Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed mainly in patients with high-risk or advanced hematologic malignancies and congenital or acquired aplastic anemias. In the context of the significant risk of graft failure after allo-HSCT from alternative donors and the risk of relapse in recipients transplanted for malignancy, the precise monitoring of posttransplant hematopoietic chimerism is of utmost interest. Useful molecular methods for chimerism quantification after allogeneic transplantation, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers. Such analyses can be performed regardless of donor's and recipient's sex. Additionally, in patients after sex-mismatched allo-HSCT, fluorescent  in situ  hybridization (FISH) can be applied.
Background
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed mainly in patients with high-risk or advanced hematologic malignancies and congenital or acquired aplastic anemias. In the context of the significant risk of graft failure after allo-HSCT from alternative donors and the risk of relapse in recipients transplanted for malignancy, the precise monitoring of posttransplant hematopoietic chimerism is of utmost interest. Useful molecular methods for chimerism quantification after allogeneic transplantation, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers. Such analyses can be performed regardless of donor's and recipient's sex. Additionally, in patients after sex-mismatched allo-HSCT, fluorescent  in situ  hybridization (FISH) can be applied.in situMethods We compared different techniques for analysis of posttransplant chimerism, namely FISH and PCR-based molecular methods with automated detection of fluorescent products in an ALFExpress DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE). We used Spearman correlation test.
Methods
We compared different techniques for analysis of posttransplant chimerism, namely FISH and PCR-based molecular methods with automated detection of fluorescent products in an ALFExpress DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE). We used Spearman correlation test.Results We have found high correlation between results obtained from the PCR/ALF Express and PCR/ABI 310 Genetic Analyzer. Lower, but still positive correlations were found between results of FISH technique and results obtained using automated DNA sizing technology.
Results
We have found high correlation between results obtained from the PCR/ALF Express and PCR/ABI 310 Genetic Analyzer. Lower, but still positive correlations were found between results of FISH technique and results obtained using automated DNA sizing technology.Conclusions All the methods applied enable a rapid and accurate detection of post-HSCT chimerism.
Conclusions
All the methods applied enable a rapid and accurate detection of post-HSCT chimerism.

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed mainly in patients with high-risk or advanced hematologic malignancies and aplastic anemias, and for some of them it is the only curative treatment. After allo-HSCT, incomplete engraftment and appearance of recipient's hematopoietic cells can lead to a coexistence of donor and host hematopoiesis – a situation known as mixed chimerism. Complete recovery of hematopoiesis of the donor origin is referred to as complete chimerism. Widely accepted molecular methods for analysis of chimerism after allo-HSCT, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers, such as variable number of tandem repeats (VNTR) or short tandem repeats (STR) [ 1 ]. Such analyses can be performed regardless of donor's and recipient's sex [ 2 ]. Additionally, in patients after sex-mismatched allo-HSCT, fluorescent  in situ  hybridization (FISH) can be applied. This technique is based on identification of Y-chromosome-specific sequences in the posttransplant sample examined. Variants of these techniques can be used for precise, quantitative assessment of the amount of donor's cells in recipient's peripheral blood and/or bone marrow after transplantation, in the long run giving a picture of the dynamics of changes in chimeric status within a hematopoietic compartment. Chimerism also reflects response to treatment [ 3 ], since it correlates with the risk of malignancy relapse. Relapse is the most frequent cause of treatment failure in recipients transplanted for hematologic malignancies, but it is still controversial if patients with mixed chimerism have an increased risk of developing relapse or graft failure. Most probably only a progressive mixed chimerism (a dynamic rise in the number of recipient's cells over time) seems to reflect a relapse or rejection. Successful outcome has been associated with a state of stable complete chimerism [ 4 ]. Here we report results of a comparison of different techniques for analysis of posttransplant chimerism: fluorescent  in situ  hybridization (FISH) and PCR-based molecular methods with fluorescent products detected in an ALF Express DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE).
Background
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed mainly in patients with high-risk or advanced hematologic malignancies and aplastic anemias, and for some of them it is the only curative treatment. After allo-HSCT, incomplete engraftment and appearance of recipient's hematopoietic cells can lead to a coexistence of donor and host hematopoiesis – a situation known as mixed chimerism. Complete recovery of hematopoiesis of the donor origin is referred to as complete chimerism.Widely accepted molecular methods for analysis of chimerism after allo-HSCT, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers, such as variable number of tandem repeats (VNTR) or short tandem repeats (STR) [ 1 ]. Such analyses can be performed regardless of donor's and recipient's sex [ 2 ]. Additionally, in patients after sex-mismatched allo-HSCT, fluorescent  in situ  hybridization (FISH) can be applied. This technique is based on identification of Y-chromosome-specific sequences in the posttransplant sample examined. Variants of these techniques can be used for precise, quantitative assessment of the amount of donor's cells in recipient's peripheral blood and/or bone marrow after transplantation, in the long run giving a picture of the dynamics of changes in chimeric status within a hematopoietic compartment. Chimerism also reflects response to treatment [ 3 ], since it correlates with the risk of malignancy relapse. Relapse is the most frequent cause of treatment failure in recipients transplanted for hematologic malignancies, but it is still controversial if patients with mixed chimerism have an increased risk of developing relapse or graft failure. Most probably only a progressive mixed chimerism (a dynamic rise in the number of recipient's cells over time) seems to reflect a relapse or rejection. Successful outcome has been associated with a state of stable complete chimerism [ 4 ].in situHere we report results of a comparison of different techniques for analysis of posttransplant chimerism: fluorescent  in situ  hybridization (FISH) and PCR-based molecular methods with fluorescent products detected in an ALF Express DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE).in situMethods Patients Investigation of hematopoietic chimerism was performed in ten children (eight girls, two boys) aged 6–16 years. They were diagnosed with acute myelogenous leukemia (n = 4), acute lymphoblastic leukemia (n = 2), chronic myelogenous leukemia (n = 1), myelodysplastic syndrome (n = 1), or Fanconi Anemia (n = 2). All recipients received hematopoietic stem cells from HLA-matched sibling donors, and sex-mismatch between donor and recipient was present in all cases. The material (peripheral blood) for hematopoietic chimerism quantification was collected in different periods after transplantation. In all 10 children reconstitution of hematopoiesis was observed. Out of 8 children transplanted for hematologic malignancies, 4 are well and alive, in complete continuous remission (CCR), while in the other 4, leukemia relapse occurred 4–23 months after transplantation. DNA isolation High-molecular-weight DNA was extracted from frozen whole blood (approximately 5 ml) or bone marrow (approximately 3–5 ml) by the standard treatment with sodium dodecyl sulfate (SDS) and proteinase K, and the salting-out method. DNA was isolated from the donors' and patients' blood samples collected before and after transplantation at various intervals in order to determine the chimeric status. Analysis of PCR products by an ABI 310 Genetic Analyzer The PCR protocol optimized for the Qiagen polymerase and the PE 9700 thermocycler was performed as described previously [ 5 ]. For fragment analysis (after capillary electrophoresis), an ABI 310 Genetic Analyzer (PE) was used [ 5 ]. All analyses were performed in the University Children's Hospital, Tuebingen. Analysis of PCR products by an ALF Express DNA Sequencer The PCR protocol optimized for the Thermal Controller MJ Research (Watertown, MA) model PTC-100™ was applied. PCR was performed in a volume of 10 μl; the PCR reaction mixture contained: 2.5 pM of each forward and reverse primer, 200 μl of each dNTP, 0.4 U Taq polymerase (Qiagen, Chatsworth, CA), 1x PCR buffer (Qiagen, Chatsworth, CA), and 40 ng of genomic DNA. Conditions for PCR were as follows: 5 min at 94°C for the first denaturation; 26 cycles of amplification with a temperature profile of 45 sec at 94°C, 1 min at 55°C, 1 min at 72°C; with additional 5 min at 72°C in the last cycle. STR loci were amplified with fluorescent PCR primers described previously [ 6 ]. Primers for microsatellite markers were labeled with Cy5 dye (TIB MOLBIOL). A 1.5-μl aliquot of PCR reaction was resuspended in 7 μl of loading solution (formamide, bromophenol blue) containing 100 bp and 300 bp internal markers. All samples, after denaturation at 95°C for 5 min, were analyzed on 6% denaturing polyacrylamide gel with 7 M urea in the sequencer. A 50–500 sizer labeled with Cy5 dye was used as an external marker (for calculation of allele sizes). Electrophoresis was carried out in 0.6xTBE buffer at 1500 V/min. The helium-neon laser was operated at a wavelength of approximately 700 nm and laser power value of 2.5 mW. Allele sizes and peak areas of fluorescent products were analyzed and calculated with the use of Fragment Manager software (Pharmacia). PCR and analysis of PCR products by the ALF Express DNA Sequencer was performed at the Institute of Human Genetics, Poznan. FISH The experiments were performed on interphase nuclei obtained by standard short culturing of fresh whole blood samples, with probes specific for chromosomes X (locus  DXZ1 ) and Y (locus  DYZ1 ). The FISH procedure according to Cytocell Ltd. was used [ 7 ]. The number of scored nuclei was 250 to 550, with a median of 300. FISH experiments were performed at the Institute of Human Genetics, Poznan. Since the material for FISH analysis was not collected in all designated periods after transplantation in some patients, FISH experiments were not performed then. Quantification of chimerism After electrophoresis in the ABI 310 Genetic Analyzer (PE), all obtained data were analyzed by GeneScan 3.1 software and then transferred to Genotyper 2.5 software [ 5 ]. All data obtained after electrophoresis of fluorescent products in the ALFExpress DNA Sequencer were transferred to Fragment Manager™ software (Pharmacia). For both, calculation of the amount of recipient's DNA was performed using the formula: % of recipient's DNA = (R1 + R2)/(D1+ D2 + R1 + R2) × 100, where: R1, R2 = peak areas of recipient's alleles; and D1, D2 = peak areas of donor's alleles. Only informative markers were used for the analysis. If donor and recipient were heterozygous but shared one allele, only the area of the non-shared alleles was considered for the analysis [ 8 ]. To make sure that quantification is accurate, we performed serial dilution experiments, where standardized mixed chimeric samples were created by mixing donor's and pretransplant recipient's DNA in a range between 0 and 100 percent. The sensitivity strongly depends on the size of alleles, the detection level was around 3–5% of patient cells. The results of chimerism detection by different methods were compared by the Spearman correlation test.
Methods
Patients Investigation of hematopoietic chimerism was performed in ten children (eight girls, two boys) aged 6–16 years. They were diagnosed with acute myelogenous leukemia (n = 4), acute lymphoblastic leukemia (n = 2), chronic myelogenous leukemia (n = 1), myelodysplastic syndrome (n = 1), or Fanconi Anemia (n = 2). All recipients received hematopoietic stem cells from HLA-matched sibling donors, and sex-mismatch between donor and recipient was present in all cases. The material (peripheral blood) for hematopoietic chimerism quantification was collected in different periods after transplantation. In all 10 children reconstitution of hematopoiesis was observed. Out of 8 children transplanted for hematologic malignancies, 4 are well and alive, in complete continuous remission (CCR), while in the other 4, leukemia relapse occurred 4–23 months after transplantation.
