Frost-bite lesions were produced in five Hanford Miniature Swine exposed to - 75 degrees C air for 1, 3, 5, 10 or 20 min. Biopsies were taken at 0, 3, 6, 12, 24 and 48 h and 1 and 2 weeks. Two hundred slides were evaluated microscopically: pyknosis, vacuolation, individualization of cells, and degeneration were graded from 0-5; 0, no change; 5, severe change. Necrosis, new epithelium, and microabscesses were recorded as present or absent. Early changes of vacuolation of keratinocytes, individualization of cells and pyknosis increased with biopsy time until 1 week, at which time more severe changes predominated. Intermediate changes of advanced degeneration and dermoepidermal microabscesses were seen at 48 h and 1 and 2 weeks. Late changes of necrosis and epithelial regeneration occurred, both separately or together in the same tissue, at 1 and 2 weeks. Regeneration occurred either as complete replacement of epithelium, or as crescents of new epithelium beneath degenerating epithelium. The results show that necrosis is a late development, so prognosis based on early skin biopsies must be guarded to unfavourable. Furthermore, these findings suggest that cold has a direct effect on epithelial cell membranes.
The distribution of copper and related changes have been studied in copper-loaded rat kidneys at the ultrastructural level by X-ray electron probe microanalysis, in order to clarify the pathogenesis of copper-induced damage and subsequent recovery in this organ. Male rats fed a high copper diet (1500 ppm) for 16 weeks were killed at intervals; their kidneys were removed and portions of kidney cortex fixed in 4% paraformaldehyde and 2% glutaraldehyde for electron microscopy: other samples were analysed for copper by AA spectrophotometry. Increasing copper accumulation was associated with progressive PCT cell disarray and characterized by irreversible nuclear damage coincident with the intranuclear accumulation of Cu, S, P, and Ca. Copper was also identified within structurally intact lysosomes associated with Zn and Fe (Type I lysosomes) or P and S (Type II lysosomes, putative Cu-MT). Subsequent copper decline and tubular recovery was associated with the facilitated lysosomal sequestration of copper and excretion of copper-containing cell products into the tubule lumina, Cu-MT and alpha-2 urinary protein-copper. The cytotoxicity of copper in the kidney, as well as the liver, is associated primarily with irreversible nuclear damage, whereas lysosomal copper sequestration protects the cell from injury.
Simian adenovirus type 7 (SA-7) was found to induce tumours originating from nerve-supporting or paraneural cells in newborn hamsters, regardless of injection site or tissues. SA-7 induces glioblastomas characterized by definite localization (subependymal regions) and its main cell type, bipolar spongioblast-like cells, in the brain of hamsters inoculated as newborns. When the eyes of newborn hamsters were directly inoculated, SA-7 failed to induce retinoblastoma (0/27), but retro or peri-bulbar SA-7 tumours frequently occurred in tissues closely related to the peripheral nerve apparatus, including the oculomotor nerve or ciliary ganglion. These tumour cells were situated like stromal cells in these nerve tissues. The histological features of the orbital tumours were similar to those of SA-7-induced subcutaneous tumours but not to brain tumours. In contrast with other hamster brain tumours induced by human adenovirus type 12 or human papova JC virus, medulloepithelioma or medulloblastoma, SA-7 induced tumours exhibit distinctive histological and localization characteristics.
The pathological effect of parainfluenza type I (Sendai virus) is known to be a bronchopneumonia, which becomes a chronic pneumonia in the immunodeficient athymic (nude) mouse. The severity of this established chronic pneumonia can be dramatically altered by providing the nude mouse with humoral monoclonal antibodies which are neutralizing, and are directed against the fusion protein, of the virus. The alveolitis, which is a significant part of the pathology, is suppressed due to a reduction (greater than 90%) in the number of virus-infected alveolar macrophages present in the alveoli. This clearly identifies the infected alveolar macrophage as the primary effector cell in the pathogenesis of alveolitis caused by parainfluenza virus type I. The implications of using virus-neutralizing monoclonal antibodies, which have little immunomodulatory toxicity, in the treatment of viral pneumonias are discussed.
