Defining the mild variant of leukocyte adhesion deficiency type II (SLC35C1-congenital disorder of glycosylation) and response to l-fucose therapy: Insights from two new families and review of the literature.

Leukocyte adhesion deficiency type II (LAD II, also known as SLC35C1-congenital disorder of glycosylation) is an autosomal recessive disorder characterized by growth and cognitive impairment, peripheral neutrophilia, recurrent infections, and the Bombay blood phenotype. A subset of patients with a milder presentation has been described with short stature and developmental delay but minimal immune and hematologic features. Some patients with LAD II benefit from oral fucose therapy, though this has not been previously studied in patients with milder disease. In this study, we describe two new patients from separate families with the milder variant of LAD II and review the published literature on this rare disorder. We demonstrate improvement in speech and cognition, CD15 expression, and core fucosylation of serum glycoproteins after 27 months of oral fucose supplementation in one patient. These patients further support the stratification of this disorder into distinct subtypes, a classical severe and an attenuated variant, and provide preliminary evidence of benefit of fucose therapy in the latter group.

Diagnostic accuracy and clinical relevance of an inflammatory biomarker panel for sepsis in adult critically ill patients.

The objective of this study was to assess the diagnostic accuracy of C-reactive protein (CRP), procalcitonin (PCT), and cellular immune markers levels in sepsis. This was a prospective observational study in adult intensive care unit (ICU) patients, between 2012 and 2014. The 8-color flow cytometric biomarker panel included CD64, CD163, and HLA-DR. Index test results were compared with sepsis, using receiver operating characteristic curve analyses. Multivariate logistic regression assessed the relationship of sets of markers with the probability of sepsis. Of 219 enrolled patients, 120 had sepsis. C-statistic was the highest for CRP (0.86) followed by neutrophil CD64 expression (0.83), procalcitonin (0.82), and Acute Physiology and Chronic Health Evaluation (APACHE) IV (0.72). After adjustment for APACHE IV, the combination of CRP, PCT, and neutrophil CD64 measure remained a significant predictor of sepsis with an excellent AUC (0.90). In a targeted ICU population at increased risk of sepsis, CRP, PCT, and neutrophil CD64 combined improve the diagnostic accuracy of sepsis.

Monocyte HLA-DR expression and neutrophil CD64 expression as biomarkers of infection in critically ill neonates and infants.

BACKGROUND: Reduced monocyte HLA-DR expression and increased neutrophil CD64 expression have been proposed as biomarkers of infection. METHODS: From 2009-2011, blood samples from neonatal intensive care unit (NICU) and pediatric intensive care unit (ICU) patients <1 y of age were collected at enrollment and during subsequent evaluation for suspected infection, if it occurred. Samples were analyzed for monocyte HLA-DR and neutrophil CD64 expression levels by flow cytometry. RESULTS: Forty-seven infants had study samples collected at enrollment; 26 infants had study samples collected at the time of a suspected infection. At enrollment, there was an inverse relationship between neutrophil CD64 expression and age (P ≤ 0.047). At the time of suspected infection, infants with an infection demonstrated a lower percentage of HLA-DR+ monocytes (P = 0.02, area under the curve (AUC) 0.78), higher percentage of CD64+ neutrophils (P = 0.009, AUC 0.81), and higher neutrophil CD64 expression levels (P = 0.04, AUC 0.75). CONCLUSION: Monocyte HLA-DR and neutrophil CD64 expression in critically ill infants are related to age and infection.

Immune monitoring using the predictive power of immune profiles.

BACKGROUND: We have developed a novel approach to categorize immunity in patients that uses a combination of whole blood flow cytometry and hierarchical clustering. METHODS: Our approach was based on determining the number (cells/µl) of the major leukocyte subsets in unfractionated, whole blood using quantitative flow cytometry. These measurements were performed in 40 healthy volunteers and 120 patients with glioblastoma, renal cell carcinoma, non-Hodgkin lymphoma, ovarian cancer or acute lung injury. After normalization, we used unsupervised hierarchical clustering to sort individuals by similarity into discreet groups we call immune profiles. RESULTS: Five immune profiles were identified. Four of the diseases tested had patients distributed across at least four of the profiles. Cancer patients found in immune profiles dominated by healthy volunteers showed improved survival (p < 0.01). Clustering objectively identified relationships between immune markers. We found a positive correlation between the number of granulocytes and immunosuppressive CD14(+)HLA-DR(lo/neg) monocytes and no correlation between CD14(+)HLA-DR(lo/neg) monocytes and Lin(-)CD33(+)HLA-DR(-) myeloid derived suppressor cells. Clustering analysis identified a potential biomarker predictive of survival across cancer types consisting of the ratio of CD4(+) T cells/µl to CD14(+)HLA-DR(lo/neg) monocytes/µL of blood. CONCLUSIONS: Comprehensive multi-factorial immune analysis resulting in immune profiles were prognostic, uncovered relationships among immune markers and identified a potential biomarker for the prognosis of cancer. Immune profiles may be useful to streamline evaluation of immune modulating therapies and continue to identify immune based biomarkers.

