The UL domain and US domain can be arranged head to tail or head to head .... After the virion has entered the cell, it is uncoated in the cytoplasm [10]. ..... macrophages and activated T cells are enclosed in the intimal mass. ..... Cytomegalovirus -infected cells in routinely prepared ..... Transplantation 1996; 61:1763-70.
9$6&8/$53$7+23+99 %) were removed. Thus, recovery of EC added to whole blood is 53 ± 16.5%, caused by losses due to isolation of the MNC fraction and EC specific staining (Table 1). EC recovery after cytocentrifugation of MNC fractions Similarly, the effect of every step on specific loss of added EC was examined for the cytocentrifugation procedure. Density centrifugation resulted in a loss of 25.8 ± 13.8% of the cells, and cytocentrifugation of the MNC fraction caused an additional loss of MNC of approximately 33%. The recovery of EC from whole blood after density centrifugation and cytocentrifugation was 43 ± 4.3%. For each sample of the MNC fraction four or more cytospots were analyzed for quantification of EC among the MNC on spot. We also determined the variance between samples and the different spots per sample. The spread in recovery of EC between spots appeared to be larger (intra-test variance 23.5%) than between different samples (inter-test variance 6.0%). Thus EC isolated from whole blood using FACS sorting or cytocentrifugation of MNC resulted in similar recovery percentages of pre-added EC from whole blood: 43 ± 4.3 % and 53 ± 16.5 % respectively.
Re cov ery in %
100
75
50
25
0
F
C
uninfe cted EC
F
C
HCM V infected EC
Figure 2 Recovery of CMV-infected or non-infected EC. One hundred non-infected EC (open bars) or CMV infected EC (hatched bars) were added to 106 MNC. No differences in recovery between CMV infected EC and non-infected EC were observed after FACS sorting (F) (P=0.239) or cytocentrifugation followed by subsequent immunofluorescent staining (C) (P=0.917) (unpaired t test). Experiments were performed in triplicate (FACS sorting of CMV-infected EC, n=2). For each sample, three or four cytospots were analyzed.
30
TABLE 1: Detection of CEC in blood of patients after FACS or after cytocentrifugation of MNC fractions Blood sample1
Amt of blood
No. of MNC in
analyzed (ml)2
MNC fraction/ ml
No. CEC detected
No. of CEC/ ml
No. of CEC /
blood
500,000 MNC
FACS
Cytospot
blood (106)
FACS
Cytospot
FACS
Cytospot
(Cytospot)
Sample 1
8.0
1.9
0.43
72
14
9.0
7.4
17.5
Sample 2
4.0
0.8
1.0
43
9
10.8
11.3
11
Sample 3
5.8
0.7
0.43
13
0
2.2
0
0
Sample 4
6.0
1.0
0.1
5
0
0.8
0
0
1
Three blood samples were obtained from a single patient at different time points (samples 1 to 3 ) and one blood sample was from another patient (sample 4).
2
The amount of blood analyzed refers to the final number of MNC analyzed. MNC were originally obtained from the indicated blood volumes.
4XDQWLILFDWLRQ RI F\WRPHJDOLF HQGRWKHOLDO FHOOV LQ 3%
CMV-infected EC The surface expression level of the antigen for MoAb E1/1 2.3 was more heterogeneous on CMV- infected EC and the mean fluorescence intensity was lower than of uninfected EC (Fig. 1). However, the expression level was sufficiently high to discriminate between FITC positive and negative cells (FITC-fluorescence is used as sortpulse). During the FACS sorting procedure or cytocentrifugation of MNC CMV-infected EC and uninfected EC behaved similarly (Fig. 2). FACS sorting of CMV- infected EC showed an extra decrease of 18% in recovery; uninfected EC did not show any decrease (Fig. 2). However, this difference was not statistically significant. Detection range Next, we studied the recovery of a dilution series of endothelial cells added to MNC varying from 5000 to 5 of EC to 1 x 106 MNC. A minimum of 5 EC was reproducibly detected using FACS sorting as well as by using cytocentrifugation and immunofluorescent staining (Fig. 3). The quantification of EC on cytospots with limited spread in recovery was only possible with a minimum of 50 EC added to 1 x 106 MNC. Below this level recovery decreased and the variation of the recovery increased (Fig. 3). Applying the FACS procedure, about 60% of the pre-added EC were recovered over the whole range of added EC tested. In addition, the recovery was also constant when 50 EC were isolated from either 1, 5 or 10 x 106 MNC (data not shown).
