Mixed chimaerism; detection and significance following BMT

Residual recipient haematopoietic cells may coexist with donor haemopoietic tissue following BMT. This is known as mixed chimaerism. The incidence of mixed chimaerism varies with the sensitivity of the detection system used; DNA based methodologies are the most sensitive. The influence of mixed chimaerism on leukaemia relapse and graft rejection is unclear. The lineages in which mixed chimaerism occurs may affect outcome.

Successful second unrelated donor BMT in a child with juvenile chronic myeloid leukaemia:documentation of chimaerism using the polymerase chain reaction

A 3-year old child with juvenile chronic myeloid leukaemia received a T cell-depleted BMT from a male unrelated donor. There was early graft failure associated with increasing splenomegaly and hypersplenism. Splenectomy was performed 53 days post-transplant and was followed by autologous marrow recovery with return of leukaemia. A second unrelated donor BMT was performed 9 months later using T cell-replete marrow from a similarly matched female donor. Grade 2 GVHD involving the skin and gut responded to treatment with steroids. Chimaerism was assessed using Y-specific polymerase chain reaction (PCR) and microsatellites. Samples taken at the time of splenectomy showed no donor marrow engraftment but there was significant engraftment in the spleen. Following the second transplant, donor-type haematopoiesis was documented using a panel of microsatellite probes. The patient remains well 6 months after transplant. Splenectomy should be considered prior to transplant in patients with significant splenomegaly and hypersplenism. Partial chimaerism in the spleen, but not bone marrow, post-BMT, has not previously been documented. PCR technology is a useful and highly sensitive way to assess chimaerism post-BMT and is informative in sex-matched cases, whilst the small amount of material required is advantageous in paediatric patients.

Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction

The influence of mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation remains unknown. Increasingly sensitive detection methods have shown that MC occurs frequently. We report a highly sensitive novel method to assess MC based on the polymerase chain reaction (PCR). Simple dinucleotide repeat sequences called microsatellites have been found to vary in their repeat number between individuals. We use this variation to type donor-recipient pairs following allogeneic BMT. A panel of seven microsatellites was used to distinguish between donor and recipient cells of 32 transplants. Informative microsatellites were subsequently used to assess MC after BMT in this group of patients. Seventeen of the 32 transplants involved a donor of opposite sex; hence, cytogenetics and Y chromosome-specific PCR were also used as an index of chimerism in these patients. MC was detected in bone marrow aspirates and peripheral blood in 18 of 32 patients (56%) by PCR. In several cases, only stored slide material was available for analysis but PCR of microsatellites or Y chromosomal material could be used successfully to assess the origin of cells in this archival material. Cytogenetic analysis was possible in 17 patients and MC was detected in three patients. Twelve patients received T-cell-depleted marrow and showed a high incidence of MC as revealed by PCR (greater than 80%). Twenty patients received unmanipulated marrow, and while the incidence of MC was lower (44%), this was a high percentage when compared with other studies. Once MC was detected, the percentages of recipient cells tended to increase. However, in patients exhibiting MC who subsequently relapsed, this increase was relatively sudden. The overall level of recipient cells in the group of MC patients who subsequently relapsed was higher than in those who exhibited stable MC. Thus, while the occurrence of MC was not indicative of a poor prognosis per se, sudden increases in the proportions of recipient cells may be a prelude to graft rejection or relapse.

Early detection of leukaemic relapse after bone marrow transplantation using the polymerase chain reaction

We report a case of acute lymphoblastic leukaemia relapsing after allogeneic bone marrow transplantation in which the polymerase chain reaction (PCR) was used to assess chimeric status. This technique demonstrated the progressive reappearance of host cells prior to clinical relapse. The relapse was of host cell origin as shown by the presence of female (recipient) metaphases containing an abnormal chromosomal marker (iso 9q) which had also been present at initial diagnosis. The emergence of host cells in this case, detected only by PCR techniques but not by cytogenetic methods, appeared to herald overt relapse. PCR analysis provides a sensitive tool for detecting a progressive rise in host cell numbers which may predict clinical relapse.

