Human bone marrow mesenchymal stem cells and chondrocytes promote and/or suppress the in vitro proliferation of lymphocytes stimulated by interleukins 2, 7 and 15.

OBJECTIVES: To investigate whether human bone marrow-derived mesenchymal stem cells (BM-MSCs) and articular chondrocytes (ACs) affect the in vitro proliferation of T lymphocytes and peripheral blood mononuclear cells (PBMCs) driven by the homeostatic interleukin (IL)2, IL7 and IL15 cytokines binding to the common cytokine receptor gamma-chain (gamma(c)) in the absence of T cell receptor (TCR) triggering. METHODS: PBMCs, total T cells and T cell subsets (CD4+ and CD8+) were stimulated with IL2, IL7 or IL15 and exposed to cultured BM-MSCs and ACs at varying cell:cell ratio either in contact or in transwell conditions. Lymphocyte proliferation was measured by (3)H-thymidine uptake or by flow cytometry of carboxyfluorescein succinimidyl ester (CFSE)-labelled lymphocytes. RESULTS: MSCs and ACs enhanced and inhibited lymphocyte proliferation depending on the extent of lymphocyte baseline proliferation and on the MSC/AC to lymphocyte ratio. Enhancement was significant on poorly proliferating lymphocytes and mostly at lower MSC/AC to lymphocyte ratio. Suppression occurred only on actively proliferating lymphocytes and at high MSC/AC to lymphocyte ratio. Neither enhancement nor inhibition required cell-cell contact. CONCLUSIONS: There is a dichotomous effect of MSCs/ACs on lymphocytes proliferating in response to the homeostatic IL2, IL7 and IL15 cytokines likely to be encountered in homeostatic and autoimmune inflammatory conditions. The effect is determined by baseline lymphocyte proliferation, cell:cell ratio and is dependent on soluble factor(s). This should be taken into account when planning cellular therapy for autoimmune disease (AD) using stromal-derived cells such as MSCs.

Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein.

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.

Phenotypical and functional characteristics of in vitro expanded bone marrow mesenchymal stem cells from patients with systemic sclerosis.

BACKGROUND: Mesenchymal stem cells (MSCs) have a potential immunomodulatory role in autoimmune disease; however, the qualitative properties and haematopoietic support capacity of MSCs derived from patients with autoimmune disease is unclear. OBJECTIVES: To further characterise phenotypically and functionally bone marrow (BM)-derived MSCs from patients with systemic sclerosis (SSc). METHODS: Key parameters of BM-derived MSC function and phenotype were assessed in 12 patients with SSc and compared with 13 healthy normal controls. The parameters included the ability to: form colony-forming unit fibroblasts (CFU-F), differentiate along the adipogenic and osteogenic lineages, express cell surface antigens defining the MSCs population, support normal haematopoiesis and suppress in vitro lymphocyte proliferation induced by either anti-CD3epsilon plus anti-CD28 monoclonal antibodies or the mixed lymphocyte reaction. RESULTS: SSc MSCs were shown to have a similar characteristic phenotype, capacities to form CFU-F and to differentiate along adipogenic and osteogenic lineages as those of healthy donor MSCs. The ability of SSc MSCs to support long-term haematopoiesis was also identical to that of controls. Both healthy donor and SSc BM MSCs reduced the proliferation of autologous and allogeneic peripheral blood mononuclear cells in a cell number dependent fashion. CONCLUSIONS: These results show that BM-derived MSCs from patients with SSc under the described culture conditions exhibit the same phenotypic, proliferative, differentiation potential and immunosuppressive properties as their healthy counterparts and could therefore be considered in an autologous setting. Further studies are needed to ensure the quality and safety of large-scale expansion of patient MSCs prior to their potential use in clinical trials.

DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum.

The Simian 11 rotavirus glycoprotein VP7 is directed to the endoplasmic reticulum (ER) of the cell and retained as an integral membrane protein. The gene coding for VP7 predicts two potential initiation codons, each of which precedes a hydrophobic region of amino acids (H1 and H2) with the characteristics of a signal peptide. Using the techniques of gene mutagenesis and expression, we have determined that either hydrophobic domain alone can direct VP7 to the ER. A protein lacking both hydrophobic regions was not transported to the ER. Some polypeptides were directed across the ER membrane and then into the secretory pathway of the cell. For a variant retaining only the H1 domain, secretion was cleavage dependent, since an amino acid change which prevented cleavage also stopped secretion. However, secretion of two other deletion mutants lacking H1 and expressing truncated H2 domains was unaffected by this mutation, suggesting that these proteins were secreted without cleavage of their NH2-terminal hydrophobic regions or secreted after cleavage at a site(s) not predicted by current knowledge.

