Immunosuppressive Effects of Mycophenolate Mofetil and Valganciclovir in Kidney Transplant Recipients

Recruitment and screening of subjects will be performed between 4 and 8 weeks after transplantation at which time consent will be obtained. Per clinical standard of care at Indiana University, VGCV is routinely stopped at 12 weeks post transplantation. At week 10 and the day of discontinuation of their prophylaxis the patients will be seen by the study physician. The subjects will also be seen at 2 and 4 weeks after discontinuing VGCV. At each study visit, the subject will have a limited history and physical (H&P) performed, one 3ml light green top (lithium heparin) tube, one 3ml dark green top (sodium heparin) tube, and one 5ml purple top (EDTA) tube will be obtained by phlebotomy during their regularly scheduled blood draw times. This blood will then be used for the Cylex® assay, Flow cytometry and MMF and GCV trough level. The patient's complete blood count and differential as well as drug levels, and medication dosages will be recorded. This information will be used for assure that Cylex® results are not being influenced by changes in immunosuppressive drug levels. The complete blood count and differential will be used to determine absolute neutrophil counts. After the week sixteen visit, the subjects will have completed the protocol. Pregnancy tests will not be performed as patients are not directly receiving any treatment secondary to this study. Flow cytometry will be performed to measure absolute CD4 counts, CD4/CD8 ratios, and look at several activation markers on lymphocytes.

Signed IRB approved informed consent must be obtained from all subjects prior to screening assessments. The following evaluations will be performed prior to initiation of phlebotomy for purposes of this study.

Medical history including age, gender, race, and renal disease, immunosuppressive regimen including dose, level and use of antibody treatment at time of transplant will be noted.

Physical examination including: vital signs, cardiac, and lung evaluation.

Laboratory testing which will be performed as part of standard of care because of the transplant are:

Hematology: complete blood count. Drug level: tacrolimus, cyclosporine and/or sirolimus.

Evaluations during treatment and post treatment

Patients will be followed by surgical co-investigators throughout the entire study.

A limited history and physical examination will be performed as indicated in the schedule of assessments.

Clinical laboratory testing Additional clinical laboratory testing beyond standard of care that will be done for this protocol includes adding a differential to the complete blood count. CBC and drug levels will be noted from already scheduled clinic appointments and post transplant protocols. Research blood levels for GCV and MPA will be run from a 3ml lithium heparin and a 5ml EDTA tube in the research laboratory or at outside laboratory.

Transplant laboratory testing On the testing days an additional 3ml sodium heparin blood tube will be drawn in phlebotomy at the same time the scheduled labs are obtained. Flow cytometric analysis will be performed at times listed above. A 5 tube panel using three color analysis will be performed using approximately 500_L of whole blood from the sodium heparinized tube. The panel will consist of the following staining protocol

Tube 1 - CD 45 FITC / CD 14 PE - Used to locate lymphocyte population as well as determine purity of specimen and monocyte contamination

Tube 2 - _1a FITC/ _1a PE/ _1a PerCP - Isotype control to check for background staining.

Tube 3 - CD 4 FITC/ CD 8 PE / CD 20 PerCP - Used to determine CD4/CD8 ratio as well as determining B cell population counts.

Tube 4 - CD 4 FITC/ CD 25 PE/ HLA - DR PerCP - Check for specific activation markers on CD 4 cells.

Tube 5 - CD 4 FITC/ CD 69 PE/ CD 3 PerCP - Used to get true CD4 counts on T-cells as well as look at another activation marker on CD4 cells.

Cylex immune assay will be run as listed above. This assay requires minimal amount of whole sodium heparinized blood and will be used from same tube as flow analyses. The whole blood is diluted with a saline base diluent. It is then stimulated overnight with phytohemagglutinin. The CD4+ or CD3+ T cells are then isolated using magnetic beads. They are lysed and ATP production is recorded using a luciferase based reaction.

Inclusion criteria

• >18 years of age
• Primary kidney transplant
• MMF included as part of baseline immunosuppression
• On valganciclovir for CMV prophylaxis. The dose will vary based on renal function but the standard dose is 900 mg per day
• No prior episodes of rejection
• On a stable dose of immunosuppressive medications -

Exclusion criteria

• Previous or multiple transplant patients (e.g. second kidney or simultaneous kidney pancreas patient)
• Uncontrolled or recurrent infections.
• Psychiatric disorder -