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Pemphigus vulgaris (PV), an autoimmune disorder characterized by blistering skin/mucus membrane lesions, is mediated by desmoglein-3 autoantibodies. We carried out a prospective clinical trial of hematopoietic stem cell transplantation (HSCT) in thymus, bone marrow (BM) and periphery to reconstitute central and peripheral arms of self-tolerance.
Eleven (M:F=5:6) patients with mean age 33.5 years and mean duration of disease 22.8 months, having painful pruritic blisters and ulcers resistant to corticosteroids, were treated with cytokine-stimulated allogeneic HSCT (mean dose: 21.8 × 108 cells/kg BW) from blood group-matched related donors. BM with mean CD34+ count 1.1% was inoculated into thymus, marrow and periphery, followed by two peripheral blood stem cell (PBSC) infusions.
Recovery began within 24 hours of HSCT and new lesions stopped after 6 months. No graft versus host disease (GvHD)/adverse effect was observed in any patient/donor. Over a mean follow-up of 8.02 years, all patients were well without recurrence/new lesions.
Drug-resistant PV can be successfully and safely treated by allogeneic HSCT.
Pemphigus vulgaris (PV) belongs to the family of pemphigus (in Greek “pemphix” means bubble/blister), a group of autoimmune blistering disorders, and was considered fatal in pre-corticosteroid era. It is mediated by autoantibodies specific for the extracellular region of desmosomal glycoprotein “desmoglein 3.” Autoimmune diseases are the result of breakdown of “self-tolerance” mechanisms that include thymic central tolerance and multiple operational mechanisms of peripheral tolerance. Hematopoietic stem cell transplantation (HSCT) has been suggested as a therapeutic strategy for autoimmune diseases. We infused allogeneic HSC in thymus, bone marrow (BM) and periphery, with an aim of reconstituting the central and peripheral arms of self-tolerance.
We conducted a prospective, single-center clinical trial between June 2001 and October 2002 to evaluate the effects of high-dose unfractionated HSCT into thymus, BM and peripheral circulation on central and peripheral arms of self-tolerance. Study protocol and consent forms were approved by Institutional Review Board.
Eleven (5 males, 6 females) patients, with a mean age of 33.5±8.8 (range: 21–50) years, with clinical and biopsy-proven PV were admitted for HSCT. All of them had painful pruritic blisters all over the body, including ulcers in oral cavities resistant to Prednisolone and topical steroid application given by their dermatologists. The average disease duration was 22.8±10.3 (range: 9–40) months [Table 1]. Donors were blood group matching relatives of patients; 3 were full-house human leukocyte antigen (HLA) match (siblings), 3 were 3/6 match (parents), 2 were 2/6 match (siblings), 1 was 1/6 match (sibling) and 2 were 0/6 match (cousins). Mean donor age was 35.1±9 (range: 21–54) years.
Our aim was to infuse 25 × 108 unfractionated HSC/kg body weight (BW) in recipients. We wanted to integrate central and peripheral arms of self-tolerance. Hence, thymic inoculation was included in this protocol. Recipients were monitored for development of skin rashes, fever and gastrointestinal symptoms of graft versus host disease (GvHD) [Figure 1].
Donor-recipient HLA typing was performed on the first day using conventional serological techniques (one-Lambda pre-dot trays for HLA-A, -B, -DR typing). Donors were administered granulocyte macrophage- colony stimulating factor (GM-CSF), 300 μg/day subcutaneously on days 2 and 3. On day 4, 300 ml BM was aspirated under local anesthesia from their posterior superior iliac crest. First, 10 ml of aspirate was concentrated and 2 ml concentrated inoculum was injected in recipient thymus. CD34+ cell count was separately performed along with total and differential cell counts. Subsequently, 100 ml was infused into sternum/iliac crests and 190 ml in the peripheral circulation of the recipients immediately by means of intravenous infusion sets without filters.
Recipients were subjected to general anesthesia for thymic inoculation of stem cells. Four-centimeter-long incision was made into right second intercostal space. After cutting all muscles, mediastinal fascia was opened and thymus was identified in retrosternal space. Then, 2 ml concentrated marrow was inoculated with 20-gauge needle. Hemostasis was checked and wound closed. BM infusion was performed using BM aspiration needles, after ensuring that the needle was in marrow.
