Reconstruction of bone marrow microenvironment for expansion of hematopoietic stem cells by a histone deacetylase inhibitor.

Ex vivo expansion of hematopoietic stem cells (HSCs) is an approach for overcoming cell insufficiency for umbilical cord blood transplantation. It was suggested that in common ex vivo cultures, the stemness specificity of HSCs is rapidly reducing due to DNA hypermethylation. Here, Nicotinamide (NAM), a DNA methyltransferase and histone deacetylase inhibitor, is used with a bioengineered Bone Marrow-like niche (BLN) for HSC ex vivo expansion. The CFSE cell proliferation assay was used for tracking HSCs division. qRT-PCR was conducted to assay the HOXB4 mRNA expression levels. The morphology of BLN-cultured cells was analyzed using scanning electron microscopy (SEM). NAM boosted the induction of HSC proliferation in the BLN group compared to the control group. In addition, the ability of HSCs to colonize was more significant in the BLN group than in the control group. Our data suggest that the presence of NAM in bioengineered niches promotes HSC proliferation. The presented approach showed that small molecules could be used in the clinical setting to overcome the limited number of CD34+ cells in cord blood units.

Identification of two novel FUT1 mutations in people with Bombay phenotype from Iran.

BACKGROUND AND PURPOSE: Bombay and Para-Bombay phenotypes are characterized by FUT1 gene mutation and lack of H antigen expression in red blood cells. ABH antigens are not present in the body secretions of Bombay individuals, while they are expressed in the secretions of para-Bombay. The aim of this study was to investigate the molecular basis of FUT1 and FUT2 genes in Iranians with the Bombay or Para-Bombay phenotype. MATERIALS AND METHODS: ABO phenotype analysis and routine serological tests were performed on 11 people with Bombay and Para-Bombay phenotypes. The coding regions of FUT1 and FUT2 genes were amplified by PCR followed by sequencing. The ABO genotypes were also determined by sequencing exons 6 and 7 of the ABO gene. RESULTS: Serological investigations confirmed the Bombay phenotype in 8 samples and the Para-Bombay phenotype in 3 samples. Family members with the Bombay phenotype had the classic c 0.725 T > G mutation in the FUT1 gene, accompanied by deletion of the FUT2 gene. Other samples had c.653 A>G, c 0.661 C>T, c 0.652 C>G, and c.722 A>C mutations in the FUT1 while FUT2 was silenced by c 0.461 G>A. CONCLUSION: In this research, we identified two novel mutations in the FUT1 gene in individuals with the Bombay phenotype. This and previous works confirm the variety of FUT1 mutations.

Mannose binding lectin-associated serine protease 2 (MASP2) gene polymorphism and susceptibility to human T-lymphotropic virus type 1 (HTLV-1) infection in blood donors from Mashhad, Iran.

Mannose binding lectin-associated serine protease 2 (MASP2) is the effector part of mannose binding lectin (MBL) that activates the complement system in an antibody-independent manner. We aimed to investigate the role of genetic polymorphisms in the MASP2 gene and susceptibility to HTLV-1 infection. A total of 172 HTLV-1 infected individuals and 170 healthy blood donors were analyzed in this case-control study. Nine single nucleotide polymorphisms (SNPs) encompassing different regions of the MASP2 gene were genotyped with a polymerase chain reaction-sequence-specific primer (PCR-SSP) assay. The relation between the SNPs genotype and the susceptibility to HTLV-1 infection was investigated with a χ2 test considering P < 0.05 as statistically significant. Two of nine tested SNPs were associated with the risk of HTLV-1 infection. The genotype TT at rs17409276 decreased the risk of HTLV-1 (P = 0.005, OR = 0.301, 95% CI = 0.124-0.728). The genotypes CC and CT at rs2273346 were also associated with a higher risk of HTLV-1 acquisition (P = 0.004, OR = 2.225, 95% CI = 1.277-3.877). These findings highlight the importance of MASP2 genetic polymorphisms in the lectin pathway of complement activation and susceptibility to HTLV-1 infection.

