Integrate CRISPR/Cas9 for protein expression of HLA-B*38:68Q via precise gene editing.

The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. We studied the expression level of a questionable (Q) HLA-B*38:68Q allele, which carries a 9-nucleotide (nt) deletion at codon 230-232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 gene editing of HLA-B*38:01:01:01 homozygous EBV B cell line resulted in one HLA-B*38:68Q/B*38:01:01:01 heterozygous and one HLA-B*38:68Q homozygous clone. Flow cytometric analysis of monoclonal anti-Bw4 antibody showed the protein expression of HLA-B*38:01:01:01 in homozygous cells was 2.2 fold higher than HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the expression of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 2.0 fold higher than HLA-B*38:68Q homozygous cells. The HLA-B*38:68Q expression was further confirmed using anti-B38 polyclonal antibody. Similarly, the expression of the HLA-B*38:01:01:01 homozygous cells was 1.5 fold higher than that of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 1.6 fold higher than that of HLA-B*38:68Q homozygous cells. The treatment of HLA-B*38:68Q homozygous cells with IFN-γ significantly increased its expression. In conclusion, we demonstrate that HLA-B*38:68Q is a low-expressing HLA allele. The CRISPR/Cas9 technology is a useful tool to induce precise gene editing in HLA genes to enable the characterization of HLA gene variants on expression and function

Not All Antibodies Are Created Equal: Factors That Influence Antibody Mediated Rejection.

Consistent with Dr. Paul Terasaki's "humoral theory of rejection" numerous studies have shown that HLA antibodies can cause acute and chronic antibody mediated rejection (AMR) and decreased graft survival. New evidence also supports a role for antibodies to non-HLA antigens in AMR and allograft injury. Despite the remarkable efforts by leaders in the field who pioneered single antigen bead technology for detection of donor specific antibodies, a considerable amount of work is still needed to better define the antibody attributes that are associated with AMR pathology. This review highlights what is currently known about the clinical context of pre and posttransplant antibodies, antibody characteristics that influence AMR, and the paths after donor specific antibody production (no rejection, subclinical rejection, and clinical dysfunction with AMR)

Clinical Utility of Microarrays: Current Status, Existing Challenges and Future Outlook

Microarray-based clinical tests have become powerful tools in the diagnosis and treatment of diseases. In contrast to traditional DNA-based tests that largely focus on single genes associated with rare conditions, microarray-based tests are ideal for the study of diseases with underlying complex genetic causes. Several microarray based tests have been translated into clinical practice such as MammaPrint and AmpliChip CYP450. Additional cancer-related microarray-based tests are either in the process of FDA review or under active development, including Tissue of Tumor Origin and AmpliChip p53. All diagnostic microarray testing is ordered by physicians and tested by a Clinical Laboratories Improvement Amendment-certified (CLIA) reference laboratory. Recently, companies offering consumer based microarray testing have emerged. Individuals can order tests online and service providers deliver the results directly to the clients via a password-protected secure website. Navigenics, 23andMe and deCODE Genetics represent pioneering companies in this field. Although the progress of these microarray-based tests is extremely encouraging with the potential to revolutionize the recognition and treatment of common diseases, these tests are still in their infancy and face technical, clinical and marketing challenges. In this article, we review microarray-based tests which are currently approved or under review by the FDA, as well as the consumer-based testing. We also provide a summary of the challenges and strategic solutions in the development and clinical use of the microarray-based tests. Finally, we present a brief outlook for the future of microarray-based clinical applications

Differences in Proinflammatory Cytokines and Monocyte Subtypes in Older as Compared With Younger Kidney Transplant Recipients.

Background:The number of elderly patients with end-stage kidney disease requiring kidney transplantation continues to grow. Evaluation of healthy older adults has revealed proinflammatory changes in the immune system, which are posited to contribute to age-associated illnesses via "inflamm-aging." Immunologic dysfunction is also associated with impaired control of infections. Whether these immunologic changes are found in older kidney transplant recipients is not currently known, but may have important implications for risk for adverse clinical outcomes. Methods:Three months after transplant, innate immune phenotype was evaluated by flow cytometry from 60 kidney transplant recipients (22 older [≥60 years] and 38 younger [<60 years old]). Multiplex cytokine testing was used to evaluate plasma cytokine levels. Younger patients were matched to older patients based on transplant type and induction immune suppression. Results:Older kidney transplant recipients demonstrated decreased frequency of intermediate monocytes (CD14++CD16+) compared with younger patients (1.2% vs 3.3%, P = 0.007), and a trend toward increased frequency of proinflammatory classical monocytes (CD14++CD16-) (94.5% vs 92.1%) (P = 0.065). Increased levels of interferon-gamma (IFN-γ) were seen in older patients. Conclusions:In this pilot study of kidney transplant recipients, we identified differences in the innate immune system in older as compared with younger patients, including increased levels of IFN-γ. This suggests that age-associated nonspecific inflammation persists despite immune suppression. The ability to apply noninvasive testing to transplant recipients will provide tools for patient risk stratification and individualization of immune suppression regimens to improve outcomes after transplantation

