The significance of the host inflammatory response on the therapeutic efficacy of cell therapies utilising human adult stem cells.

Controlling the fate of implanted hMSCs is one of the major drawbacks to be overcome to realize tissue engineering strategies. In particular, the effect of the inflammatory environment on hMSCs behaviour is poorly understood. Studying and mimicking the inflammatory process in vitro is a very complex and challenging task that involves multiple variables. This research addressed the questions using in vitro co-cultures of primary derived hMSCs together with human peripheral blood mononucleated cells (PBMCs); the latter are key agents in the inflammatory process. This work explored the in vitro phenotypic changes of hMSCs in co-culture direct contact with monocytes and lymphocytes isolated from blood using both basal and osteogenic medium. Our findings indicated that hMSCs maintained their undifferentiated phenotype and pluripotency despite the contact with PBMCs. Moreover, hMSCs demonstrated increased proliferation and were able to differentiate specifically down the osteogenic lineage pathway. Providing significant crucial evidence to support the hypothesis that inflammation and host defence mechanisms could be utilised rather than avoided and combated to provide for the successful therapeutic application of stem cell therapies.

Host bone response to polyetheretherketone versus porous tantalum implants for cervical spinal fusion in a goat model.

STUDY DESIGN: In vivo assessment of polyetheretherketone (PEEK) and porous tantalum (TM) cervical interbody fusion devices in a goat model. OBJECTIVE: Directly compare host bone response to PEEK and TM devices used for cervical interbody fusion. SUMMARY OF BACKGROUND DATA: PEEK devices are widely used for anterior cervical discectomy and fusion but are nonporous and have limited surface area for bone attachment. METHODS: Twenty-five goats underwent single-level anterior cervical discectomy and fusion and were alternately implanted with TM (n = 13) or PEEK devices (n = 12) for 6, 12, and 26 weeks. Both devices contained a center graft hole (GH), filled with autograft bone from the animal's own iliac crest. The percentage of bone tissue around the implant, percentage of the implant surface in direct apposition with the host bone, and evidence of bone bridging through the implant GH were assessed by using backscattered electron imaging. Bone matrix mineral apposition rate was determined through fluorochrome double labeling, and sections were stained for histological analysis. RESULTS: The TM-implanted animals had significantly greater volumes of bone tissue at the implant interface than the PEEK animals at all-time points. The TM animals also had a significantly greater average mineral apposition rate in the GH region at 6 and 12 weeks than the PEEK animals. No difference was observed at 26 weeks. A greater number of TM-implanted animals demonstrated connection between the autograft bone and both vertebrae compared with the PEEK implants. Histological staining also showed that the TM devices elicited improved host bone attachment over the PEEK implants. CONCLUSION: The TM implants supported bone growth into and around the implant margins better than the PEEK devices. TM's open cell porous structure facilitated host bone ingrowth and bone bridging through the device, which could be beneficial for long-term mechanical attachment and support in clinical applications.

Use of a latex biomembrane for bladder augmentation in a rabbit model: biocompatibility, clinical and histological outcomes.

PURPOSE: To investigate histological features and biocompatibility of a latex biomembrane for bladder augmentation using a rabbit model. MATERIAL AND METHODS: After a partial cystectomy, a patch of a non-vulcanized latex biomembrane (2x4 cm) was sewn to the bladder with 5/0 monofilament polydioxanone sulfate in a watertight manner. Groups of 5 animals were sacrificed at 15, 45 and 90 days after surgery and the bladder was removed. The 5-mum preparations obtained from grafted area and normal bladder were stained with hematoxylin-eosin. Immunohistochemical staining was performed with a primary antibody against alpha-actin to assess muscle regeneration. RESULTS: No death, urinary leakage or graft extrusion occurred in any group. All bladders showed a spherical shape. Macroscopically, after 90 days, the latex biomembrane was not identifiable and the patch was indistinguishable from normal bladder. A bladder stone was found in one animal (6.6%). On the 90th day, histology revealed continuity of transitional epithelium of host bladder tissue on the patch area. At this time, the muscle layers were well organized in a similar fashion to native bladder muscle layers. The inflammatory process was higher on grafted areas when compared to controls: 15 days--p < 0.0001, 45 days--p < 0.001, and 90 days--p < 0.01. The anti alpha-actin immunoexpression peaked at 45 days, when the graft was observed covered by muscle cells. CONCLUSION: The latex biomembrane is biocompatible and can be used in models for bladder augmentation in rabbits. It promotes epithelium and muscle regeneration without urinary leakage.

