Determining reference ranges for lymphocyte proliferation responses to phytohemagglutinin and Bacillus Calmette-Guérin in Iranian children.

PURPOSE: To establish reference ranges (RRs) for stimulation index of T cell proliferation triggered by phytohemagglutinin (PHA-SI) and Bacillus Calmette-Guérin (BCG-SI). METHODS: This study investigated data from 359 healthy children and 35 patients with cellular immunodeficiency as positive controls (2010-2021). We applied a colorimetric-based method (BrdU) to measure proliferation and determine the RRs at the 2.5th and 97.5th percentiles (95% confidence intervals). A cross-validation approach was performed. RESULTS: In healthy controls, the RRs for PHA-SI and BCG-SI ranged between 3 and 5.2 and 2.52 to 5.2, respectively. PHA-SI and BCG-SI were in Severe Combined Immunodeficiency (SCID) patients from 1.2 to 2.5 and 0 to 2, while in Mendelian susceptibility to mycobacterial diseases (MSMD) patients, 2.53 to 4.5 and 0.74 to 2.2, respectively. The thresholds' accuracy was checked for testing reference intervals with diagnostic effects. CONCLUSION: This study establishes PHA-SI and BCG-SI reference ranges to aid in diagnosing and treating congenital immunodeficiency diseases.

Confirmation of Hyperimmunoglobulin E Syndrome in Two Patients with an Ocular Problem: Detection of Two New DOCK8 Mutations.

Early diagnosis of primary immunodeficiencies is crucial for timely treatment and preventing unwanted complications. Next-generation sequencing (NGS) and detailed clinical and immunological evaluation can help early detect such disorders. This study aimed to confirm the diagnosis of two cases of autosomal recessive hyper-immunoglobulin E (IgE) syndrome (AR-HIES), presenting with irreversible eye involvement. Two unrelated patients with suspected AR-HIES were referred to the Immunology, Asthma and Allergy Research Institute (IAARI), Tehran, Iran. Immunological screening tests were performed for AR-HIES, which showed elevated serum IgE levels, eosinophilia, and low T-lymphocyte responses. NGS was performed, and the results were confirmed by Sanger sequencing. Sequence analysis showed a mutation in intron 17 of the dedicator of cytokinesis 8 (DOCK8) gene in the first patient, and a homozygous three base-pair deletion in exon 45 of DOCK8 in the second patient. This is the first time such mutations are reported and these variants are predicted to be damaging. Both patients suffered from persistent viral infections along with cytomegalovirus (CMV) retinitis. Suspicion of these two novel DOCK8 mutations can benefit patients presenting with recalcitrant ophthalmic viral involvements and relevant immunological test results. This would lead to earlier referrals for immunologic and genetic confirmation and thus, a more timely intervention with hematopoietic stem cell transplantation (HSCT).

Genetic Study of Hereditary Angioedema Type I and Type II (First Report from Iranian Patients: Describing Three New Mutations).

BACKGROUND: Hereditary Angioedema (HAE) is a rare autosomal dominant immunodeficiency disease with mutation in C1 inhibitor gene (SERPING1) which deficient and dysfunction of C1-INH protein result in HAE type I or type II, respectively. The present study aimed to define the genetic spectrum of HAE type I and type II among Iranian patients. METHODS: Thirty-four patients with clinical phenotype of recurrent edematous attacks in face, upper and lower limbs, hands, and upper airway entered the study. Mutations in SERPING1 were analyzed using PCR and Sanger Sequencing. In addition, Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to discover large deletions or duplications in negative screening samples by Sanger. RESULTS: Twenty-three patients were diagnosed with HAE type I and 11 with HAE type II. Fourteen distinctive pathogenic variations including five frameshift (p.G217Vfs*, p.V454Gfs*18, p.S422Lfs*9, p.S36Ffs*21, p.L243Cfs*9), seven missense (p.A2V, p.G493R, p.V147E, p.G143R, p.L481P, p.P399H, p.R466C), one nonsense (p.R494*), and one splicing defect (C.51 + 2 T˃C), which three of these mutations were identified novel. However, no mutation was found in seven patients by Sanger sequencing and MLPA. CONCLUSION: Final diagnosis with mutation analysis of HAE after clinical evaluation and assessment of C1INH level and function can prevent potential risks and life-threatening manifestations of the disorder. In addition, genetic diagnosis can play a significant role in facilitating early diagnosis, pre-symptomatic diagnosis, early diagnosis of children, asymptomatic cases, and those patients who have the borderline biochemical results of C1-INH deficiency and/or C4.

