Successful Treatment of Feline Infectious Peritonitis Disease (FIP) with Mesenchymal Stem Cells

Case History

History of feline infectious peritonitis (FIP)
In 1963, FIP was identified as a specific disease in cat, caused by a mutant feline coronavirus (FCoV) (Holzworth, 1963; Poland et al., 1996; Vennema et al., 1998; Pedersen et al., 2008). FIP can be categorized into two forms, including effusive (wet), characterized by accumulation of inflammatory fluid in the abdominal and thoracic cavities, and non-effusive (dry), which is characterized by necrotizing vasculitis and multifocal pyogranulomatous inflammation in various organs—such as the abdominal viscera, eyes, central nervous system, and lungs—along with virus-induced lymphopenia (Kennedy et al., 2020; Tasker et al., 2023). One of the outstanding features of FIP is lymphopenia, along with the apoptosis of T cells, which is associated with cytokine secretion by the virus-infected macrophages and other immune cells. The continuous replication of the virus causes an inflammatory response in FIP, leading to multiorgan failure. Currently, there is no effective licensed treatment for this disease (Takano et al., 2007; Tasker et al., 2023).
According to recent studies, there are similarities and differences between COVID-19 and FIP. These two diseases are different in terms of some pathogenic, pathological, and clinical features. However, some similar pathogenic and immunogenic events have been found in both conditions. In addition, the drug GS-441524, which is the active form of remdesivir and is recommended for the treatment of COVID-19, has also demonstrated good efficacy in treating FIP by controlling viral replication (Bearden et al., 2017; Decaro & Lorusso, 2020; Jiang et al., 2020; Mavian et al., 2020; Sironi et al., 2020; Tiwari et al., 2020; Wong et al., 2020; Malaiyan et al., 2021). Since cytokine storms are found in FIP, it is essential to consider a combination of immunomodulatory agents for treating cytokine storms and antiviral agents for inhibiting viral replication. Mesenchymal stem cells (MSCs) offer more advantages than many other anti-inflammatory agents because, in addition to their regenerative properties, they have immunomodulatory effects on both the innate and adaptive immune systems. MSCs regulate cell function and downregulate inflammatory cytokines by secreting cytokines and modulating the immune re-sponse (Iyer et al., 2020; Senegaglia et al., 2021).  

We hypothesized that stem cells in the body can simultaneously control the inflammation caused by the virus, prevent its proliferation, and repair the damaged tissues. To the authors’ knowledge, there have been no reports of the successful use of BW-MSCs in the treatment of FIP.

Clinical Presentation
Three female domestic shorthaired cats with an average age of nine months were brought to Pouya Pet Clinic with a complaint of lethargy. Other reported symptoms were recurrent fever, weight loss, and anorexia.  All cats were born on the street and had contact with feral cats. The condition of the animals at the time of presentation is shown in Table 1. 
Neurological and ocular signs, including decreased postural reflexes and ocular opacities, were observed in one cat (Table 1). 

Diagnostic Testing
FIP was diagnosed by a combination of clinical signs (weight loss, recurrent fever, ascites, decreased appetite, and anemia) and laboratory tests, including blood analysis, the Rivalta test (for effusive cases), and reverse transcription-polymerase chain reaction (RT-PCR). Radiologic and ultrasonographic studies of the thoracic and abdominal cavities were also performed to exclude other factors leading to ascites, such as cardiac failure, incarcerated hernias, or neoplastic masses. According to the results of radiology and ultrasound, free fluid was observed in the abdomen in two cases (effusive), which was interpreted as ascites (Figure 1).

No free fluid was observed in one case (none-effusive), and the finding indicated hepatomegaly (Figure 2). 

In all cases, the A/G ratio was low (with a mean of 0.46), and total protein was high (with a mean of 9.6 g/dL). Two cats had severe anemia considering Hgb and HCT levels (Table 2). 

Treatments performed 
Paracentesis was performed in cases with ascites, during which a needle was inserted into the peritoneal cavity to obtain ascitic fluid by the surgeon on the first day. 
Frozen bone marrow stem cells (BM-MSCs) were provided from Treatacaspian Co. stem cell bank with a total of 106 stem cells. For each dose administration, one vial was removed from the azoth tank. The outside of the vials was wiped with 70% ethanol, and then the cells were quickly thawed in a 37 °C water bath by gently swirling the vials. A 20 µL aliquot of cells was mixed with 20 µL of the medium and then stained with Trypan Blue for cell counting. The remaining cell suspension was transferred to a conical tube using a pipette and rinsed with 1 mL of medium (DEMEM high glucose) by centrifuging the cell suspension at 300×g for 10 minutes at room temperature. The cell pellet was resuspended by gently flicking the tube, and then 5 mL of medium was added to the conical tube (Bearden et al., 2017; Zhang et al., 2018). 
All patients received BM-MSCs on days 1, 4, 7, 14, and 21, with a dosage of 1×106 in 15 mL of normal saline, injected intravenously for one hour (Webb et al., 2013). Any reaction following the medical administration, such as skin rash or loss of appetite, was considered and noted in Table 3. 

