Immune cells and immunosuppression in a porcine bronchial model of obliterative bronchiolitis.

BACKGROUND: To study obliterative bronchiolitis (OB), we have developed a porcine heterotopic bronchial model. Allografts obliterate within 3 weeks, the immunosuppression cyclosporine (CsA)-azathioprine (AZA)-methylprednisolone (MP) delays OB, but OB is prevented when AZA is switched to 40-0-(2-hydroxyethyl)-rapamycin (RAD). To characterize our model, we studied immune cells under various immunosuppressive conditions. METHODS: The groups studied were autografts (U), allografts (A), and allografts given either CsA-RAD-MP (R), or CsA-AZA-MP (C). The implants were harvested at 3, 7, 10, 14, 21, 30, 60, and 90 days after transplantation. Epithelial damage and obliteration were graded histologically, and the number of CD4, CD8, MHC class II expressing cells, macrophages, and B lymphocytes were counted (mean+SEM)/high-power visual field. RESULTS: In group U, normal epithelium was regained with no obliteration and only few immune cells. In group A, consistent with initially acute ejection, an influx of CD4 (105+23), CD8 (166+23), and class II (92+20) cells was seen up to day 21, when total obliteration preceded by epithelial destruction had already developed. Some macrophages were seen and B cells were scarce. In group R, epithelial damage and obliteration were insignificant, but moderate numbers of CD4, CD8, and class II cells were seen. In group C, epithelial damage and obliteration were only delayed, but the immune cell response was clearly blunted. CONCLUSIONS: In our model, rejection with significant immune cell influx was still active when obliteration was total in nontreated allografts. In immunosuppressed allografts, decrease in the number of immune cells alone did prevent OB. These results support OB being T-cell mediated. RAD may have additional important effects on growth factors and proliferation in prevention of OB.

Cytomegalovirus infection, viral DNA, and immediate early-1 gene expression in rejecting rat liver allografts.

BACKGROUND: Cytomegalovirus (CMV) infection has been linked to acute and chronic rejection. We have previously shown that concomitant rat cytomegalovirus (RCMV) infection increases portal inflammation and bile duct destruction in rejecting rat liver allografts. Many of the pro-inflammatory effects of CMV have been attributed to the immediate early (IE) proteins of CMV. We wanted to investigate whether RCMV and IE-1 gene expression persist in the liver graft in our model. METHODS: Liver transplantations were performed from PVG (RT1c) into BN (RT1n) rats. One day after transplantation, the rats were infected with RCMV. No immunosuppression was given. The graft infection was studied by viral culture, immunofluorescence, DNA in situ hybridization and RT-PCR for the detection of IE-1 mRNA at various time points. RESULTS: RCMV caused an active infection from 5 days to 2 weeks after transplantation, during which infectious virus was found in the graft. Thereafter the cultures were negative. RCMV antigens and DNA were found in hepatocytes, endothelial, inflammatory, and bile duct cells during the active infection. At 4 weeks, RCMV DNA positive hepatocytes, endothelial, inflamma tory, and bile duct cells could still be found, but in much smaller quantities. IE-1 mRNA expression was, however, only detected during the active infection, not at 4 weeks postinfection. CONCLUSIONS: RCMV IE-1 expression does not persist in the graft after the active infection, although some viral DNA can be detected in the graft up to 4 weeks. In our model, the CMV-induced increase in graft damage does not seem to require the continued expression of IE-1.

Rat cytomegalovirus infection in kidney allograft recipients is associated with increased expression of intracellular adhesion molecule-1 vascular adhesion molecule-1, and their ligands leukocyte function antigen-1 and very late antigen-4 in the graft.