Cells within the central nervous system were identified as containing immunoglobulin G, A and M using immunocytochemistry in mice previously infected with Semliki Forest virus, a togavirus causing primary immune-mediated demyelination. Cells positive for these immunoglobulins were counted in cerebellar white matter, parenchyma, meninges and choroid plexus/ventricles. No positively staining cells were seen on day 6 after infection although other inflammatory cells were present at this time and virus-specific immunoglobulin was found in serum. Cells positive for IgG appeared in all areas by day 9 and remained dominant in numbers throughout. IgM-secreting cells appeared in small numbers in the parenchyma first on day 9 and subsequently in other areas, their numbers rising to a maximum on day 12 in all areas and falling thereafter. The number of IgA-secreting cells was small. They appeared by PID 12 and continued to rise on successive sampling days. Initially IgG-positive cells were seen in the perivascular cuffs but by day 12 a few had moved away from the cuffs into the adjacent parenchyma. IgG-positive cells were seen both in and away from cuffs within areas of demyelination. IgM and IgA-positive cells tended to follow the distribution of IgG-positive cells, but in fewer numbers.
The purpose of this study was to investigate the possible role of leucocytes in the pathogenesis of reperfusion-induced vasospasm of ischaemic mesenteric arteries. Scanning electron microscopy of the rat superior mesenteric artery (SMA) after 30 min of ischaemia in vivo revealed adherence of leucocytes to the vessel wall. Isolated SMA preparations were perfused with Krebs-Henseleit buffer containing noradrenaline. Infusion of homologous leucocytes resuspended in perfusate (3 x 10(6) cells/ml) into these preconstricted preparations caused a fall in resistance of 29 +/- 2%. Removal of the endothelium by collagenase treatment abolished this response. Indeed, leucocyte infusion caused an increase in resistance of 39 +/- 8% under these circumstances. Following 30 min of normothermic ischaemia, leucocyte infusion caused a transient vasodilatation of 31 +/- 4% followed by an increase of 38 +/- 11% in the perfusion resistance of isolated SMA preparations. In each case, a similar response was obtained to infusion of the cell-free supernatant. These results suggest that leucocyte activation occurs in vivo during reperfusion of the SMA after as little as 30 min of ischaemia, and that activated leucocytes can release humoral vasoactive factors which evoke an endothelium-dependent vasodilator response in normal vessels but a predominantly vasoconstrictor response following brief intervals of ischaemia.
Fischer F344 rats were given a cyclical diet of 0.06% 2-acetylaminofluorene (AAF), which progressively induced oval cell proliferation, cirrhosis and hyperplastic (or neoplastic) nodules. Primary liver tumours developed from 7 months after ceasing the diet. Liver samples taken during and after AAF administration and specimens of primary tumours were processed into frozen sections and examined microscopically for morphological changes in cell populations, stained histochemically for gamma-glutamyl transpeptidase (GGTase) and four phosphatases, and stained by the immunoperoxidase technique for the presence of antigens detected by seven anti-liver cell monoclonal antibodies and monoclonal antibodies to six oncoproteins. During and after AAF treatment several of the anti-liver antibodies revealed foci of aberrantly or heterogeneously-stained cells, although anti-oncoprotein antibodies showed no consistent changes. Foci of cells positive for GGTase and heterogeneous for adenosine triphosphatase (ATPase) were also seen. Nodules invariably showed heterogeneous antigenicity, raised GGTase and abnormal ATPase expression. Primary tumours exhibited varying degrees of positivity, negativity and heterogeneity with the anti-liver monoclonal antibodies, and all were positive for GGTase. Comparison between various parameters and different lesions showed the greatest concordance between nodules and tumours, suggesting that nodules are probably the precursors of malignant tumours in this system.
