Cecilia Johnsson

Tolerance induction ameliorates allograft vasculopathy in rat aortic transplants. Influence of Fas-mediated apoptosis.

Based on successful induction of donor-specific unresponsiveness by alloantigenic stimulation in several animal models of acute rejection, we hypothesized that similar immune manipulations would also inhibit the evolution of chronic rejection and transplant vasculopathy. To induce immune tolerance, DA rats received a PVG heart allograft and were immunosuppressed with cyclosporine for 30 d. At day 100 the animals were challenged with a PVG aortic allograft after either 1 or 18 h of cold ischemia. 8 wk after the aortic transplantation, the grafts were investigated for morphological changes, infiltrating cells, apoptosis, and Fas-Fas ligand expression. Control allografts showed advanced transplant arteriosclerosis, whereas tolerance-induced aortic allografts displayed reduced neointimal formation, less medial atrophy, fewer apoptotic cells, and fewer Fas- and FasL-expressing cells. Prolonged ischemic storage time did not profoundly alter the morphological changes of the allografts. Fas expression was found in T cells, macrophages, vascular smooth muscle cells, and endothelial cells, whereas FasL was expressed mainly by T cells and macrophages. FasL mRNA expression was evident throughout the entire allograft wall. In conclusion, induction of allospecific tolerance can effectively prevent transplant arteriosclerosis. Cold ischemia damage does not abrogate the beneficial effect of tolerance, but creates a separate identity of mainly endothelial lesions. Furthermore, Fas-mediated apoptosis appears to be involved in the pathological lesions seen in chronic rejection.

MC 1288 — A vitamin D analogue with immunosuppressive effects on heart and small bowel grafts

The vitamin D analogue MC 1288 (20-epi-1α,25-dihydroxycholecalciferol) was tested here for its possible immunosuppressive properties in vivo using different rat transplantation models. MC 1288, in a dose of 0.1 μg/kg daily, administered intraperitoneally for 10 days, was found to be effective in prolonging cardiac allograft survival. Untreated recipients rejected their grafts around day 8 while MC 1288 treatment delayed rejection until day 22 (P<0.001). Addition of the immunostimulatory drug LS-2616 (Linomide) reduced the immunosuppressive effect of MC 1288 and rejection occurred around day 11. The immunosuppressive effect of MC 1288 on rejection following small bowel transplantation was determined by measuring the amounts of hyaluronan (HA) secreted into the intestinal lumen. On day 6 post-transplantation the amounts of intraluminal HA in untreated animals was 29.2±5.3 ng/min and cm, while in MC 1288-treated animals it was just 5.0±1.6 ng/min and cm (P<0.01). We conclude that MC 1288 has immunosuppressive effects that may make it suitable for the prevention of graft rejection.

The effects of combined treatment with the novel vitamin D analogue MC 1288 and cyclosporine A on cardiac allograft survival

The efficacy of combined treatment with the novel vitamin D analogue MC 1288 (20-epi-1 alpha,25-dihydroxycholecalciferol) and cyclosporine A (CyA) in preventing rejection following organ transplantation was evaluated in the heterotopic cardiac allograft model. Wistar/Kyoto rats received hearts from PVG donors and were subsequently treated with MC 1288 and CyA in various dose combinations. Administration of MC 1288 0.1 microgram/kg together with CyA 5 mg/kg produced significantly prolonged graft survival times as compared with single therapy with MC 1288 0.1 microgram/kg (p < 0.01) or CyA 5 mg/kg (p < 0.001). MC 1288 and CyA were tested also in combination with the immunomodulating drug LS-2616, which, in several laboratories, is used as a model for the evaluation of new combinations of immunosuppressive drugs. LS-2616 abolishes the immunosuppressive effect of CyA and the transplanted hearts are thus rejected at the same time as grafts of untreated recipients, i.e. on day 8. The immunosuppressive effect of MC 1288 is also counteracted by LS-2616, but the effect is not absolute and the median graft survival time is 11.0 days. Combined treatment with MC 1288 and CyA in the presence of LS-2616 resulted in a median graft survival time of 14.0 days. The results obtained in our experiments indicate at least an additive effect of MC 1288 and CyA thus suggesting that in the future MC 1288, or other vitamin D analogues, might be used to control rejection, either alone, in combination with CyA or in combination with other immunosuppressive drugs with other mechanisms of action.