A rapid dot-blot assay to assess chimerism following sex-mismatched bone marrow transplantation

Mixed chimerism may occur more frequently than previously thought following allogeneic bone marrow transplantation and may have implications in terms of relapse, graft-versus-host disease and immune reconstitution. DNA analysis using single or multilocus polymorphic probes cannot reliably discriminate between donor and recipient cells below a level of 10%. We used probe pHY2.1, a cloned segment of tandemly repeated DNA (2000 copies) on the long arm of chromosome Y. A dot blot procedure allowed us to immobilize DNA directly from 50 microliter of peripheral blood or bone marrow. Cross-reactivity was eliminated by hybridization at conditions of extreme stringency (65 degrees C, 50% formamide). Mixing experiments detected male DNA at a level of 0.1% after 10 h exposure. Five patients were studied serially post-bone marrow transplantation. One patient showed mixed chimerism for 12 months, one had complete autologous recovery and the remaining three showed complete engraftment. All results were verified by standard karyotyping on bone marrow cells. This technique is a simple, rapid and sensitive assay for chimerism following sex mismatched bone marrow transplantation.

Calibrated integrated backscatter and myocardial fibrosis in patients undergoing cardiac surgery.

Objective - The reported association between calibrated integrated backscatter (cIB) and myocardial fibrosis is based on study of patients with dilated or hypertrophic cardiomyopathy and extensive (mean 15–34%) fibrosis. Its association with lesser degrees of fibrosis is unknown. We examined the relationship between cIB and myocardial fibrosis in patients with coronary artery disease.

Methods - Myocardial histology was examined in left ventricular epicardial biopsies from 40 patients (29 men and 11 women) undergoing coronary artery bypass graft surgery, who had preoperative echocardiography with cIB measurement.

Results - Total fibrosis (picrosirius red staining) varied from 0.7% to 4%, and in contrast to previous reports, cIB showed weak inverse associations with total fibrosis (r=−0.32, p=0.047) and interstitial fibrosis (r=−0.34, p=0.03). However, cIB was not significantly associated with other histological parameters, including immunostaining for collagens I and III, the advanced glycation end product (AGE) Nε-(carboxymethyl)lysine (CML) and the receptor for AGEs (RAGE). When biomarkers were examined, cIB was weakly associated with log plasma levels of amino-terminal pro-B-type natriuretic peptide (r=0.34, p=0.03), creatinine (r=0.33, p=0.04) and glomerular filtration rate (r=−0.33, p=0.04), and was more strongly associated with log plasma levels of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) (r=0.44, p=0.01) and soluble RAGE (r=0.53, p=0.002).

Conclusions - Higher cIB was not a marker of increased myocardial fibrosis in patients with coronary artery disease, but was associated with higher plasma levels of sVEGFR-1 and soluble RAGE. The role of cIB as a non-invasive index of fibrosis in clinical studies of patients without extensive fibrosis is, therefore, questionable.

c-Kit+ progenitors generate vascular cells for tissue-engineered grafts through modulation of the Wnt/Klf4 pathway

The development of decellularised scaffolds for small diameter vascular grafts is hampered by their limited patency, due to the lack of luminal cell coverage by endothelial cells (EC) and to the low tone of the vessel due to absence of a contractile smooth muscle cells (SMC). In this study, we identify a population of vascular progenitor c-Kit+/Sca-1- cells available in large numbers and derived from immuno-privileged embryonic stem cells (ESCs). We also define an efficient and controlled differentiation protocol yielding fully to differentiated ECs and SMCs in sufficient numbers to allow the repopulation of a tissue engineered vascular graft. When seeded ex vivo on a decellularised vessel, c-Kit+/Sca-1-derived cells recapitulated the native vessel structure and upon in vivo implantation in the mouse, markedly reduced neointima formation and mortality, restoring functional vascularisation. We showed that Krüppel-like transcription factor 4 (Klf4) regulates the choice of differentiation pathway of these cells through β-catenin activation and was itself regulated by the canonical Wnt pathway activator lithium chloride. Our data show that ESC-derived c-Kit+/Sca-1-cells can be differentiated through a Klf4/β-catenin dependent pathway and are a suitable source of vascular progenitors for the creation of superior tissue-engineered vessels from decellularised scaffolds.

