J. Ahonen

Gastric blood flow, tissue gas tension and microvascular changes during hemorrhage-induced stress ulceration in the pig

Various features of blood supply to the gastric mucosa were studied in the piglet stomach during stress ulceration induced by hemorrhagic shock. Gastric blood flow, as measured by the radioactive microsphere technique, significantly decreased during shock, but no major change occurred in the gastric function of total cardiac output. There was no difference in the magnitude of the decrease of mucosal blood flow between the nonulcerating antral mucosa and the more readily ulcerating corpus or fundic mucosa. At the same time, a significant decrease in tissue partial pressure of oxygen and increase in tissue partial pressure of carbon dioxide occurred, but again no difference was observed between the antrum and the corpus. Microangiographic studies demonstrated a clearly diminished filling of the arterial and capillary bed of the gastric mucosa during shock, suggesting intense vasoconstriction, thrombosis of the mucosal blood vessels, or both. These changes were more prominent in the corpus portion of the stomach than in the antrum. At the site of mucosal lesions, the filling defects persisted even after the shock, suggesting permanent thrombosis of the blood vessels.

Matrix Metalloproteinases 2 and 9 in Indomethacin-Induced Rat Gastric Ulcer

The restoration of functional connective tissue is a major goal of the wound healing process. The 72- and 92-kD gelatinases (MMP-2 and MMP-9) are extracellular matrix metalloproteinases (MMPs), which are known to degrade type IV and V collagens and gelatin, and have a potential role in wound healing. The spatial and temporal gelatinolytic activities of MMP-2 and MMP-9 were analyzed as a function of ulcer age, in homogenates of rat, indomethacin-induced, chronic gastric ulcers. The rats were sacrificed on 1, 3, 7, 12, 18, 24 and 28 days after subcutaneous indomethacin injections. Zymographic analyses revealed elevated activation of MMP-9 and latent and active MMP-2 in gastric ulcers, when compared to gastric tissue from non-indomethacin-treated rats. The intact tissue and tissue from ulcerous lesions contained MMP-2. The highest activity of MMP-2 was found in 3 day gastric ulcers and returned to the control level by day 24. MMP-9 was not present in the intact tissue and the highest gelatinolytic activity of MMP-9 was also observed on the 3rd day after administration of indomethacin. The activity thereafter decreased and returned to the control level by day 24. In situ hybridization was used to evaluate which cells synthesize MMP-2 and MMP-9. MMP-2 was seen mostly in fibroblast-like cells in the submucosa and MMP-9 in macrophage-like cells in the mucosa on the margins of the ulcers. Thus, we conclude that these two MMPs seem to have different functions during the gastric ulcer injury/healing process. MMP-2 may participate in the physiological turnover of the gastric extracellular matrix, whereas MMP-9 may be important in the early phase of gastric ulcer formation and also in the healing process.

Comparison of enalapril and valsartan in cyclosporine A‐induced hypertension and nephrotoxicity in spontaneously hypertensive rats on high‐sodium diet

We compared the effects of the angiotensin converting enzyme (ACE) inhibitor enalapril and the angiotensin AT1 receptor antagonist valsartan in cyclosporine A (CsA)‐induced hypertension and nephrotoxicity in spontaneously hypertensive rats (SHR). SHR (8–9 weeks old) on high‐sodium diet were given CsA (5 mg kg−1d −1 s.c.) for 6 weeks. The rats were treated concomitantly either with enalapril (30 mg kg−1d −1 p.o.) or valsartan (3 or 30 mg kg−1 d −1 p.o.). To evaluate the role of bradykinin in the action of enalapril, some rats received a bradykinin B2 receptor antagonist icatibant (HOE 140, 500 μg kg−1 d −1 s.c.) during the last 2 weeks of enalapril treatment. Blood pressure was recorded every second week by tail cuff method. Renal function was measured by serum creatinine, creatinine clearance and urinary excretion of proteins at the end of the experiment. The activity of the renal kallikrein‐kinin system was estimated by urinary kallikrein excretion. CsA caused hypertension, impaired renal function and induced morphological nephrotoxicity with glomerular damage and interstitial fibrosis. Enalapril and the lower dose of valsartan attenuated the CsA‐induced hypertension to the same extent, while the higher dose of valsartan totally abolished it. Icatibant did not reduce the antihypertensive effect of enalapril. Urinary kallikrein excretion was similar in all groups. Enalapril and valsartan equally prevented the CsA‐induced deterioration of kidney function and morphology. The renin‐angiotensin but not the kallikrein‐kinin system plays a crucial role in CsA‐toxicity during high intake of sodium in SHR.