HLA-E*01:01 allele is associated with better response to anti-HCV therapy while homozygous status for HLA-E*01:03 allele increases the resistance to anti-HCV treatments in frequently transfused thalassemia patients.

BACKGROUND: HLA-E binding to NKG2A/CD94 induces inhibitory signals that modulate NK cells cytotoxicity against infected targets. HCV-derived peptides stabilize HLA-E molecule that favours its higher expression. However, HLA-E stability and expression vary in different genotypes where the presence of HLA-E*01:03 allele is associated with higher HLA-E expression on targets that enhances NK cells inhibition and increases the chance of virus to escape from innate immune system. Here, we aimed to investigate whether HLA-E polymorphism affects HCV infection status or its treatment in major thalassemia patients who are more vulnerable to hepatitis C. METHODS AND MATERIALS: Study included 89 cases of major thalassemia positive for HCV-antibody; of those 17 patients were negative for HCV-PCR (spontaneously cleared) and 72 patients were HCV-PCR positive (persistent hepatitis under different anti-viral treatment). 16 major thalassemia patients without hepatitis, negative for HCV-antibody were also considered as patients control group. Genomic DNAs extracted from whole bloods were genotyped for HLA-E locus using a sequence specific primer-PCR strategy. RESULTS: In thalassemia patients, HLA-E*01:03 allele increased susceptibility to HCV infection [p = 0.02; 4.74(1.418-15.85)]. In addition, HLA-E*01:03/*01:03 genotype predicted more resistance to HCV treatment compared to other genotypes [p = 0.037; 3.5(1.1-11.4)]. In other words, we found that the presence of HLA-E*01:01 allele favors better response to anti-HCV therapy [p = 0.037; 3.5(1.1-11.4)]. CONCLUSION: From a mechanistic point of view, the associations between HLA-E polymorphisms and susceptibility to HCV infection or its therapeutic resistance in thalassemia patients may suggest potential roles for the innate and adaptive immune responses to this infection, which are manifested by the acts of HLA-E - NKG2A/CD94 axis in the modulation of NK cell inhibitory function as well as HLA-E associated CD8+ T cell cytolytic activity against HCV, respectively. Notably, from a clinical point of view, paying attention to these associations may not only be useful in increasing the effectiveness of current anti-HCV regimens comprising direct acting antivirals (DAAs) in more complicated patients, but may also suggest antiviral prophylaxis for patients more vulnerable to HCV infection.

An Easy Multiplex PCR-SSP Assay for the Genotyping of KEL1 and KEL2 in Multi-transfused Patients.

Kell blood group system consists of 34 antigens. KEL1 and KEL2 are the most clinically important antigens of this system, causing hemolytic disease of the fetus and newborn (HDFN) and transfusion reaction. A total of 200 samples from blood donors were tested serologically for the presence of KEL1 and KEL2 antigens on erythrocytes. Genomic DNA was analyzed by PCR-SSP method to determine the Kell genotype. A multiplex PCR-SSP assay was designed and tested to genotype KEL1/KEL2 alleles in a single reaction. PCR genotyping revealed samples as; KEL2/KEL2 (93.5%) and KEL1/KEL2 (6.5%), while no sample determined as KEL1/KEL1. A 100% concordance observed between PCR and serological results. Multiplex PCR accurately diagnosed Kell genotype. Kell blood group genotyping by PCR-SSP can be used as an alternative method, especially in multi-transfused patients where serological findings are ambiguous.

miRNA96 expression level within red blood cells is probably associated with RSL indicators during the storage of red blood cell units.

