Quantification of Active and Latent Form of Human Cytomegalovirus Infection in Umbilical Cord Blood Donors by Real-Time PCR.

BACKGROUND: Umbilical cord blood (UCB) is believed to be a highly valuable source of hematopoietic stem cells for transplantation. Objective: To investigate the prevalence of active and latent human cytomegalovirus (CMV) infection in UCB donors in Iranian population. METHODS: A total of 825 UCB samples was collected under standard procedures and analyzed for the presence of CMV DNAs in buffy coat (latent infection) and plasma (active infection). DNA was extracted from buffy coat and plasma samples separately and tested with quantitative real-time PCR. All positive samples were checked by ELISA for IgG and IgM anti-CMV antibody. RESULTS: Latent CMV infection was detected in 17 (2%) buffy coat samples with a low level of viral load, which indicated the presence of latent viral infection in donors. None of the plasma samples were found positive for CMV DNA reflecting no active infection. In the 17 positive samples, CMV viral load was 91-104 (mean: 100) copies/mL. All samples positive for viral DNA were also found positive for CMV IgG antibody by ELISA. No CMV IgM antibody was detected in positive samples. CONCLUSION: CMV is still the most important virus that infects hematopoietic stem cells and could be dangerous, especially for immunocompromized transplant recipients. We therefore suggest using real-time PCR for the detection and quantification of the viral DNA in buffy coat and plasma of UCB donors. PCR of plasma for detection of CMV and antibody assay for CMV infection add no more sensitivity for the detection of latent CMV infection in UCB donors.

Evaluation of Serum Interleukin-21 and HLA-C1 Polymorphism in Pediatrician Hematopoietic Stem Cell Transplantation for Early Diagnosis of Acute Graft-Versus-Host Disease

Background: Allogenic hematopoietic stem cell transplantation (HSCT) is a strategy used for treatment of different malignant diseases. However, success of allo-HSCT can be hampered by graft-versus-host-disease (GVHD). Natural killer (NK) cells may play an important role in activating antigen presenting cells and subsequent activation of T cells. The main purpose of this study was the evaluation of IL-21, as a blood biomarker, for early detection of acute GVHD (aGVHD) in children after HSCT and also the study of human leukocytes antigen (HLA)-C1 polymorphism, as a targeting ligand for NK cells in these patients. Methods: Fifty one children receiving HSCT were studied. Blood samples were collected at -8, 7, and 14 days of transplantation. The -8-day samples were analyzed for HLA-C1 polymorphism by PCR-sequence-specific primer technique and pre-transplantation IL-21 assay. To study the serum levels of IL-21, two blood samples were collected on days +7 and +14 and analyzed by ELISA technique. Results: The results indicated that the incidence of aGVHD in pediatric is associated with a polymorphism of HLA-C1, as alleles HLA-C01:12 (P<0.001), HLA-C01:22 (P<0.004), and HLA-C01:67 (P<0.009). On the other hand, the serum levels of IL-21 in children with aGVHD were decreased after transplantation compared to before transplantation. The serum levels of the IL-21 at 14 days after transplantation had a significant correlation with the occurrence of aGVHD (P=0.05). Conclusion: Based on the findings of this study, there is a significant correlation between HLA-C1 polymorphisms and the serum levels of IL-21 with the incidence of aGVHD.

Quantitative polymerase chain reaction for detection of human herpesvirus-7 infection in umbilical cord blood donors.

