Multidimensional Response Surface Methodology for the Development of a Gene Editing Protocol for p67phox-Deficient Chronic Granulomatous Disease.

Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34+ hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34+ cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34+ cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67phox-deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67phox expression and functional correction of CD34+-derived neutrophils from a CGD patient.

Primary Diffuse Large B Cell Lymphoma of Bone: A Single-Center Experience.

Background: Primary diffuse large B-cell lymphoma of the bone (PB-DLBCL) is a rare type of extra-nodal lymphoma. This study aimed to examine the clinical characteristics, outcomes, treatment modalities and risk of central nervous system relapse (CNSR) among adult Jordanian patients with PB-DLBCL. Methods: This retrospective study included patients aged >16 years who were diagnosed with PB-DLBCL and treated at our hospital between 2002 and 2021. Clinical characteristics, treatment modalities, outcomes and CNSR events were extracted from the hospital's data system and analysed. Patients were categorised into unifocal (UF) and multifocal (MF) PB-DLBCL groups according to the number of bone sites involved. The involvement of only one site was defined as UF, whereas the involvement of two or more sites was defined as MF. Results: In total, 12 patients were diagnosed with PB-DLBCL. Their median age was 47.5 years (range, 17-80 years). The male:female ratio was 1:1. There were eight patients in the UF PB-DLBCL group and four in the MF PB-DLBCL group. All patients received treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. In the UF PB-DLBCL group, the male:female ratio was 5:3, the median age was 41 years, and the follow-up duration was 9-135 (mean, 83.3) months. In the MF PB-DLBCL group, the male:female ratio was 1:3, the median age was 51.5 years, and the survival time was 3-11 (mean, 7) months. Three patients with vertebral UF PB-DLBCL underwent early vertebroplasty without complications. The most common site involved was the vertebral column. Most patients with UF PB-DLBCL achieved complete remission (CR), whereas no patients with MF PB-DLBCL achieved CR. Conclusion: PB-DLBCL is rare in adult Jordanian patients. UF PB-DLBCL is more common than MF PB-DLBCL. Patients with UF PB-DLBCL had a good prognosis. Patients with MF PB-DLBCL had a high international prognostic index score, risk of CNSR and short survival time.

Second Report of Chronic Granulomatous Disease in Jordan: Clinical and Genetic Description of 31 Patients From 21 Different Families, Including Families From Lybia and Iraq.

Chronic granulomatous Disease (CGD) is a rare innate immunodeficiency disorder caused by mutations in one of the six genes (CYBA, CYBB, NCF1, NCF2, NCF4, and CYBC1/EROS) encoding the superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex in phagocytes. In the Western population, the most prevalent form of CGD (about two-thirds of all cases) is the X-linked form (X-CGD) caused by mutations in CYBB. The autosomal recessive forms (AR-CGD), due to mutations in the other genes, collectively account for the remaining one-third of CGD cases. We investigated the clinical and molecular features of 22 Jordanian, 7 Libyan, and 2 Iraqi CGD patients from 21 different families. In addition, 11 sibling patients from these families were suspected to have been died from CGD as suggested by their familial and clinical history. All patients except 9 were children of consanguineous parents. Most of the patients suffered from AR-CGD, with mutations in CYBA, NCF1, and NCF2, encoding p22 phox , p47 phox , and p67 phox proteins, respectively. AR-CGD was the most frequent form, in Jordan probably because consanguineous marriages are common in this country. Only one patient from non-consanguineous parents suffered from an X910 CGD subtype (0 indicates no protein expression). AR670 CGD and AR220 CGD appeared to be the most frequently found sub-types but also the most severe clinical forms compared to AR470 CGD. As a geographical clustering of 11 patients from eight Jordanian families exhibited the c.1171_1175delAAGCT mutation in NCF2, segregation analysis with nine polymorphic markers overlapping NCF2 indicates that a common ancestor has arisen ~1,075 years ago.

Recent advances in myeloid-derived suppressor cell biology.

In recent years, studying the role of myeloid-derived suppressor cells (MDSCs) in many pathological inflammatory conditions has become a very active research area. Although the role of MDSCs in cancer is relatively well established, their role in non-cancerous pathological conditions remains in its infancy resulting in much confusion. Our objectives in this review are to address some recent advances in MDSC research in order to minimize such confusion and to provide an insight into their function in the context of other diseases. The following topics will be specifically focused upon: (1) definition and characterization of MDSCs; (2) whether all MDSC populations consist of immature cells; (3) technical issues in MDSC isolation, estimation and characterization; (4) the origin of MDSCs and their anatomical distribution in health and disease; (5) mediators of MDSC expansion and accumulation; (6) factors that determine the expansion of one MDSC population over the other; (7) the Yin and Yang roles of MDSCs. Moreover, the functions of MDSCs will be addressed throughout the text.