Patients
Investigation of hematopoietic chimerism was performed in ten children (eight girls, two boys) aged 6–16 years. They were diagnosed with acute myelogenous leukemia (n = 4), acute lymphoblastic leukemia (n = 2), chronic myelogenous leukemia (n = 1), myelodysplastic syndrome (n = 1), or Fanconi Anemia (n = 2). All recipients received hematopoietic stem cells from HLA-matched sibling donors, and sex-mismatch between donor and recipient was present in all cases. The material (peripheral blood) for hematopoietic chimerism quantification was collected in different periods after transplantation. In all 10 children reconstitution of hematopoiesis was observed. Out of 8 children transplanted for hematologic malignancies, 4 are well and alive, in complete continuous remission (CCR), while in the other 4, leukemia relapse occurred 4–23 months after transplantation.DNA isolation High-molecular-weight DNA was extracted from frozen whole blood (approximately 5 ml) or bone marrow (approximately 3–5 ml) by the standard treatment with sodium dodecyl sulfate (SDS) and proteinase K, and the salting-out method. DNA was isolated from the donors' and patients' blood samples collected before and after transplantation at various intervals in order to determine the chimeric status.
DNA isolation
High-molecular-weight DNA was extracted from frozen whole blood (approximately 5 ml) or bone marrow (approximately 3–5 ml) by the standard treatment with sodium dodecyl sulfate (SDS) and proteinase K, and the salting-out method. DNA was isolated from the donors' and patients' blood samples collected before and after transplantation at various intervals in order to determine the chimeric status.Analysis of PCR products by an ABI 310 Genetic Analyzer The PCR protocol optimized for the Qiagen polymerase and the PE 9700 thermocycler was performed as described previously [ 5 ]. For fragment analysis (after capillary electrophoresis), an ABI 310 Genetic Analyzer (PE) was used [ 5 ]. All analyses were performed in the University Children's Hospital, Tuebingen.
Analysis of PCR products by an ABI 310 Genetic Analyzer
The PCR protocol optimized for the Qiagen polymerase and the PE 9700 thermocycler was performed as described previously [ 5 ]. For fragment analysis (after capillary electrophoresis), an ABI 310 Genetic Analyzer (PE) was used [ 5 ]. All analyses were performed in the University Children's Hospital, Tuebingen.Analysis of PCR products by an ALF Express DNA Sequencer The PCR protocol optimized for the Thermal Controller MJ Research (Watertown, MA) model PTC-100™ was applied. PCR was performed in a volume of 10 μl; the PCR reaction mixture contained: 2.5 pM of each forward and reverse primer, 200 μl of each dNTP, 0.4 U Taq polymerase (Qiagen, Chatsworth, CA), 1x PCR buffer (Qiagen, Chatsworth, CA), and 40 ng of genomic DNA. Conditions for PCR were as follows: 5 min at 94°C for the first denaturation; 26 cycles of amplification with a temperature profile of 45 sec at 94°C, 1 min at 55°C, 1 min at 72°C; with additional 5 min at 72°C in the last cycle. STR loci were amplified with fluorescent PCR primers described previously [ 6 ]. Primers for microsatellite markers were labeled with Cy5 dye (TIB MOLBIOL). A 1.5-μl aliquot of PCR reaction was resuspended in 7 μl of loading solution (formamide, bromophenol blue) containing 100 bp and 300 bp internal markers. All samples, after denaturation at 95°C for 5 min, were analyzed on 6% denaturing polyacrylamide gel with 7 M urea in the sequencer. A 50–500 sizer labeled with Cy5 dye was used as an external marker (for calculation of allele sizes). Electrophoresis was carried out in 0.6xTBE buffer at 1500 V/min. The helium-neon laser was operated at a wavelength of approximately 700 nm and laser power value of 2.5 mW. Allele sizes and peak areas of fluorescent products were analyzed and calculated with the use of Fragment Manager software (Pharmacia). PCR and analysis of PCR products by the ALF Express DNA Sequencer was performed at the Institute of Human Genetics, Poznan.
Analysis of PCR products by an ALF Express DNA Sequencer
The PCR protocol optimized for the Thermal Controller MJ Research (Watertown, MA) model PTC-100™ was applied. PCR was performed in a volume of 10 μl; the PCR reaction mixture contained: 2.5 pM of each forward and reverse primer, 200 μl of each dNTP, 0.4 U Taq polymerase (Qiagen, Chatsworth, CA), 1x PCR buffer (Qiagen, Chatsworth, CA), and 40 ng of genomic DNA. Conditions for PCR were as follows: 5 min at 94°C for the first denaturation; 26 cycles of amplification with a temperature profile of 45 sec at 94°C, 1 min at 55°C, 1 min at 72°C; with additional 5 min at 72°C in the last cycle. STR loci were amplified with fluorescent PCR primers described previously [ 6 ]. Primers for microsatellite markers were labeled with Cy5 dye (TIB MOLBIOL). A 1.5-μl aliquot of PCR reaction was resuspended in 7 μl of loading solution (formamide, bromophenol blue) containing 100 bp and 300 bp internal markers. All samples, after denaturation at 95°C for 5 min, were analyzed on 6% denaturing polyacrylamide gel with 7 M urea in the sequencer. A 50–500 sizer labeled with Cy5 dye was used as an external marker (for calculation of allele sizes). Electrophoresis was carried out in 0.6xTBE buffer at 1500 V/min. The helium-neon laser was operated at a wavelength of approximately 700 nm and laser power value of 2.5 mW. Allele sizes and peak areas of fluorescent products were analyzed and calculated with the use of Fragment Manager software (Pharmacia). PCR and analysis of PCR products by the ALF Express DNA Sequencer was performed at the Institute of Human Genetics, Poznan.FISH The experiments were performed on interphase nuclei obtained by standard short culturing of fresh whole blood samples, with probes specific for chromosomes X (locus  DXZ1 ) and Y (locus  DYZ1 ). The FISH procedure according to Cytocell Ltd. was used [ 7 ]. The number of scored nuclei was 250 to 550, with a median of 300. FISH experiments were performed at the Institute of Human Genetics, Poznan. Since the material for FISH analysis was not collected in all designated periods after transplantation in some patients, FISH experiments were not performed then.
FISH
The experiments were performed on interphase nuclei obtained by standard short culturing of fresh whole blood samples, with probes specific for chromosomes X (locus  DXZ1 ) and Y (locus  DYZ1 ). The FISH procedure according to Cytocell Ltd. was used [ 7 ]. The number of scored nuclei was 250 to 550, with a median of 300. FISH experiments were performed at the Institute of Human Genetics, Poznan. Since the material for FISH analysis was not collected in all designated periods after transplantation in some patients, FISH experiments were not performed then.DXZ1DYZ1Quantification of chimerism After electrophoresis in the ABI 310 Genetic Analyzer (PE), all obtained data were analyzed by GeneScan 3.1 software and then transferred to Genotyper 2.5 software [ 5 ]. All data obtained after electrophoresis of fluorescent products in the ALFExpress DNA Sequencer were transferred to Fragment Manager™ software (Pharmacia). For both, calculation of the amount of recipient's DNA was performed using the formula: % of recipient's DNA = (R1 + R2)/(D1+ D2 + R1 + R2) × 100, where: R1, R2 = peak areas of recipient's alleles; and D1, D2 = peak areas of donor's alleles. Only informative markers were used for the analysis. If donor and recipient were heterozygous but shared one allele, only the area of the non-shared alleles was considered for the analysis [ 8 ]. To make sure that quantification is accurate, we performed serial dilution experiments, where standardized mixed chimeric samples were created by mixing donor's and pretransplant recipient's DNA in a range between 0 and 100 percent. The sensitivity strongly depends on the size of alleles, the detection level was around 3–5% of patient cells. The results of chimerism detection by different methods were compared by the Spearman correlation test.
