Expression of CD40 ligand, interferon-gamma and Fas ligand genes in endomyocardial biopsies of human cardiac allografts: correlation with acute rejection

The purpose of the present study was to investigate the expression (mRNA) of CD40 ligand (CD40L), interferon-gamma (IFN-gamma) and Fas ligand (FasL) genes in human cardiac allografts in relation to the occurrence of acute cardiac allograft rejection as well as its possible value in predicting acute rejection. The mRNA levels were determined by a semiquantitative reverse transcriptase-polymerase chain reaction method in 39 samples of endomyocardial biopsies obtained from 10 adult cardiac transplant recipients within the first six months after transplantation. Biopsies with ongoing acute rejection showed significantly higher CD40L, IFN-gamma and FasL mRNA expression than biopsies without rejection. The median values of mRNA expression in biopsies with and without rejection were 0.116 and zero for CD40L (P<0.003), 0.080 and zero for IFN-gamma (P<0.0009), and 0.156 and zero for FasL (P<0.002), respectively. In addition, the levels of IFN-gamma mRNA were significantly increased 7 to 15 days before the appearance of histological evidence of rejection (median of 0.086 in pre-rejection biopsies), i.e., they presented a predictive value. This study provides further evidence of heightened expression of immune activation genes during rejection and shows that some of these markers may present predictive value for the occurrence of acute rejection.

The 5alpha-reductase type 1, but not type 2, gene is expressed in anagen hairs plucked from the vertex area of the scalp of hirsute women and normal individuals

The aim of the present study was to determine the expression of the genes for type 1 (SDR5A1) and type 2 (SDR5A2) 5alpha-reductase isoenzymes in scalp hairs plucked from 33 hirsute patients (20 with polycystic ovary syndrome and 13 with idiopathic hirsutism) and compare it with that of 10 men and 15 normal women. SDR5A1 and SDR5A2 expression was estimated by RT-PCR using the gene of the ubiquitously expressed protein ß2-microglobulin as an internal control. The results are expressed as arbitrary units in relation to ß2-microglobulin absorbance (mean ± SEM). SDR5A2 expression was not detected in any hair samples analyzed in this study. No differences were found in SDR5A1 mRNA levels between men and normal women (0.78 ± 0.05 vs 0.74 ± 0.06, respectively). SDR5A1 gene expression in the cells of hair plucked from the scalp of normal women (0.85 ± 0.04) and of women with polycystic ovary syndrome (0.78 ± 0.05) and idiopathic hirsutism (0.80 ± 0.06) was also similar. These results indicate that SDR5A1 gene expression in the follicular keratinocytes from the vertex area of the scalp seems not to be related to the differences in hair growth observed between normal men and women and hirsute patients. Further studies are needed to investigate the expression of the 5alpha-reductase genes in other scalp follicular compartments such as dermal papillae, and also in hair follicles from other body sites, in order to elucidate the mechanism of androgen action on the hair growth process and related diseases.

Measurement of epidermal thickness in a patient with psoriasis by computer-supported image analysis

The aim of the present study was to measure full epidermal thickness, stratum corneum thickness, rete length, dermal papilla widening and suprapapillary epidermal thickness in psoriasis patients using a light microscope and computer-supported image analysis. The data obtained were analyzed in terms of patient age, type of psoriasis, total body surface area involvement, scalp and nail involvement, duration of psoriasis, and family history of the disease. The study was conducted on 64 patients and 57 controls whose skin biopsies were examined by light microscopy. The acquired microscopic images were transferred to a computer and measurements were made using image analysis. The skin biopsies, taken from different body areas, were examined for different parameters such as epidermal, corneal and suprapapillary epidermal thickness. The most prominent increase in thickness was detected in the palmar region. Corneal thickness was more pronounced in patients with scalp involvement than in patients without scalp involvement (t = -2.651, P = 0.008). The most prominent increase in rete length was observed in the knees (median: 491 µm, t = 10.117, P = 0.000). The difference in rete length between patients with a positive and a negative family history was significant (t = -3.334, P = 0.03), being 27% greater in psoriasis patients without a family history. The differences in dermal papilla distances among patients were very small. We conclude that microscope-supported thickness measurements provide objective results.

