JF Correia-Silva1, RG Resende1, MHNG Abreu2, AL Teixeira3, MM Teixeira4, H Bittencourt5, RS Gomez1 and TA Silva1*
1Department of Oral Surgery and Pathology, School of Dentistry, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
2Department of Community and Preventive Dentistry, School of Dentistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
3Department of Clinical Medicine, Universidade Federal de Minas Gerais, Faculty of Medicine Belo Horizonte, Minas Gerais, Brazil
4Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
5Hematopoietic Stem Cell Transplantation Unit, Department of Hematology, Hospital das Clínicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Received: 11 April, 2016; Accepted: 15 June, 2016; Published: 17 June, 2016
Prof. Tarcilia Aparecida Silva, Departamento de Clínica Patologia e Cirurgia, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Pampulha, Belo Horizonte, Minas Gerais 31270-901, Brazil, Tel: +55 31 3409 2478; Fax: +55 31 34092430; E-mail:
Correia-Silva JF, Resende RG, Abreu MHNG, Teixeira AL, Teixeira MM, et al. (2016) Cytokine Production and Human Cytomegalovirus Load in Allogeneic Hematopoietic Stem Cell Transplantation Outcome. Int J Immunother Cancer Res 2(1): 003-010. 10.17352/2455-8591.000008
© 2016 Correia-Silva JF et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Allogeneic HSCT; Cytokines; HCMV; Survival
allo-HSCT: allogeneic Hematopoietic Stem Cell Transplantation; GVHD: Graft-Versus-Host Disease; HCMV: Human Cytomegalovirus; IL: Interleukin; IFN: Interferon; TNF-α: Tumor Necrosis Factor-α; HLA: Human Leukocyte Antigen; HR: Hazard Ratio;
Objective: To investigate the impact of cytokine levels (IL-1β, IL-6, IL-10, IFN-γ and TNF-α) and Human cytomegalovirus (HCMV) load in the saliva and blood on the survival of allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients.
Study Design: Samples were obtained from 63 patients 7 days before and 21 days after allo-HSCT. Cytokine levels were assessed by ELISA, and HCMV load was determined by real-time PCR.
Results: The increase of IL-6 in the saliva and the reduction of IFN-γ in the blood before allo-HSCT were associated with increased risk of death. Moreover, the increase of IL-6 in the blood and of HCMV in the saliva after allo-HSCT were also associated with increased risk of death.
Conclusions: Cytokine levels and HCMV load were associated with the increased risk of death. The findings suggest a potential function of these biomarkers in the determination of allo-HSCT survival.
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed for a range of disorders, including hematological malignancy, severe aplastic anemia and genetic diseases. The attractive aspect of this therapeutic strategy is the development of potent donor T-cell-mediated immune responses that can eliminate malignant cells, which has traditionally been termed the graft versus leukemia effect. Although allo-HSCT provides the only curative therapy for many patients with malignant and non-malignant diseases, it is also associated with a high incidence of treatment-related morbidity and mortality [1,2].
Allo-HSCT recipients generally present three disturbance levels in the first weeks after allo-HSCT. These disturbances potentially can stimulate a massive release of inflammatory cytokines . First, baseline circulating inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), could be increased as a result of disease, therapy, comorbidities or ongoing infections that occur before allo-HSCT. Second, immediately after allo-HSCT, the immune dysfunction caused by the conditioning regimen, which may produce tissue injury and consequently the production of inflammatory cytokines, may affect the reconstitution of white blood cells. Third, after allo-HSCT, activated, mature donor T-cells can set off a cytokine response that in turn stimulates host T-cell responses related to the onset of acute graft-versus-host disease (aGVHD) [3,4]. Immune dysfunction following allo-HSCT involves activated T-cells secreting interferon (IFN)-γ as well as other cytokines, which activate monocytes and dendritic cells. This process appears to be associated with the induction of aGVHD, resulting in significant morbidity and mortality. In addition, a prolonged immune deficiency characterized by lymphopenia and susceptibility to infection can occur . Deregulation of inflammatory cytokines is also associated with multiple symptoms (pain, sleep disturbance and depression) during neutropenia after allo-HSCT . Interleukin-6 (IL-6) is an important pro-inflammatory cytokine involved in immune-mediated complications after allo-HSCT .