OBJECTIVE: Umbilical cord blood (UCB) has been a reasonable alternative to granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow, as a source of hematopoietic stem cells with a lower risk of graft-versus-host disease. In immunocompromised hosts after transplantation, the risk of viral infection in adults, especially with beta-herpesviruses such as human herpesvirus-7 (HHV-7), may be increased. This virus in immunocompromised patients can be reactivated from latency and converted to an active phase. Therefore, light-upon-extension real-time polymerase chain reaction (PCR) was developed to assess the prevalence and load of HHV-7 in the plasma and buffy coat of donors. METHODS: About 825 UCB samples under standard protocol from donors were collected. Then, DNA from plasma and buffy coat was extracted and quantitative real-time PCR was performed with light-upon-extension primers. RESULTS: Overall, HHV-7 was detected in 3.64% (30/825) of UCB donors. HHV-7 DNA was detected in 26 (3.2%) buffy coat samples (latent infection), and only 4 (0.48%) of them were positive for HHV-7 DNA in plasma samples (active infection); the mean HHV-7 viral load was 1.31 × 10(1) copies/mL in latent infection, and 1.94 × 10(5) copies/mL in active infection. CONCLUSIONS: We suggest that real-time PCR in plasma and buffy coat could be a useful method to detect active and latent HHV-7 infection in UCB donors and determine its role in subsequent transmission events.

Comparison of effects of age and sex on serum protein electrophoretic pattern in one-humped camels (Camelus dromedarius) in Semnan, Iran.

The aim of this study was to evaluate the influence of age and sex on the concentration of total serum protein measured by the biuret method and protein fractions determined using cellulose acetate electrophoresis in apparently healthy camels (Camelus dromedarius). Blood samples were collected from 21 camels (12 males and 9 females). The camels were further divided into two groups: 12 young camels at the age of 3 months to 2 years and 9 adult camels at the age of 3-15 years. Cellulose acetate electrophoresis of serum proteins identified five protein fractions in adult camels as young camels, these five protein fractions include albumin, α1 and α2, ß and γ-globulins. In adult camels, serum levels (g/l) of total protein, albumin, α1-globulins, α2-globulins, ß-globulins and γ-globulins were 80.9±3.10, 42.9±3.10, 1.3±0.22, 2.2±0.30, 11.8±0.30 and 22.6±0.20, respectively. However, in young camels, these levels (g/l) were 66.8±2.90, 40.2±2.40, 1.0±0.14, 2.6±0.30, 10.6±0.80 and 12.3±1.20, respectively. The albumin/globulin (A/G) ratio was 2.08±0.28 in adult camels and 3.77±0.53 in young ones. The mean serum concentrations of total protein and γ-globulins were significantly (P<0.05) higher and the A/G ratio was significantly lower in adult camels compared to young camels. The mean concentrations of γ-globulins were significantly higher and the A/G ratio was significantly (P<0.05) lower in females compared to male camels. The results of the present study indicate a significant effect of age and sex on the concentrations of some of the serum protein fractions in dromedary camels.

Generation of a dendritic cell-based vaccine in chronic lymphocytic leukaemia using CliniMACS platform for large-scale production.

We previously demonstrated that dendritic cells (DC) that have endocytosed apoptotic bodies of autologous leukemic cells (Apo-DC) can boost antileukemic T-cell responses. In this study, we report a description of the production procedure and product specification of the Apo-DC vaccine preparations for clinical use. Enriched populations of CD14+ monocytic precursors and CD19+ leukaemic cells were obtained using CliniMACS technology from a single leukapheresis product. Apoptotic bodies were obtained by irradiating (5 Gy) CD19+ selected B cells. DC were generated ex vivo by culturing monocytes with granulocyte macrophage colony-stimulating factor and interleukin-4. Following coculture with apoptotic bodies, DCs were matured with tumour necrosis factor-alpha. The mean percentage of CD14+ cells in the peripheral blood as well as in the leukapheresis product of the patients (n = 10) was approximately 2% (range, 0.8-3.3). Immunomagnetic selection using the CD14 reagent yielded a CD14+ population that was 91 +/- 2.2% (mean +/- SEM) pure. Immunomagnetic selection of CD19 expressing cells yielded a population that was 100 +/- 0.03% pure. Cell viability immediately after selection was 97% and 98% after 7 days of culture. The Apo-DC cellular vaccine product showed a mature phenotype, with a high rate of endocytosis (84%) of apoptotic leukemic B-cells. In conclusion, despite significant variability in the circulating monocyte frequency of the chronic lymphocytic leukaemia patients, our method permitted the production of a DC vaccine with high reproducibility and conforming with recommended quality standards.