Re cov ery in %
100
75
50
25
0 5
50
100
500
5000
6
EC / 1 x 10 M NC
Figure 3 Lower limit of detection. A range of 5 to 5000 EC were added to 106 MNC. Recovery was determined by FACS (hatched bars) or by cytocentrifugation and immunofluorescent staining (open bars). Experiments are performed in triplicate (where no error bar is depicted, n=1). For each sample at least three or four cytospots were analyzed
32
&KDSWHU
Quantification of CEC in PB from patients To evaluate the quantification procedure for CEC in PB, we determined CEC counts in PB from four blood samples of patients with an active CMV infection and compared detected CEC numbers after FACS sorting or cytocentrifugation. After FACS sorting of a range of 4 to 8 ml of blood (Table 2), 5 to 72 CEC were detected, equivalent of 0.8 to 9.0 CEC/ml blood (sample 4, sample 1). Counting CEC on cytospots of MNC fraction resulted in the detection of maximally 14 CEC (sample 1). Although less CEC were detected per cytospot, the recoveries of CEC per ml blood were comparable in the samples containing approximately 10 CEC per ml blood. Dextran sedimentation of granulocytes with subsequent cytocentrifuge preparation (routinely performed to monitor CMV antigenemia [29]) and immunocytochemical staining specific for endothelial cells was performed to detect numbers of CEC in PB. The same blood samples were processed as used for FACS sorting and cytocentrifugation of MNC fractions. The number of CEC per ml blood obtained by dextran sedimentation (Table 3) did not correlate with the number of CEC per ml blood FACS sorting onto slides or on cytospots of MNC fractions (Table 2).
TABLE 2: Blood sample Sample 1
Detection of CEC in PB from CMV patients by dextran sedimentation. CMVAmt of blood No. of granulo- No. of CEC No of CEC/ 2 cytes / ml of detected ml blood antigenanalyzed (ml) 6 blood (10 ) emia4 n.d.3 n.d. n.d. n.d. 3410
Sample 2
0.1
5.9
0
0
3500
Sample 3
1.0
0.5
0
0
1571
Sample 4
1.0
0.6
1
1
52
1
Samples 1 to 4 are the same as in table 1. However, CEC were isolated by dextran sedimentation. The amount of blood analyzed refers to the final number of MNC analyzed. MNC were originally obtained from the indicated blood volumes 3 n.d. : not determined. 4 Values are the numbers of pp65-positive granulocytes per 50,000 polymorphonuclear leukocytes 2
Discussion We present a three-step method to isolate and quantify cytomegalic endothelial cells from peripheral blood samples. For the development of this quantitative method we used in vitro cultured EC pre-added to different steps of the isolation procedure and cell losses during the individual isolation steps were determined. Because EC were detected only in the MNC fraction, we anticipated that these cells would behave like MNC during isolation and staining 33
4XDQWLILFDWLRQ RI F\WRPHJDOLF HQGRWKHOLDO FHOOV LQ 3%
procedures. As expected, the losses of MNC and EC were comparable. Thus, determination of MNC losses before isolation and just before sorting indicated a loss factor for EC showing that the numbers of EC in blood were approximately twice the number of detected EC. After FACS sorting and binding of sorted cells to adhesion slides almost all cells were recovered on the slide. After cytocentrifugation of the MNC fraction approximately 40 % of all cells were lost. These inevitable losses were due both to adherence of cells to the centrifugation cups and to cells being drawn into the papercards. As shown in Table 1 cell losses were negligible after binding to adhesion slides. So, determination of MNC counts prior to FACS sorting indicates losses of EC during the whole three step quantification procedure. Because functional and morphological properties of CMV-infected endothelial cells are seriously disturbed [1,2,5,9,21,22,25,26,28], isolation characteristics of CEC from patients could differ from uninfected EC. Especially expression of surface antigens is influenced by infection of EC with CMV [2,9,21,25]. Therefore, we verified the expression of the antigen of MoAb E1/1 2.3 on CMV-infected EC and confirmed staining of EC with MoAb E1/1 2.3. Although a decreased expression of this EC antigen after infection of EC with CMV was observed (Fig. 1), the MoAb could still be used as a tool for immunoaffinity methods. In addition, little or no crossreactivity with other blood cells should occur. Some monocytes stained weak positive with E1/1 2.3; however this background did not disturb our quantification because the isolated cells were also evaluated afterwards by fluorescence microscopy for the cytomegalic morphology and the fluorescent intensity. The present study with infected EC, including