Ectopic bone formation in rapidly fabricated acellular injectable dense collagen-Bioglass hybrid scaffolds via gel aspiration-ejection

Gel aspiration-ejection (GAE) has recently been introduced as an effective technique for the rapid production of injectable dense collagen (IDC) gel scaffolds with tunable collagen fibrillar densities (CFDs) and microstructures. Herein, a GAE system was applied for the advanced production and delivery of IDC and IDC-Bioglass® (IDC-BG) hybrid gel scaffolds for potential bone tissue engineering applications. The efficacy of GAE in generating mineralizable IDC-BG gels (from an initial 75-25 collagen-BG ratio) produced through needle gauge numbers 8G (3.4 mm diameter and 6 wt% CFD) and 14G (1.6 mm diameter and 14 wt% CFD) was investigated. Second harmonic generation (SHG) imaging of as-made gels revealed an increase in collagen fibril alignment with needle gauge number. In vitro mineralization of IDC-BG gels was confirmed where carbonated hydroxyapatite was detected as early as day 1 in simulated body fluid, which progressively increased up to day 14. In vivo mineralization of, and host response to, acellular IDC and IDC-BG gel scaffolds were further investigated following subcutaneous injection in adult rats. Mineralization, neovascularization and cell infiltration into the scaffolds was enhanced by the addition of BG and at day 21 post injection, there was evidence of remodelling of granulation tissue into woven bone-like tissue in IDC-BG. SHG imaging of explanted scaffolds indicated collagen fibril remodelling through cell infiltration and mineralization over time. In sum, the results suggest that IDC-BG hybrid gels have osteoinductive properties and potentially offer a novel therapeutic approach for procedures requiring the injectable delivery of a malleable and dynamic bone graft that mineralizes under physiological conditions

Transplant glomerulopathy: clinico-pathologic features

Transplant glomerulopathy is a sign of chronic kidney allograft damage. It has a distinct morphology and is associated with poor allograft survival. We aimed to assess the prevalence and clinic-pathologic features of transplant glomerulopathy, as well as determine the functional and histological implications of its severity. We performed a single-centre retrospective observational study during an eight-year period. Kidney allograft biopsies were diagnosed and scored according to the Banff classification, coupled with immunofluorescence studies. The epidemiology, clinical presentation, outcomes (patient and graft survival) and anti-HLA alloantibodies were evaluated. Transplant glomerulopathy was diagnosed in 60 kidney transplant biopsies performed for clinical reasons in 49 patients with ABO compatible renal transplant and a negative T-cell complement dependent cytotoxicity crossmatch at transplantation. The estimated prevalence of transplant glomerulopathy was 7.4% and its cumulative prevalence increased over time. C4d staining in peritubular capillaries (27.6%) was lower than the frequency of anti-HLA antibodies (72.5%), the majority against both classes I and II. Transplant glomerulopathy was associated with both acute (mainly glomerulitis and peritubular capillaritis) and chronic histologic abnormalities. At diagnosis, 30% had mild, 23.3% moderate and 46.7% severe transplant glomerulopathy. The severity of transplant glomerulopathy was associated with the severity of interstitial fibrosis. Other histological features, as well as clinical manifestations and graft survival, were unrelated to transplant glomerulopathy severity.

role of SLO in aGVHD : alterando o paradigma de allo-activação das células T, The

