Acquisition of peroxidase activity by rat alveolar macrophages during pulmonary inflammation.

The authors investigated the ability of rat alveolar macrophages to acquire peroxidase activity in the course of pulmonary inflammation. Granulomatous pulmonary inflammation was induced in bacille Calmette-Guérin (BCG)-immunized rats by intravenous injection of BCG in mineral oil. In contrast to normal alveolar macrophages, which are peroxidase-negative, alveolar macrophages lavaged from the BCG-treated rats showed significant peroxidase activity in large cytoplasmic inclusions compatible with internalized exogenous material. Alveolar macrophage uptake of intact peroxidase-positive neutrophils was also observed. Maximal numbers of peroxidase-positive alveolar macrophages were observed after the initial influx of neutrophils into the lungs, and peroxidase activity could be demonstrated in cell-free lavage fluid during the acute phase of lung injury. Normal alveolar macrophages acquired peroxidase activity after incubation with peritoneal exudate neutrophils, with purified soluble human myeloperoxidase, and with opsonized erythrocytes. It is concluded that alveolar macrophages acquire peroxidase activity from multiple sources during pulmonary inflammation. Internalization of peroxidase by the alveolar macrophage may serve to clear a potentially toxic enzyme(s) from the alveolar space and contribute to the resolution of pulmonary inflammation.     

Structural basis for self-recognition by autoimmune T-cell receptors

T-cell receptors (TCRs) recognize peptides presented by major histocompatibility complex molecules (pMHC) to discriminate between foreign and self-antigens. Whereas T-cell recognition of foreign peptides is essential for protection against microbial pathogens, recognition of self-peptides by T cells that have escaped negative selection in the thymus can lead to autoimmune disease. Structural studies of autoimmune TCR–pMHC complexes have provided insights into the mechanisms underlying self-recognition and escape from thymic deletion. Two broad categories of self-reactive TCRs can be clearly distinguished: (i) TCRs with altered binding topologies to self-pMHC and (ii) TCRs that bind self-pMHC in the canonical diagonal orientation, but where there are structural defects or suboptimal anchors in the self-ligand. For both categories, however, the overall stability of the autoimmune TCR–pMHC complex is markedly reduced compared to anti-microbial complexes, allowing the autoreactive T cells to evade negative selection, yet retain the ability to be activated by self-antigens in target organs. Additionally, the structures provide insights into TCR cross-reactivity, which can contribute to autoimmunity by increasing the likelihood of self-pMHC recognition. Efforts are now underway to understand the impact of structural alterations in autoimmune TCR–pMHC complexes on higher order assemblies involved in TCR signaling, as well as on immunological synapse formation.     

Secretion of lung fluid by the developing fetal rat alveolar epithelium in organ culture.

We studied lung explants in submersion organ culture to examine the role of the developing fetal alveolar epithelium in the production of lung fluid. Fourteen-day-gestation fetal rat lungs were grown in a collagen gel matrix supplemented with F-12 media and 10% fetal calf serum. In this model, the lung continues to grow, secrete fluid, and become progressively cystic in morphology. There is gradual thinning of the distal epithelial layer, which is lined by alveolar type II cells and their precursors. After 6 to 8 days in culture, we impaled the cyst walls with a microelectrode and continuously recorded the transepithelial potential (psi t). Stable, baseline transepithelial potentials of -1.1 to -6.2 mV (mean +/- SEM = -3.3 +/- 0.11 mV, lumen negative, n = 34) were measured in bicarbonate-buffered Ringer's solution, suggesting active electrolyte transport. When bumetanide, an inhibitor of chloride secretion in other systems, was added to the bathing solution, psi t decreased from a baseline of -3.5 +/- 0.07 mV (mean +/- SEM) to a value of -2.2 +/- 0.07 mV, suggesting chloride transport contributes to the voltage (n = 18, P less than 0.0005). Isoproterenol hyperpolarized psi t from a baseline of -4.3 +/- 1.0 mV to -6.5 +/- 1.0 mV (n = 7, P less than 0.005). 8-(4-Chlorophenylthio) adenosine 3':5'cyclic monophosphate (CPT-cAMP) plus isobutylmethylxanthine (IBMX) similarly hyperpolarized psi t from a baseline of -4.6 +/- 0.4 mV to -7.3 +/- 0.7 mV (n = 11, P less than 0.005). Addition of bumetanide after stimulation with isoproterenol or CPT-cAMP/IBMX depolarized psi t.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural analysis of fetal rat lung development.

