Pre- and Posttransplant IgA Anti-Fab Antibodies to Predict Long-term Kidney Graft Survival.

OBJECTIVES: Immunologic factors are reliable markers for allograft monitoring, because of their seminal role in rejection process. One of these factors is the immunoglobulin (Ig)A anti-Fab of the IgG antibody. This study aimed to evaluate the predictive value of pre- and posttransplant levels of this marker for kidney allograft function and survival. METHODS: Sera samples of 59 living unrelated donor kidney recipients were collected before and after transplantation (days 7, 14, and 30) and investigated for IgA anti-Fab of IgG antibody levels using enzyme-linked immunosorbent assay in relation with allograft outcome. RESULTS: Among 59 patients, 15 cases (25%) including 10 with acute rejection and 5 with chronic rejection episodes showed graft failure during a mean of 5 years of follow-up. High posttransplant levels of IgA anti-Fab antibodies were observed more frequently in patients with stable graft function (SGF) compared with patients with graft failure (P = 2 × 10(-6)). None of patients with acute or chronic rejection episodes had high levels of IgA anti-Fab antibodies at day 30 posttransplant compared with the SGF group (P = 10(-6) and P = .01, respectively). In addition, high levels of IgA anti-Fab antibody correlated with lesser concentration of serum creatinine at 1 month posttransplantation (P = .01). Five-year graft survival was associated with high levels of pre- and posttransplant IgA anti-Fab antibodies (P = .02 and P = .003, respectively). CONCLUSIONS: Our findings indicate the protective effect of higher levels of IgA anti-Fab antibodies regarding to kidney allograft outcomes and long-term graft survival.

HLA class II susceptibility pattern for type 1 diabetes (T1D) in an Iranian population.

This study aimed to determine the HLA-DRB1/HLA-DQB1 susceptibility and protection pattern for type 1 diabetes (T1D) in a population from Hamadan, north-west of Iran. A total of 133 patients with T1D were tested for HLA-DRB1 and HLA-DQB1 alleles using PCR-SSP compared to 100 ethnic-matched healthy controls. Alleles and haplotypes frequencies were compared between both groups. The most susceptible alleles for disease were HLA-DRB1*03:01, DRB1*04:02, DQB1*02:01 and DQB1*03:02, and protective alleles were HLA-DRB1*07:01, *11:01, *13:01, *14:01 and DRB1*15 and HLA-DQB1*06:01, *06:02 and *06:03. Haplotype analysis revealed that patients with T1D had higher frequencies of DRB1*03:01-DQB1*02:01 (OR = 4.86, P < 10(-7) ) and DRB1*04:02-DQB1*03:02 (OR = 9.93, P < 10(-7) ) and lower frequencies of DRB1*07:01-DQB1*02:01 (P = 0.0005), DRB1*11:01-DQB1*03:01 (P = 0.001), DRB1*13:01-DQB1*06:03 (P = 0.002) and DRB1*15-DQB1*06:01 (P = 0.001) haplotypes compared to healthy controls. Heterozygote combination of both susceptible haplotypes (DR3/DR4) confers the highest risk for T1D (RR = 18.80, P = 4 × 10(-5) ). Additionally, patients with homozygote diplotype, DR3/DR3 and DR4/DR4, showed a similar risk with less extent to heterozygote combination (P = 0.0004 and P = 0.01, respectively). Our findings not only confirm earlier reports from Iranians but also are in line with Caucasians and partly with Asians and some African patients with T1D. Remarkable differences were the identification of DRB1*04:01-DQB1*03:02, DRB1*07:01-DQB1*03:03 and DRB1*16-DQB1*05:02 as neutral and DRB1*13:01-DQB1*06:03 as the most protective haplotypes in this study.

Association of dental caries and salivary sIgA with tobacco smoking.