Investigating the Variation of TREC/KREC in Combined Immunodeficiencies.

T-cell receptor excision circles (TREC)/Kappa-deleting recombination excision circles (KREC) assay has been recently recognized for detecting patients with primary (T- and/or B-cell) immunodeficiency (PID). We aimed to investigate the alterations of these biomarkers in some combined immunodeficiency patients compared to the healthy controls in different age groups. TREC and KREC were assessed in a total of 82 PID patients, most of them with exact genetic diagnosis (3 months to 42 years); using quantitative real-time-polymerase chain reaction (PCR). Patients had a final diagnosis of common variable immunodeficiency (n=23), ataxia-telangiectasia (AT) (n=17), hyper-IgE syndrome (HIES) (7 with DOCK8 deficiency, 4 with signal transducer and activator of transcription 3 (STAT3) deficiency, and 8 children with unknown genetic defects), Wiskott-Aldrich syndrome (WAS) (n=20), purine nucleoside phosphorylase (PNP)deficiency(n=1), dedicator of cytokinesis2 (DOCK2) deficiency (n=1), recombinase activating gene1 (RAG1) deficiency (n=1). Very low to zero amounts of TREC and/or KREC were detected in 14 out of 23 cases of common variable immunodeficiency (CVID), 14 out of 17 cases of AT, 8 out of 20 cases of WAS, 6 out of 7 cases of DOCK8-deficiency patients, 4 out of 8 cases of HIES with unknown genetic defects and all patients with defects in DOCK2, PNP, and RAG1. STAT3-deficient patients were normal for both biomarkers. All patients showed a significant difference in both markers compared to age-matched healthy controls. Our findings highlight that apart from severe types of T/B cell defects, this assay can also be used for early diagnosis the patients with late-onset of disease and even PIDs without a positive family history.

Genetic Analysis of a Cohort of 275 Patients with Hyper-IgE Syndromes and/or Chronic Mucocutaneous Candidiasis.

Hyper-IgE syndromes and chronic mucocutaneous candidiasis constitute rare primary immunodeficiency syndromes with an overlapping clinical phenotype. In recent years, a growing number of underlying genetic defects have been identified. To characterize the underlying genetic defects in a large international cohort of 275 patients, of whom 211 had been clinically diagnosed with hyper-IgE syndrome and 64 with chronic mucocutaneous candidiasis, targeted panel sequencing was performed, relying on Agilent HaloPlex and Illumina MiSeq technologies. The targeted panel sequencing approach allowed us to identify 87 (32 novel and 55 previously described) mutations in 78 patients, which generated a diagnostic success rate of 28.4%. Specifically, mutations in DOCK8 (26 patients), STAT3 (21), STAT1 (15), CARD9 (6), AIRE (3), IL17RA (2), SPINK5 (3), ZNF341 (2), CARMIL2/RLTPR (1), IL12RB1 (1), and WAS (1) have been detected. The most common clinical findings in this cohort were elevated IgE (81.5%), eczema (71.7%), and eosinophilia (62.9%). Regarding infections, 54.7% of patients had a history of radiologically proven pneumonia, and 28.3% have had other serious infections. History of fungal infection was noted in 53% of cases and skin abscesses in 52.9%. Skeletal or dental abnormalities were observed in 46.2% of patients with a characteristic face being the most commonly reported feature (23.1%), followed by retained primary teeth in 18.9% of patients. Targeted panel sequencing provides a cost-effective first-line genetic screening method which allows for the identification of mutations also in patients with atypical clinical presentations and should be routinely implemented in referral centers.