Tbata modulates thymic stromal cell proliferation and thymus function.

Niche availability provided by stromal cells is critical to thymus function. Thymi with diminished function contain fewer stromal cells, whereas thymi with robust function contain proliferating stromal cell populations. Here, we show that the thymus, brain, and testes-associated gene (Tbata; also known as SPATIAL) regulates thymic epithelial cell (TEC) proliferation and thymus size. Tbata is expressed in thymic stromal cells and interacts with the enzyme Uba3, thereby inhibiting the Nedd8 pathway and cell proliferation. Thymi from aged Tbata-deficient mice are larger and contain more dividing TECs than wild-type littermate controls. In addition, thymic reconstitution after bone marrow transplantation occurred more rapidly in Rag2(-/-)Tbata(-/-) mice than in Rag2(-/-)Tbata(+/+) littermate controls. These findings suggest that Tbata modulates thymus function by regulating stromal cell proliferation via the Nedd8 pathway.

Increased T-bet+ cytotoxic effectors and type I interferon-mediated processes in chronic graft-versus-host disease of the oral mucosa.

Although chronic graft-versus-host disease (cGVHD) is a major long-term complication of allogeneic hematopoietic stem cell transplantation, little is known of its pathogenesis. We have systematically examined oral mucosa among cGVHD patients and determined that the clinical severity of oral cGVHD was correlated with apoptotic epithelial cells, often found adjacent to infiltrating effector-memory T cells expressing markers of cytotoxicity and type I cytokine polarization. Accumulation of T-bet(+) T-cell effectors was associated with both increased proliferation and the expression of the type I chemokine receptor CXCR3. Concurrently, in both infiltrating cells and keratinocytes, we observed increased expression of the CXCR3 ligand MIG (CXCL9) and interleukin-15 (IL-15), type I interferon (IFN)-inducible factors that support the migration, type I differentiation, and expansion of alloreactive effectors. In severely affected mucosa, we observed high levels of MxA, a protein specifically induced by type I IFN, and signal transducer and activator of transcription 1 (STAT1) phosphorylation, a critical step in the IFN-signaling pathway, along with increased numbers of plasmacytoid dendritic cells. These data challenge the current paradigm of cGVHD as a type II cytokine-driven disorder and support the model that oral cGVHD results from type I IFN-driven immigration, proliferation, and differentiation of T-bet(+) type I T effectors. The clinical trials are registered at http://www.clinicaltrials.gov as NCT00331968.

Interference in microsphere flow cytometric multiplexed immunoassays for human cytokine estimation.

The present study describes positive and negative interference of human cytokine measurement in multiplexed bead-based immunoassays. Significant differences in measured IL-6 and TNF-alpha values in 30 normal human plasma samples were apparent depending on whether measurements were with a 2-plex assay or embedded in a multiplex of 8-or more cytokine antibody pairs, as well as among the kits of 3-different vendors. Sample diluents containing proprietary blocking ingredients were shown to greatly affect the outcome of measured cytokine values. Additionally, recovery of IL-6 and TNF-alpha from spiked samples suggests significant negative interference from either endogenous antibodies, soluble receptors or anti-cytokine antibodies in 10% and 26% of samples, respectively. While it is evident that multiplexed immunoassays hold great promise for cytokine profiling, there are still important issues needing further study. Especially needed are universally optimized sample diluents, uniformly calibrated standards with mass values, and internal assay controls, which should greatly facilitate intralaboratory accuracy and precision and interlaboratory comparisons of cytokine measurements. Possible causes of interference and remedies are discussed.

Molecular diagnostics of inherited thrombosis.

Thrombophilia can best be defined as a disorder of coagulation that contributes to a predisposition towards thrombosis. Although the term thrombophilia has been used to describe arterial thrombosis, its most common usage has been in reference to venous thromboembolism (VTE). Thrombophilia can be a consequence of both acquired and inherited or genetic causes. Acquired causes include conditions such as surgery, cancer, and prolonged immobilization, while genetic causes have been linked to the inherited deficiencies of antithrombin, protein C, and protein S. The identification of the genetic basis of these inherited causes of thrombophilia ushered in a new way of thinking about thrombosis and the importance of its genetic component. Interest in the genetic basis of VTE was accelerated with the subsequent discovery of factor V Leiden, prothrombin G20210A, and MTHFR C677T. These single nucleotide polymorphisms (SNPs) and other genetic variants associated with VTE have become fixtures in the molecular diagnosis of inherited thrombophilia. Because of the large volume of current and anticipated future genetic testing, there has been a push to develop many different genotyping methods which are now used in both clinical and research settings. The identification of new genetic variants that may either directly or indirectly affect coagulation or the anticoagulant pathway, may greatly advance the understanding and clinical management of thrombophilia.