A indução da doença do transplante contra o hospedeiro (GVHD) depende da activação das células dadoras T pelas células do hospedeiro que apresentamantigenio (APCs). A teoria prevalecente descreve que estas interacções ocorrem nos órgãos linfáticos secundários (SLO), tais como os nóduloslinfáticos (LN), as placas de Peyer’s (PP) e o baço (SP). Esta hipótese foi testada usando ratinhos homozigóticos aly/aly (alinfoplasia) que não têm LN nem PP, usando como controlo os ratinhos heterozigóticos (aly/+) da mesma ninhada. Os dois grupos foram irradiados com dose letal após a remoção do baço aos ratinhos aly/aly (LN/PP/SP-/-), enquanto nos ratinhos aly/+ o baço foi deslocado e recolocado. Ambos receberam transplante de medula óssea (BMT) de ratinhos dadores singénicos (aly/aly, H-2b) ou de ratinhos alogénicos, com diferente complexo principal de histocompatibilidade (MHC) (BALB/c, H-2dou B10.BR, H-2k). A severidade de GVHD foi medida pela sobrevivência,e pelo sistema de pontuação, bem estabelecido, quer de doença clínica quer de doença dos órgãos alvo. Surpreendentemente, todos os ratinhos LN/PP/SP-/-sobreviveram, desenvolvendo GVHD clinicamente significativo, comparável,em severidade, com o observado nos ratinhos LN/PP/SP+/+. Além disso, asanálises histopatológicas demonstraram que os ratinhos LN/PP/SP-/-receptores de BMTdesenvolveram significativamente mais GVHD no fígado,no intestino, e na pele quando comparados com os animais singénicos decontrolo. Os ratinhos LN/PP/SP-/-desenvolveram também GVHD hepático mais severo quando comparados com os ratinhos de controlo LN/PP/SP+/+. Diferenças semelhantes foram ainda observadas, logo ao 7º dia, para o GVHDhepático entre os grupos alogénicos. Para identificar quais os órgãos extra-linfáticos do receptor que poderão servir como sítios iniciais de exposição a antigenios alogénicos, na ausência de SLO, foi examinada a expansão das células T (CD3+), a sua activação (CD69+), e a sua proliferação (CFSE) na medula óssea, 3 dias depois do BMT. Em cada caso, os ratinhos LN/PP/SP-/-transplantados com medula de dadores alogénicos apresentaram númerosabsolutos significativamente maiores quer de células, quer de divisõescelulares, se comparados com os LN/PP/SP+/+. Para garantir que as diferenças experimentais observadas nos animais aly/aly, no sistema díspar do MHC, não são apenas um fenómeno dependente da estirpe de ratinho, foramtransplantados ratinhos sem baço FucT dko (LN/PP/SP-/-), previamente tratados com o anticorpo monoclonal (mAb) anti-MadCAM-1. Após o BMT estes ratinhos apresentaram elevada pontuação clínica de GVHD, mostrando que os SLO não são necessários para a indução de GVHD. Em estudos de transplante-versus-leucemia usando hospedeiros homozigóticos (LN/PP/SP-/-) estes ratinhos morreram devido a expansão tumoral e não devido a GVHD.Estudos in vitro mostraram que a capacidade das APCs, quer das célulasdendríticas (DCs) esplénicas, quer das DCs derivadas da medula óssea, dosratinhos aly/aly e aly/+ eramcomparável. Colectivamente, estes resultados são consistentes com a noção de que os SLO não são necessários para a activação alogénica das celulas T, sugerindo que a medula óssea pode ser umlocal alternativo, embora menos eficiente, para o reconhecimento alogénico deantígenos e consequente activação das células dadoras T. Estas observações desafiam o paradigma de que os tecidos linfáticos secundários sãonecessários para a indução de GVHD.