Use of a latex biomembrane for bladder augmentation in a rabbit model: biocompatibility, clinical and histological outcomes

PURPOSE: To investigate histological features and biocompatibility of a latex biomembrane for bladder augmentation using a rabbit model. MATERIAL AND METHODS: After a partial cystectomy, a patch of a non-vulcanized latex biomembrane (2x4 cm) was sewn to the bladder with 5/0 monofilament polydioxanone sulfate in a watertight manner. Groups of 5 animals were sacrificed at 15, 45 and 90 days after surgery and the bladder was removed. The 5-µm preparations obtained from grafted area and normal bladder were stained with hematoxylin-eosin. Immunohistochemical staining was performed with a primary antibody against alpha-actin to assess muscle regeneration. RESULTS: No death, urinary leakage or graft extrusion occurred in any group. All bladders showed a spherical shape. Macroscopically, after 90 days, the latex biomembrane was not identifiable and the patch was indistinguishable from normal bladder. A bladder stone was found in one animal (6.6 percent). On the 90th day, histology revealed continuity of transitional epithelium of host bladder tissue on the patch area. At this time, the muscle layers were well organized in a similar fashion to native bladder muscle layers. The inflammatory process was higher on grafted areas when compared to controls: 15 days - p < 0.0001, 45 days - p < 0.001, and 90 days - p < 0.01. The anti alpha-actin immunoexpression peaked at 45 days, when the graft was observed covered by muscle cells. CONCLUSION: The latex biomembrane is biocompatible and can be used in models for bladder augmentation in rabbits. It promotes epithelium and muscle regeneration without urinary leakage.

Dissection of effector pathways in the host-versus-graft response to bone marrow transplantation.

We have dissected the role of the primary cytotoxic pathways Fas-FasL and perforin-granzyme in host-versus-graft reactions after allogeneic bone marrow transplantation. Sublethally irradiated female recipient mice deficient for FasL (B6.gld) or perforin (B6.prf-/-) were transplanted with bone marrow from B6 male donors. Donor engraftment in B6.prf-/- recipients was higher when compared with B6.gld, particularly when assessed by in vivo killing, demonstrating the importance of the perforin pathway over the Fas-FasL pathway. In the absence of both pathways, however, donor bone marrow engraftment was not fully restored identifying a role for an additional pathway(s) in the host-versus-graft response.

Use of porcine small intestinal submucosa in bladder augmentation in rabbit: long-term histological outcome.

AIM: To investigate long-term histological features of bladder augmentation using porcine small intestine submucosa (SIS) in a rabbit model. MATERIALS AND METHOD: Sixteen New Zealand rabbits were used. Porcine SIS was provided by a manufactured formation derived from the pig. After partial cystectomy was carried out on the bladder, a single layer of SIS (Cook-SIS Technology, Cook Biotech Incorporated, West Lafayette, IN, USA) (2 x 5 cm) was sewn to bladder with continuous 5/0 vicryl suture material in a watertight manner. Urinary diversion was not used. The rabbits were killed 12 months later and perivesical fat was removed together with bladder. The 5-microm preparations taken from the samples were stained with haematoxylin-eosin and Mason's trichrome dye. S-100 and F8 stains were also used for immunohistochemical investigations. RESULTS: The macroscopic view of bladder was normal. SIS was indistinguishable from normal bladder wall, but the region of the graft had a slight white coloration. Microscopic observations showed the continuity of transitional epithelium of host bladder tissue on SIS material. Detrusor and serosal layers were formed and these layers were indistinguishable from host bladder. Fibroblasts were scattered among the collagen fibrils. New vessel formations were present without lymphatic proliferation. Nerve regeneration was excellent. No inflammation was observed in normal and regenerated bladder wall. CONCLUSION: At the end of 12 months, the long-term histological features of bladder augmentation with porcine SIS in a rabbit model, such as presence of new vessel formations, nerve regeneration, collagen and smooth muscle regenerations, which were indistinguishable from original bladder, and the absence of inflammation, showed that SIS seems to be a viable alternative to the use of intestine in bladder augmentation.

