Genetic and functional analysis of the Nkt1 locus using congenic NOD mice: improved Valpha14-NKT cell performance but failure to protect against type 1 diabetes

Defective invariant natural killer T-cells (iNKT cells) have been implicated in the etiology of type 1 diabetes in nonobese diabetic (NOD) mice. In a genome scan of a cross between NOD and C57BL/6 mice, the most significant locus controlling the number of iNKT cells, referred to as Nkt1, was recently mapped to distal chromosome 1. Here, using congenic mice for this chromosomal segment, we definitively demonstrate the existence of Nkt1 and show that introgression of the C57BL/6 allele onto the NOD background improves both the number of iNKT cells and their rapid production of cytokines elicited by alpha-galactosylceramide treatment, explaining at least half of the difference between the NOD and C57BL/6 strains. Using new subcongenic lines, we circumscribed the Nkt1 locus to a 8.7-cM segment, between the NR1i3 and D1Mit458 markers, that notably includes the SLAM (signaling lymphocytic activation molecule) gene cluster, recently involved in murine lupus susceptibility. However, despite a significant correction of the iNKT cell defect, the Nkt1 locus did not alter the course of spontaneous diabetes in congenic mice. Our findings indicate a complex relationship between iNKT cells and autoimmune susceptibility. Congenic lines nonetheless provide powerful models to dissect the biology of iNKT cells.     

Prognostic Significance of Stringent Complete Response after Stem Cell Transplantation in Immunoglobulin Light Chain Amyloidosis

Hematologic response has emerged as a powerful prognostic factor for survival in patients with immunoglobulin light chain (AL) amyloidosis. Patients achieving a complete response (CR), based on serum and urine analysis, survive longest. However, data regarding the impact of bone marrow features post-therapy on response and survival are limited. We evaluated the impact of achieving a stringent CR (sCR), defined as undetectable bone marrow clonal plasma cells by flow cytometry, in patients with AL amyloidosis receiving an autologous stem cell transplant. A total of 573 consecutive patients transplanted for AL amyloidosis at the Mayo Clinic between April 2002 and August 2016 were included in the analysis. Of 540 patients in whom response was assessable, 220 patients (41%) achieved a CR, of whom 212 (96%) had a bone marrow biopsy at time of response assessment and were further analyzed for determination of sCR; 166 patients (78%) with a CR achieved an sCR, representing 31% of the whole cohort. Patients achieving a CR had a higher median percentage of bone marrow plasma cells (10% for CR versus 6% for sCR, P = .03), more patients with bone marrow plasma cells ≥ 10% (50% for CR versus 33% for sCR, P = .04), and were less likely to receive chemotherapy before transplantation (30% for CR versus 49% for sCR, P = .03) compared with those achieving sCR. Median overall survival for all patients achieving a CR was 175 months and was not statistically different between those achieving an sCR compared with those achieving a CR only (median not reached for sCR versus 175 months for CR, P = .65). Progression-free survival, however, was significantly shorter in patients failing to achieve an sCR (151 months for sCR versus 72 months for CR, P = .0003). Bone marrow examination post-transplant in AL amyloidosis is important and identifies patients who fail to achieve an sCR and progress earlier.     

Myocilin analysis by DHPLC in French POAG patients: increased prevalence of Q368X mutation

Primary open-angle glaucoma (POAG) is a prevalent optic neuropathy with complex genetics. A small number of patients carry a mutation in the coding region of the myocilin (MYOC) gene. The nature and the frequency of these mutations, however, vary substantially, notably with the age at onset and the ethnic origin of the patients. Here, we showed that denaturing high performance liquid chromatography (DHPLC) is an appropriate method for screening carriers of MYOC mutations. We have applied the method to a group of 237 POAG patients and 108 control subjects from France. Mutations were found in 17 (7.5%) patients and in none of the controls. A single mutation, Q368X (c.1102C>T), accounted for the majority (12/17) of these mutations, corresponding to a frequency of 5% among POAG patients, the highest ever reported for this mutation. Furthermore, analysis of allelic associations at closely linked microsatellite markers indicated that most, if not all, patients inherited Q368X from a same ancestor. Five other patients carried four distinct mutations, including N480K (c.1440C>A) (2 cases), I499F (c.1495A>T), G367R (c.1099G>A) and T438I (c.1313C>T), which is reported here for the first time. Altogether, MYOC mutations in French patients were associated with a significantly increased intraocular pressure at diagnosis. In addition, the age at diagnosis of patients with a mutation other than Q368X was significantly younger than that of Q368X carriers or of patients with a normal MYOC. Based on these observations, a screening strategy of MYOC mutations in French POAG patients is briefly outlined.     