All treated cats underwent physical examinations and blood work, consisting of complete blood count (CBC) and serum biochemistry, each week (Table 2). Three days after the first treatment, the appetite was regained, and lethargy and fever regressed in all cats. Blood samples and radiological studies were performed to monitor the condition of the cats after recovery, and the blood factors in all cats showed normal levels.
After the 21-day treatment period, no detectable ascites or abnormalities in the organs were seen on ultrasonographic and radiographic images (Figures 1 and 2). 
At the follow-up, two months after the treatment, two cats remained healthy, showing no signs of relapse, while one cat was reinfected after coming into contact with a severely diseased cat. At this stage, the continuation of the treatment with remdesivir at a dose of 30 mg/kg was continued but was not successful, and recovery was not achieved. The outcomes of this report are summarized in Tables 1, 2 and 3. 
BM-MSCs showed striking efficacy and treated cats responded to the treatment with rapid improvement of clinical signs. All cats achieved remission after 21 days of treatment. Follow-up on the cats in our report is currently ongoing, and more cats with FIP are being treated using this method. 

Study Assessments
 The main reason for the progression of FIP in patients is the immune abnormalities. When coronaviruses invade the body, they rapidly replicate and trigger the immune system to release inflammatory cells and antibodies (Akkoc, 2020; Addie et al., 2020). This leads to the immune regulatory network, to unbalanced, resulting in the release of a large number of inflammatory cytokines and cytokine storm syndrome. MSCs have immunoregulatory abilities, which can regulate both the adaptive and innate immune systems (Pinky et al., 2021; Guy et al., 2021; Chen et al., 2022). This ability may make them the most promising cell-based therapy for FIP. 
In addition to the immunomodulatory and anti-inflammatory effects of MSCs, these cells also can aid in the repair of the damaged tissues, significantly reducing the number of affected organs associated with FIP (Salari et al., 2020). The liver is one of the organs affected during FIP. Since remdesivir and GS-441524 are treatment options for FIP treat-ment, they will be metabolized in the liver and could worsen the liver function, potentially making it impossible to continue the treatment (Grein et al., 2020; Wang et al., 2020; Liu et al., 2023). Therefore, it is necessary to use a component to repair the damaged tissues during the treatment. The regenerative properties of stem cells, along with their immunomodulatory effects, make them the best option for FIP treatment with minimal side effects.
In our cell therapy results, the duration of treatment was shorter (21 days) compared to the duration of treatment with remdesivir (84 days) (Murphy et al., 2018; Pedersen et al., 2018). Since there is no access to oral remdesivir/GS-441524 for animals in the country under study, the need for daily visits for injectable medicine is eliminated. Additionally, since the majority of the infected cases are found among street cats, introducing a shorter treatment period will provide more cases with a chance to be cured. On the other hand, the costs associated with this treatment are much lower compared to the aforementioned treatment, which is considered a positive aspect of cell therapy. 
The small number of studies using MSC products in cats reveals that this treatment is still largely in the discovery phase. Cell sources, administration routes, and dosages can have different results. In the current report, BM-MSCs were used for the first time in the treatment of FIP disease. Despite the failure of adipose stem cells in some reports regarding FIP treatment, this type of stem cells, when administered at the recommended dose and method, has enabled us to achieve positive results in the treatment of FIP in cats. 

Conclusion
The results showed that the mesenchymal stem cells could cure the disease by controlling the inflammation and proliferation of the virus, as well as repairing the damaged tissues caused by the virus in the animal’s body. This makes this treatment especially superior to other routine treatments. To check the long-term effectiveness of stem cells, more cases are needed.

Ethical Considerations

Compliance with ethical guidelines
All ethical principles are considered in this article and all owners signed written informed consent before initiation of treatment.

Funding
This study did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Conceptualization, methodology and validation: All authors, investigation, resources and data curation: All authors; Writing, visualization and supervision: Samira Mohamadian; Project adminestration and funding acquisition: Samira Mohamadian, Pejman Kazerooni,  and Ali Taheri Mirghaed,

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors thanks the clinic staff for their cooperation in managing these cases.

