Jürgen Schulte Mönting

Formation of a functional thymus initiated by a postnatal epithelial progenitor cell

The thymus is essential for the generation of self-tolerant effector and regulatory T cells. Intrathymic T-cell development requires an intact stromal microenvironment, of which thymic epithelial cells (TECs) constitute a major part. For instance, cell-autonomous genetic defects of forkhead box N1 (Foxn1) and autoimmune regulator (Aire) in thymic epithelial cells cause primary immunodeficiency and autoimmunity, respectively. During development, the thymic epithelial rudiment gives rise to two major compartments, the cortex and medulla. Cortical TECs positively select T cells, whereas medullary TECs are involved in negative selection of potentially autoreactive T cells. It has long been unclear whether these two morphologically and functionally distinct types of epithelial cells arise from a common bi-potent progenitor cell and whether such progenitors are still present in the postnatal period. Here, using in vivo cell lineage analysis in mice, we demonstrate the presence of a common progenitor of cortical and medullary TECs after birth. To probe the function of postnatal progenitors, a conditional mutant allele of Foxn1 was reverted to wild-type function in single epithelial cells in vivo. This led to the formation of small thymic lobules containing both cortical and medullary areas that supported normal thymopoiesis. Thus, single epithelial progenitor cells can give rise to a complete and functional thymic microenvironment, suggesting that cell-based therapies could be developed for thymus disorders.

Intravenously Administered Immunoglobulin in the Treatment of Childhood Guillain-Barré Syndrome: A Randomized Trial

Objective. To determine the optimal treatment for childhood Guillain-Barré syndrome (GBS). Methods. We performed a randomized, multicenter study of GBS according to international diagnostic criteria. In study 1 (early treatment), children able to walk unaided for 5 meters were randomized for 1 g/kg intravenously administered immunoglobulin (IVIG) over 2 days or no treatment. The primary outcome measure was the degree of disability at nadir. In study 2 (treatment for severe GBS), children unable to walk 5 meters unaided were randomized for 1 g/kg IVIG over 2 days or 0.4 g/kg IVIG over 5 days. The primary outcome measure was the number of days needed to regain the ability to walk unaided. Children randomized for no treatment in study 1 could enter study 2 if loss of unaided walking occurred. Results. Ninety-five children with GBS were registered in 40 months. Twenty-one children were randomized in study 1 and 51 in study 2 (5 after deterioration in study 1). Twenty-eight children were not randomized for various reasons. Eleven of 21 patients in study 1 lost the ability to walk unassisted and 6 were bedridden, with no statistically significant difference between the children initially randomized for treatment versus no treatment. Recovery occurred faster in the group randomized for early treatment. In study 2, recovery did not differ significantly between the children treated for 2 days versus 5 days (median time to unaided walking: 19 days vs 13 days). Secondary transient deterioration in the disability score occurred more frequently in the group with the 2-day regimen than in the group treated for 5 days (5 of 23 patients vs 0 of 23 patients). Multivariate analysis with Cox regression showed that disease severity at the nadir was the only prognostic factor for recovery. Conclusions. Treatment with IVIG before loss of unaided walking did not give rise to a less severe course, but recovery occurred somewhat faster. However, given the small number of patients, the power of this conclusion is low. For treatment after loss of unaided walking, there was no significant difference in the effectiveness of 2 g/kg IVIG administered over 2 days versus 5 days. Early “relapses” occurred more frequently after the shorter treatment regimen.

Clinical impact of antibody formation to botulinum toxin A in children

We studied the clinical impact of neutralizing antibodies to botulinum toxin A that occurred during long‐term treatment of children between 1993 and 2001. Antibodies were found in high titers in 35 of 110 (31.8%) samples from individual patients. Antibody formation correlated with secondary nonresponse (p < 0.001). The most significant risk factors for antibody formation were the frequency of treatments (p = 0.0001) and the injection of a higher weight‐adapted maximum dose per treatment (p = 0.001).

Impaired cytokine production in whole blood cell cultures of patients with gynaecological carcinomas in different clinical stages

The production of the cytokines IFN-gamma, IL-1-alpha, IL-2 and TNF-alpha was investigated in mitogen-stimulated, whole blood cell culture from 239 untreated patients with primary gynaecological carcinomas (breast, cervix, ovary, endometrium), and 191 healthy female controls. The cytokines were measured in the 4-day post-induction supernatants by a sensitive enzymoimmunological assay. In the blood cell cultures of all four groups of cancer patients, significantly lower values of IFN-gamma (P < or = 0.001), IL-2 (P < or = 0.01) and IL-1 alpha (P < or = 0.01) were found as compared to the controls, although lymphocyte and monocyte counts were almost identical. Grouping the tumour patients into different clinical stages we could show in the four groups of carcinomas a gradual depression of the cytokine production according to growing tumour burden.