BACKGROUND: Salivary secretory IgA (sIgA) is said to play an important role in the immune response against dental caries. This study aimed to determine the salivary sIgA levels in healthy smokers and non-smokers, and its correlation with dental caries. METHODS: A total of 70 healthy subjects were selected and classified into four groups according to dental caries and tobacco smoking habits: smoking with caries (Group 1, n = 15); smoking without caries (Group 2, n = 15); non-smoking with caries (Group 3, n = 15); and non-smoking without caries (Group 4, n = 25). Salivary sIgA was measured using ELISA. The fissure and proximal caries were examined clinically and radiographically. Caries status was determined according to the decay surface index. RESULTS: Smokers showed a higher number of caries and the lowest concentration of sIgA. The highest levels of sIgA were observed in non-smoking and caries-free subjects compared to caries-active smokers (123.2 ± 19.9 vs. 13.3 ± 4.1 µg/ml respectively, p < 0.001). Also, the mean level of sIgA in Group 4 was significantly higher than Group 3 (p = 0.009). More importantly, higher and significant levels of sIgA were found in Group 3 versus Group 1 (p < 0.0001) and Group 2 (p = 0.0004). CONCLUSIONS: Our findings indicate that low concentrations of salivary sIgA are correlated with a higher prevalence of dental caries in smokers.

HLA-DRB and HLA-DQA/HLA-DQB allele and haplotype frequencies in Iranian patients with aggressive periodontitis.

BACKGROUND AND OBJECTIVE: Genetic backgrounds play a key role in susceptibility to and protection against a spectrum of periodontal diseases. Like other infectious diseases, the human leukocyte antigen (HLA) have been found to be associated with periodontitis. This study aimed to investigate differences in allele and haplotype frequencies of HLA class II antigens in a sample of Iranian patients with aggressive periodontitis compared with a healthy control group. MATERIAL AND METHODS: Fifty unrelated patients with aggressive periodontitis and 130 healthy volunteers were enrolled in this study. HLA genotyping for HLA-DRB, HLA-DQA1 and HLA-DQB1 was performed using the PCR with sequence-specific primers. Allele and haplotype frequencies were compared across groups. RESULTS: The frequencies of HLA-DQA1*03:01, HLA-DQB1*03:02 and HLA-DQB1*03:05 alleles, as well as that of the HLA-DRB1*04:01 allele, were significantly higher in patients with aggressive periodontitis compared with control subjects (p = 0.01, p = 0.04, p = 0.05 and p = 0.04, respectively). In contrast, the frequency of the HLA-DQB1*0603 allele was significantly lower in patients with aggressive periodontitis compared with control subjects (p = 0.006; odds ratio = 0.20). With regard to haplotype association, a significantly higher frequency of two haplotypes - HLA-DRB1*04:01/HLA-DQA1*03:01/HLA-DQB1*03:02 and HLA-DRB1*16:01/HLA-DQA1*01:03/HLA-DQB1*05:01 - was observed in patients with aggressive periodontitis compared with healthy controls (p = 0.01, odds ratio = 2.56 and p = 0.05, odds ratio = 5.38, respectively). CONCLUSION: These results provide additional evidence that class II HLA polymorphisms, particularly in the DQ locus, are associated with protection against and susceptibility to aggressive periodontitis.

Dynamic Changes of IFN-γ-producing Cells, TGF-ß and Their Preidctive Value in Early Outcomees of Renal Transplantation.

BACKGROUND: A growing body of evidence demonstrated an immune etiology as well as nonimmune mechanisms for episodes of clinical acute rejection and long-term allograft dysfunction. OBJECTIVE: To investigate the correlation of IFN-γ-producing cells and TGF-ß with incidence of clinical acute rejection in living-related and unrelated kidney allogarft recipients during the first post-transplant year. METHODS: This multi-center study was performed on 57 kidney allograft recipients from living-related (n=20) and unrelated (n=37) donors between April 2011 and September 2012 and who were followed prospectively for a mean period of one year. Peripheral blood samples were collected from all patients pre-transplantation and at days 14, 30 and 90 after transplantation; PBMCs were used as responding cells in enzyme-linked immunosorbent spot (ELISPOT) assay to measure the frequency of IFN-γ-producing cells after stimulation with donor lymphocytes. Additionally, TGF-ß levels were measured in cell culture supernatants of ELISPOT assay. RESULTS: During the follow-up period, 45 (79%) patients were diagnosed with stable graft function (group A); 12 (21%) experienced clinical acute rejection episodes (group B). The frequency of IFN-γ-producing cells was significantly (p<0.001) higher in the rejection group in all three times after transplantation. Also, post-transplantation comparison for TGF-ß showed a significantly (p<0.001) higher contents in group A vs. group B. Comparing the post-transplantation levels of TGF-ß and mean numbers of IFN-γ- producing cells between groups A and B demonstrated a continuous increment in TGF-ß and decreasing frequencies of IFN-γ-producing cells in group A vs. group B. CONCLUSION: Serial post-transplantation monitoring of IFN-γ-producing donor reactive cells during the first months is a clinically feasible approach for identification of kidney allogarft recipients at risk for ongoing immune-mediated graft damage and later graft loss.