Quantification of chimerism
After electrophoresis in the ABI 310 Genetic Analyzer (PE), all obtained data were analyzed by GeneScan 3.1 software and then transferred to Genotyper 2.5 software [ 5 ]. All data obtained after electrophoresis of fluorescent products in the ALFExpress DNA Sequencer were transferred to Fragment Manager™ software (Pharmacia). For both, calculation of the amount of recipient's DNA was performed using the formula:% of recipient's DNA = (R1 + R2)/(D1+ D2 + R1 + R2) × 100,where: R1, R2 = peak areas of recipient's alleles; and D1, D2 = peak areas of donor's alleles.Only informative markers were used for the analysis. If donor and recipient were heterozygous but shared one allele, only the area of the non-shared alleles was considered for the analysis [ 8 ].To make sure that quantification is accurate, we performed serial dilution experiments, where standardized mixed chimeric samples were created by mixing donor's and pretransplant recipient's DNA in a range between 0 and 100 percent. The sensitivity strongly depends on the size of alleles, the detection level was around 3–5% of patient cells.The results of chimerism detection by different methods were compared by the Spearman correlation test.Results The results of chimerism quantification with the use of an ALF Express DNA Sequencer, ABI 310 Genetic Analyzer, and FISH are compared in Table  1 . In three patients (no. 5, 6, 7) only donor's cells were detected in all post-HSCT samples and in one patient (no. 1) only recipient's cells were present in all samples examined. These results were confirmed with the use of all three methods. Three other patients (no. 2, 3, 4) exhibited mixed posttransplant chimerism according to PCR and/or FISH. In the last three patients (no.8, 9, 10), complete chimerism was detected by PCR and automated DNA sizing, but in some of their samples, low numbers of recipients' cells were detected by FISH. Table 1 Comparison of results using different DNA sizing technologies and FISH No. UPN Sex F/M Diagnosis Material examined HSCT date Days after HSCT PCR/ALF Express* PCR/ ABI 310 Genetic Analyser* FISH with probes specific to X, Y chro-mosomes* 1. 67 F AML PB 04.09.1998 542 100% 100% 100%  556 100% 100% 100% 574 100% 100% 100% 590 100% 100% 100% 639 100% 100% 100% 675 100% 100% 100% 697 100% 100% 100% 721 100% 100% 100% 750 100% 100% 100% 2. 102 M AML PB 12.10.2000 14 84% 86% 89% 19 49% 47% 51% 28 40% 40% 35% 35 47% 42% 12% 57 46% 35% 6% 3. 106 M FA PB 21.12.2000 7 100% 100% 92% 48 26% 27% 32% 91 13% 14% 9% 4. 87 F FA PB 27.12.1999 21 10% 28% ND 38 93% 94% ND 285 15% 18% ND 313 17% 19% ND 357 0% 0% ND 5. 45 F CML PB 15.11.1996 953 0% 0% ND 1030 0% 0% ND 1216 0% 0% ND 1284 0% 0% 0% 1374 0% 0% 0% 1459 0% 0% 0% 1492 0% 0% ND 6. 93 F AML PB 20.04.2000 28 0% 0% 0%  158 0% 0% 0% 277 0% 0% 0% 7. 109 F MDS PB 26.01.2001 14 0% 0% 0% 21 0% 0% ND 34 0% 0% ND 42 0% 0% ND 8. 108 F ALL PB 19.01.2001 17 0% 0% ND 21 0% 0% ND 26 0% 0% 2% 39 0% 0% ND 52 0% 0% ND 60 0% 0% ND 9. 88 F AML PB 21.01.2000 29 0% 0% ND 87 0% 0% 0% 178 0% 0% ND 267 0% 0% ND 365 0% 0% 2% 10. 95 F ALL PB 16.06.2000 27 0% 0% ND 83 0% 0% ND 200 0% 0% ND 218 0% 0% 1% ALL = acute lymphoblastic leukemia; AML = acute myelogenous leukemia; CML = chronic myelogenous leukemia; FA = Fanconi Anemia; FISH = fluorescent  in situ  hybridization; HSCT = hematopoietic stem cell transplantation; MDS = myelodysplastic syndrome; ND = no data; PB = peripheral blood; UPN = unique patient's number * results expressed as % of recipient's cells Coefficients of Spearman rank correlation between results of chimerism quantification by the three different methods are shown in Table  2 . All coefficients were statistically significant (p < 0.001). The correlation between PCR/ALF Express and PCR/ABI 310 was stronger than between FISH and both PCR methods. Table 2 Coefficients of Spearman rank correlation between results of chimerism quantification by different methods (all coefficients significant, P < 0.001). PCR/ABI 310 Genetic Analyzer FISH PCR/ALF Express 0.987 0.801 PCR/ABI 310 Genetic Analyzer 0.825 Comparison of methods used One of the advantages of application of automated DNA sizing techniques for detection of posttransplant chimerism is that the use of radioactivity is not necessary. It is arelatively simple and rapid method, consisting of two steps: polymerase chain reaction with fluorescent primers and automated detection of fluorescently labeled PCR products, separated by electrophoresis. The analysis of fluorescently labeled PCR products provides better accuracy and precision of measurement than traditional electrophoretic methods. The most time-consuming step, which might prolong the examination process, is the search for informative markers. Analysis of the chimerism status by amplification of STR loci can be performed regardless of donor's and recipient's sex. The most advantageous is the high sensitivity of the detection system used, so that only small amounts of DNA are needed. Fragment analysis in the ABI 310 Genetic Analyzer (PE) is performed after capillary electrophoresis. The examination of one sample requires about 6 hrs; 144 samples can be analyzed per day if three colors are simultaneously analyzed [ 5 ]. The advantage of this method is that thanks to the combination of three-color detection, multiple sets of samples or multiple loci for a single sample can be analyzed on one gel. It is possible to analyze more than one marker at a time after multiplex amplification of informative loci. In the ALFExpress DNA Analyzer, electrophoresis is carried out in an off-vertical gel cassette specially designed for easy and safe gel casting. The number of samples to be loaded on a gel is limited to 40 per run. The ALFwin Fragment Analyzer is used afterwards for fragment analysis. It is provided with versatile application software for the control of DNA fragment separation runs and subsequent analysis of the data. Collected data are used to accurately size PCR product peaks on the basis of external and internal standards. One analysis of 40 samples takes 7 hrs, including PCR, electrophoresis in the gel cassette, and paperwork. The appropriate assembling and cleaning of the gel cassette is critical and time consuming. It is well known that FISH is a good quantitative method of fluorescent signal detection, but requires lots of technical experience and expertise. Fluorescent  in situ  hybridization for one patient sample lasts at least 5 hrs, including preparation of interphase nuclei, hybridization with specific probes (X, Y dual-color FISH), and analysis. The high cost of the procedure is definitely a disadvantage.
Results
The results of chimerism quantification with the use of an ALF Express DNA Sequencer, ABI 310 Genetic Analyzer, and FISH are compared in Table  1 . In three patients (no. 5, 6, 7) only donor's cells were detected in all post-HSCT samples and in one patient (no. 1) only recipient's cells were present in all samples examined. These results were confirmed with the use of all three methods. Three other patients (no. 2, 3, 4) exhibited mixed posttransplant chimerism according to PCR and/or FISH. In the last three patients (no.8, 9, 10), complete chimerism was detected by PCR and automated DNA sizing, but in some of their samples, low numbers of recipients' cells were detected by FISH.Comparison of results using different DNA sizing technologies and FISHNo.UPNSex F/MDiagnosisMaterial examinedHSCT dateDays after HSCTPCR/ALF Express*PCR/ ABI 310 Genetic Analyser*FISH with probes specific to X, Y chro-mosomes*ALL = acute lymphoblastic leukemia; AML = acute myelogenous leukemia; CML = chronic myelogenous leukemia; FA = Fanconi Anemia; FISH = fluorescent  in situ  hybridization; HSCT = hematopoietic stem cell transplantation; MDS = myelodysplastic syndrome; ND = no data; PB = peripheral blood; UPN = unique patient's numberin situ* results expressed as % of recipient's cellsCoefficients of Spearman rank correlation between results of chimerism quantification by the three different methods are shown in Table  2 . All coefficients were statistically significant (p < 0.001). The correlation between PCR/ALF Express and PCR/ABI 310 was stronger than between FISH and both PCR methods.Coefficients of Spearman rank correlation between results of chimerism quantification by different methods (all coefficients significant, P < 0.001).PCR/ABI 310 Genetic AnalyzerFISHComparison of methods used One of the advantages of application of automated DNA sizing techniques for detection of posttransplant chimerism is that the use of radioactivity is not necessary. It is arelatively simple and rapid method, consisting of two steps: polymerase chain reaction with fluorescent primers and automated detection of fluorescently labeled PCR products, separated by electrophoresis. The analysis of fluorescently labeled PCR products provides better accuracy and precision of measurement than traditional electrophoretic methods. The most time-consuming step, which might prolong the examination process, is the search for informative markers. Analysis of the chimerism status by amplification of STR loci can be performed regardless of donor's and recipient's sex. The most advantageous is the high sensitivity of the detection system used, so that only small amounts of DNA are needed. Fragment analysis in the ABI 310 Genetic Analyzer (PE) is performed after capillary electrophoresis. The examination of one sample requires about 6 hrs; 144 samples can be analyzed per day if three colors are simultaneously analyzed [ 5 ]. The advantage of this method is that thanks to the combination of three-color detection, multiple sets of samples or multiple loci for a single sample can be analyzed on one gel. It is possible to analyze more than one marker at a time after multiplex amplification of informative loci. In the ALFExpress DNA Analyzer, electrophoresis is carried out in an off-vertical gel cassette specially designed for easy and safe gel casting. The number of samples to be loaded on a gel is limited to 40 per run. The ALFwin Fragment Analyzer is used afterwards for fragment analysis. It is provided with versatile application software for the control of DNA fragment separation runs and subsequent analysis of the data. Collected data are used to accurately size PCR product peaks on the basis of external and internal standards. One analysis of 40 samples takes 7 hrs, including PCR, electrophoresis in the gel cassette, and paperwork. The appropriate assembling and cleaning of the gel cassette is critical and time consuming. It is well known that FISH is a good quantitative method of fluorescent signal detection, but requires lots of technical experience and expertise. Fluorescent  in situ  hybridization for one patient sample lasts at least 5 hrs, including preparation of interphase nuclei, hybridization with specific probes (X, Y dual-color FISH), and analysis. The high cost of the procedure is definitely a disadvantage.
Comparison of methods used
One of the advantages of application of automated DNA sizing techniques for detection of posttransplant chimerism is that the use of radioactivity is not necessary. It is arelatively simple and rapid method, consisting of two steps: polymerase chain reaction with fluorescent primers and automated detection of fluorescently labeled PCR products, separated by electrophoresis. The analysis of fluorescently labeled PCR products provides better accuracy and precision of measurement than traditional electrophoretic methods. The most time-consuming step, which might prolong the examination process, is the search for informative markers.Analysis of the chimerism status by amplification of STR loci can be performed regardless of donor's and recipient's sex. The most advantageous is the high sensitivity of the detection system used, so that only small amounts of DNA are needed.Fragment analysis in the ABI 310 Genetic Analyzer (PE) is performed after capillary electrophoresis. The examination of one sample requires about 6 hrs; 144 samples can be analyzed per day if three colors are simultaneously analyzed [ 5 ]. The advantage of this method is that thanks to the combination of three-color detection, multiple sets of samples or multiple loci for a single sample can be analyzed on one gel. It is possible to analyze more than one marker at a time after multiplex amplification of informative loci.In the ALFExpress DNA Analyzer, electrophoresis is carried out in an off-vertical gel cassette specially designed for easy and safe gel casting. The number of samples to be loaded on a gel is limited to 40 per run. The ALFwin Fragment Analyzer is used afterwards for fragment analysis. It is provided with versatile application software for the control of DNA fragment separation runs and subsequent analysis of the data. Collected data are used to accurately size PCR product peaks on the basis of external and internal standards.One analysis of 40 samples takes 7 hrs, including PCR, electrophoresis in the gel cassette, and paperwork. The appropriate assembling and cleaning of the gel cassette is critical and time consuming.It is well known that FISH is a good quantitative method of fluorescent signal detection, but requires lots of technical experience and expertise. Fluorescent  in situ  hybridization for one patient sample lasts at least 5 hrs, including preparation of interphase nuclei, hybridization with specific probes (X, Y dual-color FISH), and analysis. The high cost of the procedure is definitely a disadvantage.in situDiscussion PCR-based techniques allow the relative proportions of recipient's and donor's cells in the post-HSCT period to be identified and quantified and is not only limited to sex-mismatched transplants. Although when using chimerism analysis one cannot assess whether or not the population of recipient's nucleated cells contains leukemic cells, samples taken at various intervals can show if the expansion rate of the particular population is consistent with hematologic and clinical symptoms of the disease. When it is not possible to find an informative marker for PCR amplification, only FISH analysis enables assessing the chimerism status. However, cytogenetic Y chromosome probing by FISH is limited exclusively to sex-mismatched transplantations. Results obtained with the use of ALFExpress DNA Sequencer and ABI 310 Genetic Analyzer are identical or very similar. We showed that appropriate quantitative assessment of chimerism after HSCT by using microsatellite genotyping and automated DNA sizing does not depend on the sequencer model used. The high correlation between results from the PCR/ALFExpress and PCR/ABI 310 Genetic Analyzer indicate that these two methods can be used interchangeably. The superiority of the ABI 310 Genetic Analyzer is limited to the possibility of analysis of three samples at the same time in one reaction tube and the technical ease of capillary electrophoresis with no need for the time-consuming and cumbersome use of glass plates. Lower, but still positive correlations were found between results of FISH analysis and these two methodological variants of PCR. However, in some samples analyzed with PCR, no recipient's signals were found, attesting to full donor chimerism, while at the same time residual host cells turned out to be detectable by FISH. We suggest that these results are within the range of error of the method applied.
