Closing the gap between T-cell life span estimates from stable isotope-labeling studies in mice and humans.

Quantitative knowledge of the turnover of different leukocyte populations is a key to our understanding of immune function in health and disease. Much progress has been made thanks to the introduction of stable isotope labeling, the state-of-the-art technique for in vivo quantification of cellular life spans. Yet, even leukocyte life span estimates on the basis of stable isotope labeling can vary up to 10-fold among laboratories. We investigated whether these differences could be the result of variances in the length of the labeling period among studies. To this end, we performed deuterated water-labeling experiments in mice, in which only the length of label administration was varied. The resulting life span estimates were indeed dependent on the length of the labeling period when the data were analyzed using a commonly used single-exponential model. We show that multiexponential models provide the necessary tool to obtain life span estimates that are independent of the length of the labeling period. Use of a multiexponential model enabled us to reduce the gap between human T-cell life span estimates from 2 previously published labeling studies. This provides an important step toward unambiguous understanding of leukocyte turnover in health and disease.

Homing characteristics of donor T cells after experimental allogeneic bone marrow transplantation and posttransplantation therapy for multiple myeloma.

Relapse and graft-versus-host disease remain major problems associated with allogeneic bone marrow (BM) transplantation (allo-BMT) and posttransplantation therapy in patients with multiple myeloma (MM) and other hematologic malignancies. A possible strategy for selectively enhancing the graft-versus-myeloma response and possibly reducing graft-versus-host disease is to increase the migration of alloreactive T cells toward the MM-containing BM. In the present study, we characterized the BM-homing behavior of donor-derived effector T cells in a novel allo-BMT model for the treatment of MM. We observed that posttransplantation immunotherapy consisting of donor lymphocyte infusion (DLI) and vaccination with minor histocompatibility antigen-loaded dendritic cells (DCs) was associated with prolonged survival compared with allo-BMT with no further treatment. Moreover, CD8(+) effector T cells expressing inflammatory homing receptors, including high levels of CD44, LFA-1, and inflammatory chemokine receptors, were recruited to MM-bearing BM. This was paralleled by strongly increased expression of IFN-γ and IFN-γ-inducible chemokines, including CXCL9, CXCL10, and CXCL16, especially in mice treated with DLI plus minor histocompatibility antigen-loaded DC vaccination. Remarkably, expression of the homeostatic chemokine CXCL12 was reduced. Furthermore, IFN-γ and TNF-α induced BM endothelial cells to express high levels of the inflammatory chemokines and reduced or unaltered levels of CXCL12. Finally, presentation of CXCL9 by multiple BM endothelial cell-expressed heparan sulfate proteoglycans triggered transendothelial migration of effector T cells. Taken together, our data demonstrate that both post-transplantation DLI plus miHA-loaded DC vaccination and MM growth result in an increased expression of inflammatory homing receptors on donor T cells, decreased levels of the homeostatic BM-homing chemokine CXCL12, and strong induction of inflammatory chemokines in the BM. Thus, along with increasing the population of alloreactive T cells, post-transplantation immunotherapy also might contribute to a more effective graft-versus-tumor response by switching homeostatic T cell migration to inflammation-driven migration.

A generalized mathematical model to estimate T- and B-cell receptor diversities using AmpliCot.

The efficiency of the adaptive immune system is dependent on the diversity of T- and B-cell receptors, which is created by random rearrangement of receptor gene segments. AmpliCot is an experimental technique that allows the measurement of the diversity of the T- and B-cell repertoire. This procedure has the advantage over other cloning and sequencing techniques of being time- and expense-effective. In previous studies, receptor diversity, measured with AmpliCot, has been inferred assuming a second-order kinetics model. The latter implies that the relation between diversity and concentration × time (Cot) values is linear. We show that a more detailed model, involving heteroduplex and transient-duplex formation, leads to significantly better fits of experimental data and to nonlinear diversity-Cot relations. We propose an alternative fitting procedure, which is straightforward to apply and which gives an improved description of the relationship between Cot values and diversity.

The cytotoxic protease granzyme M is expressed by lymphocytes of both the innate and adaptive immune system.