Studies of host-graft interactions in vitro.

Progenitor and stem cell transplantation represent therapeutic strategies for retinal disorders that are accompanied by photoreceptor degeneration. The transplanted cells may either replace degenerating photoreceptors or secrete beneficial factors that halt the processes of photoreceptor degeneration. The present study analyzes whether rat retinal progenitor cells differentiated into photoreceptor phenotypic cells in neurospheres have a potential to interact with rat retinal explants. Immunocytochemistry for rhodopsin and synaptophysin indicated photoreceptor cell-like differentiation in neurospheres that were stimulated by basic fibroblast growth factor and epidermal growth factor. Differentiation into neural phenotypes including photoreceptor cells was effectively blocked by an addition of leukemia inhibitory factor. Grafting of neurospheres onto retinal explants demonstrated a consistent penetration of glial cell processes into the explanted tissue. On the other hand, the incorporation of donor cells into explants was very low. A general finding was that neurospheres grafting was associated with local decrease in Müller cell activation in the explants. Further characterization of these effect(s) could provide further insight into progenitor cell-based therapies of retinal degenerative disorders.

Early renal injury after nonmyeloablative allogeneic peripheral blood stem cell transplantation in patients with chronic myelocytic leukemia.

BACKGROUND: Renal insufficiency is a common complication early after hematopoietic stem cell transplantation (HSCT). Over the past several years, significant advancement has been achieved in HSCT, especially in nonmyeloablative stem cell transplantation. Compared with traditional HSCT, nonmyeloablative HSCT employs significantly lower doses of chemoradiotherapy and lower toxicity. The current study evaluated renal insufficiency during the first 100 days in patients with chronic myelocytic leukemia (CML) who underwent nonmyeloablative allogeneic peripheral blood stem cell transplantation (allo-PBSCT) in a single center. METHODS: A total of 26 consecutive patients with CML received nonmyeloablative allo-PBSCT from 2002 to 2005 in Zhong Da Hospital. The average age of the patients was 40.2 +/- 8.2 years. Renal function was measured by serum creatinine concentration and estimated glomerular filtration rate (GFR) during the first 100 days after nonmyeloablative allo-PBSCT. Renal dysfunction was classified as follows: grade 0 (<25% decline in GFR), grade 1 (> or =25% decrease in GFR but or =twofold increase in serum creatinine but no need for dialysis) and grade 3 (> or =twofold increase in serum creatinine and need for dialysis). Acute renal failure (ARF) was defined as a doubling of baseline serum creatinine, grade 2 and grade 3. RESULTS: All the patients were successfully engrafted. Of the 26 patients, 10 (38%) patients had some degree of renal dysfunction (grade 1, 5 patients; grade 2, 4 patients; grade 3, 1 patient). They developed ARF at an average of 32.8 +/- 4.0 days after transplantation. No significant difference was observed in terms of age, baseline serum creatinine, albumin and hemoglobin between the patients with ARF and without ARF. Renal dysfunction was associated with significantly higher frequencies of sepsis and hepatic veno-occlusive disease (VOD, p < 0.01, respectively). The overall mortality rate at the end of 100 days was 19% (5/26). The mortality rate for patients with ARF was significantly higher than those without ARF (p < 0.001). CONCLUSION: During the first 100 days following nonmyeloablative allo-PBSCT in patients with CML, a 38% incidence of renal dysfunction and a 19% of ARF were found, which were much less than previous studies. Sepsis and VOD were significantly correlated with the development of renal dysfunction. Severe nephrotoxicity was associated with the increase in mortality.

Stem cells in cardiovascular disease: methods and protocols.

