ACUTE GRAFT VERSUS HOST DISEASE (GHVD).pptx

ACUTE GRAFT VERSUS HOST DISEASE (GHVD) IN RATS (pre-clinical model)

1. ACUTE GRAFT VERSUS HOST DISEASE (GHVD) IN RATS NITTAL VEKARIA M.PHARM PHARMACOLOGY (1 ST SEM)

2. INTRODUCTION: Allogeneic hematopoietic cell or bone marrow transplantation (BMT) causes graft-versus-host-disease (GVHD) GHVD is a potential risk that may occur anytime after the transplant, especially in allogenic bone marrow transplants. This condition occur when the new donor stem cells that grow and rebuild the patient’s immune system. Identify the body’s tissues and organs as foreign agents and start to attack them. GVHD condition can be classified into two categories: 1. Acute GVHD (occur after first few month after transplant) 2. Chronic GVHD

3. ANIMAL TO BE USED ARE:- Acute GVHD was induced in Lewis rats (RT1 l ) by transplantation of Dark Agouti (DA) rat (RT1 a ) bone marrow cells (6.0×10 7  cells) without immunosuppression after lethal irradiation (10 Gy ). We used the already established rat GVHD model, which involves transplantation of bone marrow cells (BMCs) from DA rats (RT1 a , RT1A a ) into lethally irradiated Lewis rat (RT1 l , RT1A l ) recipients without immunosuppression .

4. MATERIAL AND METHOD: ANIMAL: Inbred male da and lewis rat Weight: 190–220 g and 220–270 g Bone marrow transplantation: BMC suspensions were harvested from DA and Lewis rats by flushing the marrow from the femurs and tibias with cold RPMI 1640 supplemented with 2.5% fetal bovine serum and 25 mM HEPES. Recipient Lewis rats were irradiated with a dose of 10 Gy prior to BMT. After 2–3 h, 6.0×10 7  BMCs from the DA or Lewis rats were then injected into Lewis rat recipients via the tail vein.

5. RECONSTRUCTION OF TRANSPLANTED BMCs To examine the reconstruction of transplanted BMCs, blood samples were collected on days 4, 7, 14, 21, and 28 after BMT from the tail vein, to measure the number of white blood cells, and flow cytometry was conducted to assess the expression of RT1Aa , CD6+ T-cells, CD8+ T-cells, CD4+ T-cells, and CD68+ macrophages. Peripheral blood mononuclear cells were treated with anti-mouse CD16/32 Ab to block the Fc-receptors followed by direct or indirect staining of fluorochrome-conjugated antibodies. Dead cells were identified and excluded using propidium iodide. Cell suspensions were analyzed on a FACSCanto II flow cytometer

6. Systemic analysis of GVHD Systemic GVHD was evaluated using a scoring system incorporating five clinical parameters: weight loss, posture, activity, fur texture, and skin integrity. • Each parameter was graded from 0 to 2, and a clinical index was generated from the sum of these scores. • Pathological examination of skin, liver, intestine, and kidney from allogeneic BMT rats and controls. • Blood samples collected on day 28 to examine liver and renal function. • Urine collected on day 28 to examine proteinuria and urinary N-acetyl-β-D- glucosaminidase levels.

7. Study on GVHD Histological Features Skin, liver, intestine, and kidney tissues fixed in 20% buffered formalin for microscopic examination. •Tissues stained with H&E, PAS, and naphthol AS-D chloroacetate esterase for histopathological examination. • Primary antibodies used for immunohistochemistry. • Technique used to detect infiltrating CD3+ T-cells, CD8+ T-cells, and CD68+ macrophages. • Pre-incubation with pepsin for ED1+ and CD3+ detection. • Treatment with 1 mM EDTA for CD8+ detection, the section were treated with 1 mM EDTA (PH 8.0) in hot water at 90 c for 3h

8. Kidney T-cell Study Overview Examined infiltrating CD3+ T-cells, CD8+ T-cells, and CD68+ macrophages in 40 randomly selected interstitial fields. • Utilized immunofluorescence study using frozen tissues. • Evaluated deposition of IgM, IgG, and C3 using indirect method. • Identified donor type of leukocytes in kidney using double stain with FITC-conjugated anti-rat RT1Aa,b antibody and phycoerythrin-conjugated anti-rat CD45 antibody. • Identified CD8+ T-cells or CD4+ T-cells using double stain with FITC-conjugated anti-rat CD3 antibody and PE-conjugated anti-rat CD8 α or anti-rat CD4 antibody. • Evaluated expression of MHC class II in renal tubules using immunostaining with PE-conjugated anti-rat RT1B antibody. • Graded renal tubules semiquantitatively based on rat RT1B staining.