Human cytomegalovirus (HCMV) infection remains one of the most important causes of morbidity and mortality among allo-HSCT recipients [6,7]. Infection by HCMV may result not only in HCMV disease, but is also associated with other adverse effects due to its immunomodulatory actions; this might result in increased risk of bacterial, fungal and viral infections, and also of aGVHD .
Allo-HSCT outcomes could be affected by factors such as HCMV infection, recipient/donor histocompatibility, patient/donor gender, recipient age, GVHD and cytokine production [9,10]. The purpose of this study was to investigate the impact of cytokine levels (IL-1β, IL-6, IL-10, IFN-γ and TNF-α) and HCMV load in the saliva and blood, before and after allo-HSCT, on recipient survival.
Materials and Methods
The study protocol was approved by the Research Ethics Committee of the institution (Process # ETIC 097/06). Informed consent was obtained from all the patients or from parents, if the patient was less than 18 years.
Patients and samples
Sixty-three consecutive allo-HSCT patients from Hospital das Clínicas of Universidade Federal de Minas Gerais (HC-UFMG), were included in this prospective study. The patients were prepared for allo-HSCT according to the protocols of the Stem Cell Transplant Unit at HC-UFMG, which vary according to type and status of the disease. Cyclosporine, in combination with either methotrexate or mycophenolate mofetil, was used for GVHD prophylaxis. Methylprednisolone in combination with Cyclosporine was used for GVHD treatment. Antigenemia assay was used routinely for preemptive ganciclovir treatment of HCMV infection when 2 or more neutrophils’ positive nuclei for the HCMV lower matrix phosphoprotein pp65 in a cytospin preparation of 105 blood leukocytes after neutrophil recovery (‡500 neutrophils mm3) were detected, according to the protocols of the allo-HSCT Unit of HC-UFMG. This study was approved by the Institutional Ethics Committee for human subjects.
Demographic information, as well as clinical and laboratory data, were available from the database of the HC-UFMG. This clinical information includes the underlying disease, the source of the stem cells, the gender and age of the patient, the gender of the donor, the HCMV serostatus of the donor and recipient before transplantation, conditioning regimes, Human leukocyte antigen (HLA) matching and pp65 antigenemia status.
Saliva and blood samples from 63 recipients, were taken in two different stages , seven days before (stage 2) and 21 days after (stage 3) HSCT. Patients were followed for one year after allo-HSCT, or until the death of the recipient.
Enzyme-Linked Immunosorbent Assay (ELISA)
Saliva and blood from 63 patients who submitted to allo-HSCT were collected for quantification of cytokines by ELISA. Oral fluid was collected using Salivette® neutral cotton swabs (Sarstedt, Nümbrecht, Germany). Recovery of the saliva was achieved by centrifuging the container at 1500 rpm for 10 minutes. Saliva samples were diluted (1:1) in PBS (0.4 mM NaCl and 10 mM NaPO4) containing protease inhibitors (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA and 0.01 mg/mL aprotinin A) and 0.05% Tween-20, and frozen at -20°C until analysis. Four milliliters of peripheral blood were collected in anticoagulant-free tubes and centrifuged at room temperature for 30 min. The recovered serum was stored in small aliquots at -20°C until the cytokines were quantified. Concentrations of IL-1β, IL-6, IL-10, IFN-γ and TNF-α in the saliva and blood were determined using commercially available, quantitative sandwich ELISA kits (DuoSet, R&D Systems, Minneapolis, MN, USA), according to the manufacturers’ instructions. The detection ranges were 3000-46 pg/ml for IL-1β, 600-9 pg/ml for IL-6, 4000-62 pg/ml for IL-10, 1000-15 pg/ml for IFN-γ, and 1000-15 pg/ml for TNF-α. Values below the detection limits were assumed to be zero. Concentrations were expressed as pg/mL for blood. The total protein in the saliva samples was measured using the Bradford method, according to the BSA standard (Fermentas Life Sciences, Vilnius, Lithuania), and concentrations were expressed as mg/ml. Total protein concentration was used to correct the saliva cytokine values for each sample. The saliva sample values, corrected by the total protein values, were expressed in pg/mg protein.
