High frequency of donor chimerism after allogeneic transplantation of CD34+-selected peripheral blood cells

Ex vivo T cell depletion of allogeneic grafts is associated with a high (up to 80%) rate of mixed chimerism (MC) posttransplantation. The number of transplanted progenitor cells is an important factor in achieving complete donor chimerism in the T cell depletion setting. Use of granulocyte colony-stimulating factor (G-CSF) peripheral blood allografts allows the administration of large numbers of CD34+ cells. We studied the chimeric status of 13 patients who received allogeneic CD34+-selected peripheral blood progenitor cell transplants (allo-PBPCTs/CD34+) from HLA-identical sibling donors. Patients were conditioned with cyclophosphamide (120 mg/kg) and total-body irradiation (13 Gy in four fractions). Apheresis products were T cell-depleted by the immunoadsorption avidin-biotin method. The median number of CD34+ and CD3+ cells infused was 2.8x10(6)/kg (range 1.9-8.6x10(6)/kg) and 0.4x10(6)/kg (range 0.3-1x10(6)/kg), respectively. Molecular analysis of the engraftment was performed using polymerase chain reaction (PCR) amplification of highly polymorphic short tandem repeat (PCR-STR) sequences in peripheral blood samples. MC was detected in two (15%) of 13 patients. These two patients relapsed at 8 and 10 months after transplant, respectively. The remaining 11 patients showed complete donor chimerism and were in clinical remission after a maximum follow-up period of 24 months (range 6-24 months). These results were compared with those obtained in 10 patients who were treated with T cell-depleted bone marrow transplantation by means of elutriation and who received the same conditioning treatment and similar amounts of CD3+ cells (median 0.45x10(6)/kg; not significant) but a lower number of CD34+ cells (median 0.8x10(6)/kg; p = 0.001). MC was documented in six of 10 patients (60%), which was significantly higher than in the allo-PBPCT/CD34+ group (p = 0.04). We conclude that a high frequency of complete donor chimerism is achieved in patients receiving allo-PBPCT/CD34+ and that this is most likely due to the high number of progenitor cells administered.

Tyrosine kinase inhibitor insensitivity of non-cycling CD34+ human acute myeloid leukaemia cells with FMS-like tyrosine kinase 3 mutations.

The efficacy of tyrosine kinase (TK) inhibitors on non-cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non-cycling cells from a small series of human FMS-like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non-cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non-cycling cells, indicating their leukaemic origin. Significantly, the FLT3-internal tandem duplication (ITD) mutation was found in the non-cycling fraction of four out of five cases. Exposure to the FLT3-directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short-term cultures and colony-forming unit assays. Crucially, non-cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer-term cultures, TKI258-treated non-cycling cells showed no growth impairment compared to treatment-naive non-cycling cells. These findings suggest that non-cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.

Angiogenesis is an independent prognostic factor in malignant mesothelioma

Angiogenesis is essential for tumour growth beyond 1 to 2 mm in diameter. The clinical relevance of angiogenesis, as assessed by microvessel density (MVD), is unclear in malignant mesothelioma (MM). Immunohistochemistry was performed on 104 archival, paraffin-embedded, surgically resected MM samples with an anti-CD34 monoclonal antibody, using the Streptavidin-biotin complex immunoperoxidase technique. 93 cases were suitable for microvessel quantification. MVD was obtained from 3 intratumoural hotspots, using a Chalkley eyepiece graticule at × 250 power. MVD was correlated with survival by Kaplan-Meier and log-rank analysis. A stepwise, multivariate Cox model was used to compare MVD with known prognostic factors and the EORTC and CALGB prognostic scoring systems. Overall median survival from the date of diagnosis was 5.0 months. Increasing MVD was a poor prognostic factor in univariate analysis (P = 0.02). Independent indicators of poor prognosis in multivariate analysis were non-epithelial cell type (P = 0.002), performance status > 0 (P = 0.003) and increasing MVD (P = 0.01). In multivariate Cox analysis, MVD contributed independently to the EORTC (P = 0.006), but not to the CALGB (P = 0.1), prognostic groups. Angiogenesis, as assessed by MVD, is a poor prognostic factor in MM, independent of other clinicopathological variables and the EORTC prognostic scoring system. Further work is required to assess the prognostic importance of angiogenic regulatory factors in this disease. © 2001 Cancer Research Campaign.

Expansion of human hematopoietic stem/progenitor cells on decellularized matrix scaffolds

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic 'human-body niche-like' conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications.