Effects of commonly used immunosuppressants on graft-derived fibroblasts

In acute rejection of transplanted organs intragraft fibroblasts increase their production of hyaluronan. Hyaluronan has strong water binding capacity and an increased tissue content of hyaluronan thus contributes to the development of interstitial oedema. The present study examined the effects of commonly used immunosuppressants (prednisolone, cyclosporin, tacrolimus, mycophenolic acid and sirolimus) on fibroblast proliferation, hyaluronan production and cell surface receptor expression. Fibroblasts isolated from rejecting tissue and from normal, non‐transplanted tissue were studied in parallel. All substances investigated, except tacrolimus, were found to affect fibroblasts in one way or another. The most striking effect was the almost total inhibition of fibroblast proliferation in the presence of mycophenolic acid. Cyclosporin reduced the proliferation by about 50% and prednisolone had an inhibiting effect on hyaluronan production (50% reduction). These effects were observed on fibroblasts isolated from rat cardiac allografts undergoing rejection as well as on fibroblasts obtained from normal heart tissue. In contrast, sirolimus was found to stimulate the proliferation of fibroblasts from rejecting tissue (100% increase), but not that of normal fibroblasts. The majority of the fibroblasts expressed the hyaluronan receptor CD44, with a more intense expression in cultures of fibroblasts derived at rejection. None of the immunosuppressants affected the staining pattern (number of positive cells or intensity). The inhibitory effects of prednisolone, cyclosporin and mycophenolic acid on fibroblasts may contribute to the overall beneficial effects of these drugs when used for prevention or treatment of rejection.

Renomedullary and intestinal hyaluronan content during body water excess: a study in rats and gerbils

Our previous studies in rats have suggested a role for renomedullary hyaluronan (HA) in water homeostasis. The gerbil is known for its unique ability to conserve water. In the present study renal papillary and intestinal HA were compared between groups of anaesthetized gerbils and rats before and after up to 6 h of i.v. water loading. Baseline papillary HA in gerbils was only 37 % of that in the rat. Water loading in rats increased the papillary HA content. Elevation was maximal (+27 %, P < 0.05) after 2 h of water loading and then declined to control levels after 6 h of water loading (+3 %, n.s.). In contrast, the gerbil responded with a decreased papillary HA content during water loading. The depression was maximal after 2 h (‐49 %, P < 0.05) and was still 41 % below the control values after 6 h (P < 0.05). The urine flow rate increased rapidly in the rat and its maximum, 21 times above the control level (P < 0.05), occurred at the HA peak, i.e. after 2 h of water loading while in the gerbil, the urine flow rate increased slowly and slightly and was only six times above control values after 6 h of water loading (P < 0.05). The HA content along the intestine was similar in the two species: lowest in the duodenum and jejunum and highest in the distal colon. To conclude, in the rat, the elevation of papillary interstitial HA during acute water loading would counteract water reabsorption by changing the physico‐chemical characteristics of the interstitial matrix favouring rapid water diuresis. This would work as a complement to the powerful regulation by ADH. The gerbil has a diametrically different regulation of papillary HA turnover during water loading. The decreased papillary HA level during water loading and the slow and small diuretic response may represent a genetic difference in adaptation to enhance the ability to conserve water in an arid environment.

Renomedullary interstitial cells in culture; the osmolality and oxygen tension influence the extracellular amounts of hyaluronan and cellular expression of CD44

Our previous studies have suggested a role for renomedullary interstitial cells (RMICs) and renal medullary hyaluronan (HA) in water homeostasis. In the present study, cultured rat RMICs were used to examine the relationship of osmolality and oxygen tension on the extracellular amount of HA in the culture and to the cellular immunoreactivity to CD44, a HA binding protein. Under isotonic (330 mOsm(.)kg(-1) H(2)O), normoxic (20% O(2)) conditions, supernatant from sub-confluent RMICs contained 120+/-37 pg 10(4) cells(-1) 24 h(-1) of HA. Under hyperosmotic conditions (630 mOsm kg(-1) H(2)O), HA in the supernatant was decreased by 42% and under hypoosmotic conditions (230 mOsm kg(-1) H(2)O) it was doubled. Under hypoxic, iso-osmolar conditions (5% and 1% O(2), 330 mOsm kg(-1) H(2)O) this HA content was decreased by 56 and 48%, respectively, compared with normoxic, iso-osmolal conditions. Expression of CD44 on sub-confluent cells increased with increasing osmolality, as shown by immunostaining and flow cytometric analysis. The increases in CD44 from 330 to 630, 930 and 1230 mOsm kg(-1) H(2)O amounted to 5, 142 and 212%, respectively. Low oxygen tension (5% O(2)) decreased the intensity of CD44 immunofluorescence by 31%. Cell viability was similar at all conditions studied. In summary, these data indicate that cultured RMICs produce HA and are immunoreactive to CD44. In the supernatant of RMICs, the HA content decreases under hyperosmotic, hypoxic conditions. Conversely, CD44 immunoreactivity increases under hyperosmotic conditions. These results may explain our previous in vivo findings of a decreased renal papillary HA content during anti-diuresis and an increased content during water diuresis. The results support the concept that RMICs play an important role in renal water handling.

