key: cord-0009972-t5bz4kmk
authors: Macphee, Peggy J.; Dindzans, Vincent J.; Fung, Lai‐Sum; Levy, Gary A.
title: Acute and chronic changes in the microcirculation of the liver in inbred strains of mice following infection with mouse hepatitis virus type 3
date: 2005-12-05
journal: Hepatology
DOI: 10.1002/hep.1840050422
sha: f76200f5f1f1b67cc66dcff0343f36818a9c2857
doc_id: 9972
cord_uid: t5bz4kmk

The acute and chronic effects of mouse hepatitis virus type 3 on the microcirculation of the liver in both semisusceptible C3HeB/FeJ and fully resistant A/J mice were studied. In the C3HeB/FeJ mice, abnormalities of microcirculatory flow were noted as early as 12 hr after infection and by 24 hr, localized avascular foci appeared. Disturbances were characterized by granular blood flow, sinusoidal microthrombi, distortion of sinusoids by edematous hepatocytes and necrotic lesions. Following the acute infection, Day 10, two patterns of chronic disease were observed. Eighty percent of the mice developed chronic granulomatous hepatitis whereas in the remaining 20% a more severe chronic aggressive hepatitis was observed which was characterized by ongoing hepatocellular necrosis and a marked mononuclear cell infiltrate. In both cases, in vivo microcirculatory abnormalities were found predominantly around visible lesions. Onset of the microcirculatory abnormalities was found to be concomitant with a rise in monocyte related procoagulant activity. Procoagulant activity rose acutely and remained elevated throughout the chronic phase but was higher in animals with severe disease. In contrast to the above, normal blood flow and histology were seen in the resistant A/J mice at all times following infection, and procoagulant activity remained at basal levels despite active viral replication as demonstrated by immunofluorescence studies and recovery of infectious virus. These observations suggest a role for monocyte procoagulant activity in the development of microcirculatory abnormalities following mouse hepatitis virus type 3 infection which may be important in the pathogenesis of the disease.

liver (2) . The sinusoids of the simple liver acini along with the nutrient portal triad and draining terminal hepatic venule comprise the basic microvascular unit of the liver. Oxygen levels, blood composition, flow and pressure in the microcirculation contribute to a local environment which is in dynamic balance with the functionally heterogeneous hepatocytes across the simple liver acini; this is reflected by significant gradients in oxygen tension, redox state and cytoplasmic enzyme activities across zones 1 to 3 (3,4). Selective zonal injury to the hepatic parenchymal plate by toxic agents in turn results in localized zonal abnormalities of the microcirculation (5, 6) .

Infection with mouse hepatitis virus type 3 (MHV-3), a member of the single-stranded, positive polarity, RNAcontaining coronaviruses, causes a strain-dependent spectrum of liver disease in inbred mice (7) . Mice of fully susceptible strains (Balb/cJ, C57B1/6J and DBA) die of fulminant hepatitis; mice of semisusceptible strains (C3H/St, C3HeB/FeJ) develop acute hepatitis which ultimately results in chronic liver disease while resistant adult mice (A/J) develop no liver disease. Previous experiments showed that viral replication occurs to comparable levels in resistant as well as susceptible mice, hence it is apparent that viral replication alone contributes only in part to the pathogenesis of viral hepatitis (8) . Experimental evidence has suggested that host-immune defects may have a major role in determining strain-dependent susceptibility to MHV-3 infection (8) .

We have previously shown that strain-dependent susceptibility to MHV-3 correlates directly with the spontaneous T lymphocyte controlled expression of a procoagulant monokine which exhibits prothrombin cleaving activity [procoagulant activity (PCA)] (9) . A biological role for this activity in the pathogenesis of liver disease has been suggested by in uiuo microscopic observations during acute MHV-3 infection in the fully susceptible Balb/cJ strain (8) . In this system, abnormalities of the microcirculation consisting of granular blood flow and sinusoidal microthrombi preceded in uiuo viral replication by 24 hr. Subsequently, focal thrombotic and/or ischemic lesions formed and finally resulted in confluent liver necrosis.