Cytokine levels in whole blood cell cultures as parameters of the cellular immunologic activity in patients with malignant melanoma and basal cell carcinoma

For the determination of cellular immunity status, mitogen‐induced lymphocyte proliferation tests are used, along with measurements of cytokine secretion. The authors have established a test system with whole blood cell cultures in which they measured the following cytokines: alpha‐interleukin‐1 (α‐IL‐1), interleukin‐2 (IL‐2), gamma‐interferon (τ‐IFN), and alpha‐tumor necrosis factor (α‐TNF) in the supernatants by enzymoimmunologic methods. With this system, the authors tested blood samples of 72 patients with malignant melanoma, 38 patients with basal cell carcinoma, and 315 healthy control subjects. In the blood cell cultures of the patients with melanoma, significantly lower values of the lymphokines τ‐IFN and IL‐2 were found, compared with those of the control subjects, and the levels of the monokines α‐IL‐1 and α‐TNF were reduced. τ‐IFN values correlated with different clinical stages. In contrast, the patients with basal cell carcinoma had equal values for all four cytokines as an age‐matched control group. Cancer 1993; 71:231‐6.

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+ renal-cell-carcinoma-infiltrating lymphocytes.

Abstract The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3+ tumor-infiltrating lymphocytes (CD3+ TIL) and in autologous CD3+ peripheral blood lymphocytes (CD3+ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4+ TIL and CD8+ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3+ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3+ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4+ TIL and simultaneously obtained CD8+ TIL revealed a significantly higher expression of IFN in the CD8+ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8+ TIL.

Analysis of the immune reactivity of infiltrating and peripheral lymphocytes from patients with renal cell carcinoma by measuring cytokine secretion

Abstract The immunological properties of tumor-infiltrating (TIL) and peripheral blood lymphocytes (PBL) from 29 patients with renal cell carcinomas were characterized with respect to their phenotypic expression and cytokine production. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation. To eliminate all non-lymphoid cells, CD3-positive cells were specifically separated from these cell fractions with anti-CD3 magnetic beads. These pure CD3-positive PBL (CD3+PBL) and TIL (CD3+TIL) were cultured with pokeweed mitogen and the levels of the cytokines interleukin-1α (IL-1α), IL-1β, IL-2, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) measured in the 4-day post-inductional cell culture supernatants. In all cell cultures a wide range of cytokine values was found, indicating a large variation in the immunological activity of the lymphocytes of each individual. When the cell cultures of the CD3+TIL and CD3+PBL were compared in each patient similar values for IL-1α, IL-1β, IFNγ and TNFα were found. However CD3+TIL produced significantly lower levels of IL-2 than CD3+PBL upon mitogenic stimulation. This may be due to a lower CD4/CD8 ratio in the CD3+TIL as compared to the CD3+PBL. These results suggest that there are no fundamental qualitative and quantitative differences in the lymphokine-producing capacity of CD3+TIL and CD3+PBL derived from patients with renal cell carcinomas.

Cytokine production in whole blood cell cultures of patients undergoing therapy with biological response modifiers or 5-fluorouracil

We have measured the levels of interferon γ (IFNγ), tumor necrosis factor α (TNFα), interleukin-1α (IL-1α), IL-1β, and IL-2 in the whole blood cell culture supernatants of 43 tumor patients undergoing a treatment with biological response modifiers or a conventional therapy with 5-fluorouracil and leucovorin. In the blood cell cultures of the 16 patients who received 5-fluorouracil and leucovorin IFNγ levels decreased (P≤0.01) and TNFα levels rose (P≤0.05) during each therapy cycle. However, in the blood samples a declining number of total leukocytes and lymphocytes was measured (P≤0.05). Progressive disease could be correlated to a tendency towards lower IFNγ levels in the pretherapeutic cultures of these patients. The second group analyzed consisted of 8 patients receiving a low-dose IL-1β therapy. In this group we found either an unchanged or an augmented IFNγ production of the blood cells during treatment. In the group of 13 patients receiving low-dose recombinant IL-2 (≤4.5×106IU m−2 day−1) significantly increasing IFNγ levels were seen in the blood cell cultures during the therapy (P≤0.05), although total leukocyte counts decreased. In this group, 4 had stable disease for at least 2 months and 9 patients had tumor progression under therapy. In the cultures of the latter a tendency towards lower IFNγ values was found. Finally, the cytokine production in the blood cell cultures of 6 patients receiving a combination therapy of IFNα and high-dose IL-2 was studied. During this therapy a dramatically reduced production not only of IFNγ but also of all other measured cytokines was found. In this group all patients had tumor progression under therapy. It is concluded that the measurements of cytokine production in a reproducible whole blood culture system may be useful for monitoring immunological therapies and may help us to find out which doses of biological response modifiers have enhancing or suppressive effects on the functions of the immune cells.

Immunosuppressive Activity of Sera from Patients with Colorectal and Gynecological Carcinomas as Evaluated by Impaired IFN-γ, IL-1α and TNF-α Production of Human Peripheral Mononuclear Cells

Abstract The sera from patients with preoperative colorectal and gynecological carcinomas (ovarian and breast cancer) were investigated for their putative immunosuppressive activity (ISA). lSA was measured by determining the changes in the production of IFN-γ, IL-1α and TNF-α by human peripheral mononuclear cells (PBMC) from six normal donors. Phytohemagglutinin-stimulated PBMC were incubated with sera from patients with colorectal and gynecological carcinomas and healthy controls and in the 4-day post-inductional supernatants the cytokines were measurd by an enzymoimmunological assay (ELISA). Sera from patients with carcinomas significantly decreased the IFN-y production as compared to the controls. In the cultures containing sera from patients with colorectal and ovarian but not breast carcinoma, significantly lower levels of IL-1α and TNF-α were measured compared to the cultures with sera from healthy controls.