Stem cells are cells capable of proliferation, self-renewal, and differentiation into various organ-specific cell types. Stem cells are subclassified based on their species of origin (mice, rat, human), developmental stage of the species (embryonic, fetal, or adult), tissue of origin (hematopoietic, mesenchymal, skeletal, neural), and potential to differentiate into one or more specific types of mature cells (totipotent, pluripotent, multipotent). Embryonic stem (ES) cells are totipotent, primitive cells derived from the embryo that have the potential to become all specialized cell types. Conversely, adult stem cells are undifferentiated cells found in differentiated tissue that retain the potential to renew themselves and differentiate to yield organ-specific tissues. Stem cells are attractive candidates for novel therapeutics for patients with different heart diseases, including congestive heart failure, most commonly caused by myocardial infarction. The remarkable proliferative and differentiation capacity of stem cells promises an almost unlimited supply of specific cell types including viable functioning cardiomyocytes to replace the scarred myocardium following transplantation.

Relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-beta1 levels.

BACKGROUND: Transforming growth factor (TGF)-beta(1) is increased in allograft rejection and its production is associated with single nucleotide polymorphisms (SNPs). METHODS: The contribution of SNPs at codons 10 and 25 of the TGF-beta(1) gene to renal allograft damage was assessed in 6-month protocol biopsies and their association with TGF-beta(1) production. TGF-beta(1) genotypes were evaluated by polymerase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-beta(1) messenger RNA (mRNA) was measured by real-time PCR and TGF-beta(1) plasma levels were assessed by enzyme-linked immunosorbent assay. RESULTS: Eighty consecutive patients were included. Allele T at codon 10 (risk ratio, 6.7; P = 0.02) and an episode of acute rejection before protocol biopsy (risk ratio, 6.2; P = 0.01) were independent predictors of subclinical rejection (SCR). TGF-beta(1) plasma levels, but not those of TGF-beta(1) mRNA, were increased in patients with SCR (2.59 ng/mL +/- 0.91 [n = 22] vs. 2.05 ng/mL +/- 0.76 [n = 43]; P = 0.01). There was no association between allele T and TGF-beta(1) plasma or intragraft levels. CONCLUSIONS: Allele T at codon 10 of the TGF-beta(1) gene is associated with a higher incidence of SCR.

Engraftment of allogeneic hematopoietic stem cells requires both inhibition of host-versus-graft responses and 'space' for homeostatic expansion.

BACKGROUND: The establishment of host-versus-graft (HvG) tolerance is the primary aim of reduced intensity conditioning (RIC) regimens for allogeneic stem cell transplantation (SCT). It remains to be clarified to what extent recipient myeloablation is fundamental in the establishment of donor chimerism. METHODS: We have addressed this question in a murine model of RIC SCT in which the donor-recipient combination produces HvG against the male specific minor histocompatibility antigen HY. In this system engraftment can be monitored by RT-PCR and HvG effectors enumerated by tetramer analysis. RESULTS: We demonstrate that the dose of irradiation influences donor hemopoietic engraftment and affects generation of anti-donor specific T cells. Chimeric recipients do not mount a HvG immune response, becoming selectively tolerant, as demonstrated by the long term acceptance of skin grafts of donor but not third party origin. However, HvG tolerance is not sufficient to secure engraftment since, even in the absence of HvG, partial myeloablation was still required. The "space" produced by myeloablation and the consequent potential for donor cell expansion could also affect HvG tolerance, since its induction is severely impaired when donor hematopoietic cells have reduced proliferative capacity. CONCLUSIONS: We conclude that both some degree of myeloablation and HvG tolerance are required for successful engraftment, and that the capacity of donor cells to proliferate influences the induction of HvG tolerance.

A synthetic eicosanoid LX-mimetic unravels host-donor interactions in allogeneic BMT-induced GvHD to reveal an early protective role for host neutrophils.