The primary aim of this morphological investigation was to elaborate a concept allowing us to coherently define reference spaces for morphometric analysis of fetal lung development. Beyond this quantitative goal, morphological analysis of cell types, definition of compartments, and reflection about the prospective fate of their constituents provided per se new insights into the developmental processes. Lungs of rat fetuses aged 17-23 days and newborns aged 20 hours were fixed with an osmium tetroxide and glutaraldehyde mixture and their volume determined. Left lungs were embedded in Epon and investigated by light and electron microscopy. The right lung of one animal per group was embedded in methacrylate and step sections obtained to precisely locate the airways within the mesenchyme. The various cell types, their topographical relationships, and their morphological alterations with ongoing development were analyzed with regard to their prospective potentials of differentiation. The developing lung could be partitioned into four zones further subdivided into defined compartments. Zone I forms a superficial mantle around the lobes and the future acini. Consisting of primitive mesenchymal cells, it represents a zone of growth which disappears with the onset of the saccular stage. Zone II is mainly a zone of differentiation. Its interstitium stains intensely due to a dense population of dark cells. Up to gestational day 19, zone II contains future conductive airways with their vessels. After day 21, it comprises the whole prospective gas exchange region. Zones III and IV contain the elements of the airway tree and vascular system, zone IV corresponding to the most proximal generations with an adventitial layer. For all differentiation processes, a centrifugal directionality is manifested.     

Regression and spreading of self-recognition during the development of autoimmune demyelinating disease.

The autoimmune T cell repertoire in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) is characterized by CD4(+)T cells of the Th1 phenotype that recognize peptide determinants of central nervous system (CNS) myelin proteins in an MHC class II-restricted manner. Our recent studies and those performed by others have shown that progression to chronicity in EAE and MS is accompanied by a broadening of the T cell repertoire with time. This acquired neo-autoreactivity is commonly referred to as epitope spreading and is presumably the result of endogenous priming to new self-determinants during the CNS inflammation that accompanies disease onset and relapse. In the present study we extend our earlier observations by showing that disease progression in both EAE and MS is accompanied by two concurrent events, viz. (1) the spontaneous regression of the primary established autoimmune repertoire associated with disease onset, and (2) the emergence of the epitope spreading cascade associated with disease progression. Our data show that disease initiation and disease progression in both EAE and MS are typically associated with distinctly different autoimmune T cell repertoires. Our data support the view that the natural development of self-recognition during autoimmune disease may best be understood when considered in the temporal context of an 'epitope du jour' and 'moving target' perspective.     

Changes in immunological activity of rat lymphoid organs during pregnancy.

Immune reactivity in lymhoid organs of rats during the last week of syngeneic (Lewis: L x L) or allogeneic (females Lewis x males Wistar: L x W) pregnancy was compared with that found in non-pregnant animals. The thymus weight was slightly reduced and the response of thymic lymphocytes to phytohaemagglutinin and concanavalin A was slightly, but not significantly, elevated in pregnant rats. By contrast, the response of lymphoid cells from spleen and mesenteric lymph nodes to the mitogens was reduced in rats during advanced pregnancy. The immune response of lymphocytes from pregnant L x W rats to allogenic (Brown-Norway: BN) or semiallogeneic (W) irradiated cells was tested by the mixed lymphocyte culture (MLC) assay. The MLC response of para-aortic lymph nodes towards the unrelated BN cells was elevated over that of non-pregnant rats on day 15 of pregnancy. No significant enhancement was observed at the same time in the MLC response of mesenteric lymph nodes. On day 20 of pregnancy, a reduced MLC response towards BN cells was found in the mesenteric and para-aortic lymph nodes. On day 15 of pregnancy, the MLC response of mesenteric and, more markedly, that of para-aortic lymph nodes to paternal (W) cells was enhanced, as compared to that of non-pregnant rats. On day 20 of pregnancy, the response of mesenteric lymph nodes was suppressed, while the response of para-aortic lymph nodes was similar to that found in non-pregnant rats. Since the latter lymph nodes are the most directly exposed to antigenic stimulation from the uterus, it seems unlikely that the suppression in response to T-cell mitogens observed in the spleen and mesenteric lymph nodes during pregnancy accounts for the survival of the foetus. It seems more plausible that local factors in the vicinity of the uterus protect the foetus from being rejected.     