References

Addie, D. D., Curran, S., Bellini, F., Crowe, B., Sheehan, E., & Ukrainchuk, L., et al. (2020). Oral Mutian®X stopped faecal feline coronavirus shedding by naturally infected cats. Research in Veterinary Science., 130, 222-229. [DOI:10.1016/j.rvsc.2020.02.012] [PMID]

Akkoc, T. (2020) COVID-19 and Mesenchymal Stem Cell Treatment; Mystery or Not. Advances in Experimental Medicine and Biology, 1298, 167–176. [DOI:10.1007/5584_2020_557] [PMID]

Bearden, R. N., Huggins, S. S., Cummings, K. J., Smith, R., Gregory, C. A., & Saunders, W. B. (2017). In-vitro characterization of canine multipotent stromal cells isolated from synovium, bone marrow, and adipose tissue: A donor-matched comparative study. Stem Cell Research and Therapy, 8(1), 218.[DOI:10.1186/s13287-017-0639-6] [PMID]

Chen, L., Qu, J., Kalyani, F. S., Zhang, Q., Fan, L., & Fang, Y., et al. (2022) Mesenchymal stem cell-based treatments for COVID-19: status and future perspectives for clinical applications. Cellular and Molecular Life Sciences., 79(3), 142. [DOI:10.1007/s00018-021-04096-y] [PMID]

Decaro, N., & Lorusso, A. (2020) Novel human coronavirus (SARS‐CoV‐2): A lesson from animal coronaviruses. Veterinary Microbiology, 244, [DOI:10.1016/j.vetmic.2020.108693] [PMID]

Grein, J., Ohmagari, N., Shin, D., Diaz, G., Asperges, E., & Castagna, A., et al. (2020) Compassionate use of remdesivir for patients with severe Covid-19. The New England Journal of Medicine, 382(24), 2327–2336. [DOI:10.1056/NEJMoa2007016] [PMID]

Jiang, F., Deng, L., Zhang, L., Cai, Y., Cheung, C. W., & Xia, Z. (2020). Review of the clinical characteristics of coronavirus disease 2019 (COVID-19). Journal of General Internal Medicine, 35(5), 1545–1549. [DOI:10.1007/s11606-020-05762-w] [PMID]

Lam, G., Zhou, Y., Wang, J. X., & Tsui, Y. P. (2021).Targeting mesenchymal stem cell therapy for severe pneumonia patients. World Journal Stem Cells., 13(2), 139-154. [DOI:10.4252/wjsc.v13.i2.139] [PMID]

Holzworth, J. (1963) Some important disorders of cats. The Cornell Veterinarian, 53, 157–160. [PMID]

Iyer, M., Jayaramayya, K., Subramaniam, M. D., Lee, S. B., Dayem, A. A., & Cho, S. G., et al. (2020). COVID-19: An update on diagnostic and therapeutic approach. BMB Reports, 53(4), 191–205. [DOI:10.5483/BMBRep.2020.53.4.080] [PMID]

Kennedy, M. A. (2020). Feline infectious peritonitis: Update on pathogenesis, diagnostics, and treatment. The Veterinary Clinic of North America. Small Animal Practice, 50(5), 1001-1011. [DOI:10.1016/j.cvsm.2020.05.002] [PMID]

Liu, P., Jiang, J Z., Wan, X F., Hua, Y., Li, L., Zhou, J., Wang, X., Hou, F., Chen, J., Zou, J., Chen, J. (2020) Are pangolins the intermediate host of the 2019 novel coronavirus (SARS‐CoV‐2) PLoS Pathology., 16(5), e1008421. [DOI:10.1371/journal.ppat.1008421. PMID: 32407364.] [PMID]

Malaiyan, J., Arumugam, S., Mohan, K., & Gomathi Radhakrishnan, G. (2021). An update on the origin of SARS‐CoV‐2: Despite closest identity, bat (RaTG13) and pangolin derived coronaviruses varied in the critical binding site and O‐linked glycan residues. Journal of Medical Virology, 93(1), 499-505. [DOI:10.1002/jmv.26261] [PMID]

Mavian, C., Pond, S. K., Marini, S., Magalis, B. R., Vandamme, A. M., & Dellicour, S., et al. (2020). Sampling bias and incorrect rooting make phylogenetic network tracing of SARS‐COV‐2 infections unreliable. Proceedings of the National Academy of Sciences of the United States of America, 117(23), 12522–12523.[DOI:10.1073/pnas.2007295117] [PMID]

Murphy, B. G., Perron, M., Murakami, E., Bauer, K., Park, Y., & Eckstrand, C., et al. (2018). The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies. Veterinary Microbiology, 219, 226–233. [DOI:10.1016/j.vetmic.2018.04.026] [PMID]