The cytotoxic serine protease granzyme M (GrM) is one of the five human granzymes, which are mainly expressed by cytotoxic T lymphocytes and/or NK cells. Upon perforin-dependent entry into a target cell, GrM cleaves specific substrates resulting in the onset of a unique cell death mechanism. However, the role of GrM in pathophysiological conditions is not clear yet. Knowledge of the expression and regulation of GrM by lymphocyte populations is instrumental for a better understanding of the contribution of this unique granzyme in health and disease. Two previous studies demonstrated GrM protein expression by lymphocytes of the innate immune system, i.e., NK cells, NKT cells, and gammadelta T cells, whereas its expression by CD8(+) T cells remained controversial. In the present study, we have investigated the expression and regulation of GrM in lymphocyte subsets in more detail. Flow cytometry analysis with a novel specific antibody against human GrM confirmed high expression of this protease by NK cells, NKT cells, and gammadelta T cells. CD8(+) T cells also expressed GrM and comparing the naive to early effector-memory, to late effector-memory, to effector subset, this expression gradually increased during differentiation. In contrast, CD4(+) T cells hardly expressed GrM. Quantitative PCR analysis for GrM mRNA levels in the diverse lymphocyte sub-populations confirmed the FACS results. GrM protein expression by lymphocyte populations was not significantly affected by a panel of GrB-inducing cytokines, indicating that GrM expression is differentially regulated as compared to GrB. In conclusion, the human cytotoxic protease GrM is, besides by innate immune cells, also expressed by CD8(+) effector T cells, in particular by the differentiated effector CD27(-) CD45RO(-) subset. Our current findings support not only a role for GrM in the innate but also in the adaptive immune response.

Zymosan-induced generalized inflammation: experimental studies into mechanisms leading to multiple organ dysfunction syndrome.

Patients suffering from multiple organ dysfunction syndrome (MODS) comprise a heterogeneous population, which complicates research in its pathogenesis. Elucidation of the mechanisms involved in the development of MODS will ultimately necessitate the collection of tissue samples and the performance of invasive procedures. These requirements greatly reduce the possibilities for research in human subjects. Therefore, an animal model for MODS is a necessary and valuable tool. In the mid 1980s, the zymosan-induced generalized inflammation (ZIGI) model was introduced. Intraperitoneal injection of zymosan in mice or rats leads, in the course of 1 to 2 weeks, to increasing organ damage and dysfunction. The ZIGI model has been recognized as the one that best resembles human MODS and it has been used widely to study systemic inflammation in relation to organ failure. This review describes the ZIGI model and gives an overview of the results obtained.

Tissue- and time-dependent upregulation of cytokine mRNA in a murine model for the multiple organ dysfunction syndrome.

OBJECTIVE: We sought to quantitate the course of specific cytokine mRNA expression in tissues that exhibit increasing histopathological changes in time in an animal model for the multiple organ dysfunction syndrome (MODS). SUMMARY BACKGROUND DATA: The development of treatment protocols for MODS requires elucidation of the mechanisms and mediators involved. To devise logical interventions, it is necessary to collect data on cytokine expression at tissue level during the development of MODS. METHODS: Ninety-four C57BL/6 mice were given an intraperitoneal injection of 40 microg of lipopolysaccharide (LPS), followed by zymosan at a dose of 0.8 mg/g body weight 6 days later (day 0). Six additional animals did not receive zymosan and acted as controls. At several time points after zymosan injection, 6 randomly assigned, zymosan-treated animals were killed, and their livers, lungs, spleens, and kidneys were collected. mRNA expression of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, macrophage migration inhibiting factor, IL-12, interferon-gamma, and IL-10 was measured using a real-time reverse transcription-polymerase chain reaction assay. RESULTS: The injection of zymosan induced an acute peritonitis, followed by an apparent recovery. From approximately day 6 onwards, animals started to display MODS-like symptoms. During the peritonitis phase, up-regulation of cytokine mRNA was limited. During the period of apparent recovery, cytokine mRNA expression strongly increased, mostly reaching its maximum at day 9 when deterioration of the clinical condition had already set in. The up-regulation of tumor necrosis factor-alpha mRNA was most pronounced, especially in the lungs and liver. CONCLUSIONS: Interventions should preferentially be targeted against multiple cytokines and, at least in this model, there may be a treatment window well after the initial challenge.

The Ysterfontein 1 Middle Stone Age site, South Africa, and early human exploitation of coastal resources.

Human fossils and the genetics of extant human populations indicate that living people derive primarily from an African population that lived within the last 200,000 years. Yet it was only approximately 50,000 years ago that the descendants of this population spread to Eurasia, where they swamped or replaced the Neanderthals and other nonmodern Eurasians. Based on archaeological observations, the most plausible hypothesis for the delay is that Africans and Eurasians were behaviorally similar until 50,000 years ago, and it was only at this time that Africans developed a behavioral advantage. The archaeological findings come primarily from South Africa, where they suggest that the advantage involved much more effective use of coastal resources. Until now, the evidence has come mostly from deeply stratified caves on the south (Indian Ocean) coast. Here, we summarize results from recent excavations at Ysterfontein 1, a deeply stratified shelter in a contrasting environment on the west (Atlantic) coast. The Ysterfontein 1 samples of human food debris must be enlarged for a full comparison to samples from other relevant sites, but they already corroborate two inferences drawn from south coast sites: (i) coastal foragers before 50,000 years ago did not fish routinely, probably for lack of appropriate technology, and (ii) they collected tortoises and shellfish less intensively than later people, probably because their populations were smaller.