The Escherichia coli prr region encodes a functional type IC DNA restriction system closely integrated with an anticodon nuclease gene.

The prr locus was originally described as coding a ribonuclease that is activated after phage T4 infection to cut within the anticodon of a specific tRNA, inactivating protein synthesis and thus blocking phage development. Wild-type T4 phage has two genes coding the enzymes polynucleotide kinase and RNA ligase, whose only function seems to be to repair the damage done by the anticodon nuclease. As the only apparent function of the prr ribonuclease is to combat phage infection, it can be considered as an RNA-based restriction enzyme. In non-infected cells, the prr enzyme is kept inactive in a complex with three other proteins which were predicted on the basis of DNA homologies to be the subunits of a type IC DNA restriction and modification system. Unlike other type IC systems so far characterized, prr is chromosomally rather than plasmid coded. However, sequences upstream from prr also have homology with sequences from the plasmid R124 and the prophage P1. We have now investigated the prr system and shown that it is indeed a bona fide type IC system which we call EcoprrI, and which is active both in vivo and in vitro. The system is fully functional even in the absence of the anticodon nuclease and seems to be a typical type I enzyme. EcoprrI recognizes the sequence CCA(N7)RTGC. One peculiarity is that, with low efficiency, EcoprrI will recognize and methylate variants of its recognition sequence such as CCT(N7)ATGC, which is methylated in one strand of the DNA only.

Binding of proteins from embryonic and differentiated cells to a bidirectional promoter contained within a CpG island.

We have analysed binding sites of nuclear protein factors to a CpG island (HTF9), which contains the promoter for a pair of overlapping, divergently-transcribed "housekeeping" genes. Using DNaseI protection assays with extracts from a range of differentiated and undifferentiated cell lines, including mouse embryonic stem (ES) and embryonal carcinoma (EC) cells, we located multiple protein binding sites on HTF9. Most of the sites were outside the defined core promoter and could bind to previously identified transcription factors. These included constitutive, inducible and apparently tissue-specific factors in an extremely asymmetric array relative to the transcription start sites of the two genes. A number of sites showed different binding specificities or affinities in different cell types, including ES cells. However, we found no factors that were specific for both ES and EC cells, and no protein-binding site protected exclusively in undifferentiated embryonic cells.

Autologous hematopoietic stem cell transplantation for autoimmune diseases.

Experimental data and early phase I/II studies suggest that high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (HSCT) can arrest progression of severe autoimmune diseases. We have evaluated the toxicity and disease response in 473 patients with severe autoimmune disease treated with autologous HSCT between 1995 and 2003, from 110 centers participating in the European Group for Blood and Marrow Transplantation (EBMT) autoimmune disease working party database. Survival, transplant-related mortality, treatment response and disease progression were assessed. In all, 420 patients (89%; 86+/-4% at 3 years, median follow-up 20 months) were alive, 53 (11%) had died from transplant-related mortality (N=31; 7+/-3% at 3 years) or disease progression (N=22; 9+/-4% at 3 years). Of 370 patients, 299 evaluable for response (81%) showed a treatment response, which was sustained in 213 (71% of responders). Response was associated with disease (P<0.001), was better in patients who received cyclophosphamide during mobilization (relative risk (RR)3.28 (1.57-6.83)) and was worse with increasing age (>40 years, RR0.29 (0.11-0.82)). Disease progression was associated with disease (P<0.001) and conditioning intensity (high intensity, RR1; intermediate intensity, RR1.81 (0.96-3.42)); low intensity, RR2.34 (1.074-5.11)). These data from the collective EBMT experience support the hypothesis that autologous HSCT can alter disease progression in severe autoimmune disease.

[Therapeutic intensification and autologous stem cell transplantation in autoimmune diseases]. / Intensification thérapeutique et autogreffe de cellules souches hématopoïétiques pour le traitement des maladies auto-immunes.