BACKGROUND AND OBJECTIVES: Many biochemical and hematological changes occur during the storage of RBC units. Collectively, these changes are known as RSLs. Previous studies found miRNA96 as non-coding RNA that its expression level changed during RBC storage. However, its correlation with mechanical and biochemical RSL indicators is not yet determined. Therefore, this study aimed to assess possible correlations between miRNA96a and some RSLs indicators to clarify its biomarker capability for evaluating the storage quality of RBC units. MATERIALS AND METHODS: Samples were collected from ten leuko-reduced RBC units on days 0, 14, 28, and 42 of storage. miRNA96 gene expression level and RSLs indicators including hemolysis, mechanical fragility index (MFI), total antioxidant capacity (TAC), lipid peroxidation (TBARs), thiol groups, and RBC indices were measured on the days mentioned above. RESULTS: Significant correlations were found between the changes in miRNA96 expression level and the levels of hemolysis, TAC, TBARs, and MFI indices (p values < 0.05). The donors were classified into the high risk group and low risk group, according to four important characteristics and lifestyle habits (smoking, physical activity, age, and BMI). The high risk group had a significantly lower rate of hemolysis, free hemoglobin, MFI, TAC, and a higher rate of lipid peroxidation compared to low risk group (p values < 0.05). CONCLUSION: The finding suggested that upregulation of miRNA96 could prevent hemolysis of RBCs, despite the accumulation of oxidative injuries in them. The miRNA96 expression level was probably a potential predictor for mechanical and biochemical RSL indicators.

BVVL/ FL: features caused by SLC52A3 mutations; WDFY4 and TNFSF13B may be novel causative genes.

Brown-Vialetto-Van Laere (BVVL) and Fazio-Londe are disorders with amyotrophic lateral sclerosis-like features, usually with recessive inheritance. We aimed to identify causative mutations in 10 probands. Neurological examinations, genetic analysis, audiometry, magnetic resonance imaging, biochemical and immunological testings, and/or muscle histopathology were performed. Mutations in known causative gene SLC52A3 were found in 7 probands. More importantly, only 1 mutated allele was observed in several patients, and variable expressivity and incomplete penetrance were clearly noted. Environmental insults may contribute to variable presentations. Putative causative mutations in other genes were identified in 3 probands. Two of the genes, WDFY4 and TNFSF13B, have immune-related functions. Inflammatory responses were implicated in the patient with the WDFY4 mutation. Malfunction of the immune system and mitochondrial anomalies were shown in the patient with the TNFSF13B mutation. Prevalence of heterozygous SLC52A3 BVVL causative mutations and notable variability in expressivity of homozygous and heterozygous genotypes are being reported for the first time. Identification of WDFY4 and TNFSF13B as candidate causative genes supports conjectures on involvement of the immune system in BVVL and amyotrophic lateral sclerosis.

Hepcidin gene polymorphisms and iron overload in ß-thalassemia major patients refractory to iron chelating therapy.

BACKGROUND: ß Thalassemia is one of the most common groups of hereditary haemoglobinopathies. Affected people with thalassemia major are dependent on regular blood transfusion which on the long term leads to iron overload. Hepcidin is a peptide hormone and an important regulator of iron homeostasis, especially in thalassemia. Expression of this hormone is influenced by polymorphisms within the hepcidin gene, HAMP. Several studies emphasized the role of single nucleotide polymorphisms (SNPs) located in the promoter region of the gene. This study aimed to analyze the association between three SNPs in promoter of HAMP, c.-582A > G, c.-443C > T, and c.-153C > T, with iron overload in ß-thalassemia major patients. METHODS: A total of 102 samples from ß thalassemia major patients were collected. Genomic DNA was extracted and segments of DNA encompassing rs10421768 and rs142126068 were sequenced. Statistical analysis was performed by SPSS Statistics 23 using independent t test and Fisher's exact test. RESULTS: A total of 102 adult ß-thalassemia major patients were genotyped for three SNPs in the promoter region of HAMP gene by PCR and direct sequencing. Most of the patients (71.3%) were iron overloaded (based on plasma ferritin > 1000 ng/ml) in spite of receiving regular iron-chelating therapy. Our analysis revealed a statistically significant difference between the level of cardiac iron accumulation and c.-582A > G variant (p = 0.02). For c.-443C > T statistical analysis was on the edge of the significant relationship between the minor allele and serum ferritin (p = 0.058). All samples were homozygous for allele C of c.-153C > T. CONCLUSIONS: Despite chelating therapy, iron overload is still one of the main complications of thalassemia. Our findings and others emphasize the role of hepcidin -582A > G polymorphism as a key component of iron homeostasis in these patients.

Association between -221 X/Y polymorphism of mannose-binding lectin (MBL) gene and susceptibility to HTLV-1 infection among people from an endemic region in the Northeast of Iran.