Mitotic activity in adult rat brain induced by implantation of pieces of fetal rat brain and liver.

By implantation of pieces of fetal CNS into adult host brains it was found that the fetal brain tissue induced a very high mitotic activity in the adult brain at 2-4 days after implantation. The dividing cells were found within a 3-mm zone from the transplant in occipital cortex, hippocampus and brain-stem. Sham operated control animals exhibited only very few mitotic figures close to the lesion. Neither local injection of amniotic fluid nor implantation of other fetal tissues induced mitotic activity with the exception of fetal liver. Possibly, the mitogenic factor(s) released from the transplanted fetal brain is hepatic in origin.     

Maturation of immunological reactivity in the fetal lamb infected with Akabane virus

The development of cell-mediated immunological reactivity was studied in fetal lambs infected with Akabane virus. Examination of hepatic cells from fetuses between 40 and 75 days' gestation that had been infected via the transplacental route revealed inconsistent responses to Akabane, together with a uniform failure to respond to non-specific mitogens which contrasted with the behaviour of control, uninfected lambs. Following direct inoculation of fetal lambs with virus between 50 and 120 days' gestation, specific proliferative responses were observed on the part of the spleen cells from some. Direct challenge of fetal lambs of 4 months' gestation evoked cellular responses in lymph draining from the site of virus inoculation similar to those produced by challenge of adult sheep. The proliferative response of lymph-borne cells was substantially better if live, rather than inactivated, virus had been used.     

Development of angiotensin-converting enzyme in fetal rat lungs.

Angiotensin-converting enzyme (ACE) catalyzes rapid hydrolytic cleavage of angiotensin I to form angiotensin II (AII). Inasmuch as converting enzyme activity is present at birth and increases postnatally to adult values it was of interest to determine the prenatal development of ACE. Converting enzyme activity was determined in the 20,000 x g supernatant fraction of lung homogenates using hippuryl-L-histidyl-L-leucine (HHL) as substrate. Hippuric acid liberated by the hydrolysis of HHL was quantified spectrophotometrically. ACE activity was first detectable at 18 days of gestation and increased fourfold prior to birth (21 days gestation). Pulmonary ACE activity of 1-day-old animals was twice that of fetuses at day 20 of gestation; however, this increase did not appear to result from ventilation alone. The Michaelis-Menten constant for fetal ACE (2.0 mM HHL) was not different from that calculated for ACE of adult rat lungs (2.6 mM). These data were interpreted to indicate that the age-related increase in ACE activity was due to greater ACE content as opposed to further activation of preexisting enzyme. This increase in fetal ACE activity may play an important role in preparing the renin-angiotensin system for postnatal function.     

Evolution of mesenchymal cells in fetal rat lung.