THE PATHOPHYSIOLOGY of most autoimmune diseases is often poorly understood. EXPERIMENTAL CONSIDERATIONS and clinical experience suggest that high doses immunoablation followed by stem cell transplantation is a therapeutic option to consider for certain severe autoimmune disorders. THE CONCEPT OF RESTORING NORMAL IMMUNE REACTIVITY must in part br true since current results of 466 transplants (445 autologous, 21 allogeneic) patients suffering from various autoimmune diseases show a beneficial outcome in approximately 2/3 of the patients. TO IMPROVE THE EFFICACY AND SAFETY OF SUCH AN AGGRESSIVE PROCEDURE in patients with potentially affected vital organs by the underlying autoimmune disease, it is especially important to follow international consensus guidelines and to centrally collect clinical data for in depth analysis in the EBMT International Stem Cell Project for Autoimmune Disease in Basel, Switzerland. PHASE III STUDIES ARE RUNNING FOR SYSTEMIC SCLEROSIS (Astis, Autologous Stem cell Transplantation International Rheumatoid Arthritis Trial) started in 2003. A STUDY PROJECT IS PLANNED FOR MULTIPLE SCLEROSIS (Astims, Autologous Stem cell Transplantation International Multiple Sclerosis).

Consensus statement concerning cardiotoxicity occurring during haematopoietic stem cell transplantation in the treatment of autoimmune diseases, with special reference to systemic sclerosis and multiple sclerosis.

Autologous haematopoietic stem cell transplantation is now a feasible and effective treatment for selected patients with severe autoimmune diseases. Worldwide, over 650 patients have been transplanted in the context of phase I and II clinical trials. The results are encouraging enough to begin randomised phase III trials. However, as predicted, significant transplant-related morbidity and mortality have been observed. This is primarily due to complications related to either the stage of the disease at transplant or due to infections. The number of deaths related to cardiac toxicity is low. However, caution is required when cyclophosphamide or anthracyclines such as mitoxantrone are used in patients with a possible underlying heart damage, for example, systemic sclerosis patients. In November 2002, a meeting was held in Florence, bringing together a number of experts in various fields, including rheumatology, cardiology, neurology, pharmacology and transplantation medicine. The object of the meeting was to analyse existing data, both published or available, in the European Group for Blood and Marrow Transplantation autoimmune disease database, and to propose a safe approach to such patients. A full cardiological assessment before and during the transplant emerged as the major recommendation.

Ovarian follicular maturation in women. I. Intermittent versus daily administration of human menopausal gonadotropin.

This study was designed to compare the efficiency of daily intramuscular human menopausal gonadotropin (hMG) therapy to an intermittent subcutaneous regimen in women with ovulatory dysfunction. Ovarian follicular development was assessed with the use of serial ultrasound scans and serum 17 beta-estradiol (E2) levels. Ovulation was based on pregnancy and/or elevated luteal progesterone values. There were no significant differences between the two treatment regimens, based on E2 levels, follicular size, and/or number of follicles greater than 1.0 cm. Pregnancy and ovulation occurred more frequently after intramuscular hMG therapy. These results indicate that daily hMG is preferred over intermittent subcutaneous administration because of convenience.

Allogeneic hematopoietic SCT for patients with autoimmune diseases.

Allogeneic hematopoietic SCT (HSCT) has been used as treatment for single patients with autoimmune diseases (AD). To summarise currently available information, we analyzed all patients who underwent allogeneic HSCT for AD and who reported to the European Group for Blood and Marrow Transplantation (EBMT) database. Thirty-five patients receiving 38 allogeneic transplantations for various hematological and non-hematological AD were identified. Four patients had had an allogeneic HSCT for a conventional hematological indication in the past. Fifty-five per cent of the transplantation procedures led to a complete clinical response of the refractory AD and 23% to at least a partial response. The median duration of response at the last follow-up was 70.7 (15.2-130) months. Three patients relapsed at a median of 12.3 months after HSCT. Treatment-related mortality at 2 years was 22.1% (95% CI: 7.3-36.9%). Two deaths were caused by progression of AD. The probability of survival at 2 years was 70%. No single factor predicting the outcome could be identified. The retrospective nature of this study and the heterogeneous, partly incomplete data are its limitations. However, allogeneic HSCT can induce remission in patients suffering from refractory AD. These data provide the basis for carefully conducted prospective trials.

Bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donors and auto-immune disease patients reduce the proliferation of autologous- and allogeneic-stimulated lymphocytes in vitro.