BACKGROUND: The role of (MBL) gene single nucleotide polymorphisms (SNPs) has been well documented in susceptibility to several infectious diseases. This study aimed to investigate the association between two MBL promoter variants, -550 H/L and -221 X/Y, and susceptibility to HTLV-1 infection. METHODS: A total of 153 subjects infected with HTLV-1 and 169 healthy controls were recruited. SSP-PCR method was applied to genotype -550 H/L and -221 X/Y polymorphisms. Associations between genotypes or alleles and susceptibility to HTLV-1 infection were analyzed by Pearson's Chi-Square. p ≤ .05 was considered statistically significant. RESULTS: Statistical analysis revealed significant differences between the two groups in the -221 position (χ2 = 19.709; p = .000). The MBL YX genotype was significantly associated with increased susceptibility to HTLV-1 (OR = 2.73, %95 CI = 1.74-4.30). Combined genotype of the two loci showed that the HYHX genotype (OR = 2.20, 95% CI = 1.95-2.48) and LYLX (OR = 1.97, 95% CI = 1.13-3.45) were associated with an increased risk of HTLV-1 infection. CONCLUSION: Our results represent the importance of -221 X > Y variants in acquisition of HTLV-1 as this is the case for several other viral and bacterial infections.

Mutational Analysis of Bombay Phenotype in Iranian People: Identification of a Novel FUT1 Allele.

Bombay phenotype is characterized by lack of ABH antigens on RBCs and in body secretions as a result of mutations in fucosyltransferase 1 (FUT1) and fucosyltransferase 2 (FUT2) genes. The aim of this study was a mutational analysis in Iranians with this phenotype. Serological analyses including ABH and adsorption-elution tests were performed in five unrelated Bombay individuals. ABO genotyping was determined by direct sequencing. The coding regions of FUT1 and FUT2 genes were amplified and sequenced directly or after cloning into suitable vector. A novel missense FUT1 allele was detected (G1051T; G351C). Also four reported FUT1 alleles were revealed. Molecular analysis of FUT2 gene confirmed nonsecretor status in all individuals. This and our previous findings suggest the diversity and population specificity of FUT1 alleles.

Production of Universal Group O Red Blood Cells by Alpha-N-Acetylgalactosaminidase Enzyme Expressed in Pichia pastoris.

Enzymatic removal of blood groups antigens A and B is an efficient method for production of universal red blood cells. In this research, an α-N-acetylgalactosaminidase (NAGA) enzyme was expressed in Pichia pastoris for digestion of the A blood antigen. DNA sequence of the gene NAGA, originally expressed in Elizabethkingia meningosepticum (NAGA-EM), was ordered for optimization and synthesis. It was then expressed in P. pastoris (KM71H and GS115 strains). Expression of the recombinant NAGA was evaluated by dot blot, SDS-PAGE, and Western blotting. The activity of the enzyme was measured using a synthetic substrate in addition to the conversion of group A red blood cells to the O cells. Expression of NAGA-EM with an apparent molecular mass of 55 kDa was verified by dot blot, SDS-PAGE and Western blot analysis. The maximum enzyme activity in the supernatant of KM71H was higher than that in the GS115 (250 vs. 200 U/ml). Treated group A RBCs did not react with the anti-A antiserum or with the sera from individuals with blood groups B and O. The results of this study indicated that NAGA-EM is an efficient enzyme for production of universal O blood cells.

No Evidence of HTLV-II Infection Among Immonoblot Indeterminate Samples Using Nested PCR in Mashhad, Northeast of Iran.