The inflammatory effects of intradermal injections of the human recombinant cytokines interleukin 1 (IL-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF alpha) have been assessed in rabbit skin, and compared with the effects of a novel polymorphonuclear leucocyte (PMN)-stimulating activity (PSA) produced by IL-1-treated human synovial cell cultures. IL-1 (84 fmol) and GM-CSF (10 pmol) caused increases in vascular permeability with a delayed onset, as assessed by the dermal accumulation of intravenously-administered 125I-human serum albumin. These cytokines also stimulated extravascular accumulation of PMNs. In contrast, PSA-containing supernatant caused a more rapid and prolonged increase in vascular permeability and PMN accumulation. TNF alpha (84 fmol) was unable to stimulate either of these responses. The increases in vascular permeability and PMN accumulation following IL-1 administration in vivo may be a consequence of the local generation of PMN-stimulating activity by connective tissue cells, such as the activity produced by IL-1-treated synovial cell cultures that we have described.
Frost-bite lesions were produced in five Hanford Miniature Swine exposed to - 75 degrees C air for 1, 3, 5, 10, or 20 min. Biopsies were taken at 0, 3, 6, 12, 24, and 48 h, and 1 and 2 weeks. Two hundred slides were evaluated microscopically: superficial and deep hyperaemia, vascular inflammation, medial degeneration, and thrombosis were graded from 0 to 5; 0, no change; 5, severe change. Haemorrhage was recorded as present or absent. Hyperaemia was the earliest change seen, both grossly and microscopically. Leucocyte emigration and vasculitis were intermediate stages seen most commonly in the 6, 12, and 24 h biopsies. Medial degeneration and thrombosis, the most severe vascular changes, were not seen until 1-2 weeks following frost-bite injury. These findings show that the outcome of frost-bite can not be accurately predicted from early frost-bite lesions, because thrombosis and medial degeneration are not evident in early lesions.
During the development of an acute inflammatory reaction induced in the rat pleural cavity by dextran, calcium pyrophosphate, saline or phosphate buffered saline, macrophages present at a distant site (peritoneal cavity) display an increased capacity to release prostanoids: prostaglandins, prostacyclin and thromboxane. Enhanced levels of 6-keto-PGF1 alpha were observed both in peritoneal lavages (experiments in vivo) and in macrophage supernatants after 24-h culture (experiments in vitro). TXB2 levels were mainly increased in peritoneal lavages and PGE2 in culture supernatants. In vivo, levels of prostanoids in the peritoneal cavity reached a maximum 24 h after the induction of pleurisy whatever the injected substance. In vitro, amounts of arachidonic acid metabolites were highest in supernatants of cultured peritoneal macrophages harvested 72 h after the pleural injection of dextran or CaPP. These results show that the regulation of macrophage functions is closely related to prostanoid production, especially the release of PGE2 and PGI2.
The influence of previous BCG vaccination on the bactericidal activity of isoniazid and rifampicin has been studied using serial counts of viable tubercle bacilli in the spleens and lungs of mice and guinea-pigs infected intravenously with M. tuberculosis, strain H37Rv. In mice, BCG vaccination decreased the bactericidal activity of isoniazid in the spleen, but did not affect its activity in the lungs, where immunity is less strongly expressed. BCG did not influence the bactericidal activity of rifampicin in either organ. In contrast, previous BCG vaccination in the guinea-pig increased the bactericidal activity of isoniazid and rifampicin in the spleen and lungs. The differences between the animal species might result from the immune response being mainly bacteriostatic in the mouse but bactericidal in the guinea-pig.