Avaliação da angiogênese em lesões de líquen plano oral e pênfigo vulgar

Oral lichen planus and pemphigus vulgaris are chronic diseases mucous membrane immune of unknown aetiology that can be observed affecting to the oral mucous. A relevant as regards neoplasies the role angiogenesis in the inflammatory chronic disease pathogenesis as it provides a substancial interest can be considered as being an activity diseases marker; besides being through specialised research of this angiogenic process to improve of understanding pathogenic mechanism. This research proposes to assess angiogenic active through of antibody immunohistochemistry expression antiCD34 antibody in 26 OLP of reticular cases, 14 OLP erosives cases, 18 of PV cases and 15 specimens of normal oral mucosa. The result was submitted non-parametric tests of 5% significance level. It is not statistically significant correlacion was seen regarding between average vessels. However, only be effectively observed the median of OLP cases was larger than pemphigus vulgaris in fact proved average larger than oral normal mucosa (p=0,280). Regarding the microvascular count of CD34 concerning clinic form oral lichen planus (reticular and erosion) increased emphasis is more cross-border average for the form erosion clinic. Despite of the statistic tests could not be more effective (p=0,720). Even though, the results of the research is not sufficient to enable to consider of angiogenic process in the pathogenesis and lesions progression of oral lichen planus and pemphigus vulgaris, we suggest this process is present in both forms lesion, however, more studies must be made in the near future in order to prepare a well-founded proposal

RNA Loading of Leukemic Antigens into Cord Blood–Derived Dendritic Cells for Immunotherapy

The manipulation of dendritic cells (DCs) ex vivo to present tumor-associated antigens for the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTLs) attempts to exploit these cells’ pivotal role in immunity. However, significant improvements are needed if this approach is to have wider clinical application. We optimized a gene delivery protocol via electroporation for cord blood (CB) CD34+ DCs using in vitro–transcribed (IVT) mRNA. We achieved > 90% transfection of DCs with IVT-enhanced green fluorescent protein mRNA with > 90% viability. Electroporation of IVT-mRNA up-regulated DC costimulatory molecules. DC processing and presentation of mRNA-encoded proteins, as major histocompatibility complex/peptide complexes, was established by CTL assays using transfected DCs as targets. Along with this, we also generated specific antileukemic CTLs using DCs electroporated with total RNA from the Nalm-6 leukemic cell line and an acute lymphocytic leukemia xenograft. This significant improvement in DC transfection represents an important step forward in the development of immunotherapy protocols for the treatment of malignancy.

Characterisation of mesenchymal cells from osteophytes in osteoarthritis

Osteophytes form through the process of chondroid metamorphosis of fibrous tissue followed by endochondral ossification. Osteophytes have been found to consist of three different mesenchymal tissue regions including endochondral bone formation within cartilage residues, intra-membranous bone formation within fibrous tissue and bone formation within bone marrow spaces. All these features provide evidence of mesenchymal stem cells (MSC) involvement in osteophyte formation; nevertheless, it remains to be characterised. MSC from numerous mesenchymal tissues have been isolated but bone marrow remains the “ideal” due to the ease of ex vivo expansion and multilineage potential. However, the bone marrow stroma has a relatively low number of MSC, something that necessitates the need for long-term culture and extensive population doublings in order to obtain a sufficient number of cells for therapeutic applications. MSC in vitro have limited proliferative capacity and extensive passaging compromises differentiation potential. To overcome this barrier, tissue derived MSC are of strong interest for extensive study and characterisation, with a focus on their potential application in therapeutic tissue regeneration. To date, no MSC type cell has been isolated from osteophyte tissue, despite this tissue exhibiting all the hallmark features of a regenerative tissue. Therefore, this study aimed to isolate and characterise cells from osteophyte tissues in relation to their phenotype, differentiation potential, immuno-modulatory properties, proliferation, cellular ageing, longevity and chondrogenesis in in vitro defect model in comparison to patient matched bone marrow stromal cells (bMSC). Osteophyte derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses. Multipotential stem cells were identified from all osteophyte samples namely osteophyte derived mesenchymal stem cells (oMSC). Extensively expanded cell cultures (passage 4 and 9 respectively) were used to confirm cytogenetic stability and study signs of cellular aging, telomere length and telomerase activity. Cultured cells at passage 4 were used to determine 84 pathway focused stem cell related gene expression profile. Micro mass pellets were cultured in chondrogenic differentiation media for 21 days for phenotypic and chondrogenic related gene expression. Secondly, cell pellets differentiated overnight were placed into articular cartilage defects and cultured for further 21 days in control medium and chondrogenic medium to study chondrogenesis and cell behaviour. The surface antigen expression of oMSC was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSC in culture was superior to that of bMSC, and they readily differentiated into tissues of the mesenchymal lineages. oMSC also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO). Cellular aging was more prominent in late passage bMSC than in oMSC. oMSC had longer telomere length in late passages compared with bMSC, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSC and not in bMSC. In osteophyte tissues telomerase positive cells were found to be located peri vascularly and were Stro-1 positive. Eighty-four pathway-focused genes were investigated and only five genes (APC, CCND2, GJB2, NCAM and BMP2) were differentially expressed between bMSC and oMSC. Chondrogenically induced micro mass pellets of oMSC showed higher staining intensity for proteoglycans, aggrecan and collagen II. Differential expression of chondrogenic related genes showed up regulation of Aggrecan and Sox 9 in oMSC and collagen II in bMSC. The in vitro defect models of oMSC in control medium showed rounded and aggregated cells staining positively for proteoglycan and presence of some extracellular matrix. In contrast, defects with bMSC showed fragmentation and loss of cells, fibroblast-like cell morphology staining positively for proteoglycans. For defects maintained in chondrogenic medium, rounded, aggregated and proteoglycan positive cells were found in both oMSC and bMSC cultures. Extracellular matrix and cellular integration into newly formed matrix was evident only in oMSC defects. For analysis of chondrocyte hypertrophy, strong expression of type X collagen could be noticed in the pellet cultures and transplanted bMSC. In summary, this study demonstrated that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSC, oMSC maintained a higher proliferative capacity due to a retained level of telomerase activity in vitro, which may account for the relatively longer telomeres delaying growth arrest by replicative senescence compared with bMSC. oMSC behaviour in defects supported chondrogenesis which implies that cells derived from regenerative tissue can be an alternative source of stem cells and have a potential clinical application for therapeutic stem cell based tissue regeneration.