Mechanisms of immune suppression by myeloid-derived suppressor cells: the role of interleukin-10 as a key immunoregulatory cytokine.

Chronic immune activation and inflammation are unwanted consequences of many pathological conditions, since they could lead to tissue damage and immune exhaustion, both of which can worsen the pathological condition status. In fact, the immune system is naturally equipped with immunoregulatory cells that can limit immune activation and inflammation. However, chronic activation of downregulatory immune responses is also associated with unwanted consequences that, in turn, could lead to disease progression as seen in the case of cancer and chronic infections. Myeloid-derived suppressor cells (MDSCs) are now considered to play a pivotal role in the pathogenesis of different inflammatory pathological conditions, including different types of cancer and chronic infections. As a potent immunosuppressor cell population, MDSCs can inhibit specific and non-specific immune responses via different mechanisms that, in turn, lead to disease persistence. One such mechanism by which MDSCs can activate their immunosuppressive effects is accomplished by secreting copious amounts of immunosuppressant molecules such as interleukin-10 (IL-10). In this article, we will focus on the pathological role of MDSC expansion in chronic inflammatory conditions including cancer, sepsis/infection, autoimmunity, asthma and ageing, as well as some of the mechanisms by which MDSCs/IL-10 contribute to the disease progression in such conditions.

Inflammatory milieu in contrast-induced nephropathy: a prospective single-center study.

BACKGROUND: Acute kidney injury (AKI) caused by contrast exposure is a common problem, which may cause a significant increase in patients in-hospital stay and therefore the cost of treatment. This study was conducted to evaluate the role of inflammation, inflammatory markers in predicting contrast induced nephropathy (CIN). This is a prospective study that was carried out in a major tertiary referral hospital in Jordan. METHODS: Clinical data, blood and urine samples were collected from all patients admitted to the cardiology unit. All patients who agreed to participate in the study had creatinine level analysis 48-72 hours after the procedure. The CIN was defined as an increase in serum creatinine by 25% or 44 µmol/L from the baseline within 48-72 hours after the contrast administration. Patients with stage 4, 5 renal failure, patients on dialysis, and patients with recent intravenous contrast use, active infection or cancer were excluded from the study. RESULTS: Of the total 202 patients, 30 (14.8%) developed CIN. The incidence rate was 21.1% among females and 12.4% among males. In the multivariate analysis, beside eGFR, diuretics, and alkaline phosphatase, IL-33 was significantly associated with CIN, while the other cytokines did not to show this an association. CONCLUSION: Serum level of IL-33 was a significant predictor for development of CIN. Good clinical judgment and high serum levels of IL-33 may stratify patients into low and high risk for CIN.

The association of HLA-DQB1*0602 but not HLA-DRB1*15 with obstructive sleep apnea.

PURPOSE: Obstructive sleep apnea (OSA) is a sleep breathing disorder with unclear multifactorial pathogenesis. This study aimed to investigate the association between OSA and two human leukocyte antigens (HLA) alleles; DQB1*0602 and DRB1*15. METHODS: Forty patients with OSA and 40 control subjects were enrolled in the study. OSA diagnosis was made utilizing the Apnea-Hypopnea Index (AHI)≥5 in overnight polysomnography (PSG). AHI was also used to determine OSA severity. Controls were randomly selected from healthy volunteers who had a low risk for OSA, utilizing the Berlin Questionnaire. Polymerase chain reaction (PCR) using Sequence Specific Primers (PCR-SSPs) was used to determine the association between HLA (HLA-DQB1*0602 and HLA-DRB1*15) and OSA, then statistical analyses of the results were performed. RESULTS: HLA-DQB1*0602 allele was found in 85% of all OSA patients and 50% of controls (P< 0.001). In patients with severe OSA, HLA-DQB1*0602 was present in the 92.9% compared with 66.7% in non-severe OSA (P=0.05). HLA-DRB1*15 allele was found in 15% of OSA patients and 20% of controls, with no difference between the two groups (P=0.556). No statistical difference was found in HLA-DRB1*15 between severe and non-severe OSA (P=0.499). After adjusting for gender, HLA-DQB1*0602 allele was associated with increased odds of OSA (OR = 6.17, 95% CI 1.87-20.3, p = 0.003), but HLA-DRB1*15 allele was not associated with OSA (OR 0.45, 95% CI 0.12-1.73, p = 0.242). CONCLUSIONS: The presence of HLA-DQB1*0602 allele, but not HLA-DRB1*15 allele, was significantly associated with OSA.