late-stage infected EC with the owl’s eye appearance, showed a slight reduction in the recovery, which could be due to a decreased expression of the antigen for Moab E1/1 2.3 (Fig. 1). Another reason for the reduction in the recovery could be increased fragility of EC after CMV infection (30 % more cell death of CMV-infected EC compared to uninfected EC). For the development of a quantitative method to detect CEC in PB using FACS sorting the MNC fraction with EC was stained with MoAb E1/1 2.3, followed by FITC conjugated rabbit-anti-mouse Ig. Because an additional staining step involves additional washings, the recovery of EC out of the MNC fraction could be improved by using primary antibodies which are conjugated directly to a fluorescent dye, e.g. an E1/1 2.3-FITC conjugate. According to our results obtained by in vitro added EC (approximately 50 % recovery for both methods), the isolation of CEC from peripheral blood from patients by FACS sorting or by cytocentrifugation resulted in an equal yield of CEC per milliliter of blood for each method. However, after isolation of CEC from patients by FACS sorting, a five-fold greater level of CEC could be detected than by cytocentrifugation and immunofluorescent staining. The increased detection level of CEC after FACS sorting was the result of the higher number of cells that could be processed and the almost complete removal of the MNC. Consequently, a high signal (CEC) to noise (MNC) ratio was obtained, allowing for easy quantification of the sorted CEC after their binding to adhesion slides.
34
&KDSWHU
Another aspect of the comparison between FACS sorting and cytocentrifugation is the influence of clinical symptoms like leukopenia [30] or lymphocytosis [4]. Both symptoms can occur in transplant recipients after rejection therapy or due to CMV infection and result in altered MNC levels in blood. Alteration in the MNC levels will alter the number of CEC in PB per number of MNC. Until now CEC have been correlated with MNC numbers present on cytospots instead of blood volume [8-10,17]. However, CEC in PB are likely to originate from the venous vessel wall and might therefore be more indicative of vascular damage. To correct for high levels of CEC in patients caused by low MNC numbers, we argue for the relation between CEC and blood volume. Regarding a total body blood volume of approx. 5 liters, the detected concentrations of 10 CEC/ml PB actually represented 50,000 endothelial cells. No data are available about kinetics of the clearing of the circulating EC. Data obtained from CEC in PB after angioplasty showed comparable concentrations of EC in arterial as well as venous blood [6,20], suggesting that released endothelial cells might circulate. In the case of CMVinfected cytomegalic altered endothelial cells (size 30-40 µm) it is unknown whether these cells will recirculate or become trapped in capillary vessels, thus evoking disturbances in organ function. In conclusion, a reliable three step method based on FACS sorting was developed to isolate and quantify CEC from PB of patients with an active CMV infection. This method resulted in greater sensitivity than analysis of the MNC fraction on cytospots as described by us earlier [9]. Furthermore, we argue to relate this CEC count to the blood volume analyzed in order to make the results more comparable. Endothelial cells in peripheral blood are described for several pathophysiological conditions involving endothelial damage; thus our quantitative method could be applied to study the released cells caused by vascular damage. In addition, our method is useful for the detection of different kinds of rare cells circulating in the blood stream. Acknowledgements: We thank Geert Mesander for assistance with FACS sorting, Roelie van Wijk and Lucien Gjaltema for EC culture assistance, Dr M.A.GimbroneJr, Dept of Pathology, Harvard Medical School, Boston, USA for Moab E1/1 2.3 and Dr C. Sinzger for providing the endothelial adapted CMV clinical isolate TB42E. Grant support: Dutch Kidney foundation (C94.1386), European Commission Grant (ERB BMH4CT-0471 (DG12-SSMA)). References 1. Almeida GD, Porada CD, StJeor SS, Ascensao JL. Human cytomegalovirus alters interleukinproduction by endothelial cells. Blood 1994; 83:370-6. 2. Bruggeman CA, Debie WHM, Muller AD, Schutte B, van Dam-Mieras MCE. Cytomegalovirus alters the von Willebrand factor content in human endothelial cells. Thromb. Haemostas 1988; 58:264-8. 3. De Maar EF, Kleibeuker JH, Boersma-van Ek W , The TH, van Son WJ. Increased intestinal permeability during cytomegalovirus infections in renal transplants recipients. Transplant Int 1996; 9:576-80.
