Iranian Journal of Allergy, Asthma and Immunology
Volume:20 Issue: 2, Apr 2021

Containment of pandemic infections mainly depends on prompt identification of carriers, achievable through strict surveillance and truthful diagnostic testing. Although molecular identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the gold standard method, its low sensitivity and long turnaround time are among major concerns.In this retrospective single-center study, we reviewed the results of the lymphocyte and neutrophil counts of 1450 Iranian patients with coronavirus disease 2019 (COVID-19) recruited at Baqiyatallah Hospital, Tehran, Iran.Of 1450 patients, 439 cases (30.3%) were polymerase chain reaction (PCR) negative; further emphasizing that getting negative molecular testing is not as reliable as a positive result. While the lymphocyte count in cases with less than 50 years old was 1.8×103/µL (1.2-2.5), it was 1.47×103/µL (0.84-2.16) in the older group (p<0.001). Also, men experienced lower lymphocytes as compared to women (1.53×103/µL vs 1.76×103/µL; p=0.002). Of particular interest, the lymphocyte count in the PCR-negative cases was 1.77×103/µL (0.98-2.45) which was significantly higher than its count in their positive counterparts (1.53×103/µL; p=0.004). Unlike lymphocytes, sex and PCR did not significantly affect the number of neutrophils. The odds ratio for neutrophilia in patients aged older than 50, either with a negative or a positive PCR, was 2.46 and 2.23, suggesting old age as the most significant associated factor.The number of lymphocytes along with increased neutrophil count may probably serve as simple, rapid, and economical biomarkers, and are seemingly appropriate items that should be taken into account in the identification of patients with COVID-19, especially those aged more than 50.

The coronavirus disease 2019 (COVID-19) pandemic in Iran is part of the worldwide pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The present study aimed to demonstrate the clinical characteristics of patients affected by COVID-19, in our tertiary teaching hospital. Medical records and compiled data of 668 patients with suspected COVID-19 were obtained retrospectively between January to April 2020. The present study outcomes included demographic features of infected patients, underlying diseases and conditions, the relationship between the results of reverse transcription-polymerase chain reaction (RT-PCR) or CT-scan with the manifestations of the disease, mortality rate, and age distribution of fatalities among men and women. The median age of hospitalized patients was 63 years old (from 18 to 94). The patients’ chief complaints in the admission time were cough, dyspnea, fever, and gastrointestinal problems, respectively. Hospitalized patients' common comorbidities were hypertension (HTN), and cardiovascular disease (CVD) (24%), diabetes mellitus (DM) (21.5%), asthma, or chronic obstructive pulmonary disease (COPD) (6%), or other underlying diseases (15.5%). One-third of patients had no comorbidity according to the data of medical records. In hospitalized patients, 169 (84.5%) had positive RT-PCR, and 156 (78%) had positive chest CT findings. The mortality rate of males was higher than females (66.3% vs. 33.3%) and in patients with positive RT-PCR compared to patients with positive chest CT-scan findings. The majority of deaths had a history of DM or HTN/CVD in their medical records. The chief complaint of patients was cough. DM and HTN or CVD were the common underlying disease related to death in hospitalized cases. Besides, the hospitalization and mortality rate in males was higher than in females. About 87% of dead hospitalized cases had positive RT-PCR results, and this rate was 82% for chest CT results.