OBJECTIVE(S): Although HTLV-I infection is endemic in different geographical parts of the world including Northeast of Iran, there have been no documents of HTLV-II infection in this region. It is reported that one possible reason for seroindeterminate state in HTLV western blot is HTLV-II virus. This study aimed to investigate the presence of HTLV-II among blood donors with seroindeterminate western blot results. MATERIALS AND METHODS: Three ml whole blood obtained from 50 blood donors referring to Mashhad Blood Transfusion Organization who had reactive Elisa for HTLV-I and seroindeterminate HTLV western blot state. A conventional PCR was applied to detect HTLV-I provirus using specific primers while a nested PCR was designed with specific external and internal primers for the detection of HTLV-II. RESULTS: The average age of participants, 39 males and 11 females, was 37.12± 14.36 years. The average OD of the Elisa assay was 1.767± 1.195. The most common indeterminate patterns were Rgp46-II alone (n=12, 27.3%), Rgp46-I alone (n=7, 15.9%), and Rgp46-I with GD21 (n=7, 15.9%).After introducing the DNA to the PCR tests, results revealed 10 (20%) HTLV-I PCR positive samples while no HTLV-II positive sample was detected by nested PCR. There were no significant age, blood group, Optical Density of the Elisa assay, and western blot indeterminate pattern differences between HTLV-I PCR positive and negative samples. Conclusion : No HTLV-II positive sample was detected in this study which confirms the absence of HTLV-II infection in this region. However, high frequency of HTLV-I PCR positive samples among the seroindeterminate cases implies on the important role of molecular techniques for further confirmation of the infection.

Cutaneous Manifestations in HTLV-I Positive Blood Donors.

OBJECTIVE(S): Infection with the human T-cell lymphotrophic virus type-I (HTLV-I) is endemic in Mashhad, Iran. In our research we evaluated the relation between exposure to this infection and the occurrence of dermatologic manifestations. MATERIALS AND METHODS: 100 blood donors, who were seropositive but asymptomatic for infection with HTLV-I, were selected as case group. They were identified by the Blood Transfusion Organization Mashhad via the ELISA test and documented by PCR. Another 100 blood donors, that were seronegative for HTLV-I via the ELISA test and who were matched to the case group for age, gender, and existence of systemic diseases, were considered as the controls. Dermatologic evaluations and skin biopsies were performed if deemed necessary, and the results were statistically analyzed. RESULTS: 73% of the case and control groups were male, while 27% in each of these groups were female. The mean age in both groups was 40.96±11.94 years. The examination indicated that 58% of the case group and 37% of the control group had cutaneous manifestations (P<0.01). The most common diseases found in the case group were aphthous stomatitis, herpes labialis, and non-genital warts, while common diseases found in the control group were herpes labialis, aphthous stomatitis, and skin tag. The frequency of aphthous stomatitis, eczema, and non-genital warts in the case group were significantly more than the control group (P<0.05). Conclusion : Cutaneous diseases can be found more frequent in asymptomatic carriers of HTLV-I than those who are HTLV-I seronegative. The aphthous stomatitis, eczema, and non-genital warts are more prevalent in those infected by HTLV-I.

Molecular analysis of human T cell lymphotropic virus type 1 and 2 (HTLV-1/2) seroindeterminate blood donors from Northeast Iran: evidence of proviral tax, env, and gag sequences.

Human T cell lymphotropic virus type 1 and 2 (HTLV-1/2) Western blot indeterminate results are a problem for blood banks in endemic areas. To determine the prevalence of HTLV-1/2 infection among indeterminate donors, we analyzed 130 cases from Mashhad, an HTLV-1/2 endemic area in Northeast Iran. The most frequent Western blot bands were GD21 alone (37.2%) followed by rgp46-2 alone (32.1%). We further tested 40 available DNA samples of these cases by PCR for viral sequences, tax, gag, and pol, and found five cases (12.5%) to be positive for two or three HTLV-1 genes. There were no significant age, sex, and blood group differences between PCR-positive and PCR-negative cases. Among PCR-positive individuals, the most prevalent Western blot bands were variable combinations of rgp46-1, GD21, and gp21. The mean of the optical density (OD) of the enzyme-linked immunosorbent assay (ELISA) test was significantly higher in PCR-positive individuals. The frequency of the rgp46-1 band was also significantly higher in PCR-positive cases compared to PCR-negative ones. In conclusion, the majority of HTLV-indeterminate donors lack the HTLV provirus and therefore are not considered infected. However, in some cases with higher ODs in the ELISA test and seroreactivity to env proteins, rgp46-1 and GD21 in particular may be indicative of infection and need further evaluation by molecular methods.