9. Grading system: Absence of MHC class II staining -0 Mild MHC class II staining- 1 Moderate MHC class II staining-2 Marked increase of MHC class II staining- 3

10. Real-Time RT-PCR for Kidney Expression Analysis: Utilized ISOGEN to extract total renal RNA. • Purified total RNA was 1.9–2.2 of A260/A280. • Created cDNA libraries using a High Capacity cDNA Reverse Transcription kit. • Gene expression levels analyzed using quantitative RT-PCR using the THUNDERBIRD SYBR qPCR Mix. • Normalized value for mRNA expression calculated by dividing the relative quantity of relevant primers by the relative quantity of β- actin. • Quantification performed using the SDS 2.3 software program.

11. RESULT: Leukocytes in the peripheral blood after bone marrow transplantation (BMT). The study found that total white blood cell (WBC) count in peripheral blood decreased on day 4 but recovered between days 7-14 in both allogeneic and syngeneic BMT rats. Allogeneic BMT rats had higher WBC count on day 21 and decreased again on day 28, possibly due to recruitment to GVHD organs. CD6+ T-cells, CD8+ T-cells, CD4+ T-cells, and ED1+ macrophage levels also recovered between days 7 and 21.

12. Study on Body Weight and Liver Function in BMT Rats After DA to Lewis allogeneic BMT, body weight of Lewis recipient rats decreased by over 20% by day 28. • In contrast, non-BMT control rats and Lewis-to-Lewis syngeneic BMT control rats showed a gradual increase in body weight by day 28. • Macroscopic evaluations revealed dermatitis, diarrhea, liver function abnormalities, and increased serum AST and ALT levels. • Semiquantitative score of systemic acute GVHD increased by day 28. • Severe acute GVHD developed in the skin, liver, and digestive tract by day 28 in the DA-to-Lewis allogeneic BMT model. • In the Lewis-to-Lewis syngeneic BMT rats and non-BMT control rats, only few CD3+T-cells infiltrated the skin, liver, and digestive tract, and acute GVHD did not develop by day 28.

13. BODY WEIGHT AND SEMIQUANTITATIVE SCORE OF SYSTEMIC ACUTE GHVD AFTER BMT The study found that body weight decreased in allogeneic BMT rats after radiotherapy and BMT, with a significant decrease in syngeneic BMT rats between day 14 and 28. Symptoms of acute GVHD developed in allogeneic BMT rats from day 7 and did not develop in syngeneic BMT rats.

14. Allogeneic Bone Marrow Transplantation: Acute GVHD in Skin, Liver, and Intestine Macroscopic findings: Erythematous rush and alopecia on day 28 post-BMT. • Light microscopic findings: Inflammatory cells, mainly CD3+ T-cells, infiltrating the epidermis and hair follicle. • Liver: Cholangiolitis and phlebitis in portal and central veins, indicating acute GVHD. • Colon: Erosion and inflammatory cell infiltration, cryptitis, and infiltration of CD3+ T-cells, indicating acute GVHD. T

15. ACUTE GHVD IN THE SKIN, LIVER AND INTESTINE AFTER ALLOGENIC BMT The study found that body weight decreased in allogeneic BMT rats after bone marrow transplantation (BMT), with symptoms developing by day 28. Allogeneic BMT rats had a significantly lower body weight compared to syngeneic BMT rats. The study also found that acute GVHD did not develop in syngeneic BMT rats.

16. LIVER AND RENAL DYSFUNCTION AFTER ALLOGENIC BMT Liver function in allogeneic BMT rats was assessed using serum AST, ALT, and T- Bil levels. Serum AST and ALT levels increased significantly on day 28, while serum BUN and NAG levels increased, while serum Cr levels remained stable. Urine NAG levels were also increased.

17. DEVELOPMENT OF ACUTE GHVD OF THE KIDNEY Acute GVHD in skin, liver, and digestive duct leads to renal function abnormalities by day 28. • Increase in serum BUN and urinary NAG levels indicates renal dysfunction and proximal renal tubular injury. • Urinary NAG levels significantly increased in allogeneic BMT rats on day 28, even when serum creatinine levels were normal. • Urinary protein levels were not significantly different between non-BMT control rats and allogeneic BMT rats. • Pathology of the kidney with acute GVHD shows mononuclear cell infiltration to the interstitium . • Acute GVHD with mild renal inflammation characterized by infiltration of mononuclear cells to the interstitium . • Mild to moderate renal inflammation also noted peritubular capillaritis and tubulitis , acute glomerulitis, and acute endarteritis.

18. RENAL INFLAMMATION IN ACUTE GHVD AFTER ALLOGENIC BMT The study demonstrates that mild renal inflammation involves mononuclear cell infiltration into the renal interstitium , moderate inflammation expands into the peritubular interstitium , and severe inflammation results in diffuse interstitial inflammation in the renal cortex.

19. HISTOPATHOLOGICAL FEATURES IN THE KIDNEY ON 28 DAY AFTER ALLOGENIC BMT On day 28 after allogeneic bone marrow transplantation, kidney histopathological features showed tubulitis , peritubular capillaritis , acute glomerulitis, and endarteritis due to cell-mediated renal injury in acute GVHD, indicating cell-mediated renal injury.

