Cell-free carrier system for localized delivery of peripheral blood cell-derived engineered factor signaling: towards development of a one-step device for autologous angiogenic therapy.

Spatiotemporally-controlled delivery of hypoxia-induced angiogenic factor mixtures has been identified by this group as a promising strategy for overcoming the limited ability of chronically ischemic tissues to generate adaptive angiogenesis. We previously developed an implantable, as well as an injectable system for delivering fibroblast-produced factors in vivo. Here, we identify peripheral blood cells (PBCs) as the ideal factor-providing candidates, due to their autologous nature, ease of harvest and ample supply, and investigate wound-simulating biochemical and biophysical environmental parameters that can be controlled to optimize PBC angiogenic activity. It was found that hypoxia (3% O2) significantly affected the expression of a range of angiogenesis-related factors including VEGF, angiogenin and thrombospondin-1, relative to the normoxic baseline. While all three factors underwent down-regulation over time under hypoxia, there was significant variation in the temporal profile of their expression. VEGF expression was also found to be dependent on cell-scaffold material composition, with fibrin stimulating production the most, followed by collagen and polystyrene. Cell-scaffold matrix stiffness was an additional important factor, as shown by higher VEGF protein levels when PBCs were cultured on stiff vs. compliant collagen hydrogel scaffolds. Engineered PBC-derived factor mixtures could be harvested within cell-free gel and microsphere carriers. The angiogenic effectiveness of factor-loaded carriers could be demonstrated by the ability of their releasates to induce endothelial cell tubule formation and directional migration in in vitro Matrigel assays, and microvessel sprouting in the aortic ring assay. To aid the clinical translation of this approach, we propose a device design that integrates this system, and enables one-step harvesting and delivering of angiogenic factor protein mixtures from autologous peripheral blood. This will facilitate the controlled release of these factors both at the bed-side, as an angiogenic therapy in wounds and peripheral ischemic tissue, as well as pre-, intra- and post-operatively as angiogenic support for central ischemic tissue, grafts, flaps and tissue engineered implants.

Injectable system for spatio-temporally controlled delivery of hypoxia-induced angiogenic signalling.

While chronically ischaemic tissues are continuously exposed to hypoxia, the primary angiogenic stimulus, they fail to appropriately respond to it, as hypoxia-regulated angiogenic factor production gradually undergoes down-regulation, thus hindering adaptive angiogenesis. We have previously reported on two strategies for delivering on demand hypoxia-induced signalling (HIS) in vivo, namely, implanting living or non-viable hypoxic cell-matrix depots that actively produce factors or act as carriers of factors trapped within the matrix during in vitro pre-conditioning, respectively. This study aims to improve this approach through the development of a novel, injectable system for delivering cell-free matrix HIS-carriers. 3D spiral collagen constructs, comprising an inner cellular and outer acellular compartment, were cultured under hypoxia (5% O2). Cell-produced angiogenic factors (e.g. VEGF, FGF, PLGF, IL-8) were trapped within the nano-porous matrix of the acellular compartment as they radially diffused through it. The acellular matrix was mechanically fragmented into micro-fractions and added into a low temperature (5 °C) thermo-responsive type I collagen solution, which underwent a collagen concentration-dependent solution-to-gel phase transition at 37 °C. Levels of VEGF and IL-8, delivered from matrix fractions into media by diffusion through collagen sol-gel, were up-regulated by day 4 of hypoxic culture, peaked at day 8, and gradually declined towards the baseline by day 20, while FGF levels were stable over this period. Factors captured within matrix fractions were bioactive after 3 months freeze storage, as shown by their ability to induce tubule formation in an in vitro angiogenesis assay. This system provides a minimally invasive, and repeatable, method for localised delivery of time-specific, cell-free HIS factor mixtures, as a tool for physiological induction of spatio-temporally controlled angiogenesis.

Vascular tissue engineering with magnetic nanoparticles: seeing deeper.

