Daniel H. Ryan

A clinical and immunologic study of blood transfusion and postoperative bacterial infection in spinal surgery

Allogeneic blood transfusion has been implicated as an independent risk factor for postoperative bacterial infection in clinical and animal studies. The association among transfusion, quantitative immunologic factors, and infection was examined in 102 patients undergoing 109 spinal fusion procedures. In 60 procedures, patients received autologous blood only; in 24 procedures, they received at least 1 unit of allogeneic blood, and in 25 procedures, they received no transfusions. Twenty-two patients developed bacterial infections, in 8 cases while in hospital and in 14 cases after discharge. Univariate analysis revealed that patients who received any allogeneic blood and those who received no allogeneic blood differed significantly in the rate of hospital-acquired infection (20.8 vs. 3.5%), length of stay (12.3 vs. 9.7 days), days of fever greater than or equal to 38 degrees C (4.0 vs. 2.9), days on antibiotics (3.9 vs. 2.5), duration of surgery (309 vs. 231 min), blood loss (1343 vs. 887 mL), surgeon, and postoperative drop in natural killer (NK) cells (-174 vs. -42/microL). Multivariate logistic and linear regressions revealed that the number of allogeneic units transfused was the only significant predictor of in-hospital infection (p = 0.016) or days on antibiotics and length of stay. None of the clinical, surgical, or transfusion variables was significantly associated with posthospital infection, although a significantly greater drop in NK cells had occurred in patients who developed infection (p = 0.0035). These data strongly implicate allogeneic transfusion as a risk factor for in-hospital postoperative bacterial infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance

The study of human B cell tolerance has been hampered by difficulties in identifying a sizable population of autoreactive B lymphocytes whose fate could be readily determined. Hypothesizing that B cells expressing intrinsically autoreactive antibodies encoded by the VH4-34 heavy chain gene (VH4-34 cells) represent such a population, we tracked VH4-34 cells in healthy individuals. Here, we show that naive VH4-34 cells are positively selected and mostly restricted to the follicular mantle zone. Subsequently, these cells are largely excluded from the germinal centers and underrepresented in the memory compartment. In healthy donors but not in patients with systemic lupus erythematosus (SLE), these cells are prevented from differentiating into antibody-producing plasma cells. This blockade can be overcome ex vivo using cultures of naive and memory VH4-34 cells in the presence of CD70, IL-2, and IL-10. VH4-34 cells may therefore represent an experimentally useful surrogate for autoantibody transgenes and should prove valuable in studying human B cell tolerance in a physiological, polyclonal environment. Our initial results suggest that both positive and negative selection processes participate in the maintenance of tolerance in autoreactive human B cells at multiple checkpoints throughout B cell differentiation and that at least some censoring mechanisms are faulty in SLE.

Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells.

Adhesion of B cell precursors to accessory cells in the bone marrow microenvironment may be required for normal early B cell development. Human bone marrow B cell precursors adhere more avidly than mature B cells to bone marrow-derived fibroblasts. To determine the mechanism of this adhesion, expression of adhesion proteins on human B precursor cells and cell lines was measured by flow cytometry. The very late antigen (VLA) integrins VLA-4 and VLA-5 were the only adhesion proteins expressed at higher levels in B cell precursors than mature B cells. Antibodies to the alpha and beta chains of VLA-4, but not VLA-5, significantly blocked binding to bone marrow-derived fibroblasts of immature B cells and cell lines. Although fibronectin is a ligand for VLA-4, anti-fibronectin antibody and a soluble fibronectin fragment containing the VLA-4 binding domain did not block adhesion, suggesting that VLA-4 is involved in adhesion of B cell precursors, but not as a fibronectin receptor. Vascular cell adhesion molecule-1 (VCAM-1), the other known counterreceptor for VLA-4, was identified on bone marrow-derived fibroblasts, and anti-VCAM-1 significantly blocked adhesion of normal B cell precursors to bone marrow-derived fibroblasts, indicating that VLA-4/VCAM-1 interactions are important in adhesion of B cell precursors to the bone marrow microenvironment.