35
4XDQWLILFDWLRQ RI F\WRPHJDOLF HQGRWKHOLDO FHOOV LQ 3% 4.
5.
6.
7.
8.
9. 10.
11.
12.
13.
14. 15.
16.
17.
18.
19. 20. 21.
36
Einsele H, Ehninger G, Steidle M, Fischer I, Bihler S, Gerneth F, Vallbracht A, Schmidt H, Waller HD, Muller CA. Lymphocytopenia as an infavorable prognostic factor in patients with cytomegalovirus infection after bone marrow transplantation. Blood 1993; 82:1672-8. Garrett JS, Narus JC, Bohnsack JF, Carling DE, Grieves KG, Waldman WJ, Shaddy RE. Effects of cytomegalovirus infection on growth factor production in endothelial cells and fibroblasts. Pediatr. Res 1993; 38:1003-8. George F, Brisson C, Poncelet P, Laurent JC, Massot O, Arnoux O, Ambrosi P, Klein-Soyer C, Cavenaze JP. Rapid isolation of human endothelial cells from whole blood using S-Endo1 monoclonal antibody coupled to immuno-magnetic beads: Demonstration of endothelial injury after angioplasty. Thromb Haemostas 1992; 67:147-3. George F, Brouqui P, Boffa MC, Mutin M. Demonstration of Rickettsia Conorii-induced endothelial injury in vivo by measuring circulating endothelial cells, thrombomodulin, and von Willebrand factor in patients with Mediterranean Spotted Fever. Blood 1993; 82:2109-16. Gerna G, Percivalle E, Revello MG, Morini F. Correlation of quantitative human cytomegalovirus pp65-, p72- and p150- antigenemia, viremia and circulating endothelial giant cells with clinical symptoms and antiviral treatment in immunocompromised patients. Clin Diagn Virol 1993; 1:4759. Grefte A,Van der Giessen M, Van Son WJ, The TH. Circulating cytomegalovirus (CMV)-infected endothelial cells in patients with an active CMV infection. J Infect Dis 1993; 167:270-7. Grefte A, Blom N, Van der Giessen M, Van Son WJ, The TH. Ultrastructural analysis of circulating cytomegalic cells in patients with active cytomegalovirus infection: Evidence for virus production and endothelial origin. J Infect Dis. 1993; 168:1110-8. Grefte J M M, Van der Gun BFT, Schmolke S, Van der Giessen M, Van Son WJ, Plachter B, Jahn G, The TH. The lower matrix protein pp65 is the principal viral antigen present in peripheral blood leukocytes during an active cytomegalovirus infection. J Gen Virol 1992; 73: 923-32. Gross H-J, Verwer B, Houch D, Hoffman RA, Recktenwald D. Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10-7. Proc Natl Acad Sci USA 1995; 92:537-41. Jaffe EA, Nachman RL, Becher CG, Minick CR. Culture of human endothelial cells derived from umbilical veins; identification by morphologic and immunologic criteria. J Clin Invest 1973; 52:2745-56. Lefevre P, George F, Durand JM, Sampol J. Detection of circulating endothelial cells in Thrombotic Thrombocytopenic Purpura. Thromb Haemostas 1993; 69:522-32. Mazeron MC, Jahn G, Plachter B. Monoclonal antibody E-13 (M-810) to human cytomegalovirus recognizes an epitope encoded by exon 2 of the major immediate early gene. J Gen Virol 1992; 73:2699-703. Mulder AB, Blom NR, Ruiters MJH, Van der Meer J, Halie MR, Bom VJJ. Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells; reduction by using chymotrypsin instead of collagenase. Thromb Res 1995; 80:399-411. Percivalle E, Revello MG, Vago L, Gerna G. Circulating endothelial giant cells permissive for human cytomegalovirus (CMV) are detected in disseminated CMV infections with organ involvement. J Clin Invest 1993; 92:663-70. Pober JS, GimbroneJr MA, Lapiere LA, Mendrick DH, FiersW, Rothlein R, Springer TA. Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon. J Immunol 1986; 137:1893-6. Radbruch A, Recktenwald D. Detection and isolation of rare cells. Curr Op Imm 1995; 7:270-3. Sbarbati R, De Boer M, Marzilli M. Immunologic detection of endothelial cells in human whole blood. Blood 1991 77:764-9. Sedmak DD, Knight DA, Vook NC, Waldman WJ. Divergent patterns of ELAM-1, ICAM-1, and VCAM-1 expression on cytomegalovirus-infected endothelial cells. Transplant 1994; 58:1379-85.
&KDSWHU 22.
23.
24.
25.
26.
27. 28.
29.
30. 31.
Shahgasempour S, Woodroffe SB, Sullivan-Tailyour G, Garnett HM. Alterations in the expression of endothelial cell integrin receptors α5β1 and α2β1 and α6β1 after in vitro infection with a clinical isolate of human cytomegalovirus. Arch Virol 1997; 142:125-38. Sinzger C, Grefte A, Plachter B, Gouw ASH, The TH, Jahn G. Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. J Gen Virol 1995; 76:741-50. Sinzger C, Knapp J, Plachter B, Schmidt K, Jahn G. Quantification of replication of clinical cytomegalovirus isolates in cultured endothelial cells and fibroblasts by a focus expansion assay. J Virol Meth 1997; 63:103-12. Span AH, Mullers W, Miltenburg AM, Bruggeman CA. Cytomegalovirus induced PMN adherence in relation to an ELAM-1 antigen present on infected endothelial cell monolayers. Immunol 1991; 72:355-60. Span AH, Van Dam Mieras MC, Mullers W, Endert J, Muller AD, Bruggeman CA. The effect of virus infection on the adherence of leukocytes or platelets to endothelial cells. Eur J Clin Invest 1991; 21:331-8. Takahashi H, Harker LA. Measurement of human endothelial cells in whole blood. Thromb Res 1983; 31:1-12. Van Dam Mieras MC, Muller AD, van Hinsbergh VW, Mullers WJ, Bomans PH, Bruggeman CA. The procoagulant response of cytomegalovirus infected endothelial cells. Thromb Haemost 1992; 68:364-70. Van der Bij W, Torensma R, van Son WJ, Anema J, Schirm J, Tegzess AM, The TH. Rapid immunodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leucocytes. J Med Virol 1988; 25:179-88. Van Son WJ, The TH. Cytomegalovirus infection after organ transplantation: an update with special emphasis on renal transplantation. Transplant Int 1989; 2:147-64. Van Son WJ, Peset R, Duipmans JC, van der Mark ThM, The TH, Tegzess AM. Cytomegalovirus infection after renal transplantation: pulmonary dysfunction measured by decreased diffusing capacity for carbon monoxide in patients with symptomatic and asymptomatic infection. Transplantation 1987; 44:149-50.