Post-embedding immunogold electron microscopy (IAuEM) techniques utilizing low-temperature embedding in Lowicryl K4m and LR White resin were used to localize the binding sites of two sheep anti-rat glomerular basement membrane polyclonal antibodies (P1 and P2) and a sheep anti-rat lung antigen antibody. P1 localization was bilaminar in a linear pattern along the subepithelial and particularly the subendothelial aspect of the glomerular basement membrane. P2 was bound diffusely throughout the lamina densa, even at supramaximal doses. The anti-lung antibody bound in an interrupted linear pattern throughout the lung basement membrane (alveolar and capillary), and showed an intense, diffuse binding to the glomerular basement membrane. IAuEM allowed definition of the precise basement membrane binding sites of these polyclonal reagents.
Biosynthesis of interleukin-I beta (IL-I beta) by inflammatory peritoneal exudate cells (PEC) was studied in terms of biological activity (thymocyte co-mitogenic assay), IL-I beta mRNA expression (cytoplasmic slot blot analysis) and detection of immunoreactive IL-I beta molecule (immunocytochemistry). Our findings were taken to conclude that IL-I beta expression was observed only in PEC harvested from early inflammatory site and the kinetic profile of the IL-I activity during the course of the inflammation was explained by IL-I beta mRNA expression, namely de-novo synthesis by inflammatory exudate cells. With respect to a single cell level, it was definitely proved that polymorphonuclear leucocytes were the major producer of IL-I beta during the casein-induced acute inflammation in rabbits.
We have addressed the problem of distinguishing angiogenesis induced in the chick chorioallantoic membrane by injury and inflammation from angiogenesis induced by primary stimulation. Focal, slow-release application of trypsin stimulated a localized spoke-wheel pattern of vascularity. In comparison, a range of doses up to a sublethal amount of trypsin applied generally, in liquid form, resulted in no change in DNA synthesis or vessel content, despite a transient influx of inflammatory cells. This contrasts with previous work with fibrin degradation products, histamine and heparin which each produce characteristic patterns of increased DNA synthesis leading to angiogenesis in the entire 'dropped' area of the chorioallantoic membrane. Such general application, therefore, avoids the danger of misinterpretation of focal, toxic effects.
Frost-bite lesions were produced in five Hanford Miniature Swine exposed to - 75 degrees C air for 1, 3, 5, 10 or 20 min. Biopsies were taken at 0, 3, 6, 12, 24 and 48 h and 1 and 2 weeks. Two hundred slides were evaluated microscopically: pyknosis, vacuolation, individualization of cells, and degeneration were graded from 0-5; 0, no change; 5, severe change. Necrosis, new epithelium, and microabscesses were recorded as present or absent. Early changes of vacuolation of keratinocytes, individualization of cells and pyknosis increased with biopsy time until 1 week, at which time more severe changes predominated. Intermediate changes of advanced degeneration and dermoepidermal microabscesses were seen at 48 h and 1 and 2 weeks. Late changes of necrosis and epithelial regeneration occurred, both separately or together in the same tissue, at 1 and 2 weeks. Regeneration occurred either as complete replacement of epithelium, or as crescents of new epithelium beneath degenerating epithelium. The results show that necrosis is a late development, so prognosis based on early skin biopsies must be guarded to unfavourable. Furthermore, these findings suggest that cold has a direct effect on epithelial cell membranes.