The endothelium bares a paramount therapeutic and diagnostic significance in vascular disease. The current work presents a novel strategy based on the use of superparamagnetic nanoparticles to obtain an endothelial cell lining on the luminal surface of vascular conduits, which can be detected non-invasively in a clinical Magnetic Resonance Imaging (MRI) scanner. Human umbilical vein endothelial cells (HUVECs) were prelabeled with clinically approved superparamagnetic nanoparticles. Cell viability and eNOS expression were not affected by the labelling procedure. Magnetically labelled cells were delivered onto the lumen of a PTFE tubular graft by a customised electromagnet. The endothelium was detected in a 1,5T MRI scanner. Magnetic cell delivery provides an efficient technique to seed tubular scaffolds enabling the non-invasive depiction of the cells from the substrate, thus providing a reliable tool to assess the quality of cell delivery procedures.

[Umbilical cord stromal cells (UCSC). Cells featuring osteogenic differentiation potential]. / Umbilical Cord Stromal Cells (UCSC). Zellen mit osteogenem Differenzierungspotenzial.

The identification of appropriate cell types is necessary to establish cell-based therapies in regenerative medicine. These cell types must (1) be available in an appropriate amount, (2) be easy to obtain, (3) be sufficiently expandable in vitro, and (4) fit to or at least be able to differentiate into the required cell type. Since the umbilical cord is available without any intervention and represents a notable amount of tissue, we consider it to be a promising source for isolating cells for cell-based therapies. This study demonstrates that umbilical cord stromal cells (UCSC), the connective tissue cells of the umbilical cord, can be isolated in sufficient quantities and be well expanded. UCSC feature phenotypic plasticity and thus are functionally similar to stem cells. UCSC can be differentiated into cells with osteoblastic properties (expression of alkaline phosphatase, formation of bone nodules). It is concluded that the umbilical cord should no longer be regarded as valueless tissue and be unthinkingly discarded. Instead, it should be considered a valuable resource for the isolation of potent cells for cell-based therapies, especially for treatment of bone defects.

Laminin inhibits lactotroph proliferation and is reduced in early prolactinoma development.

Laminin is a component of the extracellular matrix (ECM) that regulates cell proliferation and hormone secretion. Here we describe the effects of laminin on prolactin secretion in normal and tumor cells and analyze laminin expression pattern during prolactinoma development. Prolactin secretion and cell proliferation were inhibited by laminin in GH3 cells. In contrast, no effect was observed in normal pituitary cells. Laminin showed a dynamic expression pattern during prolactinoma development, which was: (a) strong in normal pituitaries from wild type or dopamine D2 receptor deficient mice, (b) lower in pituitary hyperplasia and (c) markedly reduced in prolactinomas from D2R -/- mice. A similar gradual decrease in laminin was found by comparing normal human pituitaries, human pituitary hyperplasia and human prolactinomas. These results show dynamic changes of laminin expression during prolactinoma formation which, due to laminin action on PRL production and cell proliferation, indicate a possible role for laminin in prolactinoma development.

Retinoic acid prevents experimental Cushing syndrome.

Cushing syndrome is caused by an excess of adrenocorticotropic hormone (ACTH) production by neuroendocrine tumors, which subsequently results in chronic glucocorticoid excess. We found that retinoic acid inhibits the transcriptional activity of AP-1 and the orphan receptors Nur77 and Nurr1 in ACTH-secreting tumor cells. Retinoic acid treatment resulted in reduced pro-opiomelanocortin transcription and ACTH production. ACTH inhibition was also observed in human pituitary ACTH-secreting tumor cells and a small-cell lung cancer cell line, but not in normal cells. This correlated with the expression of the orphan receptor COUP-TFI, which was found in normal corticotrophs but not in pituitary Cushing tumors. COUP-TFI expression in ACTH-secreting tumor cells blocked retinoic acid action. Retinoic acid also inhibited cell proliferation and, after prolonged treatment, increased caspase-3 activity and induced cell death in ACTH-secreting cells. In adrenal cortex cells, retinoic acid inhibited corticosterone production and cell proliferation. The antiproliferative action and the inhibition of ACTH and corticosterone produced by retinoic acid were confirmed in vivo in experimental ACTH-secreting tumors in nude mice. Thus, we conclude that the effects of retinoic acid combine in vivo to reverse the endocrine alterations and symptoms observed in experimental Cushing syndrome.

Vascular endothelial growth factor production and regulation in rodent and human pituitary tumor cells in vitro.