OBJECTIVES: To investigate the ability of bone marrow (BM)-derived mesenchymal stromal cells (BM-MSCs) in suppressing the proliferation of stimulated lymphocytes across a range of conditions including autologous BM-MSCs derived from autoimmune disease (AD) patients. METHODS: In vitro cultures of BM-MSCs from healthy donors and AD patients were established and characterized by their differentiation potential into adipocytes and osteoblasts, and their fibroblast-colony-forming unit (CFU-F) ability and phenotype by flow cytometry. BM-MSCs (irradiated and non-irradiated) from healthy and AD patients were tested for their ability to suppress the in vitro proliferation of autologous and allogeneic peripheral blood mononuclear cells (PBMC) (from healthy donors and patients suffering from various ADs) stimulated with anti-CD3epsilon antibody alone or in combination with anti-CD28 antibody. The anti-proliferative effect of the BM-MSCs from healthy donors was tested also on transformed B-cell lines as a model of non-antigen-stimulated lymphocytes. RESULTS: BM-MSCs from healthy donors and AD patients reduced the proliferation of autologous and allogeneic PBMCs by up to 90% in a cell dose-dependent fashion. The immunosuppression was independent of the proliferation of the BM-MSCs and was also effective on already proliferating cells. It was independent also of the clinical activity of AD. An MSC dose-dependent pattern of suppression of proliferation was observed also with transformed B-cell lines, similar to that observed with proliferating PBMC. CONCLUSIONS: The BM-MSCs exhibit extensive anti-proliferative properties against lymphocytes under different conditions. This property might offer a form of immunomodulatory cellular therapy for AD patients if further confirmed in animal models.

Clinical risk assessment of organ manifestations in systemic sclerosis: a report from the EULAR Scleroderma Trials And Research group database.

BACKGROUND: Systemic sclerosis (SSc) is a multisystem autoimmune disease, which is classified into a diffuse cutaneous (dcSSc) and a limited cutaneous (lcSSc) subset according to the skin involvement. In order to better understand the vascular, immunological and fibrotic processes of SSc and to guide its treatment, the EULAR Scleroderma Trials And Research (EUSTAR) group was formed in June 2004. AIMS AND METHODS: EUSTAR collects prospectively the Minimal Essential Data Set (MEDS) on all sequential patients fulfilling the American College of Rheumatology diagnostic criteria in participating centres. We aimed to characterise demographic, clinical and laboratory characteristics of disease presentation in SSc and analysed EUSTAR baseline visits. RESULTS: In April 2006, a total of 3656 patients (1349 with dcSSc and 2101 with lcSSc) were enrolled in 102 centres and 30 countries. 1330 individuals had autoantibodies against Scl70 and 1106 against anticentromere antibodies. 87% of patients were women. On multivariate analysis, scleroderma subsets (dcSSc vs lcSSc), antibody status and age at onset of Raynaud's phenomenon, but not gender, were found to be independently associated with the prevalence of organ manifestations. Autoantibody status in this analysis was more closely associated with clinical manifestations than were SSc subsets. CONCLUSION: dcSSc and lcSSc subsets are associated with particular organ manifestations, but in this analysis the clinical distinction seemed to be superseded by an antibody-based classification in predicting some scleroderma complications. The EUSTAR MEDS database facilitates the analysis of clinical patterns in SSc, and contributes to the standardised assessment and monitoring of SSc internationally.

Autologous stem cell transplantation for progressive multiple sclerosis: update of the European Group for Blood and Marrow Transplantation autoimmune diseases working party database.

Over the last decade, hematopoietic stem cells transplantation (HSCT) has been increasingly used in the treatment of severe progressive autoimmune diseases. We report a retrospective survey of 183 multiple sclerosis (MS) patients, recorded in the database of the European Blood and Marrow Transplantation Group (EBMT). Transplant data were available from 178 patients who received an autologous graft. Overall, transplant related mortality (TRM) was 5.3% and was restricted to the period 1995-2000, with no further TRM reported since then. Busulphan-based regimens were significantly associated with TRM. Clinical status at the time of transplant and transplant techniques showed some correlations with toxicity. No toxic deaths were reported among the 53 patients treated with the BEAM (carmustine, etoposide, cytosine-arabinoside, melphalan)/antithymocyte globulin (ATG) regimen without graft manipulation, irrespective of their clinical condition at the time of the transplant. Improvement or stabilization of neurological conditions occurred in 63% of patients at a median follow-up of 41.7 months, and was not associated with the intensity of the conditioning regimen. In this large series, HSCT was shown as a promising procedure to slow down progression in a subset of patients affected by severe, progressive MS; the safety and feasibility of the procedure can be significantly improved by appropriate patient selection and choice of transplant regimen.