Lipoxin A(4) (LXA(4)) and aspirin-triggered 15-epi-LXA(4) are potent endogenous lipid mediators thought to define the inflammatory set-point. We used single prophylactic administrations of a synthetic aspirin-triggered lipoxin A(4) signal mimetic, ATLa, to probe dynamics of early host-donor interactions in a mouse model for the inflammation-associated multifactorial disease of allogeneic bone marrow transplant (BMT) -induced graft-vs.-host disease (GvHD). We first demonstrated that both host and donor are responsive to the ATLa signals. The simple and restricted regimen of a single prophylactic administration of ATLa [100 ng/mL to donor cells or 1 microg (approximately 50 microg/kg) i.v. to host] was sufficient to delay death. Clinical indicators of weight, skin lesions, diarrhea and eye inflammation were monitored. Histological analyses on day 45 post-BMT showed that the degree of cellular trafficking, particularly neutrophil infiltrate, and protection of end-organ target pathology are different, depending on whether the host or donor was treated with ATLa. Taken together, these results chart some ATLa protective effects on GvHD cellular dynamics over time and identify a previously unrecognized effect of host neutrophils in the early phase post-BMT as important determinants in the dynamics of GvHD onset and progression.-Devchand, P. R., Schmidt, B. A., Primo, V. C., Zhang, Q.-y., Arnaout, M. A., Serhan, C. N., Nikolic, B. A synthetic eicosanoid LX-mimetic unravels host-donor interactions in allogeneic BMT-induced GvHD to reveal an early protective role for host neutrophils.

Graft versus host disease in a rat small bowel transplant model after T-cell depleted donor specific bone marrow infusion

Low cytoreductive regimen of irradiation associated to unmodified bone marrow infusion (UBM) does not prevent the occurrence of graft versus host disease (GVHD) after transplant. PURPOSE: In this study we evaluated the potential advantages of a long-term immunossupression and T-cell depleted bone marrow infusion (TCDBMI) in preventing the occurrence of GVHD after small bowel transplantation (SBTx). METHODS: Heterotopic SBTX was performed with Lewis rats as recipients and DA as donors and distributed into 5 groups according to the irradiation, duration of immunossupression and the use of UBM or TCDBMI: G1 (n=6), without irradiation and G2 (n=9), G3 (n=4), G4 (n=5) and G5 (n=6) was given 250 rd of irradiation. Groups 1,2,4 and G3 and 5 were infused with 100 x 106 UBM and TCDBM respectively. Animals in G1, 2, 3 were immunossupressed with 1mg/ FK506/Kg/IM for 5 days and G4 and G5 for 15 days. Anti CD3 monoclonal antibodies and immunomagnetic beads were used for T-cell depletion.Animals were examined for rejection, GVHD, chimerism characterization and ileal and skin biopsies. RESULTS: Minimal to mild rejection was observed in all groups; however, GVHD were present only in irradiated groups. Long-term immunossupression changed the severity of GVHD in G4 and G5. Rejection was the cause of death in G1 while GVHD in G2, 3, 4 and 5, not avoided by the use of TCDBMI. Total chimerism and T-cell chimerism was statistically higher in irradiated groups when compared to G1. CONCLUSION: Extended immunossupression associated to low dose of irradiation decrease the severity of GVHD, not avoided by the use of TCDBMI.

Müller cells in long-term full-thickness retinal transplants.

Müller cells are essential in creating and maintaining intricate neuroretinal architecture. The functions of this important glial cell are not limited to mere support of the retinal neurons, but also include interaction in synaptic transmission and activation in response to retinal insult. In this study, we have examined Müller cell morphology and degree of activation in embryonic full-thickness rabbit neuroretinal grafts, which were positioned under the host retina using vitrectomy technique. After surviving 3-10 months, retinal specimens were examined with hematoxylin and eosin staining and immunohistochemical analysis of vimentin and glial fibrillary acidic protein (GFAP) expression. In the host retina covering the graft, outer layers were degenerated, and vimentin-labeled Müller cells in this area appeared short, disorganized, and displayed strong GFAP labeling. In the graft, vimentin-labeled Müller cells spanning the retinal layers in the normal manner were found. Müller cells in 3-month grafts were well labeled by GFAP, whereas in older grafts, GFAP labeling was very weak or absent. Our results suggest that Müller cells in well-laminated full-thickness retinal grafts display many of the normal morphological features and retain a normal organization even after prolonged survival times. The loss of the initial degree of Müller cell activation indicates a long-term stability of the graft. The degeneration and gliosis of the host retina covering the graft is best explained by the merangiotic nature of the rabbit retina and may limit the usefulness of the rabbit in retinal transplantation experiments.