Peripheral arterial disease as a predictor of outcome after renal transplantation

Abstract Our aim was to assess the prevalence of symptomatic and asymptomatic peripheral occlusive arterial disease (POAD) in 129 consecutive diabetic (n= 34) and non‐diabetic (n= 95) patients undergoing renal transplantation. The association of pre‐existent POAD and complaints of claudication, lower limb amputations, and graft and patient survival were evaluated during a 5‐year follow up. A questionnaire on walking capacity, ankle/brachial (ABI) and toe/brachial (TBI) pressure indices as well as the pulse volume recording (PVR) at the ankle were used to assess resting haemodynamics and the presence of POAD 4 days after the transplantation. Unquestionable ischaemia was encountered in 5 (4 %) patients all with a history of intermittent claudication and an ABI equal or below 0.77. While using assessment methods not affected by vessel calcification, i.e. toe pressures and PVR damping, a many‐fold frequency of arterial disease was observed when compared to previous studies. TBI below 0.65 was found in 11 of diabetic (32 %) and in 15 of the others (16%), and a PVR amplitude below 5 min in 28 of diabetics (82 %) and in 34 of non‐diabetics (36 %). During the 5‐year follow up, abnormal TBI and PVR values and diabetes at the time of transplantation were the greatest risk factors for proximal foot amputations. The low TBI levels also indicated a shortened patient survival. However, transplant function was not affected by the presence of abnormal haemodynamic indices at the time of transplantation.

Time-Related Effects of Cytomegalovirus Infection on the Development of Chronic Renal Allograft Rejection in a Rat Model

Cytomegalovirus (CMV) infection is a risk factor for chronic allograft rejection. The histological findings of chronic renal allograft rejection include inflammation, vascular intimal thickening, glomerulosclerosis, tubular atrophy and fibrosis. We have developed a rat model of renal transplantation in which transplants, after an early inflammatory episode, end up with chronic rejection within 60 days. During the early phase of the process in this model, CMV increased and prolonged the inflammatory response, the expression of adhesion molecules, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and their ligands, lymphocyte function antigen-1 and very late antigen-4 in the graft. Simultaneously, the production of various growth factors, such as transforming growth factor beta, platelet-derived growth factor and connective tissue growth factor was upregulated, which induce smooth muscle cell proliferation in the vascular wall and collagen synthesis by fibroblasts. Chronic rejection developed within 20 days in CMV-infected grafts. In summary, CMV infection accelerated and enhanced the early immune response, the induction of growth factors and collagen synthesis, and the development of chronic rejection in renal allografts.