The present study was designed to examine the hepatic microcirculation during both the acute and chronic phases of MHV-3 infection in mice of the semisusceptible C3HeB/FeJ strain (7, 10) . The in uiuo hepatic microcirculation was studied and compared with both the spontaneous expression of monocyte/macrophage PCA and histological parameters of liver disease.

The origin and growth of MHV-3 has been previously described (9) . MHV-3, obtained from the American Type Culture Collection, Rockville, Md. (ATTCC VR 262), was plaque-purified on monolayers of DBT cells. Stock virus was grown to a titer of 1.2 x lo7 plaque-forming units per ml in 17 CL1 cells. The virus was harvested by one cycle of freeze thawing and clarified by centrifugation at 4,500 X g for 1 hr at 4°C. Virus was assayed on monolayers of L2 cells in a standard plaque assay (9) . Viral purification was accomplished by centrifugation through polyethylene glycol and sequential potassium tartrate gradients (11) . Purified virus at a t,iter of 1.2 x lo9 plaque-forming units per ml was finally resuspended in normal saline.

A/J and C3HeB/FeJ mice, 6 to 8 weeks of age, were obtained from Jackson Laboratories, Bar Harbor, Maine. The presence of serum antibody to mouse hepatitis virus was ruled out by a standard radioimmunoassay as described previously (11) . Mice were i.p. injected with lo3 plaque-forming units of stock MHV-3 in 0.1 ml. of Dulbecco's modified Eagle's medium (DMEM). They were followed for up to 4 months and sacrificed at various time intervals. Blood was obtained by axillary bleeding; samples of liver tissue were processed for immunofluorescence, viral titers and routine H and E histology.

A heterologous antibody to MHV-3 was produced in New Zealand White male rabbits by hyperimmunization with purified MHV-3 in complete Freund's adjuvant (8) . The antiserum was found to be suitable for use a t 1/40 dilution. Immunofluorescence was studied by an indirect method using a fluorescein-labeled goat antirabbit IgG as the probe. By radioimmunoprecipitation, the antibody demonstrated predominant specificity for the nucleocapsid protein with weak E l and E2 envelope glycoprotein reactivity.

Liver tissue was homogenized in DMEM as a 10% homogenate (w/v) at 4°C. Viral titers were then determined on monolayers of L2 cells in a standard plaque assay (9) . ISOLATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMs) Heparinized blood from 10 mice for each time point was pooled and suspended in an equal volume of DMEM. Mononuclear cells were isolated over Ficoll-Hypaque gradients by centrifugation at 22°C at 2,200 X g for 12 min. Cells at the interface were collected and found to be greater than 98% mononuclear cells by cytologic examination using Giemsa stain. Viability was greater than 99% by trypan blue exclusion. Cells were washed twice and resuspended in DMEM at a concentration of 2 X lo6 mononuclear cells per ml.

Spleens were removed aseptically from C3HeB/FeJ mice, and cells were suspended in DMEM. Mononuclear cells were isolated by centrifugation over Ficoll-Hypaque at 22°C for 12 min at 2,200 x g. Cells from the interface were greater than 98% mononuclear by cytologic examination. Cells were washed twice and resuspended in DMEM at a concentration of 2 X lo6 mononuclear cells per ml.

Samples of viable cells or cells which were subjected to three cycles of freeze thawing and sonication were assayed for the capacity to shorten the spontaneous clotting time of normal citrated human plasma in a onestage clotting assay (12) . T o 0.1 ml of cellular homogenate or viable cells at 4"C, 0.1 ml of citrated normal human platelet-poor plasma or factor-deficient plasma (Helena Laboratories, Beaumont, Tex.) was added, followed by 0.1 ml of 25 mM CaC12 to start the reaction. The time for the appearance of a fibrin gel was recorded. Clotting times were converted to units of PCA by comparison to a rabbit brain thromboplastin standard (Dade Division, American Hospital Supply, Miami, Fla.) where 36 mg dry weight per ml were assigned a value 100,000 mU PCA. The assay was utilized over the range of 1 to 100,000 mU or 10' to 10' cells, and it was linear over this range with normal plasma substrate. Media with or without 10% fetal calf serum and buffers were all without activity.