Grafting of cultured microglial cells into the lesioned spinal cord of adult rats enhances neurite outgrowth.

There is contrasting in vitro and in vivo evidence regarding glial cell involvement in central nervous system (CNS) regeneration. This study has investigated the histological events that follow implantation of either microglia, mixed microglia/astrocytes, or astrocytes into the injured adult rat spinal cord. We have conducted an immunohistochemical characterization of the cellular profiles within and neuritic extension into various grafts consisting of gelfoam (GF) matrices impregnated with cultured microglia and/or astrocytes. After 2-5 weeks, prominent neuritic growth was observed into OX-42-immunoreactive (IR) microglial implants. These grafts were infiltrated by numerous host cellular elements including microvasculature and Schwann cells, and they demonstrated conspicuous laminin IR. Often, the patterns for laminin and OX-42 IR in microglial grafts were overlapping, suggesting partial expression of laminin on transplanted microglial cells. Mixed grafts of microglia and astrocytes demonstrated presence of neurites and laminin-IR elements with similar intensity as microglial grafts, while astroglial implants showed the least amount of neurite ingrowth. Some control implants consisting of cell-free GF showed marginal in-growth of neurites in areas of infiltrating OX-42-IR host cells. Collectively, our findings support a neurite growth-promoting role of activated microglia and suggest that microglia may counteract mechanisms that inhibit CNS regeneration. It remains to be determined whether the observed neurite growth-promoting effects are mediated directly by grafted and/or endogenous microglia, or whether this occurs via the recruitment of host Schwann cells.

The influence of graft-versus-host reactivity, lymphocyte depletion, and cell dose on allogeneic bone marrow engraftment.

Graft rejection has hampered the use of T cell depletion (TCD) in allogeneic bone marrow transplantation. A model of host-versus-graft (HVGR) and graft-versus-host reaction (GVHR) as two inversely related processes has been proposed. We investigated graft rejection rates in graft-versus-host-reactive and graft-versus-host-nonreactive situations in a rat and a mouse model. Model 1: LEW rats were pretreated with a fixed myeloablative dose of busulfan and increasing doses of the immunosuppressive cyclophosphamide. The animals received different doses of semiallogeneic GvH-nonreactive BM cells. Graft rejection rates were dependent on the bone marrow cell number transplanted and on the pretransplant immunosuppression. Graft rejection rates following transplantation of GvH-reactive CAP marrow and genetically GvH-nonreactive (CAP x LEW)F1 marrow were the same. In conclusion, there was no advantage with respect to engraftment for the GvH-reactive marrow. Model 2: In irradiated Balb/c mice, graft rejection rates following T cell-depleted and unmanipulated transplantation of GvH-reactive or GvH-nonreactive bone marrow grafts were identical. All experiments were done with graded numbers of BM cells and revealed a strong impact of the BM cell dose on engraftment. In our experiments the cell loss during the ex-vivo manipulation was approximately 50% and, in contrast to the clinical situation, we readjusted to the intended number after TCD. Our experiments demonstrate that neither GvHR nor T cells but the BM cell dose has a strong impact on engraftment of allogeneic bone marrow.

Preliminary results of a complete study protocol on synthetic vascular graft healing and its complications.

The microscopic and anatomic features and bacteriologic culture results of different portions of single, explanted dacron synthetic vascular grafts (SVG) were studied together with patient clinical data. With this complete study protocol a better understanding of the healing process and its associated pathology can be achieved. We studied three, amply distanced graft portions from each of five patients (15 total graft portions) undergoing revision for infectious and non-infectious reasons. We divided the SVG portions studied into a Group 1, with high degrees of graft healing and into a Group 2, with both infection-dependent, early healing complications and perigraft chronic inflammatory reaction-dependent, late healing complications. These late healing complications were found dependent upon a host vs graft reaction. This study confirmed in humans the important role of an internal and external fibrotic graft incorporation in the definitive healing of a SVG. A host vs graft reaction was suggested to be an alternative to the frequently cited low virulent infection pathogenesis of late SVG healing complications. A sure definition and treatment of late SVG healing complications will only be established by means of a complete study protocol performed on a large number of explanted SVGs.