BACKGROUND: Cytomegalovirus (CMV) infection is suggested to be a risk factor for chronic rejection. We have recently shown that rat CMV (RCMV) increases the inflammatory response and accelerates chronic rejection in a model of rat kidney allograft. In this study, the early inflammatory response and time-related expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and their ligands, leukocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), in the grafts were investigated in RCMV-infected rats and compared to noninfected rats developing chronic rejection. METHODS: Transplantations were performed in a rat strain combination of DA (RT1a)->BN (RT1n) receiving triple drug immunosuppression. One group of rats was infected with RCMV, and the other was left uninfected. The grafts were harvested at different time points after transplantation. The adhesion molecules, their ligands and activation markers, MHC class II antigens and interleukin-2-receptors (IL-2-R), were demonstrated by monoclonal antibodies and immunoperoxidase staining from frozen sections of the grafts. Graft histology was evaluated according to the Banff criteria. RESULTS: RCMV caused a significant, prolonged increase of VCAM-1 and ICAM-1 expression in the vascular endothelium compared to the noninfected grafts. Also, the number of cells expressing activation markers, LFA-1 and VLA-4 was significantly enhanced in these animals. Significantly enhanced histological changes of chronic rejection were seen in the RCMV-infected group. CONCLUSIONS: Prolonged, increased expression of ICAM-1 and VCAM-1, and increased numbers of inflammatory cells expressing their ligands in the CMV infected grafts, were associated with accelerated chronic allograft nephropathy.

Alloimmune injury preceding airway obliteration in porcine heterotopic lung implants: a histologic and immunohistologic study.

BACKGROUND: Obliterative bronchiolitis (OB), the major long-term complication of lung transplantation, has thus far lacked a good large-animal model. Our goal was to develop such a model on the basis of previous rodent models with tracheal implants. METHODS: Fragments of pulmonary tissue with structures of terminal bronchi were subcutaneously transplanted to four random-bred domestic piglets. Each animal received 10 autograft and 10 allograft implants. The histologic findings were graded from 0 to 3 for implants harvested repeatedly over 2 months. RESULTS: In autografts, partial destruction of the respiratory epithelium and gradual luminal obliteration as well as mild damage to the cartilage and the bronchial wall underwent rapid reversal after initial ischemic injury. In the allografts, epithelial destruction and gradual obliteration were total within 14 days, the difference being statistically significant (P<0.05) in both. The histologic features of the obliterative plug were similar to those of human OB. In the allografts, cartilaginous destruction and pericartilaginous inflammation increased gradually to severe levels, significantly worse than in the autografts (P<0.05). Necrosis and inflammation of the bronchial wall were also more severe in the allografts (P<0.05). CONCLUSIONS: At the end of follow-up, all autografts were vital, whereas the allografts were almost totally rejected and were without native structures. All bronchi in the allografts exhibited accelerated obliteration with histologic features characteristic of human OB, thus providing a model for research into OB and its prevention.

Direct induction of class II molecules by cytomegalovirus in rat heart microvascular endothelial cells is inhibited by ganciclovir (DHPG).

It has been demonstrated both in vivo and in vitro that cytomegalovirus regulates not only MHC class I but also class II antigen expression on vascular endothelial cells. The CMV-linked MHC induction is believed to be involved in acute rejection mechanisms and chronic vasculopathy after transplantation. In this study, we have investigated the effect of 9-(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG; ganciclovir) on CMV-induced class II expression in cultured rat heart microvascular endothelial cells. Two sets of cultured endothelial cells were infected with rat CMV. One of the sets was treated with various concentrations of DHPG and the other set was left untreated. MHC class II antigen expression on the cells was demonstrated by mAbs, by immunoperoxidase (IP) technique, and by FACS. Class II expression was maximal on CMV-infected cells 4-7 days after infection when 77.5 +/- 14.5% of the endothelial cells expressed class II in IP staining. DHPG inhibited the induction of class II, but the inhibitory effect was dependent upon the drug concentration. With increasing concentrations of DHPG (0, 1, 10, 100, and 1000 micrograms/ml), as demonstrated by IP staining (surface and intracellular expression), the frequency of class II-positive cells decreased from 77.5 +/- 14.5% to 58.0 +/- 15.0%, 36.0 +/- 23.0%, 3.0 +/- 3.0%, and 0 +/- 0%, respectively. By FACS (surface expression), the number of class II-positive cells remained somewhat lower, but decreased similarly with increasing concentrations of DHPG (from 37.8 +/- 1.1% to 6.2 +/- 3.1%) (surface expression). Also, the intensity of expression decreased concomitantly. These results suggest that DHPG inhibits CMV-induced class II expression via inhibition of CMV DNA polymerase.