Development of a dendritic cell-based vaccine for chronic lymphocytic leukemia.

Evidence for the existence of CLL-specific antigens recognized by the immune system can be gathered from the observation that many patients display monoclonal or oligoclonal expansions and skewed repertoire of T cells. In vitro functional studies have shown that tumor-specific T-cells are able to lyse the leukemic cells. Antileukemic cellular immunity may be boosted in vivo using dendritic cell-based immunotherapy. Our preclinical studies provide evidence that DC that had endocytosed apoptotic CLL cells (Apo-DC) were superior to fusion hybrids, tumor lysate or RNA in eliciting antileukemic T-cell responses in vitro. We have validated a method for enriching the small number of monocyte precursors present in the peripheral blood of CLL patients and utilize them for generating individualized, Apo-DC cellular vaccines. In most cases, a minimum of 50 x 10(6) Apo-DC could be generated, beginning with immunomagnetically enriched monocytes from a single leukapheresis product containing at least 1% CD14+ cells. Cryopreservation and thawing did not affect the phenotype or the T cell stimulatory function of Apo-DC. A phase I/II, open label clinical trial examining the feasibility, safety and immunogenicity of Apo-DC vaccination has been initiated. CLL patients receive 10(7) Apo-DC for at least five immunizations and monitored clinically and immunologically for 52 weeks. Three cohorts are accrued stepwise. Cohort I receives Apo-DC alone; Cohort II: Apo-DC+ repeated doses of low-dose GM-CSF; Cohort III: low-dose cyclophosphamide followed by Apo-DC + GM-CSF.

Idiotype vaccination in patients with myeloma reduced circulating myeloma cells (CMC).

BACKGROUND: Circulating myeloma cells (CMC), exhibiting the same immunoglobulin heavy-chain gene rearrangements as the plasma cells, are part of the myeloma clone. In this study, we evaluated the effect of idiotype (Id) vaccination on CMC. PATIENTS AND METHODS: Eleven patients were immunized with the autologous Id in combinations with granulocyte-macrophage colony-stimulating factor and interleukin 12, and followed for CMC by quantitative real-time allele-specific PCR. Id-specific T cells were monitored by proliferation assay, enzyme-linked immunospot (interferon-gamma) assay, and quantitative real-time PCR for cytokines. Regulatory T (T(reg)) cells were analyzed by flow cytometry. RESULTS: CMC were detected in 9 of 11 patients at start of vaccination. In four patients, CMC declined and two had a complete molecular remission. Further two patients had stable levels of CMC during follow-up, while in three patients CMC progressively increased. Six patients had a vaccine-induced Id-specific T-cell response. A significant correlation was observed between reduced/stable levels of CMC and the Id-specific T cells (P < 0.02). The frequency of T(reg) cells was decreased in immune responders, but increased in immune nonresponders (P < 0.05). No significant change in the serum M-protein concentration was, however, observed in any patient. CONCLUSION: Id vaccination reduced CMC, which correlated with vaccine-induced Id-specific T cells. Further studies are warranted to analyze the clinical significance of CMC and clinical effects of Id vaccination.

Generation of DC-based vaccine for therapy of B-CLL patients. Comparison of two methods for enriching monocytic precursors.