Mice were injected i.p. with 1,000 PFU of stock MHV-3 in 0.1 ml DMEM and examined at various time intervals. Control mice were injected with 0.1 ml of DMEM. Anesthesia was obtained with Nembutal (6.4 mg per 100 gm body weight) by i.p. injection. The animals were immobilized on a heated surgical table with temperature control at 37°C. Mice were intubated (PE-10 intramedic tubing, Clay Adams, NY) by tracheostomy and ventilated with room air by a volume ventilator at 50 cycles per min and 2 ml per breath. Laparotomy by midline incision was performed, and the peritoneal cavity was irrigated with Ringer's lactate solution warmed to 37°C. Tubocurare (0.02 ml of 3 mg per ml solution) was injected intramuscularly as required. The liver margin was transilluminated with a quartz rod connected to a fiberoptic light source (Intralux 150H Volpi, Zurich, Switzerland). The microcirculation was observed through an E. Leitz microscope as previously described (8) .

No abnormalities in liver histology could be found in A/J mice for up to 21 days following infection ( Figure   1A ). In contrast, at 24 hr, the livers from C3HeBIFeJ mice had focal necrotic lesions consisting of acidophilic degenerating hepatocytes and nuclear debris with a sparse inflammatory infiltrate ( Figure 2A ). By 3 days postinfection, these early lesions became larger, more numerous and were associated with an infiltrate of pyknotic polymorphonuclear leukocytes ( Figure 2C ). At 5 days, confluent necrosis was evident in some sections ( Figure 3A ). By 7 to 10 days, the lesions were densely infiltrated with intact mononuclear cells, and the adjoining liver parenchyma underwent intense hepatocyte regeneration as evidenced by many mitotic figures ( Figure  4A ). Following the acute phase of the disease (Day lo), two patterns of chronic disease were observed. The first was characterized by the appearance of epithelioid cells with large pale nuclei and pale granular cytoplasm. These appeared within the foci of necrosis by 11 days postinfection, and by Day 15, most remaining lesions were organized into granulomas consisting of centralized epit helioid cells surrounded by mononuclear cells ( of disease was observed in approximately 20% of mice. These animals developed an aggressive form of hepatitis characterized by persistent necrotic foci with a mixed inflammatory cell infiltrate, largely mononuclear, a severe terminal portal venule vasculitis and focal hepatocellular dropout ( Figure 6A ). Both of these groups of animals died within 6 to 9 months of the infection, a marked reduction from the normal 24 to 30 months.

By indirect immunofluorescence, viral antigens could be detected at 24 hr in both the fully resistant A/J mice and the semiresistant C3HeBFeJ mice. In the A/J mice, large viral deposits were found within hepatocytes, endothelial cells and Kuppfer cells ( Figure 7A ). By 48 to 72 hr, antigens were predominantly localized to the cytoplasm of the hepatocytes but some membrane deposition could be seen ( Figure 7B ). Viral antigens were detected up to 7 days following infection but were undetectable at Day 10 ( Figure 7C ).

In livers from C3HeB/FeJ mice, viral antigens were present in the cytoplasm and at the cell surface of hepatocytes at 24 hr postinfection ( Figure 8A ). By 36 to 48 hr, viral antigens were found in areas of necrosis as well as in apparently normal hepatocytes ( Figure 8B ). At 7 days, viral antigens were seen in areas of inflammation, which were characterized by a mononuclear cell infiltrate ( Figure 8C ). For up to 3 months postinfection, viral antigens were detected in both areas of inflammation and morphologically normal hepatocytes ( Figure 8D ).