Cytomegalovirus infection-enhanced allograft arteriosclerosis is prevented by DHPG prophylaxis in the rat.

BACKGROUND: Major risk factors for accelerated allograft arteriosclerosis include humoral and cellular immune response, hyperlipidemia, and viral infections. We demonstrated earlier that rat cytomegalovirus (RCMV) infection doubles smooth muscle cell proliferation and intimal thickening of rat aortic allografts. In this study, the effects of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) on RCMV-enhanced rat allograft arteriosclerosis are investigated. METHODS AND RESULTS: Aortic allografts from the DA to the WF rat strain were used. The recipients were inoculated with 10(5) plaque-forming units of RCMV 1 day after transplantation. Two groups of RCMV-infected rats were treated with DHPG with an initial dose of 20 mg/kg IP and a maintenance dose of 10 mg/kg IP twice a day for a period of 14 days. In the DHPG prophylaxis group (n = 22), the drug administration started 1 day before infection, and in the DHPG treatment group (n = 17), 7 days after infection. One group of infected rats was left untreated (n = 21). The grafts were removed 7 and 14 days and 1, 3, and 6 months after transplantation. In the DHPG prophylaxis group, no virus could be recovered by plaque assays. In the treatment group, 50% of rats were virus-positive at 1 month and 40% at 3 months. DHPG prophylaxis prevented the infiltration of inflammatory cells and their proliferation in the adventitia of RCMV-infected recipients (P < .01), with a 60% reduction in the interleukin-2 receptor expression (P < .05) and a 30% decrease in major histocompatibility complex class II expression (P = NS). DHPG prophylaxis did not significantly alter the levels of insulin-like growth factor-1, epidermal growth factor, platelet-derived growth factor-BB, transforming growth factor-beta 1, acidic fibroblast growth factor, and basic fibroblast growth factor messages in the allograft vascular wall. Early media necrosis was reduced. Arteriosclerotic alterations and proliferation of smooth muscle cells were both reduced 50% to 70% by DHPG prophylaxis (P < .05 at 3 months). The responses in the DHPG treatment group were quite similar but less impressive and statistically nonsignificant. CONCLUSIONS: We consider it likely that DHPG inhibits arteriosclerotic alterations primarily by reducing the infectious virus and thereby the inflammatory response in the allograft vascular wall; another possibility is a direct antiproliferative effect on smooth muscle cell replication. A dose-dependent inhibitory effect of DHPG on smooth muscle cell replication was recorded in an in vitro study.

Triple drug immunosuppression significantly reduces immune activation and allograft arteriosclerosis in cytomegalovirus-infected rat aortic allografts and induces early latency of viral infection.

The effect of triple drug immunosuppression (cyclosporine A 10 mg/kg/day+methylprednisolone 0.5 mg/kg/day+azathioprine 2 mg/kg/day) on rat cytomegalovirus (RCMV)-enhanced allograft arteriosclerosis was investigated applying WF (AG-B2, RT1v) recipients of DA (AG-B4, RT1a) aortic allografts. The recipients were inoculated intraperitoneally with 10(5) plaque-forming units of RCMV 1 day after transplantation or left noninfected. The grafts were removed on 7 and 14 days, and at 1, 3, and 6 months after transplantation. The presence of viral infection was demonstrated by plaque assays, cell proliferation by [3H]thymidine autoradiography, and vascular wall alterations by quantitative histology and immunohistochemistry. Triple drug immunosuppression reduced the presence of infectious virus in plaque assays and induced early latency of viral infection. It significantly reduced the peak adventitial inflammatory response (P < 0.05) and reduced and delayed intimal nuclear intensity and intimal thickening (P < 0.05) in RCMV-infected allografts. The proliferative response of smooth muscle cells was reduced by triple drug immunosuppression to 50% of that observed in nonimmunosuppressed RCMV-infected allografts, but still the proliferative peak response was seen at 1 month. Only low level immune activation, ie, the expression of interleukin-2 receptor (P < 0.05) and MHC class II, was observed under triple drug immunosuppression in the adventitia of RCMV-infected allografts, whereas there was no substantial change in the phenotypic distribution of inflammatory cells. In conclusion, although RCMV infection significantly enhances allograft arteriosclerosis also in immunosuppressed allografts, triple drug immunosuppression has no additional detrimental effect but rather a protective one on vascular wall histology. These results further suggest that RCMV-enhanced allograft arteriosclerosis may be an immunopathological condition linked to the host immune response toward the graft and/or the virus rather than a direct virus-induced phenomenon.