In Vitro Activity of Pentamidine Isethionate against Trophozoite and Cyst of Acanthamoeba.

BACKGROUND: Acanthamoebae are a causative agent of Acanthamoeba keratitis (AK) in immunocompetent individuals. Since access to propamidine isethionate (Brolene®) as a first-line treatment has been limited in recent years, in the current study, we examined the effects of pentamidine isethionate against trophozoite and cyst forms of Acanthamoeba. METHODS: This experimental study was conducted in the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, during 2019-2020. Pentamidine isethionate at concentrations of 50, 100, 200, 400, 600, 800, and 1000 µM were tested against trophozoites and cyst stages of T4 genotype, at 24- and 48-hour incubation period, and the viability was determined by trypan blue staining. In addition, the cytotoxic effect of the drug was examined in Vero cells using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The 50% inhibitory concentration (IC50) of pentamidine isethionate on trophozoite after 24 and 48h were 97.4 µM and 60.99 µM. These results on cyst after 24 and 48h were 470 µM and 175.5 µM, respectively. In MTT assay, the drug showed an inhibitory effect on Vero cell growth with IC50 values of 115.4 µM and 87.42 µM after 24h and 48h, respectively. CONCLUSION: Pentamidine isethionate exhibited an inhibitory effect on trophozoite and cyst. Given that the trophozoicidal activity of the drug is in the safe dose, it could be suggested as an alternative in patients with AK; however, further investigation is needed in an animal model to confirm the data.

Proinflammatory effects of dust storm and thermal inversion particulate matter (PM10) on human peripheral blood mononuclear cells (PBMCs) in vitro: a comparative approach and analysis.

Particulate matter (PM) as the carcinogenic air pollutants can lead to aggravated health outcomes. Epidemiological studies demonstrated that PM can be engaged in different diseases such as cardiovascular, respiratory and cancer. The in vitro secretion of proinflammatory cytokines by human peripheral blood mononuclear cells (PBMCs) has been used to assess the effects of PM with an aerodynamic diameter < 10 µm (PM10). This study compared the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1-beta (IL1-ß) secretions of PBMCs exposed to PM10 of dust storm and inversion. We collected PM10 samples during the spring and autumn seasons in two locations. Isolated PBMCs were exposed separately to 50, 150, and 300 µg/ml of different type of PM10 for 4 and 24 h. The mean concentrations of TNF-α for the PM of dust storm and inversion were 6305.61 ± 2421 and 6651.74 ± 2820, respectively. Also the mean concentrations of IL1-ß for the PM of dust storm and inversion were 556.86 ± 162 and 656.35 ± 196, respectively. Furthermore, these values for the production of IL-6 were 12,655 ± 5661 and 16,685 ± 8069, respectively. Although no significant difference was observed between the PM of dust storm and that of inversion with regard to PBMCs, the results showed a significant increase in the proinflammatory cytokine secretion of both PMs compared with the controls. Moreover, TNF-α, IL1-ß, and IL-6 secreted in cells exposed to PM10 of dust storm were about 10 times more than the controls, these values for cells exposed to PM10 of inversion were around 10, 12, and 14 times more than the controls, respectively. It can be concluded that the PM10 of both dust storm and inversion can play a significant role in proinflammatory cytokine secretion due to its harmful effect on human health. Graphical abstractThis picture shows the Proinflammatory cytokine producing potential of PM10 with two sources (dust storm and urban air pollution) in exposure with human PBMCs in vitro.

DOCK8 deficiency in six Iranian patients.

DOCK8 deficiency is a rare autosomal recessive combined immunodeficiency with high IgE level, eosinophilia, severe eczema, extensive cutaneous viral, and respiratory bacterial infections, mostly in populations with higher prevalence of consanguinity. Molecular diagnosis of this gene is a useful approach for early diagnosis and timely HSCT due to deleterious consequences.

Mannose-binding Lectin Deficiency in Patients with a History of Recurrent Infections.

