Kinetics of Torque Teno Virus Viral Load Is Associated with Infection and De Novo Donor Specific Antibodies in the First Year after Kidney Transplantation: A Prospective Cohort Study.

Torque teno virus (TTV) was recently identified as a potential biomarker for the degree of immunosuppression, and potentially as a predictor of rejection and infection in solid organ transplant patients. We evaluated TTV viral load in kidney transplant (KT) patients during the first year post-transplant to examine overall kinetics and their relationships with deleterious events, including episodes of infection and the formation of de novo donor-specific antibodies (DSAs). In a single-center, prospective observational cohort study, 81 KT patients were monitored at baseline, week 1, and month 1, 3, 6, 9 and 12, post-KT, and whenever required by clinical events. Kidney function, plasma TTV load, immunoglobulins and lymphocyte subpopulations were assessed at each time point. Twenty-six patients (32.1%) presented a total of 38 infection episodes post-KT. Induction immunosuppression with thymoglobulin, compared to basiliximab, was not associated with more infections (p = 0.8093). Patients with infectious events had lower T-cells (p = 0.0500), CD8+ T-cells (p = 0.0313) and B-cells (p = 0.0009) 1 month post-KT, compared to infection-free patients. Patients with infection also showed higher increases in TTV viral loads between week 1- month 1, post-KT, with TTV viral load variations >2.65 log10 cp/mL predicting the development of infectious events during the 12-month study period (p < 0.0001; sensitivity 99.73%; specificity 83.67%). Patients who developed de novo DSAs had lower TTV DNA viral loads at month 12 after KT, compared to patients who did not develop DSA (3.7 vs. 5.3 log10 cp/mL, p = 0.0023). Briefly, evaluating early TTV viremia is a promising strategy for defining infectious risk in the 1st year post-KT. The availability of standardized commercial real-time PCR assays is crucial to further validate this as an effective tool guiding immunosuppression prescription.

Regulatory T cells and IL-17A levels in noninfectious uveitis.

PURPOSE: Regulatory T cells (Tregs) have been intensively studied in a myriad of autoimmune diseases. As for noninfectious uveitis (NIU), results have been contradictory, and studies have failed to demonstrate a consistent reduction in Treg cell frequency in patients with active disease. The present study aims to characterize T lymphocyte subsets, including naïve and memory Tregs as well as their respective CD39 expression, in the peripheral blood of NIU patients. Inflammatory as well as suppressive cytokine profiles were also evaluated. METHODS: T cell subpopulations were evaluated by multiparametric flow cytometry using anti-CD3, anti-CD4, anti-CD45, anti-CD45RA, anti-CD197, anti-CD25, anti-CD127, and anti-CD39. Treg cells were defined as CD3 + CD4+CD25hiCD127low. A multiplex bead-based immunoassay was used to determine TNF-α, IFN-É£, IL-17A, IL-10, and TGF-ß levels. RESULTS: Twenty-nine patients with active NIU were included as well as 15 sex- and age-matched controls. There were no significant differences in T lymphocyte subsets, including Tregs, between patients and controls. However, patients with a lower grade of anterior chamber or vitreous inflammatory cellular reaction showed higher memory Treg counts than controls, with no respective increase in CD39+ expression, and a tendency for higher IL-17A levels (p = 0.06). This IL-17A elevation was present in the total NIU group (p = 0.08) as well as a positive correlation between IL-17A levels and the absolute counts of memory Tregs (p = 0.013; R = 0.465). Patients with higher IL-17A levels also showed higher serum concentrations of memory (p = 0.001) and naïve (p = 0.003) Tregs as well as elevated TNF-α (p < 0.0001) and IFN-É£ (p = 0.016) levels. Negative correlations were observed between IL-10 and TGF-ß levels and the percentages of memory (p = 0.030; R = - 0.411) and total CD39+ Tregs (p = 0.051; R = - 0.373) in the peripheral blood of NIU patients. CONCLUSION: Our results showed that total Treg levels were not reduced in patients with NIU. Further characterization of Treg subsets, including memory Tregs and respective CD39 expression, may provide additional insight on the role of Treg cells in NIU. Consistent high levels of circulating IL-17A in NIU patients are in accordance with previous studies and reinforce this cytokine's vital role in uveitis pathogenesis and its possible use as a therapeutic target.

Multicentre evaluation of stable reference whole blood for enumeration of lymphocyte subsets by flow cytometry.

BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc.

Carpobrotus edulis methanol extract inhibits the MDR efflux pumps, enhances killing of phagocytosed S. aureus and promotes immune modulation.

Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non-toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi-drug resistant cells susceptible to anticancer drugs. These non-toxic concentrations also prime THP-1 human monocyte-derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP-1 cells within 1 day of exposure to quantities normally associated with phytohaemagglutinin. The potential role of the compound(s) isolated from this plant in cancer biology is intriguing and is currently under investigation. It is supposed that the resistance modifier and immunomodulatory effect of this plant extract can be exploited in the experimental chemotherapy of cancer and bacterial or viral infections.

Intracellular activity of clinical concentrations of phenothiazines including thioridiazine against phagocytosed Staphylococcus aureus.

The effect of thioridazine (TZ) was studied on the killing activity of human peripheral blood monocyte derived macrophages (HPBMDM) and of human macrophage cell line THP-1 at extracellular concentrations below those achievable clinically. These macrophages have nominal killing activity against bacteria and therefore, would not influence any activity that the compounds may have against intracellular localised Staphylococcus aureus. The results indicated that whereas TZ has an in vitro minimum inhibitory concentration (MIC) against the strains of S. aureus of 18, 0.1 mg/l of TZ in the medium completely inhibits the growth of S. aureus that has been phagocytosed by macrophages. The latter concentration was non-toxic to macrophages, did not cause cellular expression of activation marker CD69 nor induction of CD3+ T cell production of IFN-gamma, but blocked cellular proliferation and down-regulated the production of T cell-derived cytokines (IFN-gamma, IL-5). These results suggest that TZ induces intracellular bactericidal activities independent of the capacity to generate Type 1 responses against S. aureus.