Cytogenetic findings in congenital leukemia: Case report and review of the literature

The cytogenetic findings in a five-week-old female infant with acute lymphoblastic leukemia (ALL) are reported. Markers 11q - and 19 + were observed and considered to be due to an interstitial deletion of segment 11q13 to 11q23 of chromosome #11 and an insertion of this segment into the terminal region of the short arm of #19. Previously published banded cases of leukemic infants under one year of age have been summarized. A review of the data in these 29 cases suggests that the appearance of a normal karyotype in acute leukemia of infants (less than or equal to 1 year old) is much less common than in other categories of acute leukemia. Fourteen out of 29 cases (48%) had chromosomal abnormalities involving 11q. Seven of eight ALL cases had aberrations with a breakpoint at 11q22-23; six cases had t(4;11), one case had a del(11q) and ins(19p), and another had a t(1;22;4). All of three AMMoL cases had translocations involving the long arm of #11. The percentage of patients with t(4;11) and certain translocations involving 11q in infants with ALL or AMMoL, respectively, is higher than that seen in ALL and AMMoL in general. Eleven out of 12 cases (92%) of infant acute leukemias with chromosomal abnormalities involving 11q22-23 were five months old or less.

Maturation decreases responsiveness of human bone marrow B lineage cells to stromal-derived factor 1 (SDF-1).

We compared the chemotactic responsiveness of different subsets of human B lineage cells to stromal derived factor‐1 (SDF‐1). High percentages (30–40% of input) of purified bone marrow progenitors including non‐B lineage progenitors, pro‐B cells, and pre‐B cells migrated to SDF‐1α, demonstrating that SDF‐1 is an efficacious chemoattractant of these cells. Pro‐B cells responded optimally to a lower concentration of SDF‐1 than other subsets, demonstrating that SDF‐1 is a more potent chemoattractant of this subset. A lower percentage (10–15% of input) of mature B lymphocytes migrated to SDF‐1α than pro‐B cells, demonstrating that responsiveness of B lineage cells to SDF‐1 decreases during differentiation. Inhibition by anti‐CXCR4 mAb demonstrated that migration of B lineage cells to SDF‐1 was completely dependent on CXC chemokine receptor‐4 (CXCR4). Mature B cells expressed higher levels of CXCR4 receptors than uncommitted progenitors and pro‐B cells, despite differences in responsiveness to SDF‐1. CXCR4 receptors expressed by unresponsive and SDF‐1‐responsive B cells bound SDF‐1a with similar affinities (K D= 1.7–3.3 × 10−9 M). Therefore, elements downstream from CXCR4 appear to regulate responsiveness of B cells to SDF‐1. We speculate that SDF‐1 and CXCR4 direct migration of progenitor cells in microenvironments that promote B lymphopoiesis. J. Leukoc. Biol. 66: 667–673; 1999.

Improved detection of rare CALLA-positive cells in peripheral blood using multiparameter flow cytometry

A major limitation to the detection of rare cell types in the peripheral blood using monoclonal antibodies is nonspecific binding of the antibody reagent to normal cells. Detection of rare common acute lymphoblastic leukemia antigen (CALLA)-positive cells in peripheral blood is significantly improved by using multiple flow cytometric parameters to exclude a variety of mature blood cells which may nonspecifically bind the antibody reagent. Monocytes and granulocytes are excluded by gating out cells with high 90 degrees light scatter. By gating on red fluorescence, a variety of mature cell types binding to phycoerythrin (PE)-conjugated Leu 3, Leu 2, and M3 monoclonal antibodies are also excluded. CALLA-positive lymphoblasts from 6 consecutive patients were not excluded on the basis of these parameters. Gating on log 90 degrees light scatter and log red fluorescence in this fashion reduced the incidence of nonspecific binding to peripheral blood mononuclear cells of a fluorescein-conjugated irrelevant monoclonal antibody by 98% from 308 cells per million to 5 cells per million. One CALLA-positive lymphoblast per 100,000 peripheral blood mononuclear cells could be detected in mixture experiments using this method. The normal range of CALLA-positive cells in adults is less than 16 cells per million peripheral blood mononuclear cells. This low background of CALLA-positive peripheral blood cells may permit the detection of early leukemic relapse in acute lymphoblastic leukemia by analysis of the peripheral blood. This methodology can be applied to the detection of any rare cell type by using phycoerythrin-conjugated antibodies to markers that the cell type does not possess.

Cholesteryl Ester Transfer Protein Polymorphism (TaqIB) Associates With Risk in Postinfarction Patients With High C-Reactive Protein and High-Density Lipoprotein Cholesterol Levels