Sequences at the capped 5'-ends of polyoma virus late region mRNAs: an example of extreme terminal heterogeneity.

We have localized with respect to the genomic DNA sequence the capped 5'-termini of polyoma virus late region mRNAs. A minimum of fifteen different purine termini were found within a 94 base pair region (66.36 to 68.12 map units, nt 5075-5168) immediately preceding the sequence determining the late region mRNA leader repeat (1-3). The most common termini occur at nearly every possible purine within a 25 bp sequence proximal to the leader repeat unit. These do not bear the usual positional relationship to a sequence resembling the 'TATA' box consensus. Deletion mutants lacking minor cap sites and sequences upstream from the principal cap sites were viable. A deletion mutant lacking one of the principal cap sites formed small plaques, while a slightly larger deletion further impinging on the principal cap site region was non-viable. The principal cap sites, which we assume to be transcriptional initiation points, are included in a DNaseI hypersensitive region of polyoma virus chromatin (4).

Deletion mapping of a short polyoma virus middle T antigen segment important for transformation.

Viable polyoma virus mutants were constructed that had small deletions in the early region of the genome. The deletions together removed most of the segment missing from the genome of the nontransforming mutant dl23 (N. Smolar and B. E. Griffin, J. Virol. 38:958-967, 1981). The transformation properties, as measured by colony formation in soft agar, of mutants with overlapping or contiguous deletions showed that part or all of the middle T antigen segment, consisting of the short amino acid sequence Glu4-Tyr-Met-Pro-Met, was essential for the activity of the protein in transformation. However, the segment could be deleted without significant effect on the in vitro protein kinase activity associated with the middle T antigen.

Replication and interaction of virus DNA and cellular DNA in mouse cells infected by a human adenovirus.

C57 black mouse cells infected with human adenovirus type 5 (Ad5) produced large amounts of early virus proteins, small amounts of late virus proteins and less than 0.2 infectious units (i.u.)/cell of infectious virus. Many cells died but the cultures recovered. Virus DNA and cellular DNA were synthesized. Some Ad5 DNA sedimented with cell DNA in alkaline sucrose, but virus DNA was rapidly lost from the culture after recovery and none of 28 unselected cloned survivors contained detectable amounts of virus DNA or antigens. Ad5 ts36 was temperature-sensitive for virus DNA replication in mouse cells, but ts125 was detective at 32.5 degrees C as well as at 39.9 degrees C. No difference was detected in the percentage of virus DNA that sedimented in alkali with cell DNA, in mouse cells infected by Ad5 ts+, ts36 or ts125 at 32.5 or 39.9 degrees C. All parts of the virus genome were equally represented in virus DNA that sedimented with cell DNA, in mouse cells infected by Ad5 ts+ or ts36 at either temperature.

Transcriptional 'enhancers' from SV40 and polyoma virus show a cell type preference.

Transcriptional "enhancers" have been identified in both the monkey virus SV40 and in the mouse polyoma virus. Here we report that these enhancers show a cell type preference. This was done, (i) by assaying for T antigen expression and viral DNA replication of polyoma DNA which contains its own enhancer or the enhancer of SV40 virus, and (ii) by linking either of the two enhancer elements to the rabbit beta 1-globin gene and measuring transient globin expression in mouse and primate cells. The results were consistent in all the assays: In mouse cells the polyoma enhancer is slightly more effective than the SV40 enhancer. However, in primate cells the SV40 enhancer induces a four to six fold higher level of gene expression than does the polyoma enhancer.

The high affinity binding site on polyoma virus DNA for the viral large-T protein.

In order to map the high affinity binding site for the viral large-T protein on polyoma virus DNA, we have developed an assay which does not require purified protein. It is based on the specific elution of the large-T ATPase activity from calf thymus DNA cellulose by recombinant DNA molecules including known sequences of the viral DNA. Using this assay, a high affinity binding site has been mapped on the early region side of the ori region. Binding requires the integrity of a sequence /AGAGGC/TTCC/AGAGGC/ (nucleotides 49 to 64 in the DNA sequence of the A2 strain). Similar repeats of a PuGPuGGC sequence within less than 20 bases are not found within the viral coding regions, but are strikingly common in the control regions of papovaviruses and other eukaryotic DNAs.