Saliva and blood from the first 30 patients enrolled in this study were collected for real-time PCR assays. Oral fluid was collected by swab, as previously described . Four milliliters of peripheral blood were collected in an EDTA tube for the PCR assay and stored at -20°C until processing. Total genomic DNA was extracted from saliva and whole-blood samples using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) and stored at -20°C until used. Two hundred nanograms of extracted DNA were used for HCMV real-time PCR. The HCMV-specific PCR primers used in the assay were selected from the US17 region of HCMV AD169 . Quantification of HCMV DNA was performed using SYBR Green PCR Core Reagents (PE Applied Biosystems, Foster City, CA, USA). PCR was performed with an ABI Prism 7900 instrument (PE Applied Biosystems, Foster City, CA, USA), using 96-well plates. The mean value of the duplicates was used in calculations of HCMV DNA.
Overall survival was calculated using the Kaplan-Meier method. Time to death after allo-HSCT was determined initially compared using the log-rank test for the following variables: age of patient, recipient gender, donor gender, recipient/donor gender, primary disease, stem cell source, HLA matching, clinical systemic aGVHD, pp65 antigenemia, conditioning regimen, IL-1β, IL-6, IL-10, IFN-γ and TNF-α levels before and after allo-HSCT, and HCMV load in saliva and blood before and after allo-HSCT. Based on p values lower than 0.25 the variables were included in the Cox proportional hazards models [13,14]. Four models were created. Model 1 presented the following variables: recipient/donor gender, stem cell source, clinical systemic aGVHD, and IL-6 levels in saliva before allo-HSCT. Model 2 presented the following variables: recipient/donor gender, stem cell source, and clinical systemic aGVHD. Model 3 presented the following variables: recipient/donor gender, stem cell source, clinical systemic aGVHD, and IFN-γ levels in blood before allo-HSCT. Model 4 presented the following variables: recipient/donor gender, stem cell source, clinical systemic aGVHD, and IL-6 levels in blood after allo-HSCT. Survival time after HSCT was calculated until the last follow-up or until death. The continuous variable was categorized for generating a new categorical variable with group numbers based on the median of this variable. The Kaplan-Meier curves were generated with the new categorical variables. Statistical analyses were performed using SPSS (SPSS Inc., version 16.0, Chicago, IL), and p values ≤ 0.05 were considered statistically significant.
Allo-HSCT recipients were followed from 7 days until one year after allo-HSCT, or until the death of the recipient. Of the 63 allo-HSCT recipients, 38 (60%) remained alive one year after HSCT. The survival median of the 25 deceased patients after allo-HSCT was 68 days (range 17-240). Patient and transplant characteristics, conditioning therapy, aGVHD, and survival univariate analyses are shown in Table 1.
The twenty five patients who died include the 3 patients that presented relapse. Among the transplantation-related causes: six presented GVHD, five presented bacterial infection, three presented viral infection, five presented fungal infection, two presented acute respiratory distress syndrome, and one presented acute liver insufficiency. Three recipients developed HCMV disease: two developed pneumonia and one developed enterocolitis.
The average age of the patients studied was 30 years (range 5-56). Of the 63 patients, 35 (55%) were men. Compared to female recipients, male recipients demonstrated an increased risk of death after allo-HSCT (hazard ratio (HR): 4.271; 95% CI = 1.599-11.407, p= 0.004) (Figure 1A). Transplantation for a male donor to a female recipient was associated with a reduced risk of death after allo-HSCT (HR: 0.6650; 95% CI = 0.4689-0.9431, p= 0.022) (Figure 1B). We also observed a higher incidence of death after HSCT among patients receiving peripheral blood stem cells (PBSC), a source of hematopoietic stem cells for transplant (17/26; 65%) (HR: 3.8424; 95% CI = 1.6527-8.9328, p=0.002) (Figure 1C); also, among patients developing systemic aGVHD (HR: 2.7504; 95% CI = 1.2079-6.2627, p=0.0160) (Figure 1D).
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