Serum levels of interleukin (IL)-10, IL-17, transforming growth factor (TGF)-ß1, and interferon-γ cytokines and expression levels of IL-10 and TGF-ß1 genes in renal allograft recipients after donor bone marrow cell infusion.

BACKGROUND: Cytokine storm generated by an alloimmune response after transplantation can lead to either graft survival or rejection. The aim of this study was to evaluate the serum levels of interleukin (IL)-10, IL-17, transforming growth factor (TGF)-ß1, and interferon (IFN)-γ and expression levels of IL-10 and TGF-ß1 in renal allograft recipients with or without donor bone marrow cell infusion (DBMI). METHODS: We retrospectively followed 28 living unrelated kidney recipients, including 14 with and 14 without DBMI infusion for 2 years. Also, 14 healthy subjects were included as a normal control group. PBMC gene expression analysis for mRNA levels of IL-10 and TGF-ß1 cytokines relative to ß-actin as a reference gene was performed using quantitative fluorescence real-time polymerase chain reaction at the end of 2 years posttransplantation. Also, serum levels of IL-10, TGF-ß1, IFN-γ, and IL-17 in the 3 groups were measured by enzyme-linked immunosorbent assay at the same time. RESULTS: Both patient groups showed increased gene expression and serum content of IL-10 compared with normal controls. The expression levels were only significant between control patients and normal subjects (P=.02). Serum levels of IFN-γ and IL-17 were higher in untreated patients compared with normal controls (P=.03 and P=.07, respectively). DBMI patients showed significantly lower levels of serum TGF-ß1 and IL-17 compared with normal subjects (P=.05 and P=.06, respectively). Also, infused patients showed a positive correlation between circulating levels of IL-17 and IL-10 (r=0.692; P=.006), and an inverse correlation between serum creatinine and TGF-ß1 levels (r=-0.580; P=.03). CONCLUSION: The decreased levels of inflammatory cytokines besides IL-10 with increased TGF-ß1 levels and better allograft function with improved clinical outcomes were observed among infused patients, possibly indicating immunomodulatory effects of this approach in kidney allograft patients.

Sequence-based typing of major histocompatibility complex class I chain-related gene A alleles by use of exons 2-5 information.

The polymorphic MICA (major histocompatibility complex class I chain-related gene A) (Gene ID: 100507436) gene products are a ligand of the activating natural killer cell receptor, NKG2D. Their clinical importance spans from solid organ transplantation to bone marrow transplantation and disease susceptibility. Typing of MICA genes by sequencing is hampered by an exon 5 short tandem repeat, the definition of which is critical for the final allelic and functional assignment. We present a novel sequencing approach, which uses group-specific (7T/8T) exon 5 polymerase chain reactions (PCRs) and facilitates hemizygous exon 5 MICA-PCR in approximately 70% of the tested individuals. With this method we typed the International Histocompatibility Workshop Group MICA reference panel (40 cell lines) as well as 110 healthy South German blood donors. All ambiguities, with the exception of MICA*008:01/008:04 (synonymous substitution in exon 1) and MICA*009:01/049 (nonsynonymous substitution in exon 6), could be resolved with our method. Analysis of Hardy-Weinberg equilibrium for our cohort showed no significant difference between expected and observed frequencies of MICA alleles (P = 0.6142). The three most frequent alleles in our blood donor cohort were MICA*008:01/008:04 (40.5%), MICA*002:01 (13.2%), and MICA*009:01/049 (8.6%). The 7T polymorphism was observed in 67.7% and the 8T polymorphism in 32.3% of our blood donor cohort. Individuals (24.5%) tested were homozygous. The approach described in this paper is suitable for accurate sequencing of large sample numbers, including direct readout of exon 5 sequences. It is compatible with laboratory automation and commercial human leukocyte antigen analysis software tools. It may therefore be applied in large clinical trials.