GM-CSF, 300 μg in the mornings, and granulocyte-colony stimulating factor (G-CSF), 300 μg in the evenings, were administered subcutaneously for four consecutive days to donors, from days 5 to 8. They were then subjected to leukophoresis on stem cell separator (Hemonetics, MCS 3p, USA) on day 9 and peripheral blood stem cells (PBSCs) were collected from them and immediately infused into recipient's peripheral circulation. Their CD34+ count was performed along with total and differential cell counts. This procedure was once again repeated for the next cycle and last PBSC infusion was performed on day 14. Paradigm is shown in Figure 1.
No cytoablation technique was used. All the recipients were administered Cyclosporine A (CsA), 5 mg/kg BW/day, Prednisolone, 0.4 mg/kg BW/day and anti-thymocyte globulin, 10 mg/kg BW/day on three consecutive days as an induction therapy to unfractionated HSC infusion. CsA, 3 mg/kg BW, was continued for 6 months. Prednisolone was continued in the same dose for 5 months and subsequently tapered over 6th month.
Mean thymic inoculum had total BM cell count (n×108 cells/kg BW) of 1.3±0.41 (range: 0.86–2.1) and that of inoculum of marrow plus periphery was 0.71±0.4 (range: 0.43–1.6). Mean CD34+ cell count was 1.1±0.51% (range: 0.1–1.8%).
Liver and BM functions of all patients were monitored and analyzed at weekly intervals for the first month and at monthly intervals for the next 5 months. CsA levels were monitored every fortnight by EMIT 2000 CsA assay (Syva Co., Dade Behring, USA, trough levels of CsA: 50–176 ng/ ml).
Within 24 hours of HSCT, skin lesions started regressing. Over a period of time, eruption of new lesions became lesser in number and intensity. At the end of about 6 months, new lesions stopped erupting [Figure [Figure2a2a–b]. A repeat skin biopsy was performed after clearance of lesions, which showed absence of bullous cavities and disappearance of IgG deposits [Figures [Figures3a3a–b and and4a4a–b]. Mean follow-up period was 8.02±0.47 (range 7.64–8.81) years. No GvHD was observed in any patient. Side effects of GM-CSF and G-CSF injection to donors were malaise (100%), mild pyrexia (70%) and occasional skin rashes, which responded to anti-pyretic and anti-histaminic agents. None of the donors had serious or life-threatening reactions.
PV is an interesting autoimmune disorder since it provides a model with circulating autoimmune antibodies directly pathogenic to a known specific target antigen. The pathogenic process of acantholysis occurs in specific binding sites for autoantibodies. The circulating autoantibodies, when infused in experimental mice, produce an identical skin disease. This clearly establishes the clinicopathological correlation of acantholytic site and deposition of autoantibodies.[2–4] As pemphigus is a single-organ disease with blistering as its hallmark, clinical monitoring of disease activity is simple and it is easy to assess the therapeutic response. Allogeneic rather than autologous stem cell transplantation (SCT) is required for genetically based spontaneous models of autoimmune diseases to prevent disease recurrence. This was the rationale for our strategy of allogeneic SCT in pemphigus.
Self-tolerance is the central theme for survival of an organism, induced and maintained through thymic education–selection process of T-cells. Negative selection process is one of the major operational mechanisms by which autoreactive T-cells will be eliminated through apoptosis. All the evidences available indicate that breakdown of one or more of these mechanisms results in autoimmune disorders. One of the reasons is the variability of intrathymic deletion of autoimmune T-cells. Infectious agents are also responsible for activating potentially self-reactive T-cells. We may hypothesize that immune dysregulation in genetically susceptible individual leads to escape of self-reactive T-cells into periphery. Structural similarity between the microbial and self-antigens may be precipitating activation of autoreactive T-cells (molecular mimicry) on provocation of an infectious agent.
Induction and maintenance of mechanisms for central tolerance requires the presence of autoantigens in thymus. All self-antigens do not exist in thymus. Thus, the peripheral mechanisms for self-tolerance maintenance have evolved. Multiple check points and operational mechanisms like clonal deletion, immune ignorance, and regulatory mechanisms like anergy, inhibition, suppression and deviation have developed to deal with the escaped autoreactive T-cells from thymus.
Hence, we decided to develop our own integrated approach to reconstitute the disturbed central and peripheral arms of self-tolerance with “multiple site infusion technique.”
Drug-resistant PV can be successfully treated using allogeneic unfractionated HSCT into thymus, BM and periphery. It has been found to be reproducible, safe, efficacious and cost-effective.
We are indebted to Ms. Priyadarshini Shah and Mr. Yazdi Wadia for updating patient charts and preparing the manuscript.
Source of Support: Nil
Conflict of Interest: Nil.