Discussion
PCR-based techniques allow the relative proportions of recipient's and donor's cells in the post-HSCT period to be identified and quantified and is not only limited to sex-mismatched transplants. Although when using chimerism analysis one cannot assess whether or not the population of recipient's nucleated cells contains leukemic cells, samples taken at various intervals can show if the expansion rate of the particular population is consistent with hematologic and clinical symptoms of the disease. When it is not possible to find an informative marker for PCR amplification, only FISH analysis enables assessing the chimerism status. However, cytogenetic Y chromosome probing by FISH is limited exclusively to sex-mismatched transplantations. Results obtained with the use of ALFExpress DNA Sequencer and ABI 310 Genetic Analyzer are identical or very similar. We showed that appropriate quantitative assessment of chimerism after HSCT by using microsatellite genotyping and automated DNA sizing does not depend on the sequencer model used. The high correlation between results from the PCR/ALFExpress and PCR/ABI 310 Genetic Analyzer indicate that these two methods can be used interchangeably. The superiority of the ABI 310 Genetic Analyzer is limited to the possibility of analysis of three samples at the same time in one reaction tube and the technical ease of capillary electrophoresis with no need for the time-consuming and cumbersome use of glass plates. Lower, but still positive correlations were found between results of FISH analysis and these two methodological variants of PCR. However, in some samples analyzed with PCR, no recipient's signals were found, attesting to full donor chimerism, while at the same time residual host cells turned out to be detectable by FISH. We suggest that these results are within the range of error of the method applied.Conclusions Finally, we conclude that all the methods applied enable a rapid and accurate detection of post-HSCT chimerism and with due caution can be used interchangeably.
Conclusions
Finally, we conclude that all the methods applied enable a rapid and accurate detection of post-HSCT chimerism and with due caution can be used interchangeably.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions J.J. carried out the molecular chimerism studies and drafted the manuscript. T.S. performed the statistical analysis. A.P and D.B. supplied clinical data. P.B. initiated quantitative analysis. J.W. supervised clinical part and final writing M.W. supervised laboratory part and final writing
Authors' contributions
J.J. carried out the molecular chimerism studies and drafted the manuscript.T.S. performed the statistical analysis.A.P and D.B. supplied clinical data.P.B. initiated quantitative analysis.J.W. supervised clinical part and final writingM.W. supervised laboratory part and final writingPre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background Respiratory syncytial virus (RSV) infection causes bronchiolitis in infants and children, which can be fatal, especially in immunocompromised patients. The BALB/c mouse, currently used as a model for studying RSV immunopathology, is semi-permissive to the virus. A mouse model that more closely mimics human RSV infection is needed. Since immunocompromised conditions increase risk of RSV infection, the possibility of enhancing RSV infection in the BALB/c mouse by pretreatment with cyclophosphamide was examined in this study. BALB/c mice were treated with cyclophosphamide (CYP) and five days later, they were infected with RSV intranasally. Pulmonary RSV titers, inflammation and airway hyperresponsiveness were measured five days after infection.
Background
Respiratory syncytial virus (RSV) infection causes bronchiolitis in infants and children, which can be fatal, especially in immunocompromised patients. The BALB/c mouse, currently used as a model for studying RSV immunopathology, is semi-permissive to the virus. A mouse model that more closely mimics human RSV infection is needed. Since immunocompromised conditions increase risk of RSV infection, the possibility of enhancing RSV infection in the BALB/c mouse by pretreatment with cyclophosphamide was examined in this study. BALB/c mice were treated with cyclophosphamide (CYP) and five days later, they were infected with RSV intranasally. Pulmonary RSV titers, inflammation and airway hyperresponsiveness were measured five days after infection.Results CYP-treated mice show higher RSV titers in their lungs of than the untreated mice. Also, a decreased percentage of macrophages and an increased number of lymphocytes and neutrophils were present in the BAL of CYP-treated mice compared to controls. The CYP-treated group also exhibited augmented bronchoalveolar and interstitial pulmonary inflammation. The increased RSV infection in CYP-treated mice was accompanied by elevated expression of IL-10, IL-12 and IFN-γ mRNAs and proteins compared to controls. Examination of CYP-treated mice before RSV infection showed that CYP treatment significantly decreased both IFN-γ and IL-12 expression.
Results
CYP-treated mice show higher RSV titers in their lungs of than the untreated mice. Also, a decreased percentage of macrophages and an increased number of lymphocytes and neutrophils were present in the BAL of CYP-treated mice compared to controls. The CYP-treated group also exhibited augmented bronchoalveolar and interstitial pulmonary inflammation. The increased RSV infection in CYP-treated mice was accompanied by elevated expression of IL-10, IL-12 and IFN-γ mRNAs and proteins compared to controls. Examination of CYP-treated mice before RSV infection showed that CYP treatment significantly decreased both IFN-γ and IL-12 expression.Conclusions These results demonstrate that CYP-treated BALB/c mice provide a better model for studying RSV immunopathology and that decreased production of IL-12 and IFN-γ are important determinants of susceptibility to RSV infection.
Conclusions
These results demonstrate that CYP-treated BALB/c mice provide a better model for studying RSV immunopathology and that decreased production of IL-12 and IFN-γ are important determinants of susceptibility to RSV infection.

Introduction Respiratory syncytial virus (RSV) is an important respiratory pathogen that produces an annual worldwide epidemic of respiratory illness primarily in children, but also in the elderly [ 1 , 2 ]. In the USA alone, RSV infection of children causes about 100,000 hospitalizations and 4,500 deaths annually (MMWR, 1996). RSV commonly precipitates bronchiolitis and exacerbates asthma but is also associated with severe life threatening respiratory infections in individuals with coronary artery disease or who are immunocompromised [ 3 - 6 ]. At the molecular level, RSV infection up-regulates the expression of several cytokines and chemokines, such as IL-1β, IL-6, IL-8, TNF-α, MIP1α, RANTES, and the adhesion molecule ICAM-1, ET-1, LTB4 and LTC4/D4/E4 [ 7 - 13 ]. Furthermore, elevated levels of cytokines and chemokines have been found in the nasal secretions of naturally RSV-infected children and of artificially-infected adults [ 14 - 17 ]. Defects in IL-12 and IFN-α production have been associated with severe RSV disease [ 18 ]. Despite progress in our understanding of immunopathology, the lack of a suitable animal model, with pathophysiology similar to humans, allowing appropriate virology, immunology, pathology and toxicology testing, has hindered the development of prophylactic and therapeutic interventions against RSV infection [ 19 ]. The pathology of RSV infection has been examined in a number of animals including primates, cotton rats, mice, calves, guinea pigs, ferrets and hamsters [ 19 ]. The choice of an experimental model is governed by the specific manifestation of the disease. The development of multiple animal models reflects the multifaceted nature of human RSV disease, in which clinical manifestations and sequelae depend upon age, genetic makeup, immunologic status and concurrent disease status of the individual [ 19 ]. Currently there is no single animal model that duplicates all forms of RSV disease. While cotton rats provide a good model for toxicologic evaluations, mice are considered advantageous for immunology and vaccine development. Furthermore, in mice the importance of IFN-γ, IL-6, IL-10 and IL-13 has been described [ 20 - 24 ]. The mouse provides an excellent model for human RSV infection because of the following: (a) the mouse is the best-characterized animal model, and experiments can be performed in this model in a cost- and time-effective manner, (b) a wide array of immunological reagents is available for studies in this model, and (c) the A2 strain of human RSV administered intranasally readily infects lungs of mice, and exhibits a time course of infection, pathology and resolution similar to that seen in humans [ 25 ]. Treatment of mice with the anti-RSV compound ribavirin decreases RSV titers in the lungs [ 26 ]. Depending upon the amount of RSV administered, the illness in micemay range from mild pneumonitis of the lung to weight loss [ 27 ]. Healthy BALB/c mice are semi-permissive to RSV and develop only limited inflammation and airway reactivity. Based on the reports that deficiency in IL-12 and IFN production increases severity of RSV disease, we hypothesized that rendering mice immunocompromised, will improve permissiveness to RSV and provide a better model for RSV infection. To test this hypothesis, BALB/c were treated with cyclophosphamide, infected with RSV, and characterized in terms of viral infectivity and pathology, immunology, and immunohistology. The results show that cyclophosphamide temporarily decreases IL-12 production and thus augments viral replication and the immunopathology of RSV disease.