Effect of light on ex situ production of symbiotic corals

The increasing interest in coral culture for biotechnological applications, to supply the marine aquarium trade, or for reef restoration programs, has prompted researchers to optimize coral culture protocols, with emphasis to ex situ production. When cultured ex situ, the growth performance of corals can be influenced by several physical, chemical and biological parameters. For corals harbouring zooxanthellae, light is one of such key factors, as it can influence the photosynthetic performance of these endosymbionts, as well as coral physiology, survival and growth. The economic feasibility of ex situ coral aquaculture is strongly dependent on production costs, namely those associated with the energetic needs directly resulting from the use of artificial lighting systems. In the present study we developed a versatile modular culture system for experimental coral production ex situ, assembled solely using materials and equipment readily available from suppliers all over the world; this approach allows researchers from different institutions to perform truly replicated experimental set-ups, with the possibility to directly compare experimental results. Afterwards, we aimed to evaluate the effect of contrasting Photosynthetically Active Radiation (PAR) levels, and light spectra emission on zooxanthellae photochemical performance, through the evaluation of the maximum quantum yield of PSII (Fv/Fm) (monitored non-invasively and non-destructively through Pulse Amplitude Modulation fluorometry, PAM), chlorophyll a content (also determined non-destructively by using the spectral reflectance index Normalized Difference Vegetation Index, NDVI), photosynthetic and accessory pigments, number of zooxanthellae, coral survival and growth. We studied two soft coral species, Sarcophyton cf. glaucum and Sinularia flexibilis, as they are good representatives of two of the most specious genera in family Alcyoniidae, which include several species with interest for biotechnological applications, as well as for the marine aquarium trade; we also studied two commercially important scleractinian corals: Acropora formosa and Stylophora pistillata. We used different light sources: hydrargyrum quartz iodide (HQI) lamps with different light color temperatures, T5 fluorescent lamps, Light Emitting Plasma (LEP) and Light Emitting Diode (LED). The results achieved revealed that keeping S. flexibilis fragments under the same light conditions as their mother colonies seems to be photobiologically acceptable for a short-term husbandry, notwithstanding the fact that they can be successfully stocked at lower PAR intensities. We also proved that low PAR intensities are suitable to support the ex situ culture S. cf. glaucum in captivity at lower production costs, since the survival recorded during the experiment was 100%, the physiological wellness of coral fragments was evidenced, and we did not detect significant differences in coral growth. Finally, we concluded that blue light sources, such as LED lighting, allow a higher growth for A. formosa and S. pistillata, and promote significant differences on microstructure organization and macrostructure morphometry in coral skeletons; these findings may have potential applications as bone graft substitutes for veterinary and/or other medical uses. Thus, LED technology seems to be a promising option for scleractinian corals aquaculture ex situ.

Compósitos CNTs/biocerâmico para a estimulação elétrica óssea in situ

The present thesis aims to develop a biocompatible and electroconductor bone graft containing carbon nanotubes (CNTs) that allows the in situ regeneration of bone cells by applying pulsed external electrical stimuli. The CNTs were produced by chemical vapor deposition (CVD) by a semi-continuous method with a yield of ~500 mg/day. The deposition parameters were optimised to obtain high pure CNTs ~99.96% with controlled morphologies, fundamental requisites for the biomedical application under study. The chemical functionalisation of CNTs was also optimised to maximise their processability and biocompatibility. The CNTs were functionalised by the Diels-Alder cycloaddition of 1,3-butadiene. The biological behaviour of the functionalised CNTs was evaluated in vitro with the osteoblastic cells line MG63 and in vivo, by subcutaneous implantation in rats. The materials did not induce an expressed inflammatory response, but the functionalised CNTs showed a superior in vitro and in vivo biocompatibility than the non-functionalised ones. Composites of ceramic matrix, of bioglass (Glass) and hydroxyapatite (HA), reinforced with carbon nanotubes (CNT/Glass/HA) were processed by a wet approach. The incorporation of just 4.4 vol% of CNTs allowed the increase of 10 orders of magnitude of the electrical conductivity of the matrix. In vitro studies with MG63 cells show that the CNT/Glass/HA composites guarantee the adhesion and proliferation of bone cells, and stimulate their phenotype expression, namely the alkaline phosphate (ALP). The interactions between the composite materials and the culture medium (α-MEM), under an applied electrical external field, were studied by scanning vibrating electrode technique. An increase of the culture medium electrical conductivity and the electrical field confinement in the presence of the conductive samples submerged in the medium was demonstrated. The in vitro electrical stimulation of MG63 cells on the conductive composites promotes the increase of the cell metabolic activity and DNA content by 130% and 60%, relatively to the non-stimulated condition, after only 3 days of daily stimulation of 15 μA for 15 min. Moreover, the osteoblastic gene expression for Runx2, osteocalcin (OC) and ALP was enhanced by 80%, 50% and 25%, after 5 days of stimulation. Instead, for dielectric materials, the stimulus delivering was less efficient, giving an equal or lower cellular response than the non-stimulated condition. The proposed electroconductive bone grafts offer exciting possibilities in bone regeneration strategies by delivering in situ electrical stimulus to cells and consequent control of the new bone tissue formation rate. It is expected that conductive smart biomaterials might turn the selective bone electrotherapy of clinical relevance by decreasing the postoperative healing times.

