Transfusion-associated graft-versus-host disease in an immunocompetent patient following cardiac surgery.

OBJECTIVES: We determined which of the 22 blood components obtained from unrelated donors and transfused to an apparently immunocompetent patient following open heart surgery caused transfusion-associated graft-versus-host disease (TA-GVHD). METHODS: Serologic and molecular methods were used to type the donors, the patient's family members, and the patient's postmortem tissues for HLA and a genetic marker on chromosome 17. RESULTS: Two donors were homozygous for the HLA class I antigens A1 B8, for which the patient was heterozygous. Both donors were heterozygous, not homozygous as expected, for the class II alleles. One of them had the same class II alleles as the patient (DRB1*0301, DRB3*0101/DRB1*0404, DRB4*0103). The patient's tissues were chimeric for restriction fragments at 17p13 of this donor. CONCLUSION: One-way HLA match leading to TA-GVHD can be caused by donor blood that is homozygous for class I and heterozygous for class II alleles. Two blood components given to our patient had such one-way HLA match. Class II alleles of the lymphocytes in one component were identical with those of the recipient and caused TA-GVHD. Class II alleles of the lymphocytes in the other component differed from those of the recipient and were eliminated either by the immune system of the patient or the lymphocytes that caused the TA-GVHD (graft versus graft).

The intracerebral microcirculation of the rat in hemorrhagic shock.

The intracerebral microcirculation of the isocortex was studied in unanesthetized rats under hemorrhagic shock. To observe the microvessels, three markers were separately injected intravenously during the shock period: (a) Evans blue for fluorescence microscopic visualization of the vessels. (b) India ink for gross and light microscopic evidence of retention of carbon. (c) Horseradish peroxidase (HRP) for light and electron microscopic study. Lack of spontaneous recovery from shock was associated with: (a) 55-65% blood loss, a low blood pressure (30-40 mm Hg), and a dramatic increase in pulse rate; (b) marked Evans blue fluorescence along the vessels; (c) no retention of India ink in the microcirculation; (d) peroxidase activity on the luminal surface of the endothelium. Absence of India ink in the microcirculation of the isocortex during the shock period, as shown by light and electron microscopy, suggests that there is sufficient cerebral blood flow to clear the carbon particles from the blood stream and that there are no openings greater than 30 nm in the endothelial layer allowing seepage of carbon particles through or between endothelial cells. Vascular Evans blue fluorescence and peroxidase activity were both demonstrated on the luminal surface of the endothelial cells, by light microscopy, indicating that these markers are abnormally retained. Ultrastructural demonstration of increased HRP uptake and adherence onto the endothelial cells confirms these observations. These results show that regional endothelial alterations occur in this model of hemorrhagic shock.

Morphologic and functional changes of the aortic intima during experimental hypertension.

The morphology and permeability to horseradish peroxidase of the rat aortic intima have been investigated in three experimental models of hypertension having different values of plasma renin content and plasma aldosterone level. During hypertension the aortic endothelium shows three main changes: 1) increased arithmetic mean thickness, with prominent rough endoplasmic reticulum and polyribosomes; 2) the appearance of actin microfilament bundles; and 3) increased permeability to horseradish peroxidase. These changes are not present in all models, do not appear to depend on hypertension per se, and are independent of each other. The subendothelial layer of hypertensive animals shows an increased thickness that appears to be correlated with an increase of endothelial cell volume. Our results suggest that: 1) the aortic intima reacts differently to different types of hypertension, and 2) factors other than hypertension per se play a role in the development of vascular changes observed in animals with elevated blood pressure.

Binding of anti-actin autoantibodies to platelets.

Normal platelets incubated with anti-actin autoantibodies (AAA) (from the serum of patients with chronic aggressive hepatitis) do not show binding of these antibodies as seen by indirect immunofluorescence. AAA serum does not inhibit thrombin-induced clot retraction, despite the binding of the antibodies to platelets in the clot. Similarly, AAA serum does not affect "reversible" or "irreversible" aggregation (induced by ADP, collagen or epinephrine), despite the binding of the antibodies to platelet actin under such circumstances. AAA also bind to platelets when aggregation is inhibited by EDTA. The incubation of "reversibly" aggregated platelet with AAA results in a small but definite binding of AAA to platelets. These findings suggest that during "irreversible" and/or "reversible" aggregation, changes take place at the surface of platelets which expose the antigen at the surface of the cell.

Morphological functional changes of aortic endothelium during different types of hypertension.

In rats, ligature of the aorta between the renal arteries produces hypertension which in the early phase (6-7 days) is associated with elevated plasma renin content, and later (40 days) is associated with low plasma renin. During the early phase, the endothelium of the aorta shows elevated permeability to HRP, endothelial cells are hypertrophic and contain bundles of actin microfilaments. During the late phase, permeability to HRP is normal, the endothelial cells are flat and do not contain bundles of microfilaments. Probably the endothelial cells of aorta react in different ways to various hypertensive stimuli and/or adapt to high levels of blood pressure.

Coronary microcirculatory factors and cardiac muscle cell injury.