For 12 hr following infection, virus could not be recovered from the livers of either A/J mice or C3HeB/ FeJ mice. However, by 24 hr, high titers were detected in both strains of mice ( Figure 9 ). The viral titers peaked of disease was observed in approximately 20% of mice. These animals developed an aggressive form of hepatitis characterized by persistent necrotic foci with a mixed inflammatory cell infiltrate, largely mononuclear, a severe terminal portal venule vasculitis and focal hepatocellular dropout ( Figure 6A ). Both of these groups of animals died within 6 to 9 months of the infection, a marked reduction from the normal 24 to 30 months.

By indirect immunofluorescence, viral antigens could be detected at 24 hr in both the fully resistant A/J mice and the semiresistant C3HeBFeJ mice. In the A/J mice, large viral deposits were found within hepatocytes, endothelial cells and Kuppfer cells ( Figure 7A ). By 48 to 72 hr, antigens were predominantly localized to the cytoplasm of the hepatocytes but some membrane deposition could be seen ( Figure 7B ). Viral antigens were detected up to 7 days following infection but were undetectable at Day 10 ( Figure 7C ).

In livers from C3HeB/FeJ mice, viral antigens were present in the cytoplasm and at the cell surface of hepatocytes at 24 hr postinfection ( Figure 8A ). By 36 to 48 hr, viral antigens were found in areas of necrosis as well as in apparently normal hepatocytes ( Figure 8B ). At 7 days, viral antigens were seen in areas of inflammation, which were characterized by a mononuclear cell infiltrate ( Figure 8C ). For up to 3 months postinfection, viral antigens were detected in both areas of inflammation and morphologically normal hepatocytes (Figure 8D ).

For 12 hr following infection, virus could not be recovered from the livers of either A/J mice or C3HeB/ FeJ mice. However, by 24 hr, high titers were detected in both strains of mice ( Figure 9 ). The viral titers peaked by 5 to 7 days postinfection and were no longer detected by Day 7 in A/J mice and by Day 11 in C3HeB/FeJ mice (Figure 9 ).

Previously, we have shown that PBM cells, isolated from the blood of uninfected mice and stimulated in uitro with MHV-3 responded with an increase in PCA which directly correlated with the in i h susceptibility to hepatic injury in that strain (9).

To determine whether a similar response occurs in uiuo, PBMs from A/J and C3HeB/FeJ mice were assayed directly following isolation from the blood for both viable (cell surface) and total content PCA ( Figure 10 ). Each PCA determination was made on PBMs from the pooled blood of 10 mice. Mononuclear cells from A/J and C3HeBFeJ mice which had been injected with 100 pl of DMEM as controls had a basal surface PCA of 20 mU per 10" PBMs. Following infection, there was no increase in PCA in PBMs from fully resistant A/J mice. In C:3HeB/FeJ mice, there was a greater than 10-fold increase in viable PCA by 12 hr which reached a maximum 40-fold increase over baseline at Day 4. This elevated PCA persisted for the subsequent 3 to 4 months with minor fluctuations in activity. The values of total PCA (obtained from disrupted cellular homogenates) were approximately twice those expressed at the cell surface (viable), and both activities followed parallel patterns. In order to examine the PCA response of individual mice, splenic mononuclear cells were harvested from C3HeB/FeJ mice and assayed for total content PCA ( Table 1) . Early in the course of the infection, all animals had markedly elevated PCA corresponding with the severe histologic and microcirculatory disturbances. At Days 10 to 14, two divergent patterns of PCA were observed. Although in both strains, the PCA remained elevated, animals that developed severe hepatitis (Type 1) maintained the high levels of PCA found during the acute phase of the disease whereas animals that developed chronic granulomatous hepatitis (Type 2) had PCA elevations at 5 to 10 times background but only 25 to 30% of the PCA observed in Type 1 animals. No increase in total PCA was found in PBMs or splenic macrophages from A/J mice over initial basal values.