HUB City Steps: Methods and Early Findings From a Community-Based Participatory Research Trial to Reduce Blood Pressure Among African Americans

Background: Community-based participatory research (CBPR) has been recognized as an important approach to develop and execute health interventions among marginalized populations, and a key strategy to translate research into practice to help reduce health disparities. Despite growing interest in the CBPR approach, CBPR initiatives rarely use experimental or other rigorous research designs to evaluate health outcomes. This behavioral study describes the conceptual frameworks, methods, and early findings related to the reach, adoption, implementation, and effectiveness on primary blood pressure outcomes. Methods: The CBPR, social support, and motivational interviewing frameworks are applied to test treatment effects of a two-phased CBPR walking intervention, including a 6-month active intervention quasi experimental phase and 12-month maintenance randomized controlled trial phase to test dose effects of motivational interviewing. A community advisory board helped develop and execute the culturally-appropriate intervention components which included social support walking groups led by peer coaches, pedometer diary selfmonitoring, monthly diet and physical activity education sessions, and individualized motivational interviewing sessions. Although the study is on-going, three month data is available and reported. Analyses include descriptive statistics and paired t tests. Results: Of 269 enrolled participants, most were African American (94%) females (85%) with a mean age of 43.8 (SD = 12.1) years. Across the 3 months, 90% of all possible pedometer diaries were submitted. Attendance at the monthly education sessions was approximately 33%. At the 3-month follow-up 227 (84%) participants were retained. From baseline to 3-months, systolic BP [126.0 (SD = 19.1) to 120.3 (SD = 17.9) mmHg; p \u3c 0.001] and diastolic BP [83. 2 (SD = 12.3) to 80.2 (SD = 11.6) mmHg; p \u3c 0.001] were significantly reduced. Conclusions: This CBPR study highlights implementation factors and signifies the community’s active participation in the development and execution of this study. Reach and representativeness of enrolled participants are discussed. Adherence to pedometer diary self-monitoring was better than education session participation. Significant decreases in the primary blood pressure outcomes demonstrate early effectiveness. Importantly, future analyses will evaluate long-term effectiveness of this CBPR behavioral intervention on health outcomes, and help inform the translational capabilities of CBPR efforts

Frequency of 22q11 deletions in patients with conotruncal defects

AbstractObjectives. This study was designed to determine the frequency of 22q11 deletions in a large, prospectively ascertained sample of patients with conotruncal defects and to evaluate the deletion frequency when additional cardiac findings are also considered.Background. Chromosome 22q11 deletions are present in the majority of patients with DiGeorge, velocardiofacial and conotruncal anomaly face syndromes in which conotruncal defects are a cardinal feature. Previous studies suggest that a substantial number of patients with congenital heart disease have a 22q11 deletion.Methods. Two hundred fifty-one patients with conotruncal defects were prospectively enrolled into the study and screened for the presence of a 22q11 deletion.Results. Deletions were found in 50.0% with interrupted aortic arch (IAA), 34.5% of patients with truncus arteriosus (TA), and 15.9% with tetralogy of Fallot (TOF). Two of 6 patients with a posterior malalignment type ventricular septal defect (PMVSD) and only 1 of 20 patients with double outlet right ventricle were found to have a 22q11 deletion. None of the 45 patients with transposition of the great arteries had a deletion. The frequency of 22q11 deletions was higher in patients with anomalies of the pulmonary arteries, aortic arch or its major branches as compared to patients with a normal left aortic arch regardless of intracardiac anatomy.Conclusions. A substantial proportion of patients with IAA, TA, TOF and PMVSD have a deletion of chromosome 22q11. Deletions are more common in patients with aortic arch or vessel anomalies. These results begin to define guidelines for deletion screening of patients with conotruncal defects