Cytomegalovirus infection-associated generalized immune activation in heart allograft recipients: a study of cellular events in peripheral blood and endomyocardial biopsy specimens.

An association between cytomegalovirus (CMV) infection, heart allograft rejection, and arteriosclerosis has been reported. To investigate the mechanisms of this association, the cellular immune response in peripheral blood and the inflammation in heart allografts during antigenemia were studied. CMV antigenemia occurred in 13 recipients. In recipients with severe CMV infection, a significantly weaker immune response was recorded in peripheral blood: fewer lymphoid blast cells (max. 2.4% +/- 0.4%) and large granular lymphocytes (LGL; max. 9.3% +/- 1.4%) were seen than in patients with mild or asymptomatic CMV infection (lymphoid blast cells max. 6.5% +/- 0.8% P < 0.01 and LGLs max. 20% +/- 2.3%, P < 0.05). Thus, a strong immune response with lymphoid activation was associated with clinically good outcome of CMV infection. In heart allograft histology, subendothelial inflammation of small intramyocardial vessels was a characteristic finding during CMV antigenemia compared to CMV-free recipients (at the peak P < 0.01). However, no difference in this mild and short-lived inflammatory response was observed between clinically mild or severe CMV infection. The CMV-linked generalized immune activation and inflammation of the vascular structures might contribute to the initiation of allograft vasculopathy and to the pathogenesis of chronic heart allograft rejection.

Vascular cell adhesion molecule-1 (VCAM-1) is induced during cytomegalovirus infection in vascular structures of heart allografts.

This study was designed to investigate the expression of vascular cell adhesion molecule-1 (VCAM-1) and the counter-ligand VLA-4 (CD 49d) in frozen sections of endomyocardial biopsies (EMBs) of heart allografts in relation to the onset of cytomegalovirus (CMV) infection diagnosed by CMV antigenemia. Altogether, 105 EMBs were obtained from 21 heart transplant recipients. Serial EMBs from nine patients with CMV infection, from five patients with rejection, and from seven patients with a noncomplicated postoperative course were analyzed. Associated with CMV infection, capillary expression of VCAM-1 was significantly induced when compared to control biopsies (P < 0.0001). A striking difference in the expression of VCAM-1 during rejection and CMV infection was observed: in most rejecting biopsies only a few capillaries stained faintly for VCAM-1, whereas during CMV infection, multifocal intense staining was found (P < 0.0001).

Effect of ganciclovir prophylaxis on cytomegalovirus-enhanced allograft arteriosclerosis.

Clinical and experimental studies have established the accelerating role of cytomegalovirus (CMV) infection on cardiac allograft arteriosclerosis, i.e. chronic rejection. In this study, we investigated the effect of ganciclovir prophylaxis on the development of CMV-enhanced allograft arteriosclerosis. Rat aortic allografts were done from DA donors to WF recipients and either infected with 10(5) plaque-forming units of rat CMV (RCMV) 1 day after transplantation or left uninfected. RCMV-infected allografts received ganciclovir at an initial dose of 20 mg/kg and a maintenance dose of 10 mg/kg twice a day for 14 days. Grafts were removed at 7, 14 days, and 1 and 3 months after transplantation and processed for morphometry and autoradiography. The results of this study demonstrated that ganciclovir prophylaxis significantly delays and reduces RCMV-enhanced allograft arteriosclerosis in the rat.

ICAM-1 induction on hepatocytes as a marker for immune activation of acute liver allograft rejection.

