Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell population for tissue-engineering purposes. MSCs from bone marrow (bone marrow stromal cells [BMSCs]) are negative for immunologically relevant surface markers and inhibit proliferation of allogenic T cells in vitro. Therefore, BMSCs are said to be available for allogenic cell therapy. Although the immunological characteristics of BMSCs have been the subject of various investigations, those of stem cells isolated from adipose tissue (ASCs) have not been adequately described. In addition, the influence of osteogenic differentiation in vitro on the immunological characteristics of BMSCs and ASCs is the subject of this article. Before and after osteogenic induction, the influence of BMSCs and ASCs on the proliferative behavior of resting and activated allogenic peripheral blood mononuclear cells (PBMCs) was studied as a measure of the immune response (mixed lymphocyte culture). At the same points, the expression of immunologically relevant surface markers (e.g., major histocompatibility complex (MHC)-I, MHC-II, CD40, CD40L) was measured, and correlations between the different sets of results were sought. The pattern of surface antigen expression of BMSCs is the same as that of ASCs. Analogous to BMSCs, undifferentiated cells isolated from adipose tissue lack expression of MHC-II; this is not lost in the course of the osteogenic differentiation process. In co-culture with allogenic PBMCs, both cell types fail to lead to any significant stimulation, and they both retain these characteristics during the differentiation process. BMSCs and ASCs suppress proliferation on activated PBMCs before and after osteogenic differentiation. Our results confirm that MSCs are immune modulating cells. These properties are retained even after osteogenic induction in vitro and seem to be similar in BMSCs and ASCs. Our results suggest that allogenic transplantation of BMSCs and ASCs would be possible, for example, in the context of tissue engineering.
A novel Hodgkin's disease (HD) derived cell line, L1236, was established from the peripheral blood of a patient with advanced Hodgkin's disease. Analysis of immunoglobulin (Ig) gene rearrangements revealed a biallelic Ig heavy chain and a monoallelic Ig kappa light chain gene rearrangement, pointing to a B-lymphoid origin of these cells. No DNA of Epstein-Barr virus was detected in L1236. The cells expressed the HD-associated surface antigens CD30 and CD15 as well as the transferrin receptor (CD71). Cytogenetic analysis of early passages of L1236 cells revealed a grossly disordered karyotype including cytogenetic aberrations described previously in other HD-derived cell lines. The Hodgkin/Reed-Sternberg (H-RS) cell origin of L1236 cells is further confirmed by Kanzler et al (Blood 87:3429, 1996), who found identical Ig gene rearrangement sequences in L1236 cells and H-RS cells of the same patient's bone marrow. L1236 cells expressed antigens necessary for efficient antigen presentation to T cells including HLA class I and II, B7.1 and B7.2, as well as adhesion molecules ICAM 1 and LFA 3. The cells secreted the interleukins (IL)-6, -8, -10, tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, transforming growth factor (TGF) beta, and the granulocyte-macrophage colony stimulating factor (GM-CSF). After subcutaneous inoculation into SCID mice, a necrotic regression of initially growing tumors at the injection site was followed by disseminated intralymphatic growth. Our findings, together with the results of Kanzler et al, demonstrate that H-RS cells of B-lymphoid origin were present in the peripheral blood of a patient with advanced HD. These cells exerted a malignant phenotype with regard to their in vitro and in vivo characteristics.
Ubiquitous mitochondrial creatine kinase (uMtCK), a key enzyme in energy metabolism, was identified by differential display PCR to be specifically overexpressed in L1236, the first cell line of definite Hodgkin origin. RT-PCR confirmed overexpression of uMtCK in the L1236 cell line and the absence of cytosolic B-CK, which is co-expressed with MtCK physiologically. Cyclocreatine (cCr), whose phosphorylated form is a very poor substrate for CK, inhibited proliferation of the L1236 cell line nearly entirely. This inhibition by cCr was partially reversed by competition with creatine, which by itself had no effect on proliferation of the L1236 cell line. In Hodgkin disease (HD), the telltale Hodgkin and Reed-Sternberg (H-RS) cells are surrounded by reactive tissue components. The scarcity of the malignant cells has hampered their genetic analysis. Only recently, single cell PCR analysis could show a clonal B-cell origin of the H-RS cells. 1 Sequence analysis demonstrated that these cells lost their immunoglobulin (Ig) expression due to crippling mutations acquired in the germinal centre. Normal B-cells unable to express Ig are prevented from antigenic selection and are eliminated by apoptosis. The transforming event in HD allowing the cells to escape from programmed cell death is not known. One possible candidate is Epstein-Barr virus (EBV), which is found in 50% of HD cases and has been shown to upregulate the antiapoptotic gene bcl-2 in infected cells. 2 In addition, the complex chromosomal aberrations seen in HD 3 may cause changes in gene expression resulting in resistance to apoptosis. Because of the considerable karyotypic variation from case to case, different molecular mechanisms may be involved in malignant transformation in HD and, in support of this, no HD specific genetic aberration could be identified so far.One of the mechanisms possibly involved in malignant transformation is overexpression of creatine kinase (CK). The enzyme reversibly catalyzes the transfer of a phosphate group from ATP to creatine (Cr) and occurs as different isoforms. The cytosolic braintype isoform (B-CK) is coexpressed with the ubiquitous mitochondrial isoform (uMtCK) in many cells and tissues with a high energy demand like brain, placenta, kidney, testis, sperm or endothelial cells. 4 Overexpression of CK has been demonstrated in a wide variety of solid tumors 5-8 and tumor cell lines. 9 Elevated levels of CK have been detected in the sera of patients with advanced-stage cancer and are characteristic for a poor prognosis. 6,8,10 CK catalyzes the reversible transphosphorylation between ATP and creatine: creatine ϩ ATP 7 phosphocreatine ϩ ADP.The enzyme is thought to have 2 main functions in energy metabolism. 4 It buffers the cellular ATP pool by maintaining high cytosolic concentrations of phosphocreatine (PCr), which can be used in times of high cellular energy demand for the regeneration of ATP and it maintains an energy shuttle between subcellular sites of energy supply (oxidative phosphorylation, glycolysis) and sites of ene...