Vitamin D deficiency and level of asthma control in women from North of Jordan: a case-control study.

INTRODUCTION: Reduced vitamin-D levels in patients with asthma have been associated with impaired lung function, increased airway hyper-responsiveness, and reduced glucocorticoid responsiveness. Nationwide studies revealed a considerable prevalence of vitamin-D deficiency (VDD) in Jordanian women. OBJECTIVE: A case-control study was conducted to determine the relationship between serum vitamin A and D levels and asthma among women in North of Jordan. METHODS: Sixty-eight asthmatics, age range between 14 and 65 years and 77 healthy women, age range between 19 and 51 years, were enrolled. Asthma severity was classified using Global Initiative for Asthma (GINA) guidelines and Asthma Control Test (ACT) questionnaire. Serum vitamin-A and 25-hydroxyvitamin-D (25(OH)D3) levels were measured using high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, respectively. RESULTS: The prevalence of VDD (<15 ng/ml) was higher but not statistically significant for women with asthma compared with controls (95.6% vs. 87.0%; p = 0.070). The severity of VDD correlated with the number of asthma medications (p = 0.020). 25(OH)-D3 serum levels directly correlated with asthma control level using ACT score (p = 0.012) and GINA classification (p = 0.046). After adjusting for age, the odds of having VDD for asthmatic women were 35.9 times higher than that for women with no asthma. There was no difference in serum vitamin-A level between healthy and asthmatic women (p = 0.214) and none had vitamin-A deficiency (<200 µg/dl). CONCLUSIONS: VDD is prevalent in women with asthma in northern Jordan. The severity of VDD correlated with poor asthma control and a need for more medications to control asthma. There was no association between vitamin-A and asthma.

The association between metabolic syndrome and coronary artery disease in Jordan.

BACKGROUND: Previous studies in Jordan showed a considerably high prevalence of metabolic syndrome (MeS) and its individual components. However, data about the association between MeS and coronary artery disease (CAD) in Jordan are lacking. Therefore, this study aimed to determine the association between MeS and CAD among Jordanians. METHODS: This case-control study included 269 patients diagnosed with CAD and 1026 subjects without CAD. Data collected from cases and controls included socio-demographic and clinical characteristics and anthropometric measurements. Patients were diagnosed with MeS based on the criteria of the National Cholesterol Education Program (NCEP) adult treatment panel (ATP) III. RESULTS: MeS was present in 65.1% of patients with CAD and in 44.0% of those with no CAD (p<0.0005). After adjusting for the important variables in the multivariate analysis, those with MeS were twice more likely to have CAD (OR = 1.94, 95% CI: 1.29, 2.93, p = 0.002) compared to those without MeS. The number of metabolic abnormalities was significantly associated with CAD, when it substituted MeS in the regression model, with a higher number of MeS components led to greater odds for CAD. CONCLUSIONS: The MeS, which occurs very frequently in the general population, is associated with CAD in Jordan. The identification of subjects with the MeS is conceivably useful from a clinical standpoint, as it can be anticipated that these individuals should benefit from interventions aimed at reducing cardiovascular risk.

Minocycline treatment results in reduced oral steroid requirements in adult asthma.

The tetracycline antibiotics have pleiotropic anti-inflammatory properties that may explain their therapeutic benefit in rheumatoid arthritis and acne. As these agents suppress both cellular and humoral immune responses, they may be of benefit in treating asthma and other allergic disorders. The purpose of this study was to determine whether minocycline therapy of asthma has steroid sparing effects beyond its inherent antibiotic properties. Adult asthmatic patients (n = 17) were treated with minocycline 150 mg p.o. twice daily or placebo for 8 weeks in a randomized, double-blind, placebo-controlled crossover study. Patients were evaluated for clinical improvement in oral steroid requirements, spirometry, and symptom scores (Asthma Quality of Life Questionnaire). They underwent assessment for preexisting infection (CT facial sinuses, Chlamydia pneumoniae nasopharyngeal culture, and C. pneumoniae and Mycoplasma pneumoniae serology). Minocycline use was associated with a 30% reduction in mean daily prednisone use compared with placebo (8.8 mg versus 14.4 mg, respectively; p = 0.02). Pulmonary function testing showed improvement in forced vital capacity (FVC; percent predicted; p = 0.03) and improvement in actual FVC and forced expiratory volume in 1 second (percent predicted) approached statistical significance (p = 0.05 and 0.08, respectively). Minocycline treatment was associated with significant improvement in asthma symptoms brought on by environmental triggers (p = 0.01). This preliminary study of minocycline therapy showed oral steroid-sparing properties for those with moderate persistent and severe persistent asthma.