Preservation of graft function in low-risk living kidney transplant recipients treated with a combination of sirolimus and cyclosporine

The use of sirolimus (SRL) in combination with full doses of cyclosporin A (CsA) results in reduced one-year kidney allograft function, which is associated with shorter long-term allograft survival. We determined the effect of reduced CsA exposure on graft function in patients receiving SRL and prednisone. Ninety recipients of living kidney transplants receiving SRL (2 mg/day, po) were compared to 35 recipients receiving azathioprine (AZA, 2 mg kg-1 day-1, po). All patients also received CsA (8-10 mg kg-1 day-1, po) and prednisone (0.5 mg kg-1 day-1). Efficacy end-point was a composite of biopsy-confirmed acute rejection, graft loss, or death at one year. Graft function was measured by creatinine, creatinine clearance, and graft function deterioration between 3 and 12 months (delta1/Cr). CsA concentrations in patients receiving SRL were 26% lower. No differences in one-year composite efficacy end-point were observed comparing SRL and AZA groups (18 vs 20%) or in the incidence of biopsy-proven acute rejection (14.4 and 14.3%). There were no differences in mean ± SD creatinine (1.65 ± 0.46 vs 1.60 ± 0.43 mg/dl, P = 0.48) or calculated creatinine clearances (61 ± 15 vs 62 ± 13 ml/min, P = 0.58) at one year. Mean ± SD delta1/Cr (-11 ± 17 vs -14 ± 15%, P = 0.7) or the percentage of patients with >20% (26 vs 31%, P = 0.6) or >30% delta1/Cr (19 vs 17%, P = 1) did not differ between the two groups. The use of 2-mg fixed oral doses of SRL and reduced CsA exposure was effective in preventing acute rejection and preserving allograft function.

Skin morphology of the mutant hairless USP mouse

The morphology of the skin of the mutant hairless USP mouse was studied by histological, histochemical and immunohistochemical methods and compared to the skin of BALB/c mice. Representative sections of the dorsal skin from mice of both strains aged 18 days, and 1, 3, 6, and 8 months were studied. Sections stained with hematoxylin and eosin showed cystic formations called utricles and dermal cysts in the dermis that increased in size and number during growth. Skin thickness increased significantly at 8 months. Sections stained with picrosirius and examined with polarized light, displayed different colors, suggesting different thicknesses of dermal collagen fibers (probably types I and III). Weigert, Verhoeff and resorcin-fuchsin stains revealed fibers of the elastic system. The PAS and Alcian blue methods revealed neutral and acid glycosaminoglycans in the skin ground substance of both mouse strains. Immunohistochemical staining for fibronectin and laminin did not show differences between the mutant and BALB/c mice. Mast cells stained by the Gomori method and macrophages positive for HAM 56 antibodies were observed in both mouse strains. Except for the presence of enlarged cysts in the hairless strain, no qualitative differences were found during development of the skin of BALB/c and the mutant hairless mice.

Pharmacokinetic/pharmacodynamic relationships of FTY720 in kidney transplant recipients