Angiogenesis, the formation of a new blood supply, is an essential step in tumorigenesis. Although vascular endothelial growth factor (VEGF) is known to be a very potent angiogenic factor in most solid tumors, little is known about its production and regulation in pituitary adenomas. We have investigated basal and stimulated VEGF production by rodent pituitary tumor cells (mouse corticotrope AtT20, rat lactosomatotrope GH3, mouse gonadotrope alpha T3-1 and mouse folliculostellate TtT/GF cells), and by hormone-inactive (27), corticotrope (9), lactotrope (3) and somatotrope (21) human pituitary adenoma cell cultures. All 4 pituitary cell lines secreted VEGF, which in the case of AtT20, GH3 and TtT/GF cells was inhibited by approximately 50% by dexamethasone. TtT/GF cells were the most responsive to the different stimuli used since basal values were augmented by pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), interleukin-6 (IL-6), transforming growth factor-alpha (TGF-alpha), IGF-I and the somatostatin analogue ocreotide. However, in GH3, AtT20 and alpha T3-1 cells, basal VEGF levels where not enhanced with any of the stimuli tested. The majority of the human adenomas tested (92%) basally secreted measurable VEGF which was inhibited by dexamethasone in most cases (84%). VEGF levels were increased in hormone inactive adenomas, somatotrope tumors and prolactinomas by TGF-alpha, PACAP-38, and 17 beta-estradiol, respectively. In conclusion, pituitary tumor cells are capable of producing VEGF which may be involved in tumoral angiogenesis. Our results concerning the suppression of VEGF by dexamethasone suggest that glucocorticoids may have anti-angiogenic properties and therefore therapeutic relevance for the treatment of pituitary adenomas.

Extracellular matrix components regulate ACTH production and proliferation in corticotroph tumor cells.

The extracellular matrix (ECM) conveys signals through membrane receptors called integrins producing changes in cell morphology, proliferation, differentiation and apoptosis. Previous studies suggest that the ECM plays an important role in pituitary physiology and tumorigenesis. In the present work we studied for the first time the effects of fibronectin, laminin, collagen I and collagen IV on hormone secretion and cell proliferation in the corticotroph tumor cell line AtT-20 and in normal pituitary cells, examining the signal transduction mechanisms that mediate these effects. ACTH production in AtT-20 cells was inhibited by fibronectin, laminin and collagen I. A reporter construct with the POMC promoter showed similar results, indicating that the effects of the ECM take place at the level of POMC gene transcription. In contrast, ACTH production was not significantly altered in normal pituitary cells. AtT-20 cell proliferation was stimulated by collagen IV and fibronectin, but inhibited by collagen I and laminin. In parallel, the cell morphology was modified by the ECM. We found that the production of reactive oxygen species mediate the effects of laminin and collagen IV. On the other hand, the effect of fibronectin was mimicked by beta1-integrin and Rho activation. These results show for the first time that the ECM controls ACTH biosynthesis and proliferation in corticotroph tumor cells and suggest a role for the ECM in corticotroph adenoma development.

Lipopolysaccharide directly stimulates the intrapituitary interleukin-6 production by folliculostellate cells via specific receptors and the p38alpha mitogen-activated protein kinase/nuclear factor-kappaB pathway.

Bacterial lipopolysaccharide (LPS) activates the immune system and induces increases in peripheral cytokines, which, in turn, affect the endocrine system. In particular, LPS-induced cytokines stimulate the hypothalamic-pituitary-adrenal axis to increase levels of antiinflammatory-acting glucocorticoids. In the present work, we show that LPS directly stimulates interleukin (IL)-6 release by mouse pituitary folliculostellate (FS) TtT/GF tumor cells and FS cells of mouse pituitary cell cultures. The stimulatory effect of LPS was strongly enhanced in the presence of serum, suggesting that LPS is only fully active as a complex with LPS-binding protein (LBP). Both TtT/GF cells and mouse pituitaries expressed CD14, which binds the LPS/LBP complex, and Toll-like receptor type 4, which induces LPS signals. LPS increased phospoinositol turnover in TtT/GF cells and induced phosphorylation of p38alpha mitogen-activated protein kinase and the inhibitor (IkappaB) of nuclear factor-kappa B. Nuclear factor-kappa B was activated by LPS in TtT/GF cells. Functional studies demonstrated that My4 (an antibody blocking the interaction between LPS/LBP and CD14), SB203580, (a specific inhibitor of p38alpha mitogen-activated protein kinase phosphorylation), dexamethasone, and the messenger RNA translation inhibitor cycloheximide all inhibited LPS-induced IL-6 production by TtT/GF cells and mouse pituitary FS cells. LPS-induced intrapituitary IL-6 may modulate the function of anterior pituitary cells during bacterial infection/inflammation.