I n uiuo microscopy of normal control A/J and C3HeB/ FeJ mice that had received 100 pl i.p. of DMEM revealed normal streamlined blood flow of the terminal hepatic vessels and the sinusoids ( Figure 1B ). Blood flowed from the terminal portal venules, with occasional bursts of arteriolar flow, into the proximal sinusoids. The blood could be followed as it drained into terminal hepatic venules. Liver cell cords 1 to 2 cells wide separated the sinusoids. At 12 hr postinfection in C3HeBFeJ mice, the velocity of blood flow in the terminal hepatic vessels was slowed, however, the architecture of the parenchyma was normal. Small avascular, pale-colored parenchymal lesions, spanning 2 to 3 sinusoids appeared by 24 hr ( Figure 2B ). Localized granular blood flow, caused by reduced velocity of clumped erythrocytes, was prominent in dilated sinusoids which shunted blood around the lesions. Most of the lesions were found in zone 1 (periportal) of the simple liver acinus with normal intervening parenchyma. At 48 hr, diffuse granular flow occurred in the microcirculation, and sinusoidal microthrombi could be seen ( Figure 2D) . By 3 to 4 days, the parenchymal lesions were more numerous and larger, spanning as much as a full acinus. Sinusoidal flow was disrupted by microthrombi and edematous hepatocytes, and many sinusoids were difated with large pools of stagnant blood. At 5 days, parenchymal lesions had coalesced with widespread hepatic necrosis and severe parenchymal edema ( Figure 3B ). By Days 7 to 10, the diffuse flow abnormality had resolved and many lesions demonstrated dark hemorrhagic centers ( Figure 4B ). At Day 11, small pale and more sharply circumscribed lesions were noted ( Figure  4D ). By Day 15, the lesions were further condensed and contained occasional patches of a yellow-green stained pigment. From Day 21 to 2 months, avascular foci of heaped-up tissue were scattered throughout the liver while the intervening parenchyma exhibited normal architecture and sinusoidal blood flow. Histological examination of serial sections showed these lesions to be giant cell granulomas ( Figure 5B ). In the animals that developed granulomatous hepatitis, in uiuo microcirculatory flow abnormalities were confined to the sinusoids surrounding the granulomatous lesions. This consisted largely of granular blood flow at the periphery of the granuloma, and no blood flow was observed through the granuloma itself. Histologic examination demonstrated large numbers of mononuclear cells in the sinusoids adjacent to the lesions. In approximately 20% of C3H/ eBFeJ mice, marked localized abnormalities of the liver microcirculation correlated histologically with foci of aggressive hepatitis ( Figure 6B ). These foci were composed of a central avascular core which was surrounded by pale edematous hepatocytes containing dilated and disorganized sinusoids with slow or absent blood flow. Subsequent histologic analysis of these lesions which had been studied in uiuo demonstrated large areas of necrosis with a marked predominantly mononuclear inflammatory cell infiltrate ( Figure 6A ).

The outcome of viral infection in a host is the result of a complex set of interactions between the elements of the immune system, host cells and the virus (13, 14) . A number of immune effector networks have been studied in the model of murine hepatitis virus infection; however, to date, none of these have shown good correlation with the strain dependence of disease susceptibility (15, 16) . In this paper, we present data that associate the spontaneous expression of monocyte PCA with acute and chronic liver inflammation in MHV-3-induced hepatitis. This relationship emphasizes the possible role of the coagulation cascade in the disturbances of the microcirculation that enhance liver cell necrosis. Activation of the coagulation system is a common feature of inflammation (17) (18) (19) (20) . Activated coagulation proteins serve as mediators of the inflammatory response by in turn recruiting the complement, kallikrein-kinin and fibrinolytic systems (20) . Furthermore thrombin and its activation fragment 1.2 directly elicit chemotaxis in monocytes (21) . It has been known for several years that leukocytes express a number of PCAs following stimulation both in uitro and in uiuo (20, 22) . The cellular source of this activity is the monocyte/macrophage but T lymphocytes and/or their products are necessary for the induction of these procoagulant monokines (22) . The coagulation cascade may be activated at local sites of inflammation by the procoagulant products of infiltrating leukocytes resulting in amplification of the inflammatory response with increased tissue injury.