Coronary artery ligation with or without reperfusion was carried out in Wistar rats to study the role of coronary microcirculatory factors and membrane permeability alteration of cardiac muscle cell in the evolution of cardiac muscle cell injury by using the fine structural extracellular protein tracer, horseradish peroxidase (HRP). The findings were compared with those obtained in noncoronarogenic myocardial injury models following administration of norepinephrine, a pressor, and isoproterenol, a depressor catecholamine. Following left coronary artery ligation lastingfor 10 and 20 minutes, some of the collaterals in the ischemic zone were perused by the tracer, but the numer of patent capillaries decreased during 60-min ligation. The inhomogeneous involvement of cardiac muscle cells in ischemic injury correlated well with these microcirculatory findings. In comparison to permanent ischemia, an abrupt deterioration of the cardiac muscle cell alteration occured after reperfusion with influx of HRP into the damaged cells. The binding of tracer to myofilaments was, however, a later event as compared to that seen in the catecholamine models. The latter observation implies that, in addition to microcirculatory factors, direct cardiac muscle cell stimulation should also be considered in the evolution of noncoronarogenic myocardial injury.

Contractile events during inflammation.

Contractile events during wound healing. During granulation tissue contraction, fibroblasts develop characteristics typical of smooth muscle; (1) they contain an extensive cytoplasmic fibrillar system, (2) they show immunofluorescent labeling of anti-actin antibodies, (3) there are cell and cell to stroma attachments, (4) strips of granulation tissue, when tested pharmacologically in vitro, behave similarly to smooth muscle. These data support the view that under certain conditions, fibroblasts can differentiate into a cell type structurally and functionally similar to smooth muscle and this cell, the 'myofibroblast', plays an important role in connective tissue contraction. During epithelialization, epidermal cells develop an extensive cytoplasmic contractile apparatus which has morphological and immunological characteristics similar to those of myofibroblasts. Such apparatus disappears as soon as epithelialization is completed. It is proposed that such a contractile apparatus plays a role in cell motility enabeling individual cells to rearrange themselves in an appropriate pattern. In conclusion, significant amounts of contractile proteins may be synthetized by fibroblasts and epithelial cells during wound healing and may play an important role in this process.

Cytoplasmic contractile apparatus in aortic endothelial cells of hypertensive rats.

Hypertension was produced in male Wistar rats (150 gm. body weight) by complete ligature of the aorta between the renal arteries. Electron microscopic examination revealed that 1 week later the amount of cytoplasmic microfilaments in the endothelial cells of the aortic segment above the coarctation (mean blood pressure 160 mm. Hg) was strikingly increased as compared with normal animals. The endothelial cells in the segment below the coarctation (mean blood pressure 25 mm. Hg) contained few filaments and were similar to the cells in the aortic endothelium in controls (mean blood pressure 105 mm. Hg). Microfilaments measured 40 to 70 angstrom in diameter and were mostly located close to the endothelial clefts, where they formed longitudinal bundles or a network. The bundles of microfilaments contained electron-dense areas similar to the "attachment sites" of the underlying smooth muscle. By using en face preparations of aortic endothelial cells treated with antiactin autoantibodies (AAA) followed by anti-human IgG, it was seen that in hypertensive animals the cells above the ligature were intensely fluorescent when compared with those of the aortic portion below the ligature or those of the controls. The fluorescence was abolished after incubation of the AAA sera with thrombosthenin A. The correlation between electron microscopic and immunologic findings suggests that the microfilaments present in the endothelial cells of hypertensive animals are composed, at least in part, of actin. Endothelial cells so modified may play a role in permeability or may be related to other phenomena such as electrotonic coupling and synchronized contraction of aortic cells during hypertension.

Experimental focal glomerular lesions elicited by insoluble immune complexes. Ultrastructural and immunofluorescent studies.

Injection of small amounts of ferritin intravenously, into the aorta (above the renal arteries) or into the left renal artery of rabbits hyperimmunized against this protein, results in the formation of circulating, insoluble, antigen-antibody complexes. Some of these complexes localize focally in the renal glomerular capillaries, where they elicit severe lesions. The fate of the complexes and the evolution of the lesions have been followed by immunofluorescent and electron microscopic techniques. Within a few hours, the deposition of complexes in the glomeruli resulted in a massive accumulation of neutrophils platelets, and fibrin, sometimes leading to acute focal necroses of some loops. These lesions were quite similar to those developing in the small dermal vessels during the Arthus reaction. Most of the complexes were rapidly phagocytosed and degraded by neutrophils; swelling and proliferation of endothelial and mesangial cells usually followed the acute damage and contributed to the removal of remaining complexes, cell debris, and fibrin deposits. Later, focal areas of mesangial proliferation and sclerosis were observed, containing large amounts of basement membrane-like material and sometimes of collagen fibrils; synechiae and crescent formation were noted in certain places. These observations suggest that the glomerular localization of very large, poorly soluble or insoluble immune complexes may be responsible for the focal glomerular changes seen in association with subacute bacterial endocarditis, with anaphylactoid purpura, or for some of the most severe lesions developing during chronic immune complex diseases.

Contractile apparatus in aortic endothelium of hypertensive rat.

One week after hypertension was produced in male Wistar rats (150 g body weight) by a complete aortic ligature placed between renal arteries, electron microscopic studies showed a striking increase of cytoplasmic microfilaments in the endothelial cells of the aortic segment above coarctation (mean blood pressure 160 mm Hg). These microfilaments measured 40-70 A in diameter and were located particularly close to the endothelial clefts. In "en face" preparation of aortic endothelial cells treated with antiactin autoantibodies (AAA) followed by antihuman IgG, the cells above the ligature of hypertensive animals were intensely fluorescent compared with those of the aortic portion below the ligature or that of controls. The fluorescence was abolished after incubating the AAA-containing sera with thrombosthenin-A, suggesting the presence of actin. There was also an increase transport of horseradish peroxidase and ferritin through the endothelial cell layer. While endothelial cells so modified may play a role in permeability regulation, they may also be related to such mechanisms as electronic coupling and synchronized contraction of aortic cells during hypertension.