Renal allograft immunosuppression

The long-term effects of different immunosuppressive drugs and regimens on renal allograft histology are virtually unknown. Therefore, in order to investigate the long-term effects of triple drug treatment versus different combinations of two immunosuppressive drugs on allograft histology, a prospective, randomized trial was performed. One group received triple therapy consisting of low-dose cyclosporin (CyA), azathioprine (Aza), and methylprednisolone (MP), and three groups received combinations of two drugs, i.e., Aza plus CyA, Aza plus MP, and CyA plus MP. At 2 years, there were no significant differences with regard to graft (80%) or patient (87%) survival, or to graft function between the four groups. After 2 years, a protocol core biopsy was taken of all 102 patients having a functioning graft. Of these patients, 61 (60%) were still following the original, randomized treatment protocol; in the remaining cases, changes had occurred in the original protocol and so these cases were considered drop-outs in this study. Histological specimens were examined blindly by two independent observers. Most of the 34 histological variables examined showed no changes. Diffuse fibrosis was most frequent in the CyA plus MP group (70%) and significantly more severe than in the triple therapy group. Mesangial matrix increase in glomeruli was significantly less common in the triple therapy group (8%) than in any one of the double drug combination groups (47%). Two other changes in glomeruli — Bowman capsular thickening and global glomerular sclerosis — were also less frequent in the triple therapy group. Vascular changes other than intimal proliferation (39%) and arteriosclerosis (24%) were uncommon in all groups and least frequent in the triple therapy group. Isometric vacuolation in proximal tubules was found in every group using CyA. It was least prominent in the triple therapy group and most prominent in the CyA plus MP group; it was not seen in the Aza plus MP group. Other specific findings for the groups treated with Cya could not be identified. To summarize, the changes shown were mild and rather similarly distributed in the four treatment groups. Histopathological alterations comparable with chronic rejection, i.e., persistent interstitial inflammation with pyroninophilic cells, vascular intimal proliferation, and arteriosclerosis, were seen in all groups, but these changes were least prominent in the group receiving triple therapy.

Sequential analysis of adhesion molecules and their ligands in rat renal allografts during the development of chronic rejection.

Abstract Intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) are important in endothelial cell‐leukocyte interactions. In this sequential study, the expression of ICAM‐1 and VCAM‐1 and their ligands LFA‐1 and VLA‐4 as well as major histocompatibility complex class II antigens (MHC class II), and interleukin‐2‐receptor (IL‐2R) were investigated during the development of chronic renal allograft rejection in a rat model. The time‐related expression of adhesion molecules and their ligands in the graft was correlated to the chronic allograft damage index (CADI). In association with an initial short immune activation, there was a significant ICAM‐1 and VCAM‐1 induction in the vascular endothelium and the tubular epithelium. In the interstitium, there was infiltration of lymphocytes expressing ligand molecules VLA‐4 and LFA‐1, as well as activation markers MHC class II and IL‐2R. Thereafter, the expression declined together with the increase of CADI‐values. In end‐stage chronic rejection, there was practically no expression of ICAM‐1 and VCAM‐1. In the interstitium, there were only few ligand‐expressing leukocytes. In conclusion, adhesion molecules and their ligands are involved in the induction phase of the process but no longer in the later stages of chronic rejection.

Renal allograft immunosuppression. IV. Comparison of lipid and lipoprotein profiles in blood using double and triple immunosuppressive drug combinations.

Abstract. Serum lipid and lipoprotein profiles were performed in order to investigate lipid abnormalities 2 years post‐transplantation in first cadaveric renal allograft recipients immunosuppressed with cyclosporin (CyA), azathioprine (Aza), and methylprednisolone (MP), or with any combination of two drugs. CyA was used in low doses. Total serum cholesterol, triglyceride, LDL cholesterol, HDL cholesterol, HDL2 cholesterol, HDL3 cholesterol, apolipoprotein A1, and apolipoprotein B were determined in 88 prospectively randomized patients with functioning grafts. When considering only the patients who remained on the original randomized treatment, there were no significant differences between the four groups in any of the measured variables. Mean total cholesterol was highest in the group receiving Aza and MP (6.8 mmol/l) and lowest in the group receiving triple therapy (5.8 mmol/l; NS). Mean triglyceride level was highest in the group receiving Aza and MP (2.3 mmol/1) versus 1.8–2.2 mmol/l in the groups receiving triple therapy, Aza + CyA, and CyA + MP. For all patients mean triglyceride level was highest in the group receiving Aza and MP (2.7 mmol/1) and lowest in the group receiving triple therapy (1.7 mmol/l; P<0.05). Mean HDL cholesterol ranged from 1.5 to 1.6 mmol/l in all groups. Neither CyA concentration nor CyA or MP dose correlated with cholesterol or triglyceride concentration. However, the average MP dose was twice as high in the group receiving Aza and MP as in the other two groups employing steroids. Serum cholesterol and triglyceride concentrations were related to body mass index (r= 0.28, P= 0.045 and r= 0.30, P= 0.029, respectively). Hyperlipidemia was most common in the group receiving Aza and MP, The frequency of hypercholesterolemia (serum cholesterol level > 6.5 mmol/1) was 18%, 45%, 60%, and 35% for the patients continuing with the originally randomized treatment in the groups receiving triple therapy, Aza + CyA, Aza + MP, and CyA + MP, respectively. In a normal Finnish reference population, 35% of all males and 31% of allfemaleshaveaserumcholesterollevel above 6.5 mmol/l. Thus, only patients receiving Aza and MP had a clearly higher frequency of hypercholesterolemia than that found in a normal population. Taken together, this study shows no lipid abnormalities associated with the use of low‐dose CyA for 2 years after transplantation. Hyperlipidemia occurring after transplantation is probably multifactorial and more associated with other risk factors than with the immunosuppressive therapy.

