Merrill Emmett

Protein abnormalities in adult respiratory distress syndrome, tuberculosis, and cystic fibrosis sera.

Crossed immunoelectrophoresis (X-IEP) revealed several abnormalities in serum proteins from patients with adult respiratory distress syndrome (ARDS), tuberculosis (TB), and cystic fibrosis (CF). The two quite different kinds of pulmonary disease, one acute (ARDS) and the other chronic (TB and CF) exhibited serum changes specific for each disease and abnormalities associated with inflammation and pathogenesis, in general. In ARDS sera, most proteins were extremely low, presumably due to leakage into the lungs through damaged tissue, while the acute-phase proteins, orosomucoid, α1-antitrypsin, α1antichymotrypsin, and haptoglobin, were markedly high when compared to the overall protein pattern. The extremely high α1antichymotrypsin values were not seen in corresponding TB and CF sera. Numerous TB patients had elevated α1antitrypsin, α1antichymotrypsin, and haptoglobin, but only the α1antitrypsin population mean was significantly different from normal. Gc-globulin, ceruloplasmin, and β-lipoprotein were higher and α1-lipoprotein and inter-α-trypsin inhibitor lower than normal. All other quantitative serum changes were not statistically significant. Surprisingly, all TB patients belonged to the Gc-1-1 genotype in contrast to the Gc-1-1, Gc-1–2, Gc-2-2 polymorphisms of the other populations. CF homozygote sera revealed statistically significant increases in the acute-phase proteins, α1-antitrypsin, α1-antichymotrypsin, and haptoglobin, while orosomucoid, transferrin, IgA, and IgG tended to be higher than normal. The tendency for higher levels of transferrin indicated possible iron deficiency in some patients. In contrast, prealbumin, α1lipoprotein, and inter-α-trypsin inhibitor were significantly depressed in CF patients. CF heterozygotes shared the decrease of α1-lipoprotein with the patients while exhibiting small but significant depressions of α2-macroglobulin and IgG. Though not statistically significant, lowered concentrations of α1antitrypsin were evident for the heterozygotes.

Crossed immunoelectrophoretic analysis of ARDS lavage proteins.

Serum proteins in normal and ARDS bronchoalveolar lavages were analyzed using crossed Immunoelectrophoresis. Normal lavages demonstrated relatively few proteins (albumin, α1antitrypsin, transferrin, and haptoglobin) in low concentrations. In contrast, substantial amounts of all identifiable serum proteins were detected in ARDS lavages. IgA was apparently locally produced. Two of the largest proteins, β-lipoprotein (mol wt > 2 million) and IgM (mol wt ~ 900,000) were found to be complexed as evidenced by their coprecipitation in a single spike in ARDS lavage. Electrophoretic modifications of ARDS albumin and α1antitrypsin precipitation peaks and partial identity spurring of the α1-lipoprotein peak with other precipitation loops indicated possible complex formation between these proteins and other possibly pathogenic lung fluid constituents. Similarly, modifications of orosomucoid and Gc-globulin peaks indicated possible molecular alterations resulting from interactions with other components. The relatively few protein modifications exhibited in ARDS lavages together with α1antitrypsin-protease complex formation confirm the relative absence of substantial proteolytic activity in ARDS edema fluids obtained within 12 hr of the onset of the syndrome demonstrated in previous studies.

Inhibition of Colony-Stimulating Factor (CSF) Production by Postburn Serum: Negative Feedback Inhibition Mediated by Lactoferrin

Fatal infections in severely burned patients are often preceded by a decline in the production of colony-stimulating factor (CSF) and the proliferation of granulocyte-macrophage stem cells (CFU-GM), and overwhelming sepsis is often associated with leukopenia. The underlying mechanisms accounting for these granulopoietic defects are poorly understood, but the fact that postburn serum has been shown to inhibit CSF production suggests that a humoral factor or factors may play a role. Previous work has demonstrated that plasma levels of lactoferrin (LF), a known inhibitor of CSF production, are elevated following burn injury. To determine if LF is responsible for serum-mediated inhibition of CSF production, serial plasma levels of LF were measured in 18 burn patients using an enzyme-linked immunoabsorbent assay (ELISA). LF was elevated within 24 hours of injury and was associated with an absolute granulocytosis which rapidly declined, reaching a nadir at postburn days 3 through 5. Postburn serum, especially when collected during the first 24 hours following burn injury, inhibited in vitro CSF production by normal human peripheral blood mononuclear cells. Pre-incubation of postburn serum with an LF antibody restored normal CSF production. These data suggest that LF may play an important role in the regulation of postburn granulopoiesis.

Crossed immunoelectrophoresis of rat serum

A crossed immunoelectrophoresis (X-IEP) reference profile of normal (Sprague-Dawley) rat serum antigens was developed. Twenty-three antigens were detected; 13 were identified. These included inter-alpha-trypsin inhibitor, alpha 1-antichymotrypsin, and fibronectin, which had not previously been identified in rat serum. Many of the antigens were similar electrophoretically to their analogs in human and mouse serum, but some were substantially different. Inter-alpha-trypsin inhibitor, fibronectin, and C3 globulin were more anodic, while transferrin was more cathodic. Rat haptoglobins mobility was between those of human and mouse haptoglobins. As in the mouse, but more so, alpha-macroglobulin in the rat had alpha 1 mobility rather than alpha 2 seen for this protein in human serum. Only rat alpha 1-antichymotrypsin exhibited a double peak. There were variations in haptoglobin and alpha 1-lipoprotein mobilities among different strains of rat. This establishment of a standard X-IEP map of rat serum will simultaneously permit multiple quantitative and qualitative analyses of rat serum antigens for various experimental and clinical purposes.

Differential serum protein binding of benzidine- and benzidine-congener based dyes and their derivatives

Environmental dyes and their derivatives, some of which are genotoxic, must be transported within the body to the tissues which they affect. One mechanism for this can be observed directly by crossed immunoelectrophoresis (X-IEP). Binding of these chemicals to certain serum proteins changes electrophoretic and immunoprecipitation morphology in X-IEP patterns. This is demonstrated here for four azo dyes derived from benzidine, 3,3′-dimethylbenzidine, and 3,3′-dimethoxybenzidine, and their parent aromatic amines. Direct Red 2 (a 3,3′-dimethylbenzidine-based dye), Direct Blue 15 (a 3,3′-dimethoxybenzidine-based dye), Direct Black 38 (a benzidinebased dye), and Evans Blue (a 3,3′-dimethylbenzidine-based dye) all bound to albumin, α1-lipoprotein, β-lipoprotein, and hemopexin. Direct Red 2 only slightly affected the mobilities of these proteins. Direct Blue 15 bound also to prealbumin and α1-antichymotrypsin, and degraded C3 globulin. Direct Black 38 and Evans Blue bound to numerous additional proteins. Evans Blue bound variably to proteins of sera from different individuals, suggesting that there are individual differences in serum protein binding capabilities for these chemicals. Of the three derivatives of the benzidine dyes, only 3,3′-dimethylbenzidine caused changes in X-IEP patterns, indicating its binding to the serum proteins. This chemical differentially affected sub-populations of α1-lipoprotein, either by altering its electrophoretic mobility or inhibiting its recognition by antibodies. Autoradiographic analyses demonstrated the binding of benzidine and 3,3′-dimethylbenzidine to both α1- and β-lipoproteins.