Hodgkin–Reed-Sternberg (H-RS) cells are clonal B cells carrying Ig gene rearrangements. However, in situ hybridization methods failed to demonstrate Ig gene expression in H-RS cells of classical Hodgkin’s disease (HD). Because somatic mutations rendering potentially functional Ig gene rearrangements nonfunctional were detected in some cases of the disease, it was speculated that H-RS cells in classical HD may have lost the ability to express antigen receptor as a rule. Recently, we established a novel cell line (L1236) from H-RS cells of a patient with mixed cellularity subtype of HD. L1236 cells harbor a potentially functional VH1 and a potentially functional Vκ3 gene rearrangement. However, no antibody expression was detected. To show potential reasons for this lack of Ig expression, we analyzed the genomic organization of the Ig genes and their transcription in the primary and cultivated H-RS cells of this patient. The H-RS cells were found to have switched their isotype to IgG4, confirming their mature B-cell nature. By amplifying cDNA from L1236 cells as well as from frozen biopsy material transcripts of the Vκ3 and the VH1 gene rearrangement were detected for both sources of cDNA. However, Northern blot hybridization of L1236 RNA failed to demonstrate VH1 and Vκ3 transcripts, indicating only a low level of transcription. Sequence analysis of the promoter and leader regions of the VH1 gene rearrangement from L1236 cells as well as from lymphoma-affected tissue showed a somatic mutation in the conserved octamer motif of the promoter region. Somatic mutations were also detected within the 3′ splice site of the leader intron and adjacent nucleotides in the rearranged Vκ light chain gene, leading to aberrant splicing. These mutations might prevent the generation of adequate amounts of functional Ig gene transcripts as template for translation into protein. Thus, mutations in H-RS cells that prevent Ig gene expression might also be located outside the coding region of the Ig genes.
Summary. We analysed multiple serum samples from a patient with mixed cellularity Hodgkin's disease for the Hodgkin/Reed-Sternberg cell clone-speci®c rearranged Ig gene sequence. The clone-speci®c sequence could be detected in DNA extracted from a serum sample obtained during clinical relapse but not in serum samples obtained during or after treatment following relapse.
This finding indicates the existence of a clonal H-RS cell population during the first manifestation of HD and persistence and dissemination of this clone despite aggressive treatment. Thus, in the described case the malignant nature of H-RS cells defined by dissemination and recurrence of the identical H-RS cell clone in relapsing disease is proven.
Summary Hodgkin-Reed Sternberg cells are derived from germinal centre B-cells in most cases. Somatic mutations affecting their rearranged immunoglobulin genes were detected, rendering potential functional rearrangements non-functional. Under physiological conditions such cells would be designated to undergo apoptosis within the germinal centre. In search for the specific transforming event that prevents Hodgkin-Reed Sternberg cells from programmed cell death, cytogenetic analyses were broadly performed but did not reveal specific chromosomal aberrations. Analysis of these cells on the molecular level is difficult to perform due to the scarcity of the cells in the lymphoma tissue and the given limitations of in situ studies. To overcome these limitations, the cell line L1236, known to be derived from Hodgkin-Reed Sternberg cells in situ, was chosen for allelotype analysis. Using a panel of microsatellite loci assigned to nearly all chromosomal arms, regions of loss of heterozygosity were detected on chromosomal arms 6p, 9q and 17p. The size of lost segments was estimated by amplification of additional microsatellite loci mapped to the respective regions. Further analyses of single Hodgkin-Reed Sternberg cells will reveal whether LOH affecting these regions is a recurrent event in HD and to which extent the smallest commonly affected region can be estimated.
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