the susceptible RBC, but final male and female fitness values are two times lower. In turn, susceptible RBC exhibit similar fitness to the control wild radishes. The relative fitnesses of the different types are the same whether or not they grow under competitive conditions. The consequence on fitness of the chromosome location of the transgene conferring resistance and the relevance of these results to the impact of gene flow on the environment are discussed.

The interleukin-10 promoter genotype determines clinical immune function in hemodialysis patients.

BACKGROUND: Immune dysfunction and the impaired hepatitis B vaccination response are complications of chronic renal failure that are tightly associated with inflammation induced by uremia and blood-membrane contacts. Proinflammatory cytokines, such as interleukin (IL)-6, are counter-regulated by IL-10 with a large interindividual variability. Part of the variability of cytokine production is genetically determined since polymorphisms in the cytokine gene promoters lead to high or low production. The aim of this study was to detect the genetic influence of the IL-10 promoter on immune function of chronic hemodialysis patients. METHODS: The IL-10 genotype (polymorphic bases at positions -1082 and -819) was determined in 272 chronic hemodialysis patients using highly specific PCR and related to the patients' response to a triple vaccination against hepatitis B. Secretion of IL-10 and IL-6 by peripheral blood leucocytes in vitro was determined by ELISA. RESULTS: The prevalence of the IL-10 genotypes in dialysis patients with well-preserved immune function (vaccination responders) was similar to the general population. In contrast, prevalence of the -1082G* allele (associated with high production of IL-10) was low in the nonresponders. The relative risk of vaccination nonresponse in patients homozygous for the -1082A* allele was 1.394 (95% CI, 1.091 to 1.781, P < 0.05) compared to those homozygous for -1082G*. There was no relationship between the IL-10 genotype and the type of renal disease. CONCLUSIONS: The IL-10 genotype determines IL-10 production in dialysis patients, which down-regulates uremia- and dialysis-induced chronic inflammation and helps to preserve immune defense functions.