37
UNINFECTED AND CYTOMEGALIC ENDOTHELIAL CELLS IN BLOOD DURING CYTOMEGALOVIRUS INFECTION: EFFECT OF ACUTE REJECTION
A.M. Kas-Deelen1,*, E.F. de Maar2, M.C. Harmsen1, C. Driessen1, W.J. van Son2, T.H. The1
1
Department of Clinical Immunology, 2Department of Nephrology, University Hospital Groningen, The Netherlands.
J Infect Dis (2000) 181:721-724
&KDSWHU
(QGRWKHOLDO FHOOV LQ EORRG
Abstract After transplantation cytomegalovirus infections can cause vascular damage in both the graft and the host. To study a possible relationship between the degree of vascular injury, clinical symptoms of HCMV infection and transplant rejection, we investigated the appearance and numbers of endothelial cells (EC) in blood of 54 kidney transplant recipients in a prospective clinical study. We identified two types of endothelial cells: cytomegalic endothelial cells (CEC) which were detected in patients with moderate or high HCMV antigenemia, uninfected EC were observed in patients during HCMV infection as well as without HCMV infection. The incidence of either CEC, EC or the combination of both was associated with HCMV-related clinical symptoms (P 100
16
4
10
6
13
3
3
3
Total
54
13
31
18
5
10
13
*No. positive granulocytes/50.000
&KDSWHU
70 Frequency of patients with CEC (%)
60
60
50
50
40
40
30
30
20
20
10
10
0
0
50 . 40 30
C
CEC/ml blood
150
D
EC/ml blood
100
20
50
10
20
4
15 10
2
Concentration of EC/ml per sample
Concentration of CEC/ml per sample
70
B
EC
Frequency of patients with EC (%)
A
CEC
5 0
0 1
2
3
Group
4
1
2
3
4
Group
Figure 1 Frequencies (A, B) and concentrations (C, D) of CEC (A, C) and EC (B, D) in peripheral blood of HCMV patients. The frequency is the number of patients per group with (C)EC at any moment during infection. Hatched parts of a bar represent patients with HCMV infections only and the black parts of a bar represent patients with both preceding acute rejection episodes and HCMV infection.
Twenty-eight patients had one or more rejection episodes: 13 patients experienced interstitial rejection responding to steroid treatment, 10 patients had steroid resistant interstitial rejection and 5 patients had vascular type of rejection. Episodes of vascular rejection were associated with high viral load: 0/5 in group 1 or group 2 versus 5/5 of group 3 and 4. Patients with one or more rejection episodes were equally distributed among all groups (P=0.41)(Table 1). Two distinct types of endothelial cells in peripheral blood were observed: late stage infected cytomegalic endothelial cells (CEC) and uninfected endothelial cells (EC). We never observed endothelial cells in immediate early or early stages of HCMV infection. Both CEC and EC were detectable in blood at or just after the maximum HCMV antigenemia peak. After maximum HCMV antigenemia, EC could be detected for a longer time than CEC. In 3 of 10 patients without HCMV infection, EC were demonstrated. Two of these patients had 43
(QGRWKHOLDO FHOOV LQ EORRG
EC at 15 days post transplantation, which was shortly after a rejection episode. The other patient experienced neither rejection nor HCMV infection. Remarkably, in the patients with HCMV infection, all EC were detected during HCMV antigenemia and never before HCMV infection. CEC were detected in 11/44 patients with HCMV infection (Fig. 1A) (25.0%): 8/16 patients in group 4 (50.0%) and 3/12 patients in group 3 (25.0%) (Fig. 1A). Concentrations of CEC ranged from 0.11 / ml to 30.26 / ml (median 0.89 / ml) (Fig. 1C). In 4 patients of group 4 CEC were detected at various times during HCMV antigenemia (data not shown). EC were observed in all patient categories independent of the severity of infection (Fig. 1B). The concentrations of EC ranged from 0.17 EC / ml blood to 114.05 EC / ml blood (median 2.62 EC / ml