Intercellular adhesion molecule-1 (ICAM-1) induction on hepatocytes was investigated in relation to acute liver allograft rejection, CMV infection, and systemic bacterial infections. Twenty-four liver transplant recipients underwent an episode of acute rejection, 13 developed a symptomatic clinical CMV infection, and 7 had bacterial sepsis. Seven recipients without rejection or infection complications were used as controls. All rejection episodes monitored by frequent FNABs were reversible, and lymphocyte and lymphoid blast-dominated with a with peak of inflammation (7.2 +/- 3.9 corrected increment units [CIU]). The rejections were treated with high-dose steroids, and the inflammation subsided within one week. ICAM-1 was demonstrated from fine needle aspiration biopsy (FNAB) preparations by a monoclonal antibody and immunoperoxidase staining. ICAM-1 was not detected on the hepatocytes immediately after transplantation or in control patients, but was always seen during rejection. ICAM-1 appeared 1-5 days before the onset of inflammation in FNAB. The intensity of ICAM-1 expression increased toward the peak of inflammation and subsided together with inflammation. During CMV infection a mild immune activation was seen in FNAB (peak 2.5 +/- 0.8 CIU) and in blood. An intense ICAM-1 induction also preceded the immune activation caused by CMV, and subsided slowly with successful antiviral treatment. In addition, a slight ICAM-1 induction on the hepatocytes was recorded during bacterial sepsis. ICAM-1 induction on hepatocytes appears to be linked with an early phase of immune response, and it even precedes the lymphoid activation of rejection. However, several infections, such as CMV and bacterial infections, raise an immune response and may also induce ICAM-1. In conclusion, ICAM-1 induction on hepatocytes can be considered an early, though unspecific, marker for acute liver allograft rejection.

Cytomegalovirus infection and accelerated cardiac allograft vasculopathy in human cardiac allografts.

Cardiac allograft vasculopathy is a major limiting factor of the long-term survival of heart transplant patients. An association of cytomegalovirus infection and cardiac allograft vasculopathy has been described. We analyzed 104 endomyocardial biopsy specimens obtained from 53 heart transplant recipients and correlated the histologic findings with 115 angiograms obtained from the same patients during 4 postoperative years. The frequency of vascular changes in endomyocardial biopsy specimens was significantly higher than in angiograms during the first 3 posttransplantation years (P < 0.001, P < 0.005, P < 0.03, respectively). Also, in patients with angiographically documented cardiac allograft vasculopathy, significantly higher scores of capillary and arteriolar endothelial cell accumulation and arteriolar intimal thickness were recorded when compared with the recipients with normal angiograms (P < 0.02, P < 0.05, P < 0.005, respectively). Altogether, 29 of 53 recipients underwent cytomegalovirus infection during the first posttransplant year. Cytomegalovirus infection was associated with arteriolar endothelial cell accumulation and with increased intimal thickness of intramyocardial vessels of 1-year endomyocardial biopsy specimens when compared with cytomegalovirus-free recipients (P < 0.02 and P < 0.005, respectively). After the second year, the cytomegalovirus-associated endothelial cell response subsided, but the thickness of intima had increased when compared with cytomegalovirus-free patients (P < 0.05). Thereafter, the cytomegalovirus-associated histologic changes reached a plateau. In coronary angiography, the cardiac allograft vasculopathy changes were detected in a slower pace. Thus only after 2 posttransplantation years, cytomegalovirus-associated acceleration of cardiac allograft vasculopathy was observed, compared with cytomegalovirus-free patients (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytomegalovirus infection enhances smooth muscle cell proliferation and intimal thickening of rat aortic allografts.

