T-lymphocyte and glycemic status after vitamin D treatment in type 1 diabetes: A randomized controlled trial with sequential crossover.

BACKGROUND: Type 1 diabetes mellitus (T1D) is mediated by autoaggressive T effector cells with an underlying regulatory T-cell (Treg) defect. Vitamin D deficiency is highly prevalent in T1D, which can aggravate immune dysfunction. High-dose vitamin D treatment may enhance Tregs and improve metabolism in T1D patients. METHODS: In a randomized double-blind placebo-controlled trial with crossover design, patients received either for 3 months cholecalciferol 4000 IU/d followed by 3 months placebo or the sequential alternative. Thirty-nine T1D patients (19 women and 20 men) completed the trial. RESULTS: Primary outcome was a change of Tregs, secondary HbA1C, and insulin demand. Effects were evaluated based on intra-individual changes between treatment and placebo periods for outcome measures. Exploratory analyses included vitamin D system variant genotyping and C-peptide measurements. Median 25(OH)D3 increased to 38.8 ng/ml with males showing a significantly stronger increase (p = .003). T-lymphocyte profiles did not change significantly (p > 2); however, the intra-individual change of Tregs between males and females was different with a significantly stronger increase in men (p = .017), as well as between genotypes of the vitamin D receptor (Apa, Taq, and Bsm: genotypes aa, TT, and bb; p = .004-0.015). Insulin demands declined significantly (p = .003-.039) and HbA1C improved (p < .001). Random C-peptide levels were low but rising (median, 0.125 ng/ml; range, 0.02-0.3) in 6 patients. No toxicity was observed. CONCLUSION: A daily vitamin D dose of 4000 IU for 3 months was well tolerated and enhanced Tregs in males. Glucometabolic control improved in all. Subsequent larger trials need to address ß-cell function and genotyping for individualized vitamin D doses.

Percentiles of Lymphocyte Subsets in Preterm Infants According to Gestational Age Compared to Children and Adolescents.

Preterm newborns show an increased susceptibility to infections, conceivably related to their immature immune system. To gain further knowledge about the immune development in early preterm infants, we aimed to establish references for lymphocyte subsets and compare the maturation process during hospitalization to healthy term-born children and adolescents. For this purpose, peripheral blood samples (n = 153) were collected from 40 preterm infants, gestational age (GA) 26-30 week between 2nd and 6th day of life, and were monitored in intervals of every 2 or rather 4 weeks until the end of hospitalization. Furthermore, we analysed single sample controls of 10 term neonates. We compared these data with results of a study in healthy children and adolescent (n = 176). Flow cytometry of immune cell subsets was performed as single-platform analysis using 10-colour flow cytometry. Based on preterm's age, our percentile model allows readout of absolute cell count for lymphocytes, B cells, T cells, NK cells, T8 and T4 cells. The median (minimum) value of T-, B- and NK cells after birth was 2800 (600), 790 (120) and 140 (20) cells/µl, respectively. Major differences were found in absolute cell numbers of B cells, and in the frequency of regulatory T cells, most pronounced in the earliest preterm infants (GA 26). Compared to healthy children and adolescents, preterm infants reached lymphocyte counts in between the 5th and 50th percentile when discharging the hospital. This prospective observational study provides reference percentiles for lymphocytes subsets of preterm infants. These data are conducive to interpret immunological capability of preterm infants with possible immune disorders appropriate.

No effect of antifungal compounds on functional properties of human antifungal T-helper type 1 cells.

Despite the availability of new antifungal compounds, invasive fungal disease is associated with a high mortality in hematopoietic stem cell transplant (HSCT) recipients. A growing body of evidence suggests that T lymphocytes from the T-helper type 1 (TH 1) play an important role in the antifungal host defense, and preliminary data indicate a potential benefit of infusing donor-derived antifungal TH 1 cells to HSCT patients suffering from invasive fungal disease. Unfortunately, it is unclear to date whether the function of these cells is affected by concomitantly administered antifungal agents. We therefore analyzed the effects of various concentrations of commonly used antifungal compounds such as amphotericin B, caspofungin, fluconazole, voriconazole, and posaconazole on the functional properties of cultivated human antifungal TH 1 cells. None of the antifungal compounds tested significantly influenced the secretion of interferon-γ and tumor necrosis factor-α, and only posaconazole at high concentrations slightly decreased proliferation of antifungal TH 1 cells. Our data indicate that the antifungal agents tested do not significantly affect the functional properties of antifungal TH 1 cells and can therefore be concomitantly administered.

T cells for suicide gene therapy: activation, functionality and clinical relevance.