Airborne bioaerosols and particulate matter (PM) have been associated with asthma occurrence. Due to the adverse indoor environment and the absence of any baseline data for asthmatic patients of Pakistan, this study was aimed to establish a correlation between microflora and PM of residential microenvironments of asthmatic patients. This pilot study was conducted in different residential settings of asthmatic patients registered in the Jinnah hospital, Lahore. The characterization of PM (PM01, PM2.5, PM10) and bioaerosols were carried out in the houses of fifty patients that were categorized into four groups; A-large (418.06 m2), B-medium (211 m2), C-medium (104 m2), and D-small (62.71 m2) houses. The PM concentrations were monitored; using the DustTrack8533 aerosol monitor and the bioaerosols were characterized up to the Genus; using the culture-based method and biochemical testing. The bioaerosols were sampled; using the expose plate method and were analyzed using morphological features and biochemical tests. Eleven types of fungi and seven bacterial types were found in the air samples. The tendency of asthma occurrence is linked with higher Alternaria spp and Aspergillus spp. The mean indoor readings of PM01, PM2.5, PM10 were highest in D-category (331.75, 342.5, and 502.33 respectively). Moreover, the highest bacterial 9618 CFU/m3) and fungal levels (3092 CFU/m3) were also seen in D-category. According to two-way ANOVA, bacterial concentration was significantly different among the four groups while fungi concentration was non-significant (p<0.05). Pearson correlation showed a significant positive correlation among bioaerosol counts, relative humidity, and temperature. Moreover, a positive significant correlation was also observed among PM, bioaerosols, and temperature (p<0.01). The multiple regression analysis confirms temperature as a significant predictor of bioaerosols and bacterial and fungal concentrations were observed to be a significant predictor for PM. Hence monitoring the PM levels could help in maintaining the indoor microenvironment for sensitive asthmatic patients.

Exosomes are extracellular vesicles that are involved in intracellular communication and different biological processes. Recently, the importance of microRNAs (miRNAs) in exosomes has been considered as biomarkers in asthma diagnosis. This study aimed to determine the expression of selective miRNAs from plasma-derived exosomes in moderate and severe asthmatic patients compared with healthy controls. Forty-six subjects including 22 patients with severe and mild to moderate allergic asthma and 24 healthy controls have entered this study. MiRNAs were extracted from the plasma exosomes and selective miRNAs (miR-21, miR-16, miR-Let7, miR-148a, miR-155, miR-125, miR-150, miR-146a, miR-223, miR-126) expressions levels were determined; using quantitative polymerase chain reaction (qPCR). In this study, we found a significant up-regulation of miR-223 and miR-21 in moderate asthmatic patients compared to the healthy controls (p=0.002, p=0.006). MiR-223 and miR-21 had the probability of 83% and 76% diagnosis estimation in moderate asthmatic patients respectively. Therefore, they could be used as biomarkers in these patients.  No expression of miR-125, miR-126, and miR-155 was found in plasma exosomes by qPCR in this study. The other miRNAs had no significant expression between different groups. Based on our findings,miR-223 and miR-21 may be considered biomarkers or used for targeted immunotherapies in asthma.

Sulfur Mustard (SM) induces cell injury via exerting oxidative stress, protease-anti protease imbalance, and inflammation. Inflammasome as one part of innate immunity has a critical role in the recognition of cell injuries and the initiation of the inflammation process by releasing IL-1β. Hence, the present study investigated the effects of the sub-lethal doses of 2-chloroethyl ethyl sulfide (CEES) as SM analog on the gene expression level of inflammasome-related genes as well as the potential protective effects of curcumin on this process.The effects of sub-lethal doses (500, 1000, and 2500 mM) of CEES on pulmonary epithelial cell line (A549) were determined at various time points (12, 24, and 48 h). Following the treatment of cells with CEES, the kinetic alterations of the expression levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB1), NLR family pyrin domain containing 1 (NLRP1), Caspase-1 (Casp1), and Interleukin-1β (IL-1β)  genes were analyzed; using real-time PCR. In addition, the concurrent protective effects of different doses of curcumin (20, 40, 80, and 160 mM) on modulating the effects of CEES were studied.Although it was found that the lowest sub-lethal dose of CEES (500 mM) was able to up-regulate the inflammasome-related genes, the maximum alterations occurred 48 h after the treatment with the higher sub-lethal dose (2500 mM) of CEES. The maximum alteration occurred in Casp1 (38 fold), IL-1β (19 fold), and NLRP1 (~4 fold) genes (p<0.0001). However, the NF-ĸB gene expression level did not alter following CEES exposure. Even though low doses of curcumin (20, 40, and 80 mM) were able to down-regulate the studied genes, it was found that the treatment of cells with 160 mM of curcumin for 48 h was able to normalize the expression level of all genes.The present study concludes that curcumin as an anti-inflammatory agent may have beneficial immunomodulatory effects following CEES exposure.