Objective—To investigate the roles of inflammation and a cholesteryl ester transfer protein (CETP) polymorphism potentially related to recent findings demonstrating coronary risk with increasing high-density lipoprotein cholesterol (HDL-C) level. Methods and Results—A novel graphical exploratory data analysis tool allowed the examination of coronary risk in postinfarction patients relating to HDL-C and C-reactive protein levels. Results demonstrated a high-risk subgroup, defined by high HDL-C and C-reactive protein levels, exhibiting larger HDL particles and lower lipoprotein-associated phospholipaseA2 levels than lower-risk patients. Subgroup CETP-associated risk was probed using a functional CETP polymorphism (TaqIB, rs708272). In the high-risk subgroup, multivariable modeling revealed greater risk for B2 allele carriers (less CETP activity) versus B1 homozygotes (hazard ratio, 2.41; 95% CI, 1.04 to 5.60; P=0.04). Within the high-risk subgroup, B2 allele carriers had higher serum amyloid A levels than B1 homozygotes. Evidence also demonstrates that CETP genotypic differences in HDL subfraction distributions regarding non–HDL-C and lipoprotein-associated phospholipaseA2 may potentially relate to impaired HDL remodeling. Conclusion—Postinfarction patients with high HDL-C and C-reactive protein levels demonstrate increased risk for recurrent events. Future studies should aim at characterizing altered HDL particles from such patients and at elucidating the mechanistic details related to inflammation and HDL particle remodeling. Such patients should be considered in drug trials involving an increase in HDL-C level.

Polymorphism in the Angiotensinogen Gene, Hypertension, and Ethnic Differences in the Risk of Recurrent Coronary Events

The M235T mutation of the human angiotensinogen gene has been shown to be associated with elevated circulating angiotensinogen concentrations and essential hypertension. The frequencies of the 235T allele are significantly different in black and white subjects. We analyzed the independent contribution of the angiotensinogen M235T mutation to the development of recurrent coronary events (coronary-related death, nonfatal myocardial infarction, or unstable angina) in a cohort of 916 black (n=145) and white (n=771) postmyocardial infarction patients who were prospectively studied during an average follow-up of 28 months. The frequency of the 235T allele was significantly higher among black (82%) than among white (44%) patients (P<0.001). There was no evidence for Hardy–Weinberg disequilibrium. During follow-up, 41 cardiac events (28%) occurred in blacks and 197 (26%) in whites (P=0.49). Multivariate Cox proportional hazards regression analysis demonstrated that 235T homozygosity was independently associated with increased risk of coronary events among black (hazard ratio: 2.37; P=0.04) but not white (hazard ratio: 0.93; P=0.68) patients, with a significant ethnic-related interaction effect (P for the difference=0.04). Among hypertensive black patients, the TT genotype was associated with a 3.3-fold (P=0.02) increase in the risk of coronary events. Our findings suggest that homozygosity for the 235T mutation in the angiotensinogen gene is an independent risk factor for coronary events in black postmyocardial infarction patients. The presence of hypertension significantly augments the risk associated with this genetic mutation.

Novel Compound Heterozygous Mutations in the KCNQ1 Gene Associated with Autosomal Recessive Long QT Syndrome (Jervell and Lange-Nielsen syndrome)

Background: The Jervell and Lange‐Nielsen syndrome (JLNS) is the autosomal recessive form of long QT syndrome (LQTS)—a familial cardiac disorder that causes syncope, seizures, and sudden death from ventricular arrhythmias, specifically torsade de pointes. JLNS is associated with sensorineural deafness and has been shown to occur with homozygous mutations in KCNQ1 or KCNE1 in JLNS families in which QTc prolongation is inherited as a dominant trait. This study investigated the molecular pathology of a family with clinical evidence of JLNS.

Thrombospondin-4 polymorphism (A387P) predicts cardiovascular risk in postinfarction patients with high HDL cholesterol and C-reactive protein levels

Few studies are available in human populations investigating involvement of vascular inflammation and oxidative stress-related dysfunctional transformation of high-density lipoprotein (HDL) in establishing cardiovascular disease (CVD) risk. To this end, the current work investigated a subgroup of post-infarction patients at high-risk for recurrent events defined by high levels of HDL cholesterol (HDL-C) and concurrently high levels of C-reactive protein (CRP). Thrombospondin-4 (TSP-4), a matricellular protein of vessel walls associated with inflammation, was investigated in terms of CVD risk using multivariable modelling with a well-characterised functional genetic polymorphism of THBS4 (A387P, rs1866389) along with previously demonstrated risk-related functional genetic polymorphisms of CYBA (C242T, rs4673) and CETP (TaqIB, rs708272), and a set of blood markers. Results revealed risk-association for the gain-of-function P-allele of the THBS4 polymorphism (hazard ratio 2.00, 95% confidence interval 1.10-3.65, p=0.024). Additionally, von Willebrand factor was associated with D-dimer levels in the higher-risk P allele patients suggestive of a connection between endothelial dysfunction and thrombogenesis. In conclusion, TSP-4, a matricellular protein involved in regulating vascular inflammation, plays a role in establishing recurrent coronary risk in post-infarction patients with high levels of HDL-C and CRP. Further studies should focus on additional effects of vascular inflammatory processes on anti-atherogenic functionality of HDL particles.