In order to control graft-versus-host disease after donor lymphocyte infusion, T cells can be retrovirally transduced with a suicide gene. However, the immune competence of activated T cells appears compromised, responsible for reduced alloreactivity. The present study compared different activation protocols using soluble or bead-coupled antibodies regarding T-cell subtype expansion capacity and functionality. T cells were purified on a laboratory and clinical scale using both CD3 and CD4/CD8 antibodies for selection, leading to a mean purity of 96%. Transductions were performed with a GMP-grade CD34/HSV-TK vector. Activation with soluble CD3/CD28-antibodies +1000 U/ml IL-2 induced a 50-fold expansion of T cells over 14 days, whereas T cells activated with bead-coupled antibodies only expanded 2-4-fold restricted to the first week. Apart from using soluble antibodies, proliferation was highly IL-2 dependent. Expansion of CMV-specific T cells coincided with the expansion of whole CD3(+) cells. Soluble antibodies and higher IL-2 concentrations preferentially stimulated CD8(+) T cells, while bead-coupled antibodies +20 U/ml IL-2 preserved the CD4/CD8 ratio. Irrespective of the activation protocol, there was a shift from a naive to memory phenotype. When activated with soluble antibodies, mainly CD8(+) T cells were transduced. Furthermore, Th1/Th2 cytokine secretion was reduced. In contrast, CD4(+)/CD8(+) T cells activated with bead-coupled antibodies were rather homogenously transduced and cytokine secretion did not appear to be affected.

Treatment of relapse after allogeneic stem cell transplantation in children and adolescents with ALL: the Frankfurt experience.

Therapy for post-transplant relapse of paediatric ALL is limited. Standardised curative approaches are not available. We hereby describe our local procedure in this life-threatening situation. A total of 101 ALL patients received their first allogeneic stem cell transplantation (SCT) in our institution. After relapse, our primary therapeutic goal was to cure the patient with high-dose chemotherapy or specific immunotherapy (HDCHT/SIT) followed by a second SCT from a haploidentical donor (transplant approach). If this was not feasible, low-dose chemotherapy and donor lymphocyte infusions (LDCHT+DLI) were offered (non-transplant approach). A total of 23 patients suffered a post-transplant relapse. Eight patients received HDCHT/SIT, followed by haploidentical SCT in 7/8. Ten received LDCHT+DLI. The eight patients treated with a second transplant and the ten treated with the non-transplant approach had a 4-year overall survival of 56% and 40%, respectively (P=0.232). Prerequisites for successful treatment of post-transplant relapse by either a second transplant or experimental non-transplant approaches are good clinical condition and the capacity to achieve haematological remission by the induction treatment element.

Dendritic cell reconstitution is associated with relapse-free survival and acute GVHD severity in children after allogeneic stem cell transplantation.

DCs are potent APCs and key regulators of innate and adaptive immunity. After allo-SCT, their reconstitution in the peripheral blood (PB) to levels similar to those in healthy individuals tends to be slow. We investigate the age- and sex-dependant immune reconstitution of myeloid (mDC) and plasmacytoid DC (pDC) in the PB of 45 children with leukaemia or myelodysplastic syndrome (aged 1-17 years, median 10) after allo-SCT with regard to relapse, acute GVHD (aGVHD) and relapse-free survival. Low pDC/µL PB up to day 60 post SCT are associated with higher incidence of moderate or severe aGVHD (P=0.035), whereas high pDC/µL PB up to day 60 are associated with higher risk of relapse (P<0.001). The time-trend of DCs/µL PB for days 0-200 is a significant predictor of relapse-free survival for both mDCs (P<0.001) and pDCs (P=0.020). Jointly modelling DC reconstitution and complications improves on these simple criteria. Compared with BM, PBSC transplants tend to show slower mDC/pDC reconstitution (P=0.001, 0.031, respectively), but have no direct effect on relapse-free survival. These results suggest an important role for both mDCs and pDCs in the reconstituting immune system. The inclusion of mDCs and pDCs may improve existing models for complication prediction following allo-SCT.

Multivariate analyses of immune reconstitution in children after allo-SCT: risk-estimation based on age-matched leukocyte sub-populations.

The speed of immune recovery after allo-SCT is of central importance to overcome infectious complications and relapse. To evaluate the immune reconstitution of pediatric patients concerning overall survival, we developed a three-component multivariate model and generated a reference domain of ellipsoidal shape on the basis of normal leukocyte subtype values of 100 healthy children and adolescents. The leukocyte subtypes include absolute nos. of leukocytes, CD14(+) monocytes, lymphocytes, CD3(+) T cells, CD3(+)CD4(+) helper T cells, CD3(+)CD8(+) cytotoxic T cells, CD3(-)CD56(+) natural killer-cells and CD19(+) B cells, all of which are correlated, thus, requiring the application of multivariate as opposed to multiple univariate modeling. According to their immune reconstitution, 32 pediatric patients post allo-SCT were classified into low-risk and high-risk groups on the basis of our new model. Therefore, we evaluated if the patients reached the ellipsoid of normal leukocyte sub-population values post SCT. We detected a significantly higher number of long-time survivors among the low-risk group compared with the high-risk group at days 200 (P=0.001) and 300 (P<0.0001). This is superior to our previously published univariate analysis. Combined with the clinical observation, a classification into risk groups based on an extended patient cohort may represent a predictor for complications.