Organ damage in zymosan-induced multiple organ dysfunction syndrome in mice is not mediated by inducible nitric oxide synthase.

OBJECTIVE: To examine the role of inducible nitric oxide synthase (iNOS) in the development of the multiple organ dysfunction syndrome (MODS) in a murine model by using either a selective iNOS inhibitor or iNOS knockout mice. DESIGN: Prospective randomized laboratory study. SETTING: Central animal laboratory and experimental laboratory. SUBJECTS: Fifty inbred C57BL/6 mice, 39 iNOS knockout (-/-) mice, and 30 wild-type (+/+) mice, 7-9 wks old, weighing 20-25 g. INTERVENTIONS: Mice received an aseptic intraperitoneal injection of 40 microg of lipopolysaccharide followed by zymosan at a dose of 1 mg/g of body weight 6 days later (day 0). In experiment 1, C57BL/6 mice additionally received intraperitoneal injections with 5 mg of aminoguanidine or saline every 12 hrs, from 4 days after the injection of zymosan onward. In experiment 2, both iNOS-/- mice and corresponding wild-type (iNOS+/+) mice were treated with lipopolysaccharide and zymosan. MEASUREMENTS AND MAIN RESULTS: In all animals, the injection of zymosan induced an acute peritonitis, followed by an apparent recovery. From approximately day 6 onward, animals entered the third-MODS-like-phase, indicated by weight loss, a decrease in body temperature, and significant mortality rates. Quantitative reverse transcriptase polymerase chain reaction and immunochemistry revealed a strongly increased expression of iNOS messenger RNA and iNOS protein in livers of mice in the last phase. However, neither the in vivo administration of aminoguanidine to C57BL/6 mice nor the complete absence of iNOS enzyme (iNOS-/- mice) had a beneficial effect on survival rate, body temperature, or body weight. In addition, relative lung, liver, and spleen weights and lung scores were not different between experimental groups. CONCLUSIONS: The current results strongly argue against an essential and causative role of iNOS in the development of organ damage in our murine model of MODS.

Improved survival of TNF-deficient mice during the zymosan-induced multiple organ dysfunction syndrome.

The purpose of the study was to investigate the course of the zymosan-induced multiple organ dysfunction syndrome (MODS) in the absence of tumor necrosis factor (TNF) in a murine model. Tumor Necrosis Factor-alpha-lymphotoxin-a knockout (TNF/LT-/-) mice (n = 36) and wild-type (TNF/LT+/+) mice (n = 36) received 40 microg of lipopolysaccharide (LPS) intraperitoneally followed by zymosan at a dose of 1 mg/g body weight 6 days later (day 0). Animals were monitored daily for body weight and temperature and clinical symptoms. At day 22, most of the surviving mice were killed to examine organ weight and histology. A small number of animals were followed until day 48. In all animals, zymosan induced an acute sterile peritonitis phase followed by an apparent recovery. From day 8 onwards the TNF/LT+/+ mice entered a third-MODS-like-phase, characterized by loss of body weight, decreased body temperature, and significant mortality. At day 22, survival in the TNF/LT-/- mice (92%) was significantly (P = 0.01) higher than in the TNF/LT+/+ mice (60%). In addition, average body temperature and average relative (vs. weight at day 0) body weight were higher in the TNF/LT-/- mice than in the TNF/LT+/+ mice (35.9 degrees C and 100% vs. 33.3 degrees C and 84%, respectively). However, at this time point, surviving animals from both groups showed similar and significant organ damage, indicated by an increase in absolute and relative (vs body weight) weight of lung, spleen, and liver (liver only in the TNF/LT-/- mice). Moreover, histopathological examination of organs from the surviving animals showed a similar degree of microscopic damage in both groups. Interestingly, besides mononuclear cells, inflammatory infiltrates in lungs and livers of TNF/LT+/+ but not of TNF-/- mice contained neutrophils. In conclusion, TNF-deficient mice exhibit significantly improved morbidity and mortality during zymosan-induced MODS. However, the absence of TNF does not completely protect against MODS in this murine model.