Retinoic acid stimulates meningioma cell adhesion to the extracellular matrix and inhibits invasion.

Meningiomas are tumours derived from the arachnoid and pia mater. During embryogenesis, these membranes develop from the migrating craniofacial neural crest. We have previously demonstrated that meningiomas have characteristic features of embryonic meninges. Craniofacial neural crest derivatives are affected during normal development and migration by retinoic acid. We speculated, therefore, that meningioma cell migration and invasion would be affected in a similar way. In this study we investigated the mechanisms of invasion and migration in meningiomas and the effects of retinoic acid (RA). We found that low doses of RA inhibit in vitro invasion in meningioma cells, without affecting cell proliferation or viability. The matrix metalloproteinases MMP-2 (72 kDa gelatinase) and MMP-9 (92 kDa gelatinase), which play a key role in invasion in other tumours, are not affected by RA. RA inhibits cell migration on collagen I and fibronectin. A possible mechanism for these effects is provided by the fact that RA strongly stimulates adhesion of meningioma cells to extracellular matrix substrates. As in vitro invasion, migration and decreased adhesion to the extracellular matrix correlate with the clinical manifestation of tumour invasion, we conclude that RA induces a non-invasive phenotype in meningioma cells.

Interleukin-1 beta enhances interleukin-1 receptor antagonist content in human somatotroph adenoma cell cultures.

In addition to the well-known modulation of immune and inflammatory responses, the interleukin-1 (IL-1) system has been shown to be involved in the regulation of anterior pituitary hormone secretion and growth. We previously demonstrated that IL-1 receptor antagonist (IL-1ra) is expressed in human pituitary adenomas cultured in vitro. In the present study, we investigated the regulation of IL-1ra protein by IL-1 beta (1-100 U/mL) in human somatotroph adenomas (n = 9) cultured for 12-48 h. IL-1 beta significantly enhanced the concentration of IL-1ra dose dependently in the somatotroph adenoma cell lysates, whereas IL-1ra concentrations remained unchanged in the culture supernatants. Furthermore, basal IL-1ra concentrations were significantly higher in the cell lysates compared with the corresponding culture supernatants. The regulation of IL-1ra in somatotroph adenoma cells is different from human cultured monocytes, in which IL-1 beta significantly stimulated IL-1ra secretion into the culture supernatants, and no change of intracellular IL-1ra content was observed. Incubation of the somatotroph adenoma cells with 100 U/mL IL-1 beta did not result in a change of GH concentrations in the culture supernatants. Enhancement of intracellular IL-1ra protein by IL-1 beta may represent a mechanism intrinsic to somatotroph adenoma cells to counterregulate the response to IL-1 beta on hormone secretion or cellular growth.

Inhibition of lipopolysaccharide-induced monocyte interleukin-1 receptor antagonist synthesis by cortisol: involvement of the mineralocorticoid receptor.

Glucocorticoids, as a part of their physiological role in the control of inflammatory and immune processes, suppress the expression of interleukin-1 (IL-1) and other cytokines. Human monocyte IL-1 receptor antagonist (IL-1ra) messenger ribonucleic acid (mRNA) expression and protein secretion are inhibited by dexamethasone. We have now further studied the regulation of IL-1ra by the major physiological human glucocorticoid, cortisol. We found that cortisol incubation induced a decrease in IL-1ra mRNA expression and a significant inhibition of IL-1ra protein secretion in cell cultures of human peripheral monocytes stimulated with the bacterial endotoxin lipopolysaccharide (LPS). Oral administration of 276 mumol cortisol to normal subjects also decreased LPS-induced IL-1ra synthesis in cultured monocytes. By coincubating the monocytes with either the mineralocorticoid antagonist spironolactone or the glucocorticoid receptor antagonist RU 38486, the in vitro cortisol-induced inhibition of LPS-stimulated IL-1ra secretion was partially reversed. The mineralocorticoid aldosterone exerted a significant decrease in LPS-induced monocyte IL-1ra secretion in vitro, which was blocked by coincubation with spironolactone. In addition, the expression of mineralocorticoid receptor mRNA in human monocytes was observed by PCR of reversed transcribed RNA. Our results further indicate that corticosteroids physiologically control the IL-1/IL-1ra system during inflammatory or immune processes. Moreover, we provide evidence that, in addition to a glucocorticoid receptor-mediated effect, the mineralocorticoid receptor is involved in the inhibition of monocyte IL-1ra secretion by cortisol.