The nucleotide-binding oligomerization domain 2 (NOD2) is the key regulator of inflammatory responses and has been involved in the pathogenesis of rheumatoid arthritis (RA). Laboratory and in silico evaluations have demonstrated that some polymorphisms in 3ˊUTR of NOD2 gene could influence the secondary structure of this region and similarly thermodynamic features of hybridization site and finally deregulate the expression of NOD2. In the current study, for the first time, we evaluated the possible association between single nucleotide polymorphisms (SNPs) rs3135500 and rs3135499 in the NOD2 gene with RA risk in the Iranian population. One hundred and fifteen patients with RA and 120 healthy subjects were recruited in this case-control study. Genotyping of rs3135500 and rs3135499 polymorphisms were accomplished using the real‑time polymerase chain reaction high resolution melting (HRM) method. We found a substantial association of AA and AG genotypes in rs3135500 with the risk of RA (AA vs GG; OR=5.547; 95%CI [2.564-11.999]; p<0.001 and AG vs GG; OR=2.179; 95%CI [1.145-4.147]; p=0.017). Moreover, in the patient group, there was a significant relationship between the increased concentration of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) with rs3135500 (A allele) (p<0.05). However, there were no important associations between rs3135499 with the risk of RA (p>0.05). However, we found a noteworthy association of the C allele in rs3135499 with an increased level of CRP in patients (p>0.05). Our findings propose a considerable association between NOD2 polymorphisms with increased risk of RA and disease activity.

Gluten sensitivity contributes to various degrees of neurological manifestations and neurodegenerative immunological changes. We investigated the experimental features of anti-gliadin immune responses in the central nervous system (CNS) of mice. Female C57BL6 mice were divided into three groups. Mice immunized with complete Freund's adjuvant (CFA) or gliadin emulsified in CFA, and the control group received phosphate-buffered saline (PBS). Immunohistochemistry, hematoxylin-eosin, and Luxol fast blue staining were performed on the sections. The serum levels of interleukin (IL)-17 and interferon-gamma (IFN-γ) were measured using enzyme-linked immunosorbent assay (ELISA). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the mRNA levels of chemokine (C-X-C motif) ligand-2 (CXCL-2), C-C motif chemokine ligand-2 (CCL-2), and CXCL-10.  In gliadin+CFA immunized mice, the microscopic lesions included perivascular edema, focal-microgliosis, and acute neuronal necrosis in the cortex, subcortical, Purkinje cell layer, and ventral horn of the spinal cord. While extravasation of anti-IgG antibodies and selective targeting of Purkinje cells were observed in gliadin+CFA immunized mice. A significant increase in serum IL-17 and IFN-g levels (p<0.05), as well as expression of CXCL-2, CCL-2, and CXCL-10 in mice immunized with gliadin+CFA, were monitored versus controls. Our findings indicated that the immune responses directed against gliadin peptides might contribute to blood-brain barrier breakdown, extravasation of serum anti-IgG, gliosis, and acute neuronal necrosis in the cortex and cerebellar Purkinje cells. Anti-IgG antibodies may cause extravasation of blood-born anti-gliadin antibodies and selective targeting of Purkinje cells observed in mice immunized with peptide tryptic (pt) -gliadin in CFA.

Dendritic cells (DCs) play key roles in regulating the immune response using the specialized function of processing and presenting antigens. Prolactin (PRL), a hormone produced by the pituitary gland, participates in DC maturation and function. The present study was aimed to determine the frequencies of peripheral blood DC subpopulations of myeloid DC (MDC) and plasmacytoid DC (PDC) in hyperprolactinemic (HPRL) women compared to normal healthy volunteers. This study was conducted on 70 women, including 35 HPRL patients and 35 matched healthy controls, whose PRL serum levels were in the normal range (lower than 25 ng/mL). Serum thyroid-stimulating hormone (TSH) levels were measured in both groups as an indicator of normal thyroid function. The electrochemiluminescence immunoassay method was applied to measure the serum levels of TSH and PRL. The frequencies of MDC and PDC in the peripheral blood samples of both groups were determined by flow cytometry. The mean serum PRL levels in the HPRL patients and healthy individuals were 46.41±21.96 and 13.75±11.19, respectively (p<0.0001); however TSH levels in both groups were similar and within the normal range (0.4–4.5 mIU/mL) (p=0.2). The frequencies of both MDC and PDC subpopulations in the peripheral blood of HPRL patients were significantly lower than they were in the healthy controls. However, the ratio of MDCs/PDCs in HPRL patients was not significantly different between the two groups (p=0.8). Our study revealed that an increased level of serum PRL may lead to a reduction in the number of MDC and PDC subpopulations. These results could help clarify the complex relationship between the immune system and the neuroendocrine axis and may be of potential use in understanding the pathogenesis of endocrine and immune disorders.