Introduction
Respiratory syncytial virus (RSV) is an important respiratory pathogen that produces an annual worldwide epidemic of respiratory illness primarily in children, but also in the elderly [ 1 , 2 ]. In the USA alone, RSV infection of children causes about 100,000 hospitalizations and 4,500 deaths annually (MMWR, 1996). RSV commonly precipitates bronchiolitis and exacerbates asthma but is also associated with severe life threatening respiratory infections in individuals with coronary artery disease or who are immunocompromised [ 3 - 6 ]. At the molecular level, RSV infection up-regulates the expression of several cytokines and chemokines, such as IL-1β, IL-6, IL-8, TNF-α, MIP1α, RANTES, and the adhesion molecule ICAM-1, ET-1, LTB4 and LTC4/D4/E4 [ 7 - 13 ]. Furthermore, elevated levels of cytokines and chemokines have been found in the nasal secretions of naturally RSV-infected children and of artificially-infected adults [ 14 - 17 ]. Defects in IL-12 and IFN-α production have been associated with severe RSV disease [ 18 ].Despite progress in our understanding of immunopathology, the lack of a suitable animal model, with pathophysiology similar to humans, allowing appropriate virology, immunology, pathology and toxicology testing, has hindered the development of prophylactic and therapeutic interventions against RSV infection [ 19 ]. The pathology of RSV infection has been examined in a number of animals including primates, cotton rats, mice, calves, guinea pigs, ferrets and hamsters [ 19 ]. The choice of an experimental model is governed by the specific manifestation of the disease. The development of multiple animal models reflects the multifaceted nature of human RSV disease, in which clinical manifestations and sequelae depend upon age, genetic makeup, immunologic status and concurrent disease status of the individual [ 19 ]. Currently there is no single animal model that duplicates all forms of RSV disease. While cotton rats provide a good model for toxicologic evaluations, mice are considered advantageous for immunology and vaccine development. Furthermore, in mice the importance of IFN-γ, IL-6, IL-10 and IL-13 has been described [ 20 - 24 ]. The mouse provides an excellent model for human RSV infection because of the following: (a) the mouse is the best-characterized animal model, and experiments can be performed in this model in a cost- and time-effective manner, (b) a wide array of immunological reagents is available for studies in this model, and (c) the A2 strain of human RSV administered intranasally readily infects lungs of mice, and exhibits a time course of infection, pathology and resolution similar to that seen in humans [ 25 ]. Treatment of mice with the anti-RSV compound ribavirin decreases RSV titers in the lungs [ 26 ]. Depending upon the amount of RSV administered, the illness in micemay range from mild pneumonitis of the lung to weight loss [ 27 ].Healthy BALB/c mice are semi-permissive to RSV and develop only limited inflammation and airway reactivity. Based on the reports that deficiency in IL-12 and IFN production increases severity of RSV disease, we hypothesized that rendering mice immunocompromised, will improve permissiveness to RSV and provide a better model for RSV infection. To test this hypothesis, BALB/c were treated with cyclophosphamide, infected with RSV, and characterized in terms of viral infectivity and pathology, immunology, and immunohistology. The results show that cyclophosphamide temporarily decreases IL-12 production and thus augments viral replication and the immunopathology of RSV disease.Materials and Methods Animals Female six-week old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in a pathogen-free environment. All procedures were reviewed and approved by the University of South Florida Committee on Animal Research. Cyclophosphamide treatment Cyclophosphamide (CYP; Sigma, St. Louis, MO) was administered to mice intraperitoneally (i.p.) at a single dose of 100 mg per kg five days prior to RSV infection. RSV infection, weight determination and tissue collection The A2 strain of human RSV (American Type Culture Collection, Manassas, VA) was propagated in Hep-2 cells (ATCC) in a monolayer culture as previously described (Behera et al., 1998). Mice were infected intranasally with 5 × 10 5  PFU of RSV in a volume of 50 μl five days after treatment with CYP. One set of animals was monitored for weight loss at days 5, 10, 15 and 22 following CYP treatment (0, 5, 10 and 17 days after RSV infection). A second set of animals was sacrificed five days after infection and their lungs were removed for determination of RSV titers, cytokine levels and histopathology. RSV plaque assay HEp-2 cells (5 × 10 5 /well) in 6-well plates were infected with 5 × 10 5  pfu RSV per well for 2 hours at 37°C. The RSV was removed and the wells were overlaid with 1.5 ml of growth medium containing 0.8% methylcellulose. The cells were then incubated at 37°C for 72 hours, after which the overlay was removed. Following incubation, the cells were fixed in cold 80% methanol for 3 hours, blocked with 1 % horse serum in PBS at 37°C for 30 min, then incubated with anti-RSV monoclonal antibody (NCL-RSV 3, Vector Laboratories, Burlingame, CA) diluted 1:400 for 1 hour at 37°C. Secondary antibody staining and substrate reactions were performed using the Vectastain ABC Kit (Vector Laboratories) and diaminobenzidine in H 2 O 2  (Pierce, Rockford, IL) was used as a chromagen. The plaques were enumerated by microscopy and the results were expressed as mean ± standard error of the mean. Determination of airway hyperresponsiveness (AHR) AHR was measured in unrestrained mice using a whole body plethysmograph (Buxco, Troy, NY), as previously described (Schwarze et al., 1997) and expressed as enhanced pause (Penh). Groups of mice (n = 4) were exposed for 5 min to nebulized PBS and subsequently to increasing concentrations (6, 12, 25 and 50 mg/ml) of nebulized methacholine (MCh; Sigma, St, Louis, MO) in PBS using an ultrasonic nebulizer. After nebulization, recordings were taken for 5 minutes. Penh values were averaged and expressed as a percentage of baseline Penh values obtained following PBS exposure. Immunohistochemical analysis Mouse lungs were rinsed with intratracheal injections of PBS then perfused with 10 % neutral buffered formalin. Lungs were removed, paraffin-embedded, sectioned at 20 μm and stained with hematoxylin and eosin (H & E). A semi-quantitative evaluation of inflammatory cells in the lung sections was performed as previously described (Kumar et al., 1999). Whole lung homogenates were prepared using a TissueMizer and assayed for cytokines IL-10, IL-12 and IFN-γ by ELISA (R & D Systems, Minneapolis MN), following the manufacturer's directions. The results are expressed as cytokine amount in picograms per gram of lung (pg/g). Detection of RSV and cytokines in the lungs by RT-PCR Total cellular RNA was isolated from lung tissue using TRIZOL reagent (Life Technologies, Gaithersburg, MD). Forward and reverse primers used were as follows: RSV-N forward: 5'-GCG ATG TCT AGG TTA GGA AGA A-3'; reverse: 5'-GCT ATG TCC TTG GGT AGT AAG CCT-3'; mouse IFN-γ Forward: 5'-GCT CTG AGA CAA TGA ACG CT-3'; reverse: 5'-AAA GAG ATA ATC TGG CTG TGC-3'; mouse IL-10 forward: 5'-GGA CTT TAA GGG TTA CTT GGG TTG CC-3'; reverse: 5'-CAT TTT GAT CAT CAT GTA TGC TTC T-3'; mouse IL-12 forward: 5'-CAG TAC ACC TGC CAC AAA GGA -3'; reverse: 5'-GTG TGA CCT TCT CTG CAG ACA -3' and β-actin forward: 5'-GAC ATG GAG AAG ATC TGG CAC-3'; reverse: 5'-TCC AGA CGC AGG ATG GCG TGA -3'. All PCRs were denatured at 95°C for 1 min, annealed at 56°C for 30 sec, and extended at 72°C for 1 min for 25–35 cycles. All amplifications were done in triplicate and repeated three times. The PCR products were separated by agarose gel electrophoresis and quantified using Advanced Quantifier Software (BioImage, Ann Arbor, MI). Cell enumeration of bronchoalveolar lavage fluid Bronchoalveolar lavage (BAL) fluid was collected and differential cell counts were performed as previously described (Kumar et al., 1999). Briefly, BAL was centrifuged and the cell pellet was suspended in 200 μl of PBS and counted using a hemocytometer. The cell suspensions were then centrifuged onto glass slides using a cytospin centrifuge at 1000 rpm for 5 min at room temperature. Cytocentrifuged cells were air dried and stained with a modified Wright's stain (Leukostat, Fisher Scientific, Atlanta, GA) which allows differential counting of monocytes and lymphocytes. At least 300 cells per sample were counted by direct microscopic observation. Statistical analysis Values for all measurements were expressed as mean ± SD or SEM. The data were analyzed by ANOVA. Paired and unpaired results were compared by a Wilcoxon rank sum test or Mann-Whitney test respectively. Differences between groups were considered significant at  p  < 0.05.
Materials and Methods
Animals Female six-week old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in a pathogen-free environment. All procedures were reviewed and approved by the University of South Florida Committee on Animal Research.
Animals
Female six-week old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in a pathogen-free environment. All procedures were reviewed and approved by the University of South Florida Committee on Animal Research.Cyclophosphamide treatment Cyclophosphamide (CYP; Sigma, St. Louis, MO) was administered to mice intraperitoneally (i.p.) at a single dose of 100 mg per kg five days prior to RSV infection.
Cyclophosphamide treatment
Cyclophosphamide (CYP; Sigma, St. Louis, MO) was administered to mice intraperitoneally (i.p.) at a single dose of 100 mg per kg five days prior to RSV infection.RSV infection, weight determination and tissue collection The A2 strain of human RSV (American Type Culture Collection, Manassas, VA) was propagated in Hep-2 cells (ATCC) in a monolayer culture as previously described (Behera et al., 1998). Mice were infected intranasally with 5 × 10 5  PFU of RSV in a volume of 50 μl five days after treatment with CYP. One set of animals was monitored for weight loss at days 5, 10, 15 and 22 following CYP treatment (0, 5, 10 and 17 days after RSV infection). A second set of animals was sacrificed five days after infection and their lungs were removed for determination of RSV titers, cytokine levels and histopathology. RSV plaque assay HEp-2 cells (5 × 10 5 /well) in 6-well plates were infected with 5 × 10 5  pfu RSV per well for 2 hours at 37°C. The RSV was removed and the wells were overlaid with 1.5 ml of growth medium containing 0.8% methylcellulose. The cells were then incubated at 37°C for 72 hours, after which the overlay was removed. Following incubation, the cells were fixed in cold 80% methanol for 3 hours, blocked with 1 % horse serum in PBS at 37°C for 30 min, then incubated with anti-RSV monoclonal antibody (NCL-RSV 3, Vector Laboratories, Burlingame, CA) diluted 1:400 for 1 hour at 37°C. Secondary antibody staining and substrate reactions were performed using the Vectastain ABC Kit (Vector Laboratories) and diaminobenzidine in H 2 O 2  (Pierce, Rockford, IL) was used as a chromagen. The plaques were enumerated by microscopy and the results were expressed as mean ± standard error of the mean.
RSV infection, weight determination and tissue collection
The A2 strain of human RSV (American Type Culture Collection, Manassas, VA) was propagated in Hep-2 cells (ATCC) in a monolayer culture as previously described (Behera et al., 1998). Mice were infected intranasally with 5 × 10 5  PFU of RSV in a volume of 50 μl five days after treatment with CYP. One set of animals was monitored for weight loss at days 5, 10, 15 and 22 following CYP treatment (0, 5, 10 and 17 days after RSV infection). A second set of animals was sacrificed five days after infection and their lungs were removed for determination of RSV titers, cytokine levels and histopathology.RSV plaque assay HEp-2 cells (5 × 10 5 /well) in 6-well plates were infected with 5 × 10 5  pfu RSV per well for 2 hours at 37°C. The RSV was removed and the wells were overlaid with 1.5 ml of growth medium containing 0.8% methylcellulose. The cells were then incubated at 37°C for 72 hours, after which the overlay was removed. Following incubation, the cells were fixed in cold 80% methanol for 3 hours, blocked with 1 % horse serum in PBS at 37°C for 30 min, then incubated with anti-RSV monoclonal antibody (NCL-RSV 3, Vector Laboratories, Burlingame, CA) diluted 1:400 for 1 hour at 37°C. Secondary antibody staining and substrate reactions were performed using the Vectastain ABC Kit (Vector Laboratories) and diaminobenzidine in H 2 O 2  (Pierce, Rockford, IL) was used as a chromagen. The plaques were enumerated by microscopy and the results were expressed as mean ± standard error of the mean.
RSV plaque assay
HEp-2 cells (5 × 10 5 /well) in 6-well plates were infected with 5 × 10 5  pfu RSV per well for 2 hours at 37°C. The RSV was removed and the wells were overlaid with 1.5 ml of growth medium containing 0.8% methylcellulose. The cells were then incubated at 37°C for 72 hours, after which the overlay was removed. Following incubation, the cells were fixed in cold 80% methanol for 3 hours, blocked with 1 % horse serum in PBS at 37°C for 30 min, then incubated with anti-RSV monoclonal antibody (NCL-RSV 3, Vector Laboratories, Burlingame, CA) diluted 1:400 for 1 hour at 37°C. Secondary antibody staining and substrate reactions were performed using the Vectastain ABC Kit (Vector Laboratories) and diaminobenzidine in H 2 O 2  (Pierce, Rockford, IL) was used as a chromagen. The plaques were enumerated by microscopy and the results were expressed as mean ± standard error of the mean.Determination of airway hyperresponsiveness (AHR) AHR was measured in unrestrained mice using a whole body plethysmograph (Buxco, Troy, NY), as previously described (Schwarze et al., 1997) and expressed as enhanced pause (Penh). Groups of mice (n = 4) were exposed for 5 min to nebulized PBS and subsequently to increasing concentrations (6, 12, 25 and 50 mg/ml) of nebulized methacholine (MCh; Sigma, St, Louis, MO) in PBS using an ultrasonic nebulizer. After nebulization, recordings were taken for 5 minutes. Penh values were averaged and expressed as a percentage of baseline Penh values obtained following PBS exposure.
