HD Ochs

We examined the role of the neutrophil membrane antigen complex designated CDw18 (LFA-1/Mac-1/p150, 95) in human peripheral blood neutrophil adherence to cultured human umbilical vein endothelial cells (HEC) pretreated with lipopolysaccharide (LPS), interleukin 1 (IL 1), or recombinant tumor necrosis factor-alpha (rTNF-alpha). Pretreatment of HEC with LPS produced a dose-and time-dependent increase in subsequent neutrophil adherence (7 +/- 1% adherence to untreated HEC vs 38 +/- 3% adherence to HEC pretreated for 4 hr with LPS 150 ng/ml; mean +/- SE of 22 experiments: p less than 0.001). This effect was observed in primary and passaged HEC, but not in bovine aortic endothelial cells or human dermal fibroblasts. The LPS-induced activity appeared to be associated with the HEC surface, since it was not removed by washing and was not detected in the supernatant medium. Inhibition of RNA or protein synthesis during pretreatment of HEC with LPS prevented induction of the adherence-promoting activity. Pretreatment of HEC with IL 1 and rTNF-alpha produced a similar protein synthesis-dependent increase in neutrophil adherence to HEC. Coincubation of neutrophils with murine monoclonal antibody (MoAb) 60.3, an antibody directed to the CDw18 complex, produced a 70 +/- 4% inhibition of neutrophil adherence to LPS-pretreated HEC, 59 +/- 5% inhibition of adherence to IL 1-pretreated HEC, and 65 +/- 11% inhibition of adherence to rTNF-alpha-pretreated HEC (means +/- SE of 18, seven, and five experiments, respectively). Notably, MoAb 60.3 did not completely inhibit neutrophil adherence to pretreated HEC, although it completely inhibited adherence to untreated HEC when neutrophils were activated directly with phorbol ester. Similarly, the adherence of neutrophils from a patient with an inherited deficiency of the CDw18 complex to LPS-, IL 1-, and rTNF-alpha-pretreated HEC was markedly reduced compared with normal neutrophils (5 to 11% adherence with CDw18-deficient neutrophils vs 43 to 54% adherence with normal neutrophils), but adherence to pretreated HEC was still significantly greater than adherence to HEC that were not pretreated (2% adherence). We conclude that LPS, IL 1, and rTNF-alpha induce synthesis of an endothelial cell-surface factor(s) that promotes neutrophil adherence primarily by a mechanism involving the CDw18 complex. It thus appears that the CDw18 complex is important for augmented neutrophil adherence to endothelium in vitro whether the is stimulated directly by inflammatory mediators or indirectly by endothelial-dependent mechanisms.

We are reporting a male patient who suffered from chronic granulomatous disease associated with cytochrome b-245 deficiency and McLeod red cell phenotype, Duchenne muscular dystrophy, and retinitis pigmentosa. On cytogenetic analysis, he seemed to have a very subtle interstitial deletion of part of band Xp21. Since it was impossible to know whether this material was truly deleted or inserted elsewhere in the genome, somatic cell and molecular studies were carried out. In somatic cell hybrids, the deleted X chromosome was isolated on a Chinese hamster background. Southern blot analysis with 20 single-copy probes, that had been mapped to the X short arm, led to the discovery of one (probe 754) that is missing from this patient's X chromosome and also from his total DNA. This proves that he, indeed, has a deletion rather than a balanced insertion. The results provide cytological mapping information for the X-linked phenotypes present in this patient. Furthermore, probe 754 recognizes a restriction fragment length polymorphism of high frequency that makes it the most powerful probe currently available for linkage studies with X-linked muscular dystrophy.

We have previously described two patients with a congenital defect in neutrophil function characterized by an inability to form pus. The patients' neutrophils lack a membrane glycoprotein of mol wt 150,000 daltons (GP-150) on analysis by SDS-PAGE. This glycoprotein is part of a membrane antigen complex recognized by the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produces defects in chemotaxis and phagocytosis in vitro similar to those observed in the patients. Since neutrophil adherence to vascular endothelium is prerequisite to neutrophil emigration in vivo, we examined the interaction of the patients' neutrophils and normal neutrophils treated with MoAb 60.3 with cultured endothelium. Adherence was determined as the percentage of 51Cr-labeled purified peripheral blood neutrophils which remained adherent to plastic wells or endothelial monolayers after a 45-minute incubation at 37 degrees C. The percentage of neutrophils from patient 1 remaining adherent to uncoated, fibronectin-coated, or laminin-coated plastic was similar to that observed in normal neutrophils (55% to 84% adherence with normal neutrophils v 73% to 78% adherence with the patient's neutrophils and 63% to 82% adherence with MoAb 60.3-treated normal neutrophils). The adherence of the neutrophils from patient 1 and MoAb 60.3-treated normal neutrophils to human or bovine endothelium in serum-free medium was also not significantly different from that observed in normal neutrophils (less than 10% adherence with normal, MoAb 60.3-treated, and patient neutrophils). In medium containing 10% autologous or heterologous human plasma, however, the adherence of neutrophils from patient 1 or MoAb 60.3-treated normal neutrophils to endothelial monolayers was significantly reduced (35% +/- 7% of normal neutrophils in seven experiments). Although phorbol myristate acetate (PMA) (10 ng/mL) and calcium ionophore A23187 (10(-5) mol/L) markedly increased the adherence of normal neutrophils to endothelial monolayers in serum-free medium (40% to 85% adherence), neither agent increased the adherence of the neutrophils from patient 1 or normal neutrophils treated with MoAb 60.3 (less than 5% adherence). The adherence of PMA-activated neutrophils from patient 2 to endothelial monolayers was also markedly decreased when compared with that of normal neutrophils. Postsecretory cell-free supernatants from PMA-activated normal neutrophils failed to augment adherence of neutrophils from patient 1 (less than 5% adherence).(ABSTRACT TRUNCATED AT 400 WORDS)

One-hundred fifty-three recipients of HLA-identical sibling marrow transplants for aplastic anemia or hematologic malignancy were injected with bacteriophage phi X174 (phage), pneumococcal polysaccharide antigen (PPA), or keyhole limpet hemocyanin (KLH). Antibody levels were determined several times in the 6 wk after injection. Multiple regression techniques were used to determine what factors played significant roles in the antibody response. The most significant factors were the time elapsed from transplantation, chronic graft-versus-host disease (GVHD), and antithymocyte globulin (ATG) treatment. All patients had low antibody responses to all antigens in the first 180 days from transplant. Beyond 180 days patients without chronic GVHD showed antibody responses indistinguishable from those of normal donors. However, patients with chronic GVHD had the following impairments: (1) primary response to phage, (2) conversion from IgM to IgG in secondary response to phage, (3) secondary response to KLH, and (4) response to PPA. ATG treatment given to patients either prophylactically or therapeutically for acute GVHD was followed by lower primary responses to phage in the first 180 days and poor ability to switch from IgM to IgG antibody in the secondary response beyond 180 days postgrafting. Other factors did not yield additional significant information about ability to predict antibody responses including diagnosis, conditioning regimen, treatment in or out of laminar air flow rooms, transplantation, pretransplant refractoriness of the recipient to platelet transfusions from random donors, donor age or donor sex, and steroid administration for treatment for prevention of GVHD. The data indicate that, given enough time after transplantation, the ability to produce normal antibody function recovers except in those patients experiencing chronic GVHD.