Despite the unparalleled success of anti-CD20-targeted immunotherapy, the currently available mAbs are not sufficiently efficacious in the treatment of lymphoma. 1F5 is one of a panel of anti-CD20 mAbs that was used in the B-cell lymphoma serotherapy. Despite the efficacy of murine 1F5 mAbs in lymphoma patients, the 1F5 chimeric antibodies with human effector functionality are yet to be approved and widely used in the treatment of lymphoma. In this study, the conversion of 1F5 mAb from mouse IgG2a to human-mouse chimeric IgG1 was achieved and the chimeric antibody was partially characterized. We constructed the 1F5 chimeric mouse-human anti-CD20 antibody genes using an efficient Splicing by overlap extension-polymerase chain reaction (SOE-PCR) technique and cloned the chimeric heavy and light genes in pBudCE4.1mammalian expression vector, followed by purification of the expressed chimeric 1F5 mAbs using affinity chromatography. Our investigation also included the biological properties of purified chimeric antibodies. The generated 1F5 chimeric mAbs mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against Raji and Daudi Burkitt's lymphoma cell lines, which were comparable with rituximab and exhibit superior reduction in cell viability in vitro, compared to rituximab. The current study indicated that the generated chimeric 1F5 mAbs has potential CDC and ADCC activity which was comparable with rituximab whereas it exhibits a superior reduction in cell viability, compared to rituximab. Our work contributes to future studies involving in vivo biological functions and the application of the 1F5 chimeric antibody.

Conditioned medium (CM) derived from mesenchymal stem cells (MSCs) contains bioactive molecules including microRNAs (miRs) that could be a potential tool for controlling cancer cells' behavior. Due to the properties of CM, this study assesses the effects of miR-34a related MSC-CM on tumor behavior through the evaluation of migration, invasion, apoptosis, and PDL1 expression in breast cancer cell lines. The miR-34a overexpression vector or scramble control was produced using lentiviral vectors, DNA cloning, and the transfection of the HEK-293T cell line. It was then transduced into human adipose-derived mesenchymal stem cells (hAD-MSCs). MSC-CMs were collected and added onto MDA-MB-231 cell lines. The functional evaluations were performed by transwell, wound healing, and Annexin V/PI methods on the treated MDA-MB-231 cell lines. The PDL1 expression was also assessed by Real-time PCR and western blot. The findings of this study showed that ectopic miR‑34a expression was significantly upregulated in manipulated hASC with miR-34a (p<0.0001). Treatment of MDA-MB-231 cell line with miR-34a-hAD-MSC-CM, scramble-hAD-MSC-CM, or hAD-MSC-CM displayed not only a reduction in the number of migrated or invaded cells (p=0.01) but also an increase in the apoptotic cells in the test group (p=0.02) when compared to the control groups. It also showed down-regulation in the gene (p=0.05) and protein expression levels of PDL1 in the test group. The results of the present study showed that simultaneous application of miR-34a and MSC-CM can be considered as a new method for changing the cancerous microenvironment; and therefore, as a potential strategy in breast cancer therapy.