BACKGROUND: The generation of Ag-loaded DC under good manufacturing practice (GMP) conditions is logistically challenging and further compounded when the starting precursors need to be purified from B-CLL patients who have overwhelming numbers of circulating B-CLL cells and decreased numbers of monocytes. METHODS: We have previously demonstrated that DC with endocytosed B-CLL apoptotic bodies are powerful stimulators of anti-leukemic T cells. In this study we compared counterflow elutriation and immunomagnetic separation for enriching monocyte precursors, and evaluated the feasibility of generating DC from B-CLL patients and the effects of cryopreservation. RESULTS: Monocyte yield from a single leukapheresis product of a B-CLL patient varied from 1 x 108 to 10 x 108 total cells, from which 40-200 x 106 mature DC could be produced. Adequate numbers of monocytes could not be enriched from one patient with 0.2% monocytes in the leukapheresis product, and the target of 50 x 106 DC was barely achieved in another patient with 0.9% monocytes in the pheresed cells. These results suggested that successful production of DC is dependent on a minimum frequency of 1% CD14(+) monocytes in the leukapheresis product. Cryopreservation of tumor cell-loaded DC yielded a recovery rate of 86+/-4.4% upon thawing, with a total viability of 90+/-2.8%. Most importantly, cryopreserved Ag-loaded DC retained their morphology, phenotype and function. DISCUSSION: The results demonstrate that adequate numbers of functional DC required for clinical therapy can be generated from patients who have >1% of CD14(+) monocytes in the leukapheresis product. Moreover, Ag-loaded DC can be cryopreserved and recovered without significant change in phenotype or function.

Association between a high BK virus load in urine samples of patients with graft-versus-host disease and development of hemorrhagic cystitis after hematopoietic stem cell transplantation.

BK virus (BKV) load in urine alone or in combination with acute graft-versus-host disease (GVHD) was correlated to development of hemorrhagic cystitis (HC). BKV load in combination with acute GVHD discriminated the best, while BKV and viral load alone, but not GVHD, still showed predictive ability for HC.

Apoptotic tumor cells are superior to tumor cell lysate, and tumor cell RNA in induction of autologous T cell response in B-CLL.

B cell chronic lymphocytic leukemia (B-CLL) is a chronic leukemia manifested by increased numbers of B cells in circulation. The slow, smouldering nature of the disease in a significant proportion of the cases makes it an ideal target for immunotherapy. Dendritic cell (DC)-based immunotherapy is emerging as an exciting modality with significant clinical potential. In this study, three strategies for delivering antigens to DC, namely apoptotic bodies (Apo-DC), tumor lysates, and tumor RNA were studied in an autologous setting. In all six CLL patients, Apo-DC induced higher HLA-restricted, T cell responses than DC pulsed with tumor lysate or RNA. Real-time PCR confirmed higher expression of genes for IL-2 and IFN-gamma in T cells stimulated with Apo-DC. Concurrently, no IL-10 and low IL-4 responses indicated that the immune response was primarily of the Th1 type. Enzyme-linked immunospot assay revealed high IFN-gamma secretion by T cells when Apo-DC was used to stimulate autologous T cells in all patients. Our data suggest that cellular vaccines with DC loaded with apoptotic bodies may be a suitable approach for immunotherapy of B-CLL.

Dendritic cells loaded with apoptotic tumour cells induce a stronger T-cell response than dendritic cell-tumour hybrids in B-CLL.

Dendritic cells (DC) are professional (specialised) antigen-presenting cells that can capture antigen from apoptotic tumour cells and induce MHC class I- and II-restricted responses. Also, DC fused with tumour cells may be effective for immune response induction. Both cell preparations may be considered as vaccine candidates in a therapeutic approach. We examined autologous T-cell activation by DC that had endocytosed leukaemic B-cell apoptotic bodies (Apo-DC) and compared it to the T-cell stimulatory capacity of DC that were fused with tumour cells. Following incubation, 22.6+/-6.2 (mean+/-s.e.m.) of DC had endocytosed leukaemic cells, while the frequency of DC-leukaemic cell hybrids was 10.5+/-2.6%. Apo-DC and hybrid cells both demonstrated the ability to stimulate a tumour-specific T-cell immune response in vitro. A T-cell proliferation response was also observed in four out of five CLL patients when using Apo-DC. However, fusion hybrids lacked the ability to elicit a proliferative response. Apo-DC also induced an IFN-gamma response, as did hybrid cells. The cytokine response induced by Apo-DC was significantly higher than that induced by fusion (P<0.05). This study shows that endocytosed apoptotic tumour cells induced a significantly stronger T-cell response than DC hybrids; and as such should be a better candidate for vaccine production.