Previous studies indicate that the subendothelium of rabbit aortae de-endothelialized with a balloon catheter rapidly becomes covered with a monolayer of platelets; after 60 min few additional platelets accumulate and although most platelets are lost from the injured surface by 4 days, there is a substantial delay before re-endothelialization. We examined the dynamics of platelet accumulation on rat aortae de-endothelialized with a balloon catheter to determine if the response to this type of injury is similar to rabbit aortae. When 51Cr-platelets were injected prior to aortic de-endothelialization, 25,500 +/- 2,750 platelets/mm2 accumulated on rat subendothelium in the first 15 min. After 60 and 92 h, fewer platelets remained on the surface (13,740 +/- 2,400 and 5,020 +/- 1,330 platelets/mm2, respectively). When 51Cr-platelets were injected into rats 30 min after injury, platelet accumulation in a 30-min period was 8,610 +/- 1,230 platelets/mm2. By 4 days rat aortae did not accumulate newly injected platelets significantly in a 30-min period, but in a 24-h period 20,600 +/- 3,490 platelets/mm2 accumulated. Morphologically, the non-endothelialized areas of rat aortae were almost completely covered with platelets 4 days after injury. Fourteen days after injury, rat aortae did not accumulate newly injected platelets and, morphologically, no platelets were present on the surface which was almost re-endothelialized. Thus, in rats, as with rabbits, platelets rapidly accumulate on de-endothelialized aortae and the ability to attract newly introduced platelets is considerably reduced shortly after injury. In contrast to rabbits, however, de-endothelialized aortae in rats remain attractive to new platelets up to 4 days following injury, but less so than at the time of injury. Also, in contrast to rabbits, 14 days after injury to rat aortae the surface is almost completely re-endothelialized. Thus, there are species differences in platelet interactions with de-endothelialized vessels.
Polymorphonuclear leucocyte (PMN) stimulation is known to generate oxygen free radicals. Exogenous oxygen free radicals, generated by xanthine and xanthine oxidase, have been implicated in the decrease of cardiac contractility. It is possible that PMN have increased capacity to release oxygen free radicals in failing heart. It was, therefore, decided to investigate PMN chemiluminescence (oxygen free radicals) from blood in dogs with heart failure due to chronic volume overload. The dogs were divided into two groups: (A) normal, six dogs; (B) dogs with mitral insufficiency (MI) of 6-9 months' duration, six dogs. Haemodynamic parameters were recorded to assess cardiac failure. Mixed venous blood was collected to measure PMN chemiluminescence. Stimulation of PMN was initiated by addition of opsonized zymosan and chemiluminescence was monitored using a luminometer. The haemodynamic parameters in dogs with MI showed that these dogs had left ventricular failure. The peak chemiluminescent activity of PMN in blood of dogs with left ventricular failure was approximately four times that in the blood from normal dogs. This increase in chemiluminescence reflects an increase in the generation of oxygen free radicals from PMN in dogs with chronic heart failure. The decrease in the myocardial contractility in cardiac failure might be due to an increase in the oxygen free radicals produced by the PMN.
Changes in the lipids of tissues from mice infected with bacille Calmette-Guérin (BCG) have been detected by gas-liquid chromatography. Infection with BCG resulted in (1) an increase in the polyunsaturated to saturated fatty acid ratio of phospholipids and (2) a decrease in the total triacylglycerol fatty acid content of spleen, liver and peritoneal macrophages. The alteration in fatty acid composition was significant in the phosphatidylethanolamine fraction of the phospholipids. The relation of these findings to an increased sensitivity to bacterial endotoxins is discussed.
A polyclonal anti-cytokeratin antibody has been used to examine the expression of this intermediate filament both during normal development in the rat and in a variety of pathological states in the rat and mouse. Bile duct proliferation induced by the administration of alpha-naphthylisothiocyanate (ANIT) as well as the oval cell proliferation induced by 3'-methyl-4-dimethylaminoazobenzene (3-MeDAB) have been used to examine the expression of the rodent cytokeratins in the proliferating cells regarded as being of bile duct origin. Examples of cholangiofibrosis and cholangiocarcinomas were also examined for evidence of cytokeratin expression using this antibody, as well as proliferations of a morphological intermediate type between epithelial and mesenchymal. In all cases we have been able to demonstrate continuity of phenotypic expression of the cytokeratins recognized by this antibody in cells which are recognized as bile duct in origin, even where their morphological appearance does not resemble an epithelial cell type. Because this antibody can be used on formalin-fixed, paraffin-processed tissues, after trypsin treatment, it is proposed that it can be used routinely in the toxicological evaluation (even retrospectively) of bile duct related proliferations and tumours.