Pedersen, N. C., Allen, C. E., & Lyons, L. A. (2008). Pathogenesis of feline enteric coronavirus infection. Journal of Feline Medicine and Surgery, 10(6), 529–541. [DOI:10.1016/j.jfms.2008.02.006] [PMID]

Pedersen, N. C., Perron, M., Bannasch, M., Montgomery, E., Murakami, E., & Liepnieks, M., et (2019). Efficacy and safety of the nucleoside analog GS-441524 for treatment of cats with naturally occurring feline infectious peritonitis. Journal of Feline Medicine and Surgery, 21(4), 271–281. [DOI:10.1177/1098612X19825701] [PMID]

Pinky, Gupta, S., Krishnakumar, V., Sharma, Y., Dinda, A. K., & Mohanty, S. (2021). Mesenchymal stem cell derived exosomes: a nano platform for therapeutics and drug delivery in combating COVID-1. Stem Cell Reviews and Reports, 17(1), 33–43. [DOI:10.1007/s12015-020-10002-z] [PMID]

Poland, A. M., Vennema, H., Foley, J. E., & Pedersen, N. C. (1996) Two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus. Journal of Clinical Microbiology, 34(12), 3180–3184. [DOI:10.1128/jcm.34.12.3180-3184.1996]

Salari, V., Mengoni, F., Del Gallo, F., Bertini, G., & Fabene, P. F. (2020). The anti-inflammatory properties of mesenchymal stem cells in epilepsy: Possible treatments and future perspectives. International Journal of Molecular Sciences, 21(24), 9683. [DOI:10.3390/ijms21249683] [PMID]

Senegaglia, A. C., Rebelatto, C. L. K., Franck, C. L., Lima, J. S., Boldrini-Leite, L. M., & Daga, D. R., et al. (2021). Combined use of tocilizumab and mesenchymal stromal cells in the treatment of severe covid-19: Case report. Cell Transplantation, 30, [DOI:10.1177/09636897211021008] [PMID]

Sironi, M., Hasnain, S. E., Rosenthal, B., Phan, T., Luciani, F., & Shaw, M. A., et al. (2020). SARS‐CoV‐2 and COVID‐19: A genetic, epidemiological, and evolutionary perspective. Infection, Genetics and Evolution, 84, [DOI:10.1016/j.meegid.2020.104384] [PMID]

Takano, T., Hohdatsu, T., Hashida, Y., Kaneko, Y., Tanabe, M., & Koyama, H. (2007). A “possible” involvement of TNF-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis. Veterinary Microbiology, 119(2-4), 121–131. [DOI:10.1016/j.vetmic.2006.08.033] [PMID]

Tasker, S., Addie, D. D., Egberink, H., Hofmann-Lehmann, R., Hosie, M. J., & Truyen, U., et al. (2023). Feline infectious peritonitis: European advisory board on cat diseases guidelines. Viruses, 15(9), 1847. [DOI:10.3390/v15091847] [PMID]

Tiwari, M., & Mishra, D. (2020). Investigating the genomic landscape of novel coronavirus (2019‐nCoV) to identify non‐synonymous mutations for use in diagnosis and drug design. Journal of Clinical Virology, 128, [DOI:10.1016/j.jcv.2020.104441] [PMID]

Vennema, H., Poland, A., Foley, J., & Pedersen, N. C. (1998). Feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses. Virology, 243(1), 150–157. [DOI:10.1006/viro.1998.9045] [PMID]

Wang, Y., Zhang, D., Du, G. (2020) Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial. 395(10236):1569-1578. [DOI:10.1016/S0140-6736(20)31022-9] [PMID]

Webb, T. L., Quimby, J. M., & Dow, S. W. (2012). In vitro comparison of feline bone marrow-derived and adipose tissue-derived mesenchymal stem cells. Journal of Feline Medicine and Surgery, 14(2), 165-168. [DOI:10.1177/1098612X11429224] [PMID]

Wong, G., Bi, Y. H., Wang, Q. H., Chen, X. W., Zhang, Z. G., & Yao, Y. G. (2020). Zoonotic origins of human coronavirus 2019 (HCoV‐19/SARS‐CoV‐2): Why is this work important. Zoological Research, 41(3), 213–219. [DOI:10.24272/j.issn.2095-8137.2020.031] [PMID]

Zhang, C., Lee, H. J., Shrivastava, A., Wang, R., McQuiston, T. J., Challberg, S. S., Pollok, B. A., & Wang, T. (2018). Long-term in vitro expansion of epithelial stem cells enabled by pharmacological inhibition of PAK1-ROCKMyosin II and TGF-β signaling. Cell Reports, 25(4),1109-1123.e5. [DOI:10.1016/j.celrep.2018.09.072] [PMID]