Mannose-binding lectin (MBL) is a protein of innate immune system that is involved in opsonization and complement activation. MBL deficiency is associated with predisposition to infectious diseases; however subnormal levels are also seen in healthy subjects. The aim of this study was to investigate the prevalence and clinical manifestation of MBL deficiency in patients with increased susceptibility to infection. We studied the MBL serum concentration of 104 patients with a history of recurrent and/or severe infections referred to Immunology, Asthma and Allergy Research Institute (IAARI) in order to evaluate the primary immunodeficiency (PID). The distribution of MBL deficiency in these patients and 593 healthy subjects of previous study were analyzed. The frequency of individuals with MBL deficiency was significantly higher in patients with recurrent and/or severe infections (13.5% [14/104]) compared with healthy subjects (4.7% [28/593]; p=0.001; OR 3.1, 95% CI 1.5-6.1). However, in 10.9% (7/64) of patients with recurrent infections without any immunodeficiency background, the MBL deficiency was detected. On the whole, our findings indicate an association between MBL deficiency and increased susceptibility to infections.

The prevalence of Selective Immunoglobulin M Deficiency (SIgMD) in Iranian volunteer blood donors.

BACKGROUND: Selective Immunoglobulin M Deficiency (SIgMD) is known as a rare primary immunodeficiency characterized by an isolated deficiency of serum IgM. Other immunoglobulin levels and T-cell immunity are usually normal; although IgE may be elevated. SIgMD can be asymptomatic or with various bacterial and viral infections. It can also be associated with autoimmune diseases or malignancies. In the present study, we report for the first time, the prevalence of SIgMD in Iranian healthy adult population. MATERIALS AND METHODS: A total of 3436 healthy donors were examined in the study; from August, 2006 to April, 2008. Serum IgM concentration was measured using the nephelometric method. We considered serum IgM less than 30 mg/dl as IgM deficiency. RESULTS: Among 3436 participants, 65% were male and 34% were female; aging from 17 to 72 years (38.18±10.78). Thirteen individuals were detected as IgM deficient subjects with the male to female ratio of 11/2, the prevalence of 0.37% and the frequency of 1/265. The mean serum IgM level was 24±4.56 (16-29 mg/dl) in these cases. Among 13 IgM-deficient subjects, 7 cases were available for evaluating the clinical manifestations. In addition to atopic dermatitis which was the most common symptom in these patients, others were allergic rhinitis, food allergy, urinary tract infection and skin fungal infection. Two patients had no history of infectious disease or atopic conditions. CONCLUSION: In the present study we could determine the prevalence of SIgMD in our adult population (0.37%). The most common comorbid condition was atopy. Neither severe or life-threatening infections, nor autoimmune diseases (based on their history; the antibody screening was not performed as part of this study) or malignancies were found in these patients. Further evaluation is recommended to elucidate the prevalence of SIgMD among patients with recurrent infections.

Type I and Type II Hereditary Angioedema: Clinical and Laboratory Findings in Iranian Patients.

BACKGROUND: Hereditary angioedema (HAE) is a rare autosomal dominant disorder characterized by C1-INH (C1 esterase inhibitor), low serum levels (type I), dysfunction (type II) or normal serum levels and function (type III), which lead to subcutaneous and submucosal edema attacks. The aim of this study was to investigate the demographic, clinical and laboratory findings of Iranian patients with HAE. METHODS: The patients with a history or symptoms of angioedema who were referred to Immunology, Asthma and Allergy Research Institute (IAARI) between Jan 2006 and Jan 2014, were assessed based on a specific questionnaire and laboratory evaluation. The patients with a definite diagnosis of HAE type I and type II were entered into this study. RESULTS: Among 51 patients, 63.3% were diagnosed with HAE type I and 36.7% with HAE type II. Fifteen patients were under 18 years and 36 were adults. The mean age of symptoms onset and diagnosis were 12.33 ± 10.20 years and 24.48 ± 14.64 years, respectively. The mean delay of diagnosis was 11.02 ± 11.60 years. The most commonly involved locations of edema were hands, face and genitalia. Moreover, laryngeal edema was observed in 61.2% of patients, which led to death in two patients during this study. CONCLUSION: Hereditary angioedema is a life threatening disease with considerable morbidity and mortality. The outcomes of this study can be used to inform clinicians and health care providers about HAE, which can help earlier diagnosis and better management of the patients, specifically in life threatening attacks.