Natural killer (NK) cell therapy has proven to be a promising approach for the treatment of malignancies. Osaki method for ex-vivo autologous NK cell expansion has been recently introduced in Japan. To start clinical trial phase I at Shiraz University of Medical Sciences in collaboration with the Japanese group, this preclinical setting study aimed to evaluate the proliferative efficacy of the method, the activation status of expanded autologous NK cells, and the likely unwanted contamination of the final cell product.Peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy individuals' peripheral blood and transferred directly to the specified initial culture bag containing anti-CD52 and anti-CD3 and Interleukin (IL)-2. The cells were cultured for 14-17 days in an incubator, during which the cells received condition media, and underwent several passages into bigger culture bags. All the procedures were carried out in a cleanroom and associated facilities. Before and after activation PBMCs were analyzed for their phenotype and cytotoxic activity; using flow cytometry and cytokine release assay.Our results indicated that NK (CD3-CD16+/-CD56+) cells were expanded 510-fold on average (range 200-1100 fold), and the purity of NK cells per whole lymphocytes exceeded 68%. The expanded cells were highly lytic as indicated by in-vitro cytotoxic assay, with a strong expression of Natural killer group 2 member D (NKG2D) and CD16. The prepared final cell products were negative for HCV, HBV, HIV, mycoplasma, and endotoxin.In the preclinical phase, large numbers of activated and un-contaminated NK cells from healthy individuals' peripheral blood were successfully generated. The method seems to provide ample clean cell product with no contamination and has the potential to be used for NK cell therapy in future clinical trials, suitable to be infused back to the donors in phase I clinical trial.

Coronavirus disease 2019 (COVID-19) is a worldwide public health problem that has attracted much attention due to its clinical findings. Measurement of IgG and IgM antibodies is of great importance for researchers and it will help to develop a new diagnostic and therapeutic method in clinical care.In this cross-sectional study, we aim to measure the IgG and IgM antibody levels in 401 suspected COVID-19 volunteers. We also measure the time duration for the appearance of IgG and IgM antibodies from the onset of symptoms to sampling time.Of 401 participants enrolled in the study, 255 (63.59%) were healthy, 79 (19.70%) were a carrier, 59 (14.71%) were cured and 8 (1.99%) were borderline. Of 142 subjects diagnosed with COVID-19, 41 (28.87%) presented with gastrointestinal (GI) symptoms, 83 (58.45%) had no GI symptoms, and 18 (12.68%) were asymptomatic.According to our findings, the measurement of IgG and IgM antibodies will provide the tool for the diagnosis of COVID-19 and significantly boost research into novel diagnostic and therapeutic approaches.

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare primary immunodeficiency disorder characterized by recurrent infections and low immunoglobulin levels due to variable combined immunodeficiency, and centromeric region instability, and facial dysmorphism. We describe a 12-year-old boy with recurrent respiratory tract infections, facial anomalies, scoliosis, and psychomotor retardation. He had recurrent pneumonia with low serum IgG and IgM levels during infancy and preschool age. Later at the age of 10, he developed recurrent ear infections. An IgA and IgM deficiency was found accompanied by a normal B-cell and T-cell count as well as an impaired candida-induced T-cell proliferation. Further evaluations revealed a missense mutation in the DNMT3B gene on chromosome 20. Chromosomal analysis showed a sunburst multi-radial feature on chromosome 1, which is a hallmark of ICF syndrome. The genetic mutation and chromosomal abnormality along with clinical findings are compatible with the diagnosis of ICF syndrome. To the best of our knowledge, this is the first time that scoliosis is observed in an ICF patient. The additional variable clinical symptoms in the case were the presence of spastic gait as well as hypogammaglobulinemia with immunoglobulin isotype switch at different ages.

Generalized bullous fixed drug eruption (GBFDE) is a specific variant of fixed drug eruption that belongs to severe cutaneous adverse reactions (SCARs) and its diagnosis is based mainly on clinical course and especially on the reoccurrence of typical bullous lesions in previous and new sites after re-administration of the offending drug. We present a well-documented case of fluconazole-induced GBFDE, with a positive patch test to fluconazole (30% weight/volume preparation) and clinical tolerance to itraconazole proven by negative oral provocation. Even in SCARs, patch testing represents a useful diagnostic tool, while oral provocation remains the gold standard in cases that an alternative but the chemically relevant drug must be administered.