Occurrence of stem-pitting strains of Citrus tristeza virus in Croatia

Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 43°30'N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Neretva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field- infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kaštela, Split, Metković (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof greenhouse at 21 to 33° C.

Enzymatic grafting: A novel approach to develop multifunctional materials of interest

Today more than 99% of plastics are petroleum-based because of the availability and cost of the raw material. The durability of disposed plastics contributes to the environmental problems as waste and their persistence in the environment causes deleterious effects on the ecosystem. Environmental pollution awareness and the demand for green technology have drawn considerable attention of both academia and industry into biodegradable polymers. In this regard green chemistry technology has the potential to provide solution to this issue. Enzymatic grafting has recently been the focus of green chemistry technologies due to the growing environmental concerns, legal restrictions, and increasing availability of scientific knowledge. Over the last several years, research covering various applications of robust enzymes like laccases and lipases has been increased rapidly, particularly in the field of polymer science, to graft multi-functional materials of interest. In principle, enzyme-assisted grafting may modify/impart a variety of functionalities to the grafted composites which individual materials fail to demonstrate on their own. The modified polymers through grafting have a bright future and their development is practically boundless. In the present study series of graft composites with poly(3-hydroxybutyrate) (P(3HB) as side chain and cellulose as a backbone polymer were successfully synthesised by introducing enzymatic grafting technique where laccase and lipase were used as model catalysts [1-3]. Subsequently, the resulting composites were removed from the casting surface under ambient environment and characterised by Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and X-ray diffraction (XRD) in detail. Moreover, the thermo-mechanical behaviours of the grafted composites were investigated by differential scanning calorimetry (DSC) and dynamic mechanical analyser (DMA) measurements, respectively. In addition, hydrophobic and hydrophilic characteristics of the grafted polymers were studied through drop contour analysis using water contact angle (WCA). In comparison to the individual counterparts improvement was observed in the thermo- mechanical properties of the composites to varied extent. The tensile strength, elongation at break, and Young’s modulus values of the composites reached their highest levels in comparison to the films prepared with pure P(3HB) only which was too fragile to measure any of the above said characteristics. Interestingly, untreated P(3HB) was hydrophobic in nature and after lipase treatment P(3HB) and P(3HB)-EC-based graft composite attained higher level of hydrophilicity. This is a desired characteristic that enhances the biocompatibility of the materials for proper cell adhesion and proliferation therefore suggesting potential candidates for tissue engineering/bio-medical type applications [3]. The present research will be a first step in the biopolymer modification. To date no report has been found in literature explaining the laccase/lipase assisted grafting of P(3HB) [1-3]. The newly grafted composites exhibit unique functionalities with wider range of potential applications in bio-plastics, pharmaceutical, and cosmetics industries, tissue engineering, and biosensors. [1] H.M.N. Iqbal, G. Kyazze, T. Tron and T. Keshavarz, Cellulose 21, 3613-3621 (2014). [2] H.M.N. Iqbal, G. Kyazze, T. Tron and T. Keshavarz, Carbohydrate Polymers 113, 131-137 (2014). [3] H.M.N. Iqbal, G. Kyazze, T. Tron and T. Keshavarz, Polymer Chemistry In-Press, DOI: 10.1039/C4PY0 0857J (2014).