Inbred DA (AG-B4, RT1a) and WF (AG-B2, RT1v) rats were used as donors and recipients of aortic allografts. The recipient rats were inoculated i.p. either on day 1 (early infection) or on day 60 (late infection) with 10(5) plaque-forming units of rat cytomegalovirus (RCMV). The control rats were left noninfected. The presence of viral infection was demonstrated by plaque assays from biopsies of the salivary glands, liver, and spleen at sacrifice. The rats received 300 microCi[3H]thymidine by i.v. injection 3 h before sacrifice, and the grafts were removed at various time points for histology, immunohistochemistry, and autoradiography. RCMV infection significantly enhanced the generation of allograft arteriosclerosis. Infection at the time of transplantation had two important effects. First, the infection was associated with an early, prominent inflammatory episode and proliferation of inflammatory cells in the allograft adventitia. Second, the viral infection doubled the proliferation rate of smooth muscle cells and the arteriosclerotic alterations in the intima. In late infection the impact of RCMV infection on the allograft histology was nearly nonexistent. RCMV infection showed no effect in syngeneic grafts. These results suggest that early infection is more important to the generation of accelerated allograft arteriosclerosis than late infection, and that an acute alloimmune response must be associated with virus infection, to induce accelerated allograft arteriosclerosis. RCMV-infected aortic allografts, as described here, provide the first experimental model to investigate the interaction between the virus and the vascular wall of the transplant.

Cytomegalovirus induces class II expression in rat heart endothelial cells.

An association between cytomegalovirus infection, cardiac allograft rejection, and atherosclerosis has been described. It has been suggested that cytomegalovirus induces major histocompatibility complex antigen expression in the graft and may trigger rejection. The induction of major histocompatibility complex antigens is thought to be mediated by interferon-gamma produced by activated T cells during the infection. To study whether cytomegalovirus infection induces major histocompatibility complex class II antigen expression in heart endothelium, cultured rat heart endothelial cells were infected with rat cytomegalovirus. The infection was shown by cytopathic effect and immunofluorescence using monoclonal cytomegalovirus-specific antibodies. Major histocompatibility complex class II antigen expression was analyzed before and during cytomegalovirus infection by two different methods, by a fluorescence-activated cell sorter and immunoperoxidase techniques using monoclonal antibodies. Uninfected endothelial cell cultures were treated with interferon-gamma and used as positive controls of class II induction. Induction of class II antigens was recorded in cytomegalovirus-infected endothelial cell cultures, and during the course of infection the class II expression increased toward the appearance of cytopathic effect. In uninfected cells, class II was induced by interferon-gamma, but this induction could be inhibited by adding antiinterferon-gamma antibody to the cultures. However, anti-interferon-gamma did not inhibit the induction of class II caused by cytomegalovirus. In conclusion, cytomegalovirus induced major histocompatibility complex class II antigen expression in rat heart endothelial cells in vitro. This induction of class II was independent of interferon-gamma and was caused by the virus itself. Direct induction of class II antigens by cytomegalovirus in heart endothelium may also be involved in rejection mechanisms in vivo.

Quantitation of cytomegalovirus infection-associated histologic findings in endomyocardial biopsies of heart allografts.

To investigate whether histologic changes in heart allografts may be associated with cytomegalovirus infection, 46 heart transplant recipients were monitored for cytomegalovirus. Altogether, 762 endomyocardial biopsy specimens were analyzed. The histologic changes were semiquantitatively scored from mild to severe, and special attention was paid to inflammatory infiltrates and vascular changes. Cytomegalovirus infection occurred in 27 of 46 patients, shown by cytomegalovirus-antigenemia test. The endomyocardial biopsy findings were investigated in relation to cytomegalovirus-antigenemia. In the acute phase of cytomegalovirus infection during antigenemia, an inflammatory infiltrate, subendothelial lymphocytosis, was a characteristic finding in the endomyocardial biopsy specimens. An intense arteriolar endothelial cell proliferation and thickening of intima occurred. Long-term histologic findings with two years follow-up revealed a cytomegalovirus-associated enhanced vascular intimal thickening that narrowed the lumen of small intramyocardial arterioles. Acute rejection episodes were diagnosed in 15 of 27 patients with cytomegalovirus and in 9 of 19 patients free of cytomegalovirus. The inflammatory infiltrate of acute rejection was more peripheral to the vessels and did not obscure the findings characteristic to cytomegalovirus infection. The arteriolar endothelial proliferation and intimal thickening were significantly more prominent in cytomegalovirus infection than in biopsy specimens from patients with rejection only. In long-term follow-up, arteriolar endothelial proliferation declined, but the intimal thickness persisted and increased. The increase was significantly higher in patients with cytomegalovirus than in patients with rejection. In conclusion, an inflammatory response in vessel walls with alterations of small intramyocardial arterioles leading to narrowing of the vascular lumen of the graft was associated with cytomegalovirus infection in heart transplant patients.