Determination of airway hyperresponsiveness (AHR)
AHR was measured in unrestrained mice using a whole body plethysmograph (Buxco, Troy, NY), as previously described (Schwarze et al., 1997) and expressed as enhanced pause (Penh). Groups of mice (n = 4) were exposed for 5 min to nebulized PBS and subsequently to increasing concentrations (6, 12, 25 and 50 mg/ml) of nebulized methacholine (MCh; Sigma, St, Louis, MO) in PBS using an ultrasonic nebulizer. After nebulization, recordings were taken for 5 minutes. Penh values were averaged and expressed as a percentage of baseline Penh values obtained following PBS exposure.Immunohistochemical analysis Mouse lungs were rinsed with intratracheal injections of PBS then perfused with 10 % neutral buffered formalin. Lungs were removed, paraffin-embedded, sectioned at 20 μm and stained with hematoxylin and eosin (H & E). A semi-quantitative evaluation of inflammatory cells in the lung sections was performed as previously described (Kumar et al., 1999). Whole lung homogenates were prepared using a TissueMizer and assayed for cytokines IL-10, IL-12 and IFN-γ by ELISA (R & D Systems, Minneapolis MN), following the manufacturer's directions. The results are expressed as cytokine amount in picograms per gram of lung (pg/g).
Immunohistochemical analysis
Mouse lungs were rinsed with intratracheal injections of PBS then perfused with 10 % neutral buffered formalin. Lungs were removed, paraffin-embedded, sectioned at 20 μm and stained with hematoxylin and eosin (H & E). A semi-quantitative evaluation of inflammatory cells in the lung sections was performed as previously described (Kumar et al., 1999). Whole lung homogenates were prepared using a TissueMizer and assayed for cytokines IL-10, IL-12 and IFN-γ by ELISA (R & D Systems, Minneapolis MN), following the manufacturer's directions. The results are expressed as cytokine amount in picograms per gram of lung (pg/g).Detection of RSV and cytokines in the lungs by RT-PCR Total cellular RNA was isolated from lung tissue using TRIZOL reagent (Life Technologies, Gaithersburg, MD). Forward and reverse primers used were as follows: RSV-N forward: 5'-GCG ATG TCT AGG TTA GGA AGA A-3'; reverse: 5'-GCT ATG TCC TTG GGT AGT AAG CCT-3'; mouse IFN-γ Forward: 5'-GCT CTG AGA CAA TGA ACG CT-3'; reverse: 5'-AAA GAG ATA ATC TGG CTG TGC-3'; mouse IL-10 forward: 5'-GGA CTT TAA GGG TTA CTT GGG TTG CC-3'; reverse: 5'-CAT TTT GAT CAT CAT GTA TGC TTC T-3'; mouse IL-12 forward: 5'-CAG TAC ACC TGC CAC AAA GGA -3'; reverse: 5'-GTG TGA CCT TCT CTG CAG ACA -3' and β-actin forward: 5'-GAC ATG GAG AAG ATC TGG CAC-3'; reverse: 5'-TCC AGA CGC AGG ATG GCG TGA -3'. All PCRs were denatured at 95°C for 1 min, annealed at 56°C for 30 sec, and extended at 72°C for 1 min for 25–35 cycles. All amplifications were done in triplicate and repeated three times. The PCR products were separated by agarose gel electrophoresis and quantified using Advanced Quantifier Software (BioImage, Ann Arbor, MI).
Detection of RSV and cytokines in the lungs by RT-PCR
Total cellular RNA was isolated from lung tissue using TRIZOL reagent (Life Technologies, Gaithersburg, MD). Forward and reverse primers used were as follows: RSV-N forward: 5'-GCG ATG TCT AGG TTA GGA AGA A-3'; reverse: 5'-GCT ATG TCC TTG GGT AGT AAG CCT-3'; mouse IFN-γ Forward: 5'-GCT CTG AGA CAA TGA ACG CT-3'; reverse: 5'-AAA GAG ATA ATC TGG CTG TGC-3'; mouse IL-10 forward: 5'-GGA CTT TAA GGG TTA CTT GGG TTG CC-3'; reverse: 5'-CAT TTT GAT CAT CAT GTA TGC TTC T-3'; mouse IL-12 forward: 5'-CAG TAC ACC TGC CAC AAA GGA -3'; reverse: 5'-GTG TGA CCT TCT CTG CAG ACA -3' and β-actin forward: 5'-GAC ATG GAG AAG ATC TGG CAC-3'; reverse: 5'-TCC AGA CGC AGG ATG GCG TGA -3'. All PCRs were denatured at 95°C for 1 min, annealed at 56°C for 30 sec, and extended at 72°C for 1 min for 25–35 cycles. All amplifications were done in triplicate and repeated three times. The PCR products were separated by agarose gel electrophoresis and quantified using Advanced Quantifier Software (BioImage, Ann Arbor, MI).Cell enumeration of bronchoalveolar lavage fluid Bronchoalveolar lavage (BAL) fluid was collected and differential cell counts were performed as previously described (Kumar et al., 1999). Briefly, BAL was centrifuged and the cell pellet was suspended in 200 μl of PBS and counted using a hemocytometer. The cell suspensions were then centrifuged onto glass slides using a cytospin centrifuge at 1000 rpm for 5 min at room temperature. Cytocentrifuged cells were air dried and stained with a modified Wright's stain (Leukostat, Fisher Scientific, Atlanta, GA) which allows differential counting of monocytes and lymphocytes. At least 300 cells per sample were counted by direct microscopic observation.
Cell enumeration of bronchoalveolar lavage fluid
Bronchoalveolar lavage (BAL) fluid was collected and differential cell counts were performed as previously described (Kumar et al., 1999). Briefly, BAL was centrifuged and the cell pellet was suspended in 200 μl of PBS and counted using a hemocytometer. The cell suspensions were then centrifuged onto glass slides using a cytospin centrifuge at 1000 rpm for 5 min at room temperature. Cytocentrifuged cells were air dried and stained with a modified Wright's stain (Leukostat, Fisher Scientific, Atlanta, GA) which allows differential counting of monocytes and lymphocytes. At least 300 cells per sample were counted by direct microscopic observation.Statistical analysis Values for all measurements were expressed as mean ± SD or SEM. The data were analyzed by ANOVA. Paired and unpaired results were compared by a Wilcoxon rank sum test or Mann-Whitney test respectively. Differences between groups were considered significant at  p  < 0.05.
Statistical analysis
Values for all measurements were expressed as mean ± SD or SEM. The data were analyzed by ANOVA. Paired and unpaired results were compared by a Wilcoxon rank sum test or Mann-Whitney test respectively. Differences between groups were considered significant at  p  < 0.05.pResults Cyclophosphamide treatment augments RSV infection in mice Groups of mice were injected i.p. with a single dose of CYP or PBS and five days later infected with RSV. Five days after infection, RSV titers in one group of mice were measured by plaque assay of lung homogenates (Fig.  1A ). The mice pretreated with CYP produced significantly more ( p  < 0.01) RSV plaques compared to the PBS control group. Weight loss is a clinical correlate of RSV infection, therefore weights were measured in a parallel group of mice on day 5, 10, 15, and 22 after CYP treatment (day 0, 5, 10 and 17 after RSV infection) (Fig.  1B ). CYP treatment alone resulted in a weight loss or reduced weight gain compared to PBS, but the RSV-infected, CYP-treated mice lost significantly more weight than those exposed to RSV alone p < 0.05; †.p < 0.01(vs. RSV) or PBS (p < 0.01 vs. Control). Pretreatment with CYP resulted in increased weight loss in the RSV-infected mice through day 15 and reduced weight gain at day 22 indicating that cyclophosphamide treatment exacerbated the pathology of RSV infection. Figure 1 (A) CYP increases RSV titer in the lungs of BALB/c mice.  Mice were treated with CYP (100 mg/kg, i.p.) or PBS and 5 days later infected with RSV (50 μl i.n. twice, 10 6  PFU/ mouse). Animals were sacrificed on day 4 and RSV titers were measured in whole lung homogenates by RSV plaque assay. (n = 4 for each group; § P < 0.01 vs PBS group).  (B) Cyclophosphamide affects body weight.  Mice (n = 4) were infected with RSV alone or were treated with CYP (100 mg/kg i.p.) prior to infection. Body weights were measured on day 1, 5, 10, 15, and 22 after treatment. Bars represent means ± SEM. (* P < 0.05; †. P < 0.01 vs RSV); ‡ P < 0.05; § P < 0.01 vs control). Cyclophosphamide pretreatment increases RSV-inducible lung inflammation To examine whether CYP treatment increases inflammatory effects in the lungs of RSV-infected mice, we determined airway hyperresponsiveness (AHR), cellular infiltration into the lung and lung histopathology. Groups of BALB/c mice either infected with RSV alone or treated with cyclophosphamide (CYP) prior to infection were lavaged and cells in the fluid were centrifuged onto slides. BAL cells were stained with Leukostat. Cells were counted from 4 different slides from each group in a blinded fashion. Cell counts were plotted as percentage of total cells (Fig.  2A ). There was a decrease in the number of macrophages and increases in lymphocyte and neutrophil numbers following RSV infection that was enhanced by prior treatment with CYP. To analyze the extent of lung pathology, lungs were paraffin embedded, sectioned and stained with hemotoxylin-eosin (HE). The lung sections from RSV-infected, CYP-treated mice (Fig.  2C , a & b) showed significantly greater inflammation than lungs from mice given RSV alone (Fig.  2C , c & d). The RSV-infected groups showed greater inflammation than uninfected control mice (Fig.  2C  e & f). Figure 2 (A) BAL cell differential of RSV-infected mice . Mice were treated with cyclophosphamide (CYP) or vehicle 5 days before infection with RSV. Animals were sacrificed on day 4 postinfection and BAL was performed. Following cytocentrifugation, BAL cells were stained with Leukostat and counted from 4 different slides from each group in a blinded fashion. Cell counts as percentage of total were plotted.  (B) Measurement of airway hyperrresponsiveness (AHR) . Mice treated as above were tested for AHR by methacholine challenge in a plethysmograph. AHR is expressed as PENH, percent of control.  (C) Lung histopathology.  Mice were infected with RSV alone  (C and D)  or treated with cyclophosphamide  (A and B)  prior to RSV infection. The third group of mice was not exposed to RSV  (E and F).  Animals were sacrificed on day 5 and their lungs removed and sectioned. Paraffin-embedded lung sections were stained with hematoxylin-eosin. Increased RSV infection in CYP-treated BALB/c mice is associated with increased production of immunoregulatory cytokines To examine the cytokine profile in lungs from RSV-infected mice with or without CYP treatment, the gene expression of IL-10, IFN-γ and IL-12 was measured by RT-PCR. Gel profiles and densitometric analyses are shown in Fig.  3A  and  3B . The results show that mice infected with RSV after CYP treatment have increased mRNA expression for all of three cytokines compared to control mice or mice infected with RSV alone. To determine whether mice treated with CYP and infected with RSV do produce more of these proteins, cytokines were measured by ELISA on homogenates prepared from whole lungs. IL-10 (p < 0.05 vs. PBS), IL-12 (p < 0.05 vs. RSV) and IFN-γ (p < 0.05 vs. PBS) levels were significantly higher in the CYP-treated group than the control group (Fig.  4A–C ). Figure 3 Detection of RSV and cytokines in the lungs of BALB/c mice. (A)  RSV-N and IL-10, IL-12, IFN-γ and β-actin were checked by RT-PCR. Mice were infected with RSV alone or treated with CYP prior to infection. The third group was uninfected (PBS) as control. Animals were sacrificed on day 5, their lungs removed and RNA was isolated and used in RT-PCR assay.  (B) Densitometric analysis of the band densities from part A.  Relative intensity refers to the ratio of the intensity of each cDNA product to that of β-actin. Figure 4 CYP-treated BALB/c mice produce higher levels of Th1 and Th2 cytokines . Mice were infected with RSV alone or were treated with cyclophosphamide prior to infection. The third group of mice received no virus (PBS only) as control. Animals were sacrificed on day 5 and their lungs removed. Whole lung homogenates were prepared and cyrtokines were measured by ELISA. Results are given as mean ± SEM (n = 4 for each group).  (A)  CYP-pretreated mice produce higher IL-10 in the lungs. (‡ P < 0.05 vs. PBS).  (B)  IL-12 was higher in CYP-treated mice. (§ P < 0.01 vs. PBS. * P < 0.05 vs. RSV).  (C)  IFN-γ was higher in CYP-treated mice. (* P < 0.05 vs. RSV; ‡ P < 0.05 vs. PBS). Cyclophosphamide treatment causes a transient reduction in IL-12 and IFN-γ expression To examine the mechanism underlying the increased RSV infection in CYP-treated animals, the levels of two cytokines that exert antiviral activity, IL-12 and IFN-γ was measured after treatment with CYP (Fig.  5 ). Mice were sacrificed on days 1, 2, 4, 6 after treatment and IL-12 and IFN-γ protein levels were measured in whole lung homogenates by ELISA. Untreated BALB/c mice were used as controls. Treatment with CYP gradually decreased both IL-12 (p < 0.05) and IFN-γ (p < 0.01 vs. Control) until day 4. These results show that decreased production of IL-12 and IFN-γ may play a role in the observed increase in RSV infection in CYP-treated mice. Figure 5 Changes in IL-12 and IFN-γ levels over time in CYP-treated mice.  Mice were treated with cyclophosphamide at 100 mg/kg i.p. Animals were sacrificed on day 1, 2, 4, 6 after treatment and their lungs removed. Whole lung homogenates were prepared and IL-12  (A)  and IFN-γ  (B)  were measured by ELISA. Untreated mice were used as control. Results are shown as mean ± SEM (n = 2; ‡ P < 0.05; § P < 0.01 vs. control).