Expression and localization of endothelin-1 and endothelin receptors in human meningiomas. Evidence for a role in tumoral growth.

In addition to its well-known homoeostatic actions in the cardiovascular system, ET-1 has been shown to constitute a potent growth regulatory peptide in various tissues. We have studied the expression of ET-1 and its receptors (ET-Ar and ET-Br) in human meningiomas (n = 35) as well as their involvement in cellular growth. By PCR of reverse-transcribed RNA we detected ET-1 mRNA in 91% (32 of 35), ET-Ar mRNA in 82% (29 of 35), and ET-Br mRNA in 42% (15 of 35) of human meningiomas examined. The localization of ET-1 mRNA, ET-Ar mRNA, and ET-1 peptide in tumoral cells was observed by in situ hybridization and immunohistochemistry, whereas ET-Br mRNA was expressed at low level only in cells belonging to blood vessels. In addition, we found that ET-1 stimulated [3H] thymidine incorporation in primary cell cultures of 20 meningiomas and that this effect could be blocked by BQ-123, a specific antagonist for ET-Ar. In contrast, RES-701-3, an antagonist of ET-Br, did not block the proliferative effect of ET-1. In conclusion, our data provide evidence that ET-1 constitutes an important growth factor for meningiomas acting via ET-Ar. We can hypothesize that ET-1, acting in concert with other growth factors and cytokines, is involved in the meningioma tumorigenesis.

Cellular localization of endothelin receptor mRNAs (ETA and ETB) in brain tumors and normal human brain.

We performed in situ hybridization (ISH), using radioactively labeled deoxyoligonucleotides, to localize endothelin (ET) receptor subtype mRNAs (ETA and ETB) in a series of meningiomas in comparison to normal human meninges. Our examination also addressed the cerebellum adjacent to the meninges investigated. Tumor cells of all meningiomas examined showed strong hybridization signals for ETA receptor mRNA, whereas for ETB receptor mRNA only weak hybridization signals could be detected in endothelial cells of tumor blood vessels and in some cell clusters. mRNAs for neither ET receptor were detectable in normal leptomeninges. In human cerebellum we found ETA receptor mRNA only in large blood vessels; ETB receptor mRNA was intensely expressed in Bergmann glial cells. Neither subtype was detectable in neurons.

Expression of interleukin-1 receptor antagonist in human pituitary adenomas in vitro.

The production of cytokines and their receptors in the pituitary gland as well as receptor-mediated cytokine effects on pituitary function have been demonstrated. We have investigated whether the naturally occurring interleukin-1 receptor antagonist (IL-1ra), which has been shown to block IL-1 biological actions during inflammatory processes, could be expressed in human pituitary adenomas (n = 16) cultured in vitro. By polymerase chain reaction of reverse-transcribed RNA we detected IL-1ra messenger RNA in cultures of all types of pituitary adenomas under basal conditions as well as after stimulation of the cells with endotoxin or phorbol myristate acetate. The amplified complementary DNA fragment was identical to the fragment observed when RNA from purified human monocytes was subjected to reverse transcription polymerase chain reaction. In addition, we provide evidence that the IL-1ra messenger RNA detected in human pituitary adenomas corresponds to the intracellular IL-1ra variant. By using specific primers for the monocyte/macrophage marker CD14 as a control, we could exclude a contamination by monocytes or macrophages in the cell cultures of pituitary adenomas as a source of IL-1ra expression. Immunofluorescence studies showed the presence of cellular IL-1ra protein in the pituitary adenoma cultures and the colocalization with hormone-producing cells in GH- and ACTH-secreting adenomas. Production of IL-1ra within the anterior pituitary may act as a protective mechanism, modulating the sensitivity of pituitary cells to circulating or intrinsically produced IL-1 during inflammatory or tumoral processes.

Endothelin expression in normal human anterior pituitaries and pituitary adenomas.