The correlation between symptomatic CMV infection and CMV antigenemia in heart allograft recipients.

Previous studies have demonstrated that CMV-specific antigens detected from peripheral blood leukocytes correlate with active CMV infection in transplant patients. However, the clinical diagnosis of CMV infection is difficult, and the significance of a positive blood finding is unclear, while CMV antigenemia and viremia may also occur in asymptomatic patients. To investigate the clinical significance of CMV antigenemia after heart transplantation, 68 heart allograft recipients were monitored weekly. Altogether 501 blood specimens were analyzed. CMV was demonstrated in blood leukocytes by a monoclonal antibody and immunoperoxidase staining, and the antigenemia level was expressed as CMV positive cells/50,000 leukocytes. CMV antigenemia occurred in 28/68 patients, and 12 of them developed a symptomatic infection. Of all blood specimens 88/501 were CMV positive, and 30 of them related to the clinical manifestation of CMV. When antigenemia level exceeded > 100/50,000, a significant correlation between antigenemia and CMV-related clinical manifestation was reached (P < 0.001). Of the 28 antigenemia positive patients 16 never developed any clinical signs of CMV infection. Their maximal antigenemia level was low (median 23, range 30-90) compared with those with clinical manifestation (median 500, range 30-1000) (P < 0.002). In conclusion, high antigenemia levels (> 100/50,000) correlate with clinical manifestations of CMV infection. Patients with lower levels (< 100/50,000) do not necessarily ever develop a symptomatic infection. Quantitative monitoring of CMV antigenemia may, thus, be helpful in the clinical diagnosis of CMV infection in heart transplant patients.

Cytomegalovirus infection accelerates cardiac allograft vasculopathy: correlation between angiographic and endomyocardial biopsy findings in heart transplant patients.

In order to determine the impact of cytomegalovirus (CMV) infection on cardiac allograft vasculopathy (CAV), we quantitated angiograms and endomyocardial biopsy (EMB) specimens obtained from 53 heart transplant recipients. CMV infection was particularly associated with the development of discrete stenosis in major branch vessels (P < 0.03). Also, the number of diffusely affected vessel segments was significantly higher in CMV patients than in CMV-free recipients after the 2nd postoperative year (P < 0.05). The EMB histology correlated well with angiography. Significantly higher levels of arteriolar endothelial cell proliferation and intimal thickness were recorded in biopsies of CMV patients than in those of CMV-free recipients during the 1st postoperative year (P < 0.02 and P < 0.005, respectively). The CMV-associated vascular changes in EMB histology clearly preceded angiographically detectable CAV findings. Taken together, CMV infection accelerated heart allograft arteriosclerosis. The histological changes appeared prior to changes detected by coronary angiography. The CMV effect was particularly pronounced during the first 2 post-transplant years but leveled off thereafter. Thus, CMV-accelerated allograft arteriosclerosis may be linked in particular with early graft loss of CMV-infected heart transplant recipients.

Cytomegalovirus infection of human kidney cells in vitro.

To study which structures of a kidney allograft are the main targets for cytomegalovirus (CMV), human glomerular epithelial and mesangial cells, as well as tubular epithelial and endothelial cells were isolated by steel meshes of different pore sizes and enzymatic treatments. The various cultured cell types were characterized by morphology and specific antibodies. Human CMV was inoculated onto cell monolayers using two different culture methods: conventional tissue culture and rapid shell vial culture. To analyze whether CMV had a direct effect on the immunologic properties of kidney parenchymal cells, MHC class I and class II antigen expression was estimated before and after the infection. CMV infected all kidney cells identically. All cells expressed class I strongly after the infection, but they were class I positive prior to infection. Class II antigens were not expressed on the cell surface either before or after the infection. In conclusion, human kidney cells of glomerular, tubular and vascular origin were all infected by CMV without any difference. CMV had no significant direct effects on the antigenic properties of the cells.