Ex vivo PKH26-labelling of lymphocytes for studies of cell migration in vivo

A prerequisite for studies of cell migration is that the cells of interest can be appropriately labelled and subsequently easily traced. The use of radioisotopes or fluorescent substances that bind covalently to the cell surface, e.g. fluorescein isothiocyanate (FITC) or rhodamine isothiocyanate (RITC), have limitations such as rapid loss of the labelling, toxicity and interference with cell surface molecules. In the present work the authors labelled rat spleen lymphocytes with the fluorescent labelling molecule PKH26, which is incorporated into the lipid bilayer of cytoplasmic membranes. The labelled lymphocytes were injected intravenously into syngeneic recipients and 2 or 6 days later the lymphocytes were detected in various organs by using flow cytometry and fluorescence microscopy. As could be expected, the lymphocytes homed to lymphoid tissues, preferably the spleen, and no labelled cells were found in non‐lymphoid organs such as the heart and the kidney. Membrane labelling proved to be intense, uniform and stable and PKH26 positive cells were easily detectable in fractions less than 0.2% in peripheral blood and the various tissues after 6 days of in vivo circulation. Thus, the PKH26 dye appears to be suitable for labelling cell populations used in the study of cell migration in vivo, both under normal conditions and when specific immunological processes are taking place, such as graft rejection and tumour growth.

Hormonal regulation of renomedullary hyaluronan

Aim:  Hyaluronan (HA) is involved in renomedullary water handling through its water‐binding capacity. This study addressed the effect of hormones involved in regulating fluid‐electrolyte homeostasis on renomedullary HA content in vivo and in vitro.

Comparison of ultrasmall superparamagnetic iron oxide particles and low molecular weight contrast agents to detect rejecting transplanted hearts with magnetic resonance imaging.

Purpose:This study compared 3 different contrast agents for magnetic resonance imaging (MRI) to determine whether their rate of permeability could discriminate between acutely rejecting and nonrejecting transplanted hearts. Materials and Methods:Heterotopic heart transplantation in rats was performed and animals were divided into allogeneic and syngeneic groups. At the sixth postoperative day, MRI was performed with 2 ultrasmall superparamagnetic iron oxide particles of different sizes, unformulated NC 100150 and AMI-227, besides gadodiamide. Results:For unformulated NC100150, the signal intensity between the groups differed at all times (P < 0.0001); for AMI-227, all times but 3 (P < 0.05). With low molecular weight gadolinium chelate, no discrimination between rejecting and nonrejecting groups could be made. Conclusion:Acutely rejecting and nonrejecting transplanted hearts can be discriminated from each other using the difference in permeability assessed by blood pool agents and MRI. With first pass of gadolinium chelate no discrimination could be made between rejecting and nonrejecting grafts.

Successful retransplantation of mouse-to-rat cardiac xenografts under immunosuppressive monotherapy with cyclosporine

The present study was undertaken to investigate whether retransplantation with a second xenograft, from the same species as the primary graft, is possible to achieve using only moderate immunosuppression. Heterotopic mouse-to-rat cardiac transplantations were performed, and the recipients were treated with 15-deoxyspergualin (DSG) and cyclosporine (CsA) at high doses for days -1 to 4 and at moderate doses for days 5 to 28. From day 29 and onward, the immunosuppressive protocol consisted of daily oral administration of CsA 10 mg/kg as monotherapy. Animals that had beating grafts when DSG treatment was stopped were retransplanted 56-154 days after the primary transplantation, either with a vascularized graft (heart) or with nonvascularized graft (pancreatic islets), under continued therapy with CsA. Six of 10 secondary cardiac xenografts functioned for more than 50 days and were harvested beating after 60-100 days. In contrast, nonimmunosuppressed or DSG-treated rats are known to reject a second cardiac mouse graft hyperacutely. The unresponsiveness was confined to cardiac tissue, as the pancreatic islets, transplanted under the kidney capsule, were totally rejected after 14 days. Long-term functioning cardiac xenografts, primary and secondary, had a well-preserved morphology, and infiltrating mononuclear cells were found just in the periphery of the grafts. A majority of these cells were macrophages expressing the ED1, but not the ED2, antigen. No deposition of IgG or complement was seen in any of the graft vessels, whereas a slight deposition of IgM was observed in some vessels of both primary and secondary grafts. In conclusion, we have demonstrated that unresponsiveness can be induced by effective immunosuppression of the recipient at the time of the initial transplantation, so that retransplantation with a second xenograft can be performed successfully under single-drug immunosuppressive therapy with CsA.