blood)(Fig. 1D). Patients with rejection episodes had endothelial cells in blood (EC, CEC, or both) during HCMV infection more often (66.7%), than patients without rejection (15.0%)(P+E@9FDS ,QWKLVUHDFWLRQLVWKHUHDFWLRQUDWHRI&2ZLWKKHPRJORELQ +E DWWKHDYHUDJHQRUPDO+E concentration (9 mmol/l). [Hb] is the hemoglobin concentration as a fraction of the average normal Hb concentration. Values were expressed as percentages of those predicted, with the predicted values being taken from Cotes et al. [11] and Quanjer et al. [12]. Patients were monitored for HCMV pp65-antigenemia twice a week. This test was performed according the procedure recently reviewed for standardization [1]. No HCMV prophylaxis such as ganciclovir, acyclovir or hyperimmune gammaglobulin was given. Eight patients received ganciclovir because of clinical symptoms associated with rising HCMV antigenemia values or preemptive because of anti-rejection treatment. CEC in blood was studied at approximately 15 days after transplantation (i.e. before infection) and weekly during HCMV infection. This was continued until the HCMV antigenemia was negative (n=4) or less than 5/50.000 cells (n=5). CEC in blood were analyzed as described recently [6]. In brief, mononuclear cells (MNC) were isolated by density centrifugation using Lymfoprep (Nycomed Pharma AS, Oslo, 77
&2 GLIIXVLRQ DQG &(&
Norway). 1 x 105 MNC were cytocentrifuged on a slide. The cytospots were stained by indirect immunofluorescence with the following antibodies: C10/C11 directed against HCMV pp65 and E1/1 2.3 directed to a 90kD cell surface antigen of endothelial cells [13]. Four cytospots were analyzed if the concentration of MNC/ml blood was 1.5 x 106 or less, otherwise 6 to 8 cytospots were analyzed. The number of analyzed slides represented a detection limit of 20 CEC/ml blood in 95% of all samples. Statistical analysis was performed using Student’s t-test for paired and unpaired samples. Results In this study CO diffusion was determined in fifteen patients (nine male / six female) with active HCMV infection. The median age was 42 years (range 18 - 63 years). Eleven patients had a primary infection (positive donor organ, negative recipient) and the remaining four patients had secondary infections (positive - positive combination). Nine patients had clinical symptoms such as fever, malaise, leukocytopenia, thrombocytopenia and elevated liver enzymes. Nine out of the fifteen patients were studied for CEC (Table 1). Table 1: Differences in pulmonary CO diffusion before and during HCMV infection A + CEC (n=4)
- CEC (n=5)
û.&2F PHDQ± SD)
22.28 ± 22.0
23.0 ± 25.0
N.S.
û9FDS PHDQ± SD)
17.03 ± 18.9
19.94 ± 18.0
N.S.
û'P PHDQ± SD)
28.7 ± 23.85
17.56 ± 13.8
N.S.
HCMV-Ag ≥ 100* (n=8)
HCMV-Ag ≤ 100* (n=7)
û.&2F PHDQ± SD)
26.01 ± 6.12
25.0 ± 10.52
N.S.
û9FDS PHDQ± SD)
21.01 ± 11.89
31.67 ± 21.25
N.S.
û'P PHDQ± SD)
29.48 ± 20.33
6.69 ± 12.26
P < 0.05
B
CEC were observed in four out of these nine patients. In two patients CEC were observed once, in one patient twice and in the last patient even times during the course of HCMV antigenemia. The CEC concentrations ranged from 0.22 CEC/ml to 30.26 CEC/ml (median 1.28 CEC/ml). The decrease in KCOc observed during HCMV infection was not different between patients with or without CEC (22.28 ± 22.0 versus 23.0 ± 25.0, p=ns). The decrease in Vcap and Dm showed no difference either (17.03 ± 18.9 versus 19.94 ± 18.0, p=ns and 28.7 ± 23.85 versus 17.56 ± 13.8, p=ns, respectively) (Table 1A).
78
&KDSWHU
The CO diffusion was analyzed in relation to the severity of infection. Patients with high HCMV antigenemia (≥ 100 pp65+ PMNs/50.000) showed a higher decrease of Dm than patients with low or moderate HCMV levels (≤ 100 pp65+ PMNs/50.000) (29.48 ± 20.33 versus 6.69 ± 12.26, p