Results
Cyclophosphamide treatment augments RSV infection in mice Groups of mice were injected i.p. with a single dose of CYP or PBS and five days later infected with RSV. Five days after infection, RSV titers in one group of mice were measured by plaque assay of lung homogenates (Fig.  1A ). The mice pretreated with CYP produced significantly more ( p  < 0.01) RSV plaques compared to the PBS control group. Weight loss is a clinical correlate of RSV infection, therefore weights were measured in a parallel group of mice on day 5, 10, 15, and 22 after CYP treatment (day 0, 5, 10 and 17 after RSV infection) (Fig.  1B ). CYP treatment alone resulted in a weight loss or reduced weight gain compared to PBS, but the RSV-infected, CYP-treated mice lost significantly more weight than those exposed to RSV alone p < 0.05; †.p < 0.01(vs. RSV) or PBS (p < 0.01 vs. Control). Pretreatment with CYP resulted in increased weight loss in the RSV-infected mice through day 15 and reduced weight gain at day 22 indicating that cyclophosphamide treatment exacerbated the pathology of RSV infection. Figure 1 (A) CYP increases RSV titer in the lungs of BALB/c mice.  Mice were treated with CYP (100 mg/kg, i.p.) or PBS and 5 days later infected with RSV (50 μl i.n. twice, 10 6  PFU/ mouse). Animals were sacrificed on day 4 and RSV titers were measured in whole lung homogenates by RSV plaque assay. (n = 4 for each group; § P < 0.01 vs PBS group).  (B) Cyclophosphamide affects body weight.  Mice (n = 4) were infected with RSV alone or were treated with CYP (100 mg/kg i.p.) prior to infection. Body weights were measured on day 1, 5, 10, 15, and 22 after treatment. Bars represent means ± SEM. (* P < 0.05; †. P < 0.01 vs RSV); ‡ P < 0.05; § P < 0.01 vs control).
Cyclophosphamide treatment augments RSV infection in mice
Groups of mice were injected i.p. with a single dose of CYP or PBS and five days later infected with RSV. Five days after infection, RSV titers in one group of mice were measured by plaque assay of lung homogenates (Fig.  1A ). The mice pretreated with CYP produced significantly more ( p  < 0.01) RSV plaques compared to the PBS control group. Weight loss is a clinical correlate of RSV infection, therefore weights were measured in a parallel group of mice on day 5, 10, 15, and 22 after CYP treatment (day 0, 5, 10 and 17 after RSV infection) (Fig.  1B ). CYP treatment alone resulted in a weight loss or reduced weight gain compared to PBS, but the RSV-infected, CYP-treated mice lost significantly more weight than those exposed to RSV alone p < 0.05; †.p < 0.01(vs. RSV) or PBS (p < 0.01 vs. Control). Pretreatment with CYP resulted in increased weight loss in the RSV-infected mice through day 15 and reduced weight gain at day 22 indicating that cyclophosphamide treatment exacerbated the pathology of RSV infection.pCyclophosphamide pretreatment increases RSV-inducible lung inflammation To examine whether CYP treatment increases inflammatory effects in the lungs of RSV-infected mice, we determined airway hyperresponsiveness (AHR), cellular infiltration into the lung and lung histopathology. Groups of BALB/c mice either infected with RSV alone or treated with cyclophosphamide (CYP) prior to infection were lavaged and cells in the fluid were centrifuged onto slides. BAL cells were stained with Leukostat. Cells were counted from 4 different slides from each group in a blinded fashion. Cell counts were plotted as percentage of total cells (Fig.  2A ). There was a decrease in the number of macrophages and increases in lymphocyte and neutrophil numbers following RSV infection that was enhanced by prior treatment with CYP. To analyze the extent of lung pathology, lungs were paraffin embedded, sectioned and stained with hemotoxylin-eosin (HE). The lung sections from RSV-infected, CYP-treated mice (Fig.  2C , a & b) showed significantly greater inflammation than lungs from mice given RSV alone (Fig.  2C , c & d). The RSV-infected groups showed greater inflammation than uninfected control mice (Fig.  2C  e & f). Figure 2 (A) BAL cell differential of RSV-infected mice . Mice were treated with cyclophosphamide (CYP) or vehicle 5 days before infection with RSV. Animals were sacrificed on day 4 postinfection and BAL was performed. Following cytocentrifugation, BAL cells were stained with Leukostat and counted from 4 different slides from each group in a blinded fashion. Cell counts as percentage of total were plotted.  (B) Measurement of airway hyperrresponsiveness (AHR) . Mice treated as above were tested for AHR by methacholine challenge in a plethysmograph. AHR is expressed as PENH, percent of control.  (C) Lung histopathology.  Mice were infected with RSV alone  (C and D)  or treated with cyclophosphamide  (A and B)  prior to RSV infection. The third group of mice was not exposed to RSV  (E and F).  Animals were sacrificed on day 5 and their lungs removed and sectioned. Paraffin-embedded lung sections were stained with hematoxylin-eosin.
Cyclophosphamide pretreatment increases RSV-inducible lung inflammation
To examine whether CYP treatment increases inflammatory effects in the lungs of RSV-infected mice, we determined airway hyperresponsiveness (AHR), cellular infiltration into the lung and lung histopathology. Groups of BALB/c mice either infected with RSV alone or treated with cyclophosphamide (CYP) prior to infection were lavaged and cells in the fluid were centrifuged onto slides. BAL cells were stained with Leukostat. Cells were counted from 4 different slides from each group in a blinded fashion. Cell counts were plotted as percentage of total cells (Fig.  2A ). There was a decrease in the number of macrophages and increases in lymphocyte and neutrophil numbers following RSV infection that was enhanced by prior treatment with CYP. To analyze the extent of lung pathology, lungs were paraffin embedded, sectioned and stained with hemotoxylin-eosin (HE). The lung sections from RSV-infected, CYP-treated mice (Fig.  2C , a & b) showed significantly greater inflammation than lungs from mice given RSV alone (Fig.  2C , c & d). The RSV-infected groups showed greater inflammation than uninfected control mice (Fig.  2C  e & f).Increased RSV infection in CYP-treated BALB/c mice is associated with increased production of immunoregulatory cytokines To examine the cytokine profile in lungs from RSV-infected mice with or without CYP treatment, the gene expression of IL-10, IFN-γ and IL-12 was measured by RT-PCR. Gel profiles and densitometric analyses are shown in Fig.  3A  and  3B . The results show that mice infected with RSV after CYP treatment have increased mRNA expression for all of three cytokines compared to control mice or mice infected with RSV alone. To determine whether mice treated with CYP and infected with RSV do produce more of these proteins, cytokines were measured by ELISA on homogenates prepared from whole lungs. IL-10 (p < 0.05 vs. PBS), IL-12 (p < 0.05 vs. RSV) and IFN-γ (p < 0.05 vs. PBS) levels were significantly higher in the CYP-treated group than the control group (Fig.  4A–C ). Figure 3 Detection of RSV and cytokines in the lungs of BALB/c mice. (A)  RSV-N and IL-10, IL-12, IFN-γ and β-actin were checked by RT-PCR. Mice were infected with RSV alone or treated with CYP prior to infection. The third group was uninfected (PBS) as control. Animals were sacrificed on day 5, their lungs removed and RNA was isolated and used in RT-PCR assay.  (B) Densitometric analysis of the band densities from part A.  Relative intensity refers to the ratio of the intensity of each cDNA product to that of β-actin. Figure 4 CYP-treated BALB/c mice produce higher levels of Th1 and Th2 cytokines . Mice were infected with RSV alone or were treated with cyclophosphamide prior to infection. The third group of mice received no virus (PBS only) as control. Animals were sacrificed on day 5 and their lungs removed. Whole lung homogenates were prepared and cyrtokines were measured by ELISA. Results are given as mean ± SEM (n = 4 for each group).  (A)  CYP-pretreated mice produce higher IL-10 in the lungs. (‡ P < 0.05 vs. PBS).  (B)  IL-12 was higher in CYP-treated mice. (§ P < 0.01 vs. PBS. * P < 0.05 vs. RSV).  (C)  IFN-γ was higher in CYP-treated mice. (* P < 0.05 vs. RSV; ‡ P < 0.05 vs. PBS).