FTY720 is a new and effective immunosuppressive agent, which produces peripheral blood lymphopenia through a lymphocyte homing effect. We investigated the relationship between the dose of FTY720 or blood concentration (pharmacokinetics, PK) and peripheral lymphopenia (pharmacodynamics, PD) in 23 kidney transplant recipients randomized to receive FTY720 (0.25-2.5 mg/day) or mofetil mycophenolate (2 mg/day) in combination with cyclosporine and steroids. FTY720 dose, blood concentrations and lymphocyte counts were determined weekly before and 4 to 12 weeks after transplantation. The effect of PD was calculated as the absolute lymphocyte count or its reductions. PK/PD modeling was used to find the best-fit model. Mean FTY720 concentrations were 0.36 ± 0.05 (0.25 mg), 0.73 ± 0.12 (0.5 mg), 3.26 ± 0.51 (1 mg), and 7.15 ± 1.41 ng/ml (2.5 mg) between 4 and 12 weeks after transplantation. FTY720 PK was linear with dose (r² = 0.98) and showed low inter- and intra-individual variability. FTY720 produced a dose-dependent increase in mean percent reduction of peripheral lymphocyte counts (38 vs 42 vs 56 vs 77, P < 0.01, respectively). The simple Emax model [E = (Emax * C)/(C + EC50)] was the best-fit PK/PD modeling for FTY720 dose (Emax = 87.8 ± 5.3% and ED50 = 0.48 ± 0.08 mg, r² = 0.94) or concentration (Emax = 78.3 ± 2.9% and EC50 = 0.59 ± 0.09 ng/ml, r² = 0.89) vs effect (% reduction in peripheral lymphocytes). FTY720 PK/PD is dose dependent and follows an Emax model (EC50 = 0.5 mg or 0.6 ng/ml). Using lymphopenia as an FTY720 PD surrogate marker, high % reductions (~80%) in peripheral lymphocytes are required to achieve best efficacy to prevent acute allograft rejection.

Heterotopic transplantation of glycerin-preserved trachea: effect of respiratory epithelium desquamation on acute rejection

An effective preservation method and decreased rejection are essential for tracheal transplantation in the reconstruction of large airway defects. Our objective in the present study was to evaluate the antigenic properties of glycerin-preserved tracheal segments. Sixty-one tracheal segments (2.4 to 3.1 cm) were divided into three groups: autograft (N = 21), fresh allograft (N = 18) and glycerin-preserved allograft (N = 22). Two segments from different groups were implanted into the greater omentum of dogs (N = 31). After 28 days, the segments were harvested and analyzed for mononuclear infiltration score and for the presence of respiratory epithelium. The fresh allograft group presented the highest score for mononuclear infiltration (1.78 ± 0.43, P <= 0.001) when compared to the autograft and glycerin-preserved allograft groups. In contrast to the regenerated epithelium observed in autograft segments, all fresh allografts and glycerin-preserved allografts had desquamation of the respiratory mucosa. The low antigenicity observed in glycerin segments was probably the result of denudation of the respiratory epithelium and perhaps due to the decrease of major histocompatibility complex class II antigens.

Prostanoids modulate inflammation and alloimmune responses during graft rejection

Acute rejection of a transplanted organ is characterized by intense inflammation within the graft. Yet, for many years transplant researchers have overlooked the role of classic mediators of inflammation such as prostaglandins and thromboxane (prostanoids) in alloimmune responses. It has been demonstrated that local production of prostanoids within the allograft is increased during an episode of acute rejection and that these molecules are able to interfere with graft function by modulating vascular tone, capillary permeability, and platelet aggregation. Experimental data also suggest that prostanoids may participate in alloimmune responses by directly modulating T lymphocyte and antigen-presenting cell function. In the present paper, we provide a brief overview of the alloimmune response, of prostanoid biology, and discuss the available evidence for the role of prostaglandin E2 and thromboxane A2 in graft rejection.