The pattern of spontaneous monocyte PCA observed in these experiments in the A/J and C3HeBFeJ mice is in agreement with the previously described direct correlation of monocyte PCA with susceptibility to liver disease following MHV-3 infection of PBM with MHV-3 in uitro (8, 9) . In the C3HeB/FeJ mice, a constant proportion (50%) of PCA was expressed at the cell surface, consistent with the physiologic importance of PCA in situ. During the early stage of the infection, splenic macrophage PCA did not discriminate between those mice that develop chronic aggressive hepatitis or chronic granulomatous hepatitis. Only by Day 10 could distinct patterns of PCA response be identified, and these correlated with the severity of the histologic lesion ( Table   1 ). As the mice studied are inbred strains, it is apparent that factors other than genetic are involved in the evolution of the immune-inflammatory process. Other investigators have shown that diet, age, sex, stress and temperature affect immune responses to infectious processes (23). However, in studies using recombinant inbred strains of mice, we have shown that PCA and susceptibility/resistance to MHV-3 infection are genetically linked and are controlled by two independent non-H-2linked genetic loci (Dindzans, V. et al., Hepatology 1984; 4: 1053, Abstract). Determinations of P-glucuronidase, 5'nucleotidase and elastase in macrophages from MHV-3 infected and control animals did not correlate with susceptibility to infection and resultant hepatitis (24) and thus, monocyte PCA is not simply a nonspecific marker of inflammation.

The immunofluorescence studies and viral titers in the resistant A/J mice demonstrate that permissiveness to viral replication alone cannot explain the disease observed. In addition, the abnormalities in the microcirculation following MHV-3 infection were detected prior to viral replication. Evidence for a role of the microcirculation in the pathogenesis of acute hepatitis has been generated by in uiuo microscopic studies of the liver during toxic hepatitis. Severe flow abnormalities, consisting of sinusoidal constriction and dilation, granular blood flow, microhemorrhages and microthrombi, are prominent features of toxic hepatitis (25) . During hepatitis caused by toxic agents, microcirculatory abnormalities begin in a zonal distribution with the most severe changes occurring in areas of greatest hepatocellular damage ( 5 6 , 26) .

The earliest abnormality seen following MHV-3 infection in uiuo was granular blood flow, a relatively nonspecific finding whose mechanism has been attributed to several factors (27, 28) . These include: hypotension, neurally mediated vasoconstriction and vasodilation, humoral mediators (histamine, bradykinin) of vasodilation, hemoconcentration due to capillary permeability in-creases and changes in the surface charge of erythrocytes due to fibrin adsorption. Studies using fluorescent-labeled proteins during the acute phase of ally1 formate hepatitis revealed increased sinusoidal permeability within 10 min of administration of the toxin, coincident with the appearance of granular flow (6) . Similar abnormalities were detected in MHV-3-infected C3HeB/FeJ mice, suggesting similar pathophysiological mechanisms may be operative. In the acute phase of MHV-3 infection, viral antigens were diffusely deposited in the liver, and extensive microcirculatory disturbances were observed. In contrast, during the chronic phase, viral antigens were localized in focal deposits. Thus, continued specific stimulation of PCA in the chronic phase may result in localized microcirculatory disturbances confined to the areas of antigen deposition. Since it is known that PCA directly correlates with in vitro and in uivo measures of delayed cutaneous h-ypersensitivity (231, it is conceivable that PCA exerts its influence through both cellular and humoral immune mechanisms. This could occur by the production of lymphokines and/or the recruitment of activated T cells macrophages and NK cells into the chronically affected area. Finally, since PCA is a potent serine protease, it may have effects through its proteolytic action on substrates other than prothrombin.

As an example, one of the earliest pathologic features of experimental allergic hepatitis is the deposition of immune complexes within the sinusoids (32). This is followed by thrombotic occlusion of sinusoids with resultant focal coagulative necrosis (32). A similar pathologic mechanism has been proposed for fulminant hepatitis B (33). We have previously reported that immune complexes stimulate the production of PCA, thus asso-ciating the production of immune coagulants with liver injury (24, 34) .

In this paper, we present further evidence suggesting a link between viral induction of monocyte/macrophage PCA and disturbances in the microcirculation in liver disease.

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