The extended clinical phenotype of 64 patients with dedicator of cytokinesis 8 deficiency.

BACKGROUND: Mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency (CID) also classified as autosomal recessive (AR) hyper-IgE syndrome (HIES). Recognizing patients with CID/HIES is of clinical importance because of the difference in prognosis and management. OBJECTIVES: We sought to define the clinical features that distinguish DOCK8 deficiency from other forms of HIES and CIDs, study the mutational spectrum of DOCK8 deficiency, and report on the frequency of specific clinical findings. METHODS: Eighty-two patients from 60 families with CID and the phenotype of AR-HIES with (64 patients) and without (18 patients) DOCK8 mutations were studied. Support vector machines were used to compare clinical data from 35 patients with DOCK8 deficiency with those from 10 patients with AR-HIES without a DOCK8 mutation and 64 patients with signal transducer and activator of transcription 3 (STAT3) mutations. RESULTS: DOCK8-deficient patients had median IgE levels of 5201 IU, high eosinophil levels of usually at least 800/µL (92% of patients), and low IgM levels (62%). About 20% of patients were lymphopenic, mainly because of low CD4(+) and CD8(+) T-cell counts. Fewer than half of the patients tested produced normal specific antibody responses to recall antigens. Bacterial (84%), viral (78%), and fungal (70%) infections were frequently observed. Skin abscesses (60%) and allergies (73%) were common clinical problems. In contrast to STAT3 deficiency, there were few pneumatoceles, bone fractures, and teething problems. Mortality was high (34%). A combination of 5 clinical features was helpful in distinguishing patients with DOCK8 mutations from those with STAT3 mutations. CONCLUSIONS: DOCK8 deficiency is likely in patients with severe viral infections, allergies, and/or low IgM levels who have a diagnosis of HIES plus hypereosinophilia and upper respiratory tract infections in the absence of parenchymal lung abnormalities, retained primary teeth, and minimal trauma fractures.

The diagnosis of hyper immunoglobulin e syndrome based on project management.

Hyperimmunoglobulin E Syndrome (HIES) is a complex primary immunodeficiency characterized by both immunologic and non-immunologic manifestations. High serum IgE level, eosinophilia, eczema, recurrent skin and lung infections constitute the immunologic profile of HIES, whereas characteristic facial appearance, scoliosis, retained primary teeth, joint hyperextensibility, bone fractures following minimal trauma and craniosynostosis are the main non-immunologic manifestations. The diagnosis of HIES cannot be made by routine immunologic tests. As the main characteristic laboratory abnormalities of this syndrome are highly elevated serum IgE levels and eosinophilia; both features have a broad spectrum of differential diagnosis. The purpose of this essay was presenting the best way for diagnosis management of HIES. Based on the genetic reports of patients of the Center for Chronic Immunodeficiency (CCI) as a single center experience, and applying project management (PM) in health care research projects, we sought the best way for a rapid diagnosis of HIES. The combination of project management principles with immunologic and genetic knowledge to better define the laboratory and clinical diagnosis lead to an improvement of the management of patients with HIES. These results are shown in one "Decision Tree" which is based on 342 genetic reports of the CCI during the past ten years. It is necessary to facilitate the diagnostic analysis of suspected HIES patients; applying project management in health care research projects provides a better and more accurate diagnosis eventually leading to a better patients' care. This Abstract was presented at 16th Biennial Meeting of the European Society for Immunodeficiencies (ESID 2014), Prague, Czech Republic.

The Effect of IL-22 and IL-28 in Induction of Type 1 Regulatory T (Tr1) Cells.