20. THE INFILTRATING CELLS IN THE KIDNEY AND THE MHC CLASS II EXPRESSIONS IN RENAL TUBULES. The study found that in allogeneic bone marrow transplantation rats, CD3+ T-cells and ED1+ macrophages infiltrated the kidney on day 28. The number of these cells increased significantly compared to non-transplanted and syngeneic control rats. Additionally, the expression of MHC class II in renal tubules increased in allogeneic BMT rats.

21. INFILTERATING CELLS IN THE KIDNEY IN ACUTE GHVD AFTER ALLOGENIC BMT The study found that CD8+ T-cells infiltrated the kidney in acute GVHD after allogeneic bone marrow transplantation (BMT). CD4+ T-cells were also noted in inflammation, indicating both class I-restricted and class II-restricted T cell-mediated reactions in renal acute GVHD. Most CD45+ leukocytes were expressed in the kidney, suggesting donor-type leukocyte infiltration.

22. REAL TIME RT-PCR ANALYSIS OF CYTOKINES IN THE KIDNEY AFTER BMT IFN-γ and TNF-α expression was significantly up-regulated in kidneys of allogeneic and syngeneic BMT rats on day 28, with no significant difference in IL-4 and IL-17 expressions.

23. DISCUSSION: GVHD is a major complication after Hematologic Cancer Treatment (HCT), with the kidney being a direct target organ. Pathological examination revealed infiltration of mononuclear cells, CD3+ T-cells, CD8+ T-cells, CD4+ T-cells, and macrophages in the renal interstitium , leading to peritubular capillaritis , tubulitis , acute glomerulitis, and endarteritis. Increased urinary NAG level was an early marker of acute GVHD in the kidney, while serum Cr and urinary protein levels remained stable. Increased urinary NAG level was an early marker of acute GVHD in the kidney, while serum Cr and urinary protein levels remained stable. Acute GVHD in rat BMT model differs from clinical human GVHD after HCT, as human GVHD is induced in rich stem cell transplantation with close HLA matching between donor and recipient. Acute GVHD is a major cause of morbidity and mortality after allogeneic HCT, responsible for 15–40% of mortality.

24. Acute GVHD can contribute to renal dysfunction indirectly through nephrotoxicity induced by a calcineurin inhibitor used in prophylaxis against GVHD, severe GVHD with diarrhea and dehydration, and cytomegalovirus reactivation Acute renal disorders after HCT are mediated by multiple factors, including high-dose chemotherapy, rapid cytolysis of tumor and normal marrow, infusion of cryopreserved marrow or blood progenitor cells, post-transplantation infections, and antimicrobials used for prophylaxis and treatment of infections. Recent studies suggest that GVHD can involve renal insufficiency, with membraneanous nephropathy after HCT possibly associated with chronic GVHD. In autopsy cases after HCT, allogeneic HCT recipients with severe GVHD tended to have tubulitis and peritubular capillaritis.Study on Acute Gout-Respiratory Disease (GVHD) in Allogeneic BMT Rats Significant infiltration of donor leukocytes in the kidney was found in allogeneic BMT recipients with systemic acute GVHD. Infiltration of CD3+ T-cells, CD8+ T-cells, CD4+ T-cells, and macrophages mediated renal inflammation with peritubular capillaritis , tubulitis , acute glomerulitis, and endarteritis.

25. Acute GVHD in the kidney was similar to pathological findings in allogeneic renal transplantation. Expression of MHC class II in renal tubules significantly increased in acute renal GVHD, indicating T cell-mediated immunologic injury of renal tubules and renal microvasculature. GVHD is caused by host-reactive T-cells derived from the donor bone marrow or from the peripheral blood that contaminates the donor bone marrow during its preparation. CD8+ cytotoxic T-cells and CD4+ helper T-cells were identified as key players mediating GVHD pathogenesis. Both cellular factors (donor T-cells and macrophages) and soluble factors (cytokines) play a role in the development of acute GVHD. Th1 cytokines (IFN-γ, IL-2, and TNF-α) have been implicated in the pathophysiology of acute GVHD. Expressions of IFN-γ and TNF-α mRNA increased in the kidney of allogeneic BMT rats compared with those in syngeneic BMT control rats. IL-4, one of the Th2 cytokines, was not significantly different between allogeneic and syngeneic BMT rats, possibly associated with the absence of antibody-mediated immune injury.

26. Acute renal GVHD is a significant complications post-HCT. Serum BUN and urinary NAG levels increase in acute renal GVHD. Inflammatory damage to renal tubules may be linked to increased urinary NAG levels. Urgent need for further studies to understand GVHD occurrence, correlation between GVHD and urinary NAG levels, and effective pre-emptive therapy.