The frequency of DRB1*1454 in South German Caucasians.

Human leukocyte antigen (HLA)-DRB1*1454 differs from HLA-DRB1*1401 in only one position in exon 3. Before description of this polymorphism both alleles were routinely typed as HLA-DRB1*1401. This prompted our study on relative frequency of these alleles. We determined relative frequencies of DRB1*1454 and DRB1*1401 by sequence-based tying (SBT) in 106 samples and found DRB1*1454 in 87.9% and DRB1*1401 in 12.1% of previously DRB1*1401/54 ambiguous tested individuals. Population frequencies were estimated using data from the local bone marrow donor database. Phenotype and genotype frequency of DRB1*1454 and DRB1*1401 were 5.592%, 0.2828, and 0.773%, 0.00391, respectively. The corresponding figures for DRB1*1404 were 0.238% and 0.00119. Other DRB1*14 alleles were very rare. The most frequent haplotype was DRB1*1454-DRB3*0202-DQB1*0503. Although DRB1*1454 is almost exclusively associated with DRB3*0202, DRB1*1401 is linked with either DRB3*0201 or DRB3*0202. HLA-DRB1*1454 is the most common DRB1*14 allele among German Caucasians and should be considered as a preferred allele over DRB1*1401.

TH1/TH2 cytokines and soluble CD30 levels in kidney allograft patients with donor bone marrow cell infusion.

OBJECTIVE: We investigated the relevance of donor bone marrow cell infusion (DBMI) and serum levels of interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and soluble CD30 (sCD30) in kidney recipients. PATIENTS AND METHODS: We analyzed the allograft outcomes correlated with sCD30, IFN-gamma, and IL-10 levels using pre- and posttransplantation sera from 40 live donor renal transplants (20 patients with DBMI [2.1 x 10(9) +/- 1.3 x 10(9) mononuclear cells/body] and 20 controls). RESULTS: Patients with acute rejection episodes (ARE)-3/20 DBMI and 6/20 controls-showed increased sCD30 and IFN-gamma as well as decreased IL-10 posttransplantation compared with nonrejectors. Significant differences were observed for sCD30 and IFN-gamma levels: 59.54 vs 30.92 ng/mL (P = .02) and 11.91 vs 3.01 pg/mL (P = .01), respectively. Comparison of pre- and posttransplant levels of IFN-gamma, IL-10, and sCD30 in ARE patients showed higher levels in posttransplant sera except for IFN-gamma in controls (6.37 vs 11.93; P = .01). Increased IFN-gamma and IL-10 were correlated with rejection (r = .93; P = .008). sCD30 correlated with serum creatinine among ARE patients in control and DBMI groups (r = .89; P = .019; and r = 1.00; P < .0001, respectively). CONCLUSIONS: Higher levels of sCD30, IFN-gamma, and IL-10 posttransplantation in rejecting patients provided evidence for coexistence of cellular and humoral responses in ARE. There appeared to be a down-regulatory effect of infusion on alloresponses.

Genetic association of interleukin-4, interleukin-10, and transforming growth factor-beta gene polymorphism with allograft function in renal transplant patients.

Despite advances in immunosuppressive therapy in the past decade, allograft rejection remains the primary cause for kidney graft failure. Cytokines are known to be important mediators in renal allograft outcome. The aim of the present study was to ascertain whether interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-beta cytokine gene polymorphisms contributed to kidney graft outcome. We evaluated single nucleotide polymorphism in IL-4 (-1098G/T, -590C/T, -33C/T), IL-10 (-1082A/G, -819C/T, -592A/C), and TGF-beta (codon 10 and 25) in 100 renal transplant recipients and 139 normal healthy control using polymerase chain reactions based on sequence-specific primers. Recipients were clinically characterized as rejection episode (RE) versus stable graft function (SGF). The results showed the frequencies of IL-4 -33 T allele in the RE, SGF, and control group to be 7%, 73%, and 28%, respectively. IL-10 -592 A allele frequency was 39% in RE, 26% in SGF, and 28% in the control group. TGF-beta codon 10 T allele was 39% in RE, 35% in SGF, and 53% in control group. In conclusion, this study suggested that some cytokine gene alleles reflected SGF among kidney transplant recipients.