Cytokines have been introduced as critical inducers in the development of Th subpopulations.Cytokines like IL-10 are involved in inducing regulatory T cells such as Type 1 regulatory T (Tr1) cells cells. IL-22 is a member of IL-10 family of cytokines, and IL-28A is a member of IFN-γ family. In this study, cord blood mononuclear cells (CBMC) from normal healthy individuals were isolated by Ficoll and then naïve T cells were purified by CD4+CD25+ Regulatory T cell Isolation kit. The effect of these two cytokines on production of IL-5, TGF-ß, IL-10, IL-4 and IFN-γ cytokines from cord blood T cells was investigated to identify Tr1 cells as well as Th1 and Th2 polarization. Flow cytometric analysis showed that IL-28A and IL-22 were not effective in expression of IL-5 and TGF-ß either alone or in synergy, but in view of IL-10, IL-4 and IFN-γ, the results showed that IL-22 increased IL-10 and IL-4 but had a decreasing effect on IFN-γ. The results showed that IL-28A was not effective in increasing or decreasing the level of IL-10, IL-4 and IFN-γ. Therefore, according to these results, IL-22 and IL-28A were not effective in inducing Tr1 cells.

Development of a new immunochromatographic assay using gold nanoparticles for screening of IgA deficiency.

A new competitive immunochromatography (ICG) strip test based on polyclonal antibody (pAb) conjugated with gold nanoparticles (NPs) was developed and its applications for primary screening of immunoglobulin (Ig) A in serum were evaluated. Nanocolloidal gold as the detection reagent, with an average particle diameter of 20 nm, was synthesized and labelled pAb. The antibody-nanocolloidal gold probe was applied on the conjugate pad, and human IgA was immobilized on a nitrocellulose membrane as the capture reagent to prepare the ICG strip test. It took only 10 minutes to accomplish a semi-quantitative detection of serum IgA in this assay. In the optimized investigational conditions, the ICG strip test could distinguish human serum IgA in the range from 1 to 270 ng/mL with a detection limit of 5 ng/mL. The reliability of testing procedures was examined by performing the ICG strip test with 11 serum samples and comparing the results with those obtained via enzyme-linked immunosorbent assay (ELISA). The ICG strip was sufficiently sensitive and accurate for a rapid screening of IgA in human serum.

Polymorphisms and serum level of mannose-binding lectin: an Iranian survey.

Mannose-binding lectin (MBL) is a Ca⁺² -dependent collagenous lectin, that is produced by liver and mediates innate immune responses by opsonization of pathogens. The serum level of MBL varies widely among healthy individuals, ranging from 0.05 µg/ml (or lower) to over 5 µg/ml, mainly depending on genetic variation. This study has examined promoter and exon 1 of mbl2 genotype among 117 Iranian healthy blood donors. MBL Single Nucleotide Polymorphisms (SNPs) were genotyped using polymerase chain reaction (PCR), and serum levels of MBL were quantified using a double-antibody enzyme linked immunosorbent assay (ELISA). Results of this study showed that there are two promoter polymorphisms at -550 (H/L variants) and -221 (Y/X variants) positions, and three polymorphisms in exon 1 at codon 52 (D Allele), 54 (B Allele), and 57 (C Allele) in this population. B allele was significantly correlated with the lowest serum MBL level. Our results also showed that the most frequent genotype was HYA/LXA, and the genotype that associated with the highest serum level of MBL was HYA/HYA. The genotype that causes lowest MBL production in Iranian population was LYB/LXA. These results showed some differences compared to that of the other populations. To verify the originality of these differences we may need to extend the study to a larger samples of respective populations; meanwhile the importance of a new mutation, nucleotide 101 of MBL2 exon1, reported in the current study should be taken in considerations in terms of its possible pathobiological effects in following studies.

Mannan-binding lectin serum levels in 593 healthy Iranian children and adults.