Exploratory calcineurin inhibitor-free regimens in living-related kidney transplant recipients

Chronic allograft nephropathy is among the major causes of graft loss even in low-risk kidney transplant recipients and correlates with acute nephrotoxic events during the first year post-transplant. Therefore, calcineurin inhibitor-free regimens may improve patient and graft survival among recipients of living-related kidney transplants. To confirm this hypothesis, we evaluated the efficacy and safety of two calcineurin inhibitor-free regimens in 92 low-risk recipients of one-haplotype living-related kidney transplants. Immunosuppression consisted of tacrolimus, azathioprine and prednisone (group I, GI, N = 38), 2 doses of daclizumab, mycophenolate mofetil (MMF), and prednisone (GII, N = 33) and 2 doses of daclizumab, MMF, sirolimus and prednisone (GIII, N = 21). At 12 months, treatment failure (biopsy-confirmed acute rejection, graft loss or death) was higher in GII compared to GIII and GI (54.5 vs 24.0 vs 13.1%, P < 0.01, respectively). In patients of black ethnicity the incidence of acute rejection was 25 vs 83.3 vs 20% (P = 0.055), respectively. Patient and graft survival was comparable. There were no differences in mean creatinine or calculated creatinine clearance at 12 months. Overall incidence of post-transplant diabetes mellitus (3.3%) and cytomegalovirus disease (4.3%) was similar in all groups. Further development of effective calcineurin inhibitor-free regimens should exclude patients of black ethnicity and may need full-induction therapy, perhaps with depleting agents, and concentration-controlled use of sirolimus and MMF.

Proximal tubular dysfunction as an indicator of chronic graft dysfunction

New strategies are being devised to limit the impact of renal sclerosis on graft function. Individualization of immunosuppression, specifically the interruption of calcineurin-inhibitors has been tried in order to promote better graft survival once chronic graft dysfunction has been established. However, the long-term impact of these approaches is still not totally clear. Nevertheless, patients at higher risk for tubular atrophy and interstitial fibrosis (TA/IF) development should be carefully monitored for tubular function as well as glomerular performance. Since tubular-interstitial impairment is an early event in TA/IF pathogenesis and associated with graft function, it seems reasonable that strategies directed at assessing tubular structural integrity and function would yield important functional and prognostic data. The measurement of small proteins in urine such as α-1-microglobulin, N-acetyl-beta-D-glucosaminidase, alpha/pi S-glutathione transferases, β-2 microglobulin, and retinol binding protein is associated with proximal tubular cell dysfunction. Therefore, its straightforward assessment could provide a powerful tool in patient monitoring and ongoing clinical assessment of graft function, ultimately helping to facilitate longer patient and graft survival associated with good graft function.

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Studentt-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Lyophilized allografts without pre-treatment with glutaraldehyde are more suitable than cryopreserved allografts for pulmonary artery reconstruction

Various methods are available for preservation of vascular grafts for pulmonary artery (PA) replacement. Lyophilization and cryopreservation reduce antigenicity and prevent thrombosis and calcification in vascular grafts, so both methods can be used to obtain vascular bioprostheses. We evaluated the hemodynamic, gasometric, imaging, and macroscopic and microscopic findings produced by PA reconstruction with lyophilized (LyoPA) grafts and cryopreserved (CryoPA) grafts in dogs. Eighteen healthy crossbred adult dogs of both sexes weighing between 18 and 20 kg were used and divided into three groups of six: group I, PA section and reanastomosis; group II, PA resection and reconstruction with LyoPA allograft; group III, PA resection and reconstruction with CryoPA allograft. Dogs were evaluated 4 weeks after surgery, and the status of the graft and vascular anastomosis were examined macroscopically and microscopically. No clinical, radiologic, or blood-gas abnormalities were observed during the study. The mean pulmonary artery pressure (MPAP) in group III increased significantly at the end of the study compared with baseline (P=0.02) and final [P=0.007, two-way repeat-measures analysis of variance (RM ANOVA)] values. Pulmonary vascular resistance of groups II and III increased immediately after reperfusion and also at the end of the study compared to baseline. The increase shown by group III vs group I was significant only if compared with after surgery and study end (P=0.016 and P=0.005, respectively, two-way RM ANOVA). Microscopically, permeability was reduced by ≤75% in group III. In conclusion, substitution of PAs with LyoPA grafts is technically feasible and clinically promising.