Endothelins (ETs) are important regulators of growth and function in many endocrine tissues. This study was designed to verify the expression of ETs in a series of normal human pituitaries and pituitary adenomas. We examined 13 normal pituitaries and 58 pituitary adenomas for the presence of immunoreactive (ir) ET-1 and ET-3. No ir-ET-1 was detected in any of the 13 normal pituitaries, whereas ir-ET-3 was observed in 4 of 13 (31%) cases. In contrast, 48% (28 of 58) of pituitary adenomas display immunoreactivity for ET-1, whereas 31% (18 of 58) show immunoreactivity for ET-3. With respect to the type of tumors, staining was as follows: nonfunctioning adenomas: ET-1, 14 of 33; ET-3, 9 of 33; somatotropinomas: ET-1, 8 of 16; ET-3, 6 of 16; corticotropinomas: ET-1, 5 of 5; ET-3, 2 of 5; and prolactinomas; ET-1, 1 of 4; ET-3, 1 of 4. Using double immunostaining, we found the colocalization of ET-3 in normal pituitaries and of ET-1 and ET-3 in pituitaries adenomas in each hormone-secreting cell. In Cushing adenomas, ET-1 was coexpressed in corticotropic cells in all 5 cases (100%). In the same tumors, by reverse transcriptase polymerase chain reaction, we investigated the presence of ET-1 and ET-3 messenger ribonucleic acids and found that they are expressed, respectively, in 18 of 21 and 7 of 11 tumors examined. Our findings demonstrate that pituitary adenomas frequently display ET-1 as well as ET-3 immunoreactivity, in contrast to normal pituitaries, in which only ET-3 was found.(ABSTRACT TRUNCATED AT 250 WORDS)

High frequency vibration induced gating of subcortical and cortical median nerve somatosensory evoked potentials: different effects on the cervical N13 and on the P13 and P14 far-field SEP components.

Subcortical and cortical somatosensory evoked potentials (SEP) to median nerve stimulation were recorded before, during and after high frequency (270 Hz) vibration of the fingers 1-3 in 8 healthy subjects. A marked decrease of the amplitude of all potentials was observed. The attenuation of the sensory nerve action potential (SNAP) of the median nerve and the attenuation of SEP components N9, N11 and N13 showed no differences, while the attenuation of the subcortical P14 component was significantly higher. This is in accordance with a generator of the cervical N13 in the interneurons beside the lemniscal pathway. The cortical N20 (post-rolandic) was significantly more decreased in amplitude than P14 while P22 (pre-rolandic) remained reduced in amplitude like P14. An increased latency of the far-field subcortical P14 was observed, while P13 recorded in the same montage remained unchanged in latency. These findings suggest different generators of these peaks. A generator of P14 above the nucleus cuneatus is confirmed. A presynaptic generator of P13 is suspected.

[Dynamic properties of the ipsilateral tactile-elicited stapedius muscle reflex in probands with normal hearing]. / Untersuchungen über die dynamischen Eigenschaften des ipsilateral taktil ausgelösten Stapediusmuskelreflexes an Hörgesunden.

We tested 94 male and female persons with normal hearing. From these persons we elicited from the external ear canal the ipsilateral tactile stapedius muscle reflex and examined its dynamic properties. The electric stimulus consisted of a series of electric impulses of a duration of 30 msec lasting for 1 sec. The intensity ranged from 2.0-8.0 mA increasing in steps of 0.5 mA. Statistical evaluation showed a significant reflex dependence on stimulus intensity and sex of the volunteers but not on the volunteer's age. Starting with a stimulus intensity of 3.5 mA all tested persons showed good reflex response. The average latency between the start of the stimulus and the beginning of the reflex was 130-200 msec diminishing with growing stimulus intensity. The best parameters describing the dynamic properties of the reflex seem to be the latencies L 1 and L 3 at a stimulus intensity of 3.5 - 6.0 mA.

[Long-term changes in the immune system following cytostatic polychemotherapy in patients with testicular tumors in remission]. / Langzeitveränderungen des Immunsystems nach zytostatischer Polychemotherapie bei Patienten mit Hodentumoren in Remission.

Patients who received combination chemotherapy for metastatic nonseminomatous testicular cancer showed a decreased Con A induced suppressor cell function. Additionally 15% of the patients who received ifosfamid had a decreased IgM-serum concentration, an increased number of macrophages and a decreased proliferation rate in MLC.