Mannan-binding lectin (MBL) is a vital protein of innate immune system and has two critical functions: complement activation through the lectin pathway and opsonization. MBL deficiency has been classified as the most common inherited immunodeficiency known in humans (about 30% of the population), and is associated with predisposition to infections and high risk of some autoimmune diseases. The purpose of this study was to determine the profile of MBL serum level in Iranian healthy population in association with sex and age groups for the first time. We studied the serum concentration of MBL in 593 Iranian healthy cases: 340 males and 235 females in 4 different age groups by using enzyme-linked immunosorbent assay. The mean serum levels of MBL were 3.854 ± 2.77 µg/ml at the age of less than 6 months, 4.147 ± 3.54 µg/ml at 6 months to 2 years of age, 4.410 ± 3.09 µg/ml at 2-6 years and 2.207 ± 1.73 µg/ml in adults. There was significant differences in the mean concentration of MBL among different age groups of children and also between children and adults (p<0.05). No association was observed between sex and MBL concentrations. MBL serum levels of Iranian population seem to be different from some of other populations which may be explained by genetic variations. The MBL values in this study can be used as a normal reference range for future studies in Iranian population.

Normal range determination of lymphocytes subsets in normal adults in Iran.

Immunophenotyping of lymphocytes is very essential for evaluation of immune system. Due to the effect of environmental factors and ethical diversity on immune system, establishment of an internal normal range of lymphocyte subsets is a necessity for each population. The aim of this study was to determine the normal range of T and B lymphocytes, and NK cells in normal Iranian adults. Two hundred and thirty three Iranian normal adult volunteers took part in this study. Complete Blood Count (CBC) was performed for them with Sysmex (KX21) and cells with CD3, CD4, CD8, CD19 and CD16/56 surface markers were simultaneously detected by flow cytometry method with FACstar system. Their percentile and absolute count were determined.The volunteers were 150 male and 83 female. Mean percentages of lymphocyte subpopulation were: CD3 (67.66 ±7.76), CD19 (14.41±5.09), CD4 (39.22±6.7), CD8 (25.42 ±5.4) and CD16/56 (10.14±6.42). Also, their mean absolute count of lymphocyte bearing CD3, CD19, CD4 and CD8 were 1,504±505/µl, 332±186/µl, 827±313/µl and 522±185/µl, respectively.Our results are comparable with similar Asian results from other Asian population, but are different from European population, we therefore conclude that it is necessary for each laboratory to establish an internal normal range for the lymphocytes bearing above- mentioned markers.

Cytotoxicity of human cord blood natural killer cells is enhanced by recombinant interleukin-15.

Hematopoietic cord blood (CB) stem cell transplantation has more advantages to other cell sources because of lower Graft Versus Host Disease (GVHD). Interleukin-15(IL-15) is an immunoregulatory cytokine, known to enhance cytolytic function of cord Natural Killer (NK) cells. The aim of this study was to investigate the effect of IL-15 on NK cytotoxicity simultaneously in different cell death stages. We compared the ability of IL-15 to enhance the NK cytotoxicity of CB in comparison to adult blood Mononuclear Cells (MNCs) against K562 target cells by co-staining with AnnexinV-FITC and Propidium Iodide after 3.5 h incubation at 37 °C and 5% CO2 by using flow cytometric method. We also evaluated phenotypic changes after treatment by IL-15 in both cell sources. Our results indicated that CB samples had lower level of apoptosis, while necrosis was negligible; also by escalating Effector: Target (E: T), we got higher level of apoptosis and necrosis in peripheral blood (PB). NK activity of cord and adult MNCs was enhanced by incubation with IL-15 (10 ng/ml) for 72 h with significantly higher results of PB in comparison to CB (p<0.0001). Moreover, IL-15 increased the percentage of CD3-/CD56+ and CD25+ cells after 72 h incubation. Results showed incubation with human recombinant (hr) IL-15 for 3 days increased NK activity. Taken together, these results indicated that NK cytotoxicity of CB MNCs could be augmented by human recombinant (hr) IL-15, but this activity did not reach to same level of PB counterparts. We established that CD25 expression on CB MNCs could be increased with IL-15, in 72-hour cultures, but to a lesser degree compared to that on corresponding adult PB MNCs.