Renal allograft immunosuppression: II. A randomized trial of withdrawal of one drug in triple drug immunosuppression

Abstract. A prospective randomized study was conducted to evaluate the impact of four different conversion protocols on graft outcome in long‐term follow‐up. Between January 1986 and May 1987, 128 patients with first cadaveric kidney allografts were randomized at the time of transplantation to four treatment groups of 32 patients each, to be assigned 10 weeks post‐transplantation. During the first 10 weeks, all patients received triple therapy with low‐dose azathioprine (Aza), cyclosporin (CyA), and methylprednisolone (MP). After 10 weeks, one group continued with triple therapy (group A) while the three other groups received different combinations of two drugs, namely, Aza and CyA (group B), Aza and MP (group C), or CyA and MP (group D). Withdrawal of MP (group B) or especially of CyA (group C) was associated with 4/29 (14%) and 10/28 (36%) acute rejection episodes, respectively, for 60 days after conversion. All rejections were mild and reversible. There were no rejections after Aza withdrawal or in the group that continued on triple therapy during the corresponding time period. The most common reason for dropping out after withdrawal, for those patients who could not continue on the originally randomized medication, was azathioprine intolerance (n= 12). Five patients were switched back to triple therapy after CyA withdrawal due to rejection. Steroid intolerance was rare and CyA in low doses was very well tolerated. At 1 year there were no statistically significant differences in graft survival between groups A, B, C, and D‐81 %, 88%, 88%, and 88%, respectively‐or in patient survival‐88%, 88%, 88%, and 97%, respectively. For those patients continuing with the originally randomized treatment protocol, there were no differences in patient or graft survival either, the means being 91% and 89%, respectively. The most common cause of death after withdrawal was cardiovascular in nature, and there were no more fatal infections under triple drug treatment than with double drug regimens. There were no statistically significant differences in mean serum creatinine values at 1 year. The median serum creatinine values for groups A, B, C, and D were 112, 132, 133, and 133 μmol/l, respectively. At 1 year the mean CyA dose in the groups that continued with CyA was 3. 5–4. 2 mg/kg per day and CyA concentrations were equal.

Detection of cytomegalovirus by the early-antigen immunofluorescence test versus conventional tissue culture

The two methods commonly used to diagnose cytomegalovirus (CMV) infections, conventional tissue culture and detection of early CMV nuclear antigen by immunofluorescence from cell culture, were performed in parallel on 597 clinical specimens. CMV was detected by the early-antigen test in 108 samples, of which 102 (94 %) were detected 1 to 3 days after inoculation. Of these 108 CMV-positive specimens, seven were negative on conventional culture. Two samples negative in the early-antigen test were positive on conventional culture. Thus, CMV was detected in 110 specimens. A cytopathic effect in conventional tissue culture occurred 9 to 42 days after inoculation. The diagnosis of CMV infection was possible by the conventional method 29.6 ± 12.7 days and by early-antigen immunofluorescence 1.9 ± 1.5 days after obtaining the specimen. The rapid early-antigen test was slightly more sensitive than culture, and fewer samples were lost due to bacterial or fungal infections during incubation. Detection of CMV by conventional culture usually requires several weeks and provides a diagnosis only retrospectively. The main advantage of the early-antigen test is that a virologically proven diagnosis of CMV infection is available at an early stage.