Increased RSV infection in CYP-treated BALB/c mice is associated with increased production of immunoregulatory cytokines
To examine the cytokine profile in lungs from RSV-infected mice with or without CYP treatment, the gene expression of IL-10, IFN-γ and IL-12 was measured by RT-PCR. Gel profiles and densitometric analyses are shown in Fig.  3A  and  3B . The results show that mice infected with RSV after CYP treatment have increased mRNA expression for all of three cytokines compared to control mice or mice infected with RSV alone. To determine whether mice treated with CYP and infected with RSV do produce more of these proteins, cytokines were measured by ELISA on homogenates prepared from whole lungs. IL-10 (p < 0.05 vs. PBS), IL-12 (p < 0.05 vs. RSV) and IFN-γ (p < 0.05 vs. PBS) levels were significantly higher in the CYP-treated group than the control group (Fig.  4A–C ).Cyclophosphamide treatment causes a transient reduction in IL-12 and IFN-γ expression To examine the mechanism underlying the increased RSV infection in CYP-treated animals, the levels of two cytokines that exert antiviral activity, IL-12 and IFN-γ was measured after treatment with CYP (Fig.  5 ). Mice were sacrificed on days 1, 2, 4, 6 after treatment and IL-12 and IFN-γ protein levels were measured in whole lung homogenates by ELISA. Untreated BALB/c mice were used as controls. Treatment with CYP gradually decreased both IL-12 (p < 0.05) and IFN-γ (p < 0.01 vs. Control) until day 4. These results show that decreased production of IL-12 and IFN-γ may play a role in the observed increase in RSV infection in CYP-treated mice. Figure 5 Changes in IL-12 and IFN-γ levels over time in CYP-treated mice.  Mice were treated with cyclophosphamide at 100 mg/kg i.p. Animals were sacrificed on day 1, 2, 4, 6 after treatment and their lungs removed. Whole lung homogenates were prepared and IL-12  (A)  and IFN-γ  (B)  were measured by ELISA. Untreated mice were used as control. Results are shown as mean ± SEM (n = 2; ‡ P < 0.05; § P < 0.01 vs. control).
Cyclophosphamide treatment causes a transient reduction in IL-12 and IFN-γ expression
To examine the mechanism underlying the increased RSV infection in CYP-treated animals, the levels of two cytokines that exert antiviral activity, IL-12 and IFN-γ was measured after treatment with CYP (Fig.  5 ). Mice were sacrificed on days 1, 2, 4, 6 after treatment and IL-12 and IFN-γ protein levels were measured in whole lung homogenates by ELISA. Untreated BALB/c mice were used as controls. Treatment with CYP gradually decreased both IL-12 (p < 0.05) and IFN-γ (p < 0.01 vs. Control) until day 4. These results show that decreased production of IL-12 and IFN-γ may play a role in the observed increase in RSV infection in CYP-treated mice.Discussion The main focus of this study has been to establish and characterize an immunocompromised mouse model for studying RSV infection. Mice are only semi-permissive to RSV infection, yet can serve as a useful model for immunological studies. Compared to the traditional BALB/c mouse model, the use of cyclophosphamide to create an immunocompromised condition provides an effective means of augmenting RSV replication and disease. Pretreatment of BALB/c mice with CYP results in RSV titers in the lungs of these immunocompromised mice that are increased significantly compared to the group infected with RSV without cyclophosphamide treatment. These results are consistent with an earlier study in the cotton rat model [ 28 ]. In another study, a high titer RSV inoculum (10 7  PFU/ml) was administered intranasally to old mice and resulted in clinical illness and appreciable pathology in the lung [ 25 ], while mice inoculated with 10 6  PFU/ml, or less, did not exhibit symptoms of illness. Most studies using mice as models employ lower doses of RSV because RSV infection in humans, which induces a pneumonia-like pulmonary inflammation, occurs typically at sub-clinical RSV doses. The loss of body weight in CYP-treated mice following RSV infection compared to untreated control mice confirmed that CYP-treatment increased the susceptibility to RSV infection at lower inocula. This finding that permissiveness to RSV can be augmented by rendering mice immunocompromised is significant, as it increases the utility of the mouse model for RSV infection. Consistent with increased RSV infection, the cellular population in BAL fluid was altered. Especially significant are the increases in lymphocytes and neutrophils in CYP-treated mice compared to controls. This data is in agreement with previous reports showing that RSV infection increased lymphocyte infiltration in the lung [ 2 ]. Along with increased cellular infiltration, lung pathology, particularly epithelial denudation and goblet cell hyperplasia, is also markedly increased. Expression of IL-10, IL-12 and IFN-γ was examined to determine if RSV-induced changes in the levels of these cytokines in the lungs of CYP-treated mice played a role in the increased lung immunopathology. RSV-infected CYP-treated mice exhibited significantly increased expression of these cytokines at the protein and mRNA level in agreement with previous observations that RSV infection induced enhanced expression of Th1 and Th2 cytokines [ 22 , 29 ]. Other studies have shown that IFN-γ can induce production of IL-12 in a self-activating loop, by activating macrophages which produce IL-12 [ 30 ]. Although CYP treatment is known to induce an immunosuppressed condition, the mechanism is unclear. The results of cytokine analysis on days 1 to 5 after cyclophosphamide treatment indicated that IL-12 and IFN-γ were reduced and the reduction was highest on days four and two, respectively. These results suggest that the ability of cells to produce these cytokines at the time of RSV infection is an important determinant of the magnitude of infection in terms of increased RSV replication and titer. Impairment in IL-12 and IFN-γ production at the key moment of acute infection leads to rapid viral replication and subsequent pathology. In a previous report we demonstrated the importance of IFN-γ by artificially increasing IFN-γ levels and showing that viral titers were decreased because of the induction of 2'-5'oligoadenylate synthetase which activates RNase L to degrade viral RNA [ 31 ]. Also, studies in humans have suggested that individuals lacking IFN-α or IL-12 are at a higher risk of severe RSV disease. Thus, down-regulation of the production of these cytokines is a likely factor underlying the observed enhancement of RSV infection by cyclophosphamide treatment. In conclusion, the results of this study demonstrate that cyclophosphamide treatment of BALB/c mice renders them more susceptible to RSV infection as revealed by increased RSV titers in the lung and decreased body weight. The mechanism of this increase in infection involves transient down regulation of IFN-γ and IL-12 induced by cyclophosphamide treatment.
Discussion
The main focus of this study has been to establish and characterize an immunocompromised mouse model for studying RSV infection. Mice are only semi-permissive to RSV infection, yet can serve as a useful model for immunological studies. Compared to the traditional BALB/c mouse model, the use of cyclophosphamide to create an immunocompromised condition provides an effective means of augmenting RSV replication and disease. Pretreatment of BALB/c mice with CYP results in RSV titers in the lungs of these immunocompromised mice that are increased significantly compared to the group infected with RSV without cyclophosphamide treatment. These results are consistent with an earlier study in the cotton rat model [ 28 ]. In another study, a high titer RSV inoculum (10 7  PFU/ml) was administered intranasally to old mice and resulted in clinical illness and appreciable pathology in the lung [ 25 ], while mice inoculated with 10 6  PFU/ml, or less, did not exhibit symptoms of illness. Most studies using mice as models employ lower doses of RSV because RSV infection in humans, which induces a pneumonia-like pulmonary inflammation, occurs typically at sub-clinical RSV doses. The loss of body weight in CYP-treated mice following RSV infection compared to untreated control mice confirmed that CYP-treatment increased the susceptibility to RSV infection at lower inocula. This finding that permissiveness to RSV can be augmented by rendering mice immunocompromised is significant, as it increases the utility of the mouse model for RSV infection.Consistent with increased RSV infection, the cellular population in BAL fluid was altered. Especially significant are the increases in lymphocytes and neutrophils in CYP-treated mice compared to controls. This data is in agreement with previous reports showing that RSV infection increased lymphocyte infiltration in the lung [ 2 ]. Along with increased cellular infiltration, lung pathology, particularly epithelial denudation and goblet cell hyperplasia, is also markedly increased.Expression of IL-10, IL-12 and IFN-γ was examined to determine if RSV-induced changes in the levels of these cytokines in the lungs of CYP-treated mice played a role in the increased lung immunopathology. RSV-infected CYP-treated mice exhibited significantly increased expression of these cytokines at the protein and mRNA level in agreement with previous observations that RSV infection induced enhanced expression of Th1 and Th2 cytokines [ 22 , 29 ]. Other studies have shown that IFN-γ can induce production of IL-12 in a self-activating loop, by activating macrophages which produce IL-12 [ 30 ].Although CYP treatment is known to induce an immunosuppressed condition, the mechanism is unclear. The results of cytokine analysis on days 1 to 5 after cyclophosphamide treatment indicated that IL-12 and IFN-γ were reduced and the reduction was highest on days four and two, respectively. These results suggest that the ability of cells to produce these cytokines at the time of RSV infection is an important determinant of the magnitude of infection in terms of increased RSV replication and titer. Impairment in IL-12 and IFN-γ production at the key moment of acute infection leads to rapid viral replication and subsequent pathology. In a previous report we demonstrated the importance of IFN-γ by artificially increasing IFN-γ levels and showing that viral titers were decreased because of the induction of 2'-5'oligoadenylate synthetase which activates RNase L to degrade viral RNA [ 31 ]. Also, studies in humans have suggested that individuals lacking IFN-α or IL-12 are at a higher risk of severe RSV disease. Thus, down-regulation of the production of these cytokines is a likely factor underlying the observed enhancement of RSV infection by cyclophosphamide treatment.In conclusion, the results of this study demonstrate that cyclophosphamide treatment of BALB/c mice renders them more susceptible to RSV infection as revealed by increased RSV titers in the lung and decreased body weight. The mechanism of this increase in infection involves transient down regulation of IFN-γ and IL-12 induced by cyclophosphamide treatment.Competing Interests The author(s) declare that they have no competing interests.
Competing Interests
The author(s) declare that they have no competing interests.Authors' Contributions XK, BAL and cell enumeration; GH, data analysis; GP, AHR, RT-PCR; MK, RSV infection and assay, tissue collection, RT-PCR; AB, cyclophosphamide treatment, tissue collection, ELISAs; TSR, immunohistochemistry; JZ, cell culture and virus preparation; RFL, experimental design and analysis; SSM, project design, experimental analysis and data interpretation.
Authors' Contributions
XK, BAL and cell enumeration; GH, data analysis; GP, AHR, RT-PCR; MK, RSV infection and assay, tissue collection, RT-PCR; AB, cyclophosphamide treatment, tissue collection, ELISAs; TSR, immunohistochemistry; JZ, cell culture and virus preparation; RFL, experimental design and analysis; SSM, project design, experimental analysis and data interpretation.