Phylogenetic and evolutionary analyses of dengue viruses isolated in Jakarta,Indonesia

Dengue has affected Indonesia for the last five decades and become a major health problem in many cities in the country. Jakarta, the capital of Indonesia, reports dengue cases annually, with several outbreaks documented. To gain information on the dynamic and evolutionary history of dengue virus (DENV) in Jakarta, we conducted phylogenetic and evolutionary analyses of DENV isolated in 2009. Three hundred thirty-three dengue-suspected patients were recruited. Our data revealed that dengue predominantly affected young adults, and the majority of cases were due to secondary infection. A total of 171 virus isolates were successfully serotyped. All four DENV serotypes were circulating in the city, and DENV-1 was the predominant serotype. The DENV genotyping of 17 isolates revealed the presence of Genotypes I and IV in DENV-1, while DENV-2 isolates were grouped into the Cosmopolitan genotype. The grouping of isolates into Genotype I and II was seen for DENV-3 and DENV-4, respectively. Evolutionary analysis revealed the relatedness of Jakarta isolates with other isolates from other cities in Indonesia and isolates from imported cases in other countries. We revealed the endemicity of DENV and the role of Jakarta as the potential source of imported dengue cases in other countries. Our study provides genetic information regarding DENV from Jakarta, which will be useful for upstream applications, such as the study of DENV epidemiology and evolution and transmission dynamics.     

MASS-FIX for the detection of monoclonal proteins and light chain N-glycosylation in routine clinical practice: a cross-sectional study of 6315 patients

Immunoenrichment-based matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), termed MASS-FIX, offers several advantages over immunofixation for the detection and isotyping of serum monoclonal protein, including superior sensitivity and specificity, the ability to differentiate therapeutic monoclonal antibodies, and the rapid identification of light chain (LC) N-glycosylation. We identified 6315 patients with MASS-FIX performed at our institution since 2018. Of these, 4118 patients (65%) with a wide array of plasma cell disorders (PCD), including rare monoclonal gammopathies of clinical significance, had a positive MASS-FIX. Two-hundred twenty-one (5%) of the MASS-FIX positive patients had evidence of LC N-glycosylation, which was more commonly identified in IgM heavy chain isotype, kappa LC isotype, and in diagnoses of immunoglobulin light chain (AL) amyloidosis and cold agglutinin disease (CAD) compared to other PCD. This cross-sectional study describes the largest cohort of patients to undergo MASS-FIX in routine clinical practice. Our findings demonstrate the widespread utility of this assay, and confirm that LC N-glycosylation should prompt suspicion for AL amyloidosis and CAD in the appropriate clinical context.Subject terms: Myeloma, Translational research     

Coexisting and clonally identical classic hodgkin lymphoma and nodular lymphocyte predominant hodgkin lymphoma

We report a case of concurrent nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and classic Hodgkin lymphoma (cHL), of nodular sclerosis subtype, in an otherwise healthy 24-year-old man with a strong family history of cHL. The patient was found to have a parotid mass, which was diagnosed as NLPHL, and a thymic mass diagnosed as cHL, of nodular sclerosis subtype concurrently. The lesion in the parotid showed features typical of NLPHL by morphology and immunophenotype. The LP cells were positive for PAX5, CD20, Oct2, weakly positive for CD30, and negative for CD15. The thymic lesion, diagnosed as cHL, of nodular sclerosis subtype, showed prominent bands of fibrosis and Hodgkin/Reed-Sternberg and lacunar cells positive for CD30 and CD15. These cells were variably positive for CD20 and negative for Oct2. PAX5 was weakly positive. Immunoglobulin gene rearrangement studies by polymerase chain reaction were carried out on microdissected Hodgkin/Reed-Sternberg and LP cells, which were shown to have identically sized peaks. NLPHL and cHL are 2 distinct diseases and are almost never seen concurrently. We present a case in which polymerase chain reaction analysis indicated that the tumor cells of these 2 distinct entities were clonally identical.