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.

Proof of concept : A bioinformatic and serological screening method for identifying new peptide antigens for Chlamydia trachomatis related sequelae in women

This study aimed to identify new peptide antigens from Chlamydia (C.) trachomatis in a proof of concept approach which could be used to develop an epitope-based serological diagnostic for C. trachomatis related infertility in women. A bioinformatics analysis was conducted examining several immunodominant proteins from C. trachomatis to identify predicted immunoglobulin epitopes unique to C. trachomatis. A peptide array of these epitopes was screened against participant sera. The participants (all female) were categorized into the following cohorts based on their infection and gynecological history; acute (single treated infection with C. trachomatis), multiple (more than one C. trachomatis infection, all treated), sequelae (PID or tubal infertility with a history of C. trachomatis infection), and infertile (no history of C. trachomatis infection and no detected tubal damage). The bioinformatics strategy identified several promising epitopes. Participants who reacted positively in the peptide 11 ELISA were found to have an increased likelihood of being in the sequelae cohort compared to the infertile cohort with an odds ratio of 16.3 (95% c.i. 1.65 – 160), with 95% specificity and 46% sensitivity (0.19-0.74). The peptide 11 ELISA has the potential to be further developed as a screening tool for use during the early IVF work up and provides proof of concept that there may be further peptide antigens which could be identified using bioinformatics and screening approaches.

Divergent outcomes following transcytosis of IgG targeting intracellular and extracellular chlamydial antigens

Antibodies can play a protective but non-essential role in natural chlamydial infections dependent on antigen specificity and antibody isotype. IgG is the dominant antibody in both male and female reproductive tract mucosal secretions, and is bi-directionally trafficked across epithelia by the neonatal Fc receptor (FcRn). Using physiologically relevant pH-polarized epididymal epithelia grown on Transwells®, IgG specifically targeting an extracellular chlamydial antigen; the Major Outer Membrane Protein (MOMP), enhanced uptake and translocation of infection at pH 6-6.5 but not at neutral pH. This was dependent on FcRn expression. Conversely, FcRn-mediated transport of IgG targeting the intracellular chlamydial inclusion membrane protein A (IncA), induced aberrant inclusion morphology, recruited autophagic proteins independent of lysosomes, and significantly reduced infection. Challenge of female mice with MOMP-specific IgG-opsonized C. muridarum delayed infection clearance but exacerbated oviduct occlusion. In male mice, MOMP-IgG elicited by immunization afforded no protection against testicular chlamydial infection, whereas; the transcytosis of IncA-IgG significantly reduced testicular chlamydial burden. Together these data show that the protective and pathological effects of IgG are dependent on FcRn-mediated transport as well as the specificity of IgG for intracellular or extracellular antigens.

Human CD141(+) (BDCA-3)(+) dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141(+) DC subset. CD141(+) DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-beta, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c(+) DC subset. Polyinosine-polycytidylic acid (poly I:C)-activated CD141(+) DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8(+) cytotoxic T lymphocytes than poly I:C-activated CD1c(+) DCs. Importantly, CD141(+) DCs, but not CD1c(+) DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141(+) DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8 alpha(+) DC subset. The data demonstrate a role for CD141(+) DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Identification of novel antigens in blood vessels in rectovaginal endometriosis

To identify specific markers of rectovaginal endometriotic nodule vasculature, highly enriched preparations of vascular endothelial cells and pericytes were obtained from endometriotic nodules and control endometrial and myometrial tissue by laser capture microdissection (LCM), and gene expression profiles were screened by microarray analysis. Of the 18 400 transcripts on the arrays, 734 were significantly overexpressed in vessels from fibromuscular tissue and 923 in vessels from stromal tissue of endometriotic nodules, compared with vessels dissected from control tissues. The most frequently expressed transcripts included known endothelial cell-associated genes, as well as transcripts with little or no previous association with vascular cells. The higher expression in blood vessels was further corroborated by immunohistochemical staining of six potential markers, five of which showed strong expression in pericytes. The most promising marker was matrix Gla protein, which was found to be present in both glandular epithelial cells and vascular endothelial cells of endometriotic lesions, although it was barely expressed at all in normal endometrium. LCM, combined with microarray analysis, constitutes a powerful tool for mapping the transcriptome of vascular cells. After immunohistochemical validation, markers of vascular endothelial and perivascular cells from endometriotic nodules could be identified, which may provide targets to improve early diagnosis or to selectively deliver therapeutic agents.