The evolution of connective tissue cells in the developing fetal rat lung is studied under the electron microscope from the 15th until the 21st day of gestation and is compared to the evolution of epithelial cells. Three successive types of stem cells ("mesocytoblasts") are present during the first stages of lung development studied (15 to 18 days of gestation). These stem cells appear to be able to differentiate into fibroblasts or into smooth muscle cells, according to their localization along the broncho-alveolar tubule. Myoblasts are situated near the bronchial epithelium, whereas fibroblasts occur under the alveolar epithelium. Epithelo-mesenchymal interactions are assumed to play a role in this differentiation process. Synthesis of both, collagen and elastic fibers and of cytoplasmic filaments by fibroblasts as well as by myoblasts reveal the multiple potentialities of the mesenchymal stem cell and suggest a common origin. The early fibroblast in characterized by long cytoplasmic processes which contain numerous cytofilaments, and by the presence of collagen fibers in the vicinity of the cell. Later on, (20 days of gestation) the mature fibroblast of the lung mesenchyme shows areas of RER, glycogen and lipidic vacuoles in its cytoplasm. Cytofilaments are numerous within very long cytoplasmic processes and elastic and collagen fibers are very frequent beside the cytoplasmic membrane. The earliest fibroblast differentiation occurs under the epithelium of primitive respiratory bronchioles, which indicate the limit between the bronchial and the alveolar territories. Later on, differentiating fibroblasts are found throughout the whole alveolar walls. Connective tissue cells other than mesenchymal stem cells, fibroblasts or myoblasts are observed during lung development. Vacuolar cells, similar to Hofbauer cells, transiently appear on the 16th day of gestation. On the 20th and the 21st day macrophage-like cells are present in the septal space of the alveolar wall. The absence of intermediate stages of differentiation and parallel evolution of blood cells suggest that those connective tissue cells are differentiated elsewhere and have then migrated from blood into lung mesenchyme. No cell death has been observed in the developing lung.     

Metabolic activation of the fetal rat brain

In the present study, we tried to examine whether the 2-deoxy-D-[14C]glucose (2-DG) technique is useful to investigate the metabolic activity of fetal rat brain in the maternal uterus. The result shows that metabolic mapping of the fetal brain was clearly obtained by means of the 2-DG technique.     

Pathological and immunological profiles of rat tuberculosis

To investigate the pathological and immunological profiles of rat tuberculosis, Lewis female rats were infected aerially with Mycobacterium tuberculosis. Histopathology, immunological profiles of mononuclear cells from M. tuberculosis-infected rat lung tissue, and the expression patterns of cytokine and iNOS mRNAs were examined over time. M. tuberculosis induced granulomatous lesions in the lungs, spleen, lymph nodes and liver, but these lesions lacked central necrosis. Multinucleate giant cells were observed in late-phase tuberculosis. CD4(+) and CD8(+) T cells increased with time and reached a peak 5 weeks after infection, decreasing gradually thereafter. ED1 antigen, suggestive of alveolar macrophages, was expressed at a high level in early phase tuberculosis and remained at the same level even in the late phase. OX62 antigen increased gradually and reached a peak 5 weeks after infection. Interferon-gamma, tumour necrosis factor-alpha and iNOS mRNAs were expressed strongly over time, but their expression decreased 12 weeks after infection. Because rat tuberculosis is very similar to murine tuberculosis and it is easy to obtain mononuclear cells from M. tuberculosis-infected rat lung tissue, the rat tuberculosis model appears to be suitable for immunological studies in vivo.     

胎儿脑电图用于产时胎儿宫内窘迫诊断的研究

目的观察分娩时胎儿脑电图波形变化,探讨直接胎儿脑电图可以用于分娩时监测,提供一种新的产时胎儿宫内窘迫的诊断方法。方法采用自制的经阴道胎儿脑电电极对65例产妇在第一产程末至第二产程结束前进行直接胎儿脑电图监测,观察脑电图波形变化;比较胎儿缺氧组和正常组胎儿脑电图波形异常率;并根据分娩时胎心监护、羊水性状和新生儿Apgar评分等结果把研究对象分为正常组和胎儿缺氧组两组,将两组脑电图结果进行比较;再根据胎儿脐动脉血血气分析结果把研究对象分为胎儿酸中毒组和正常组,将两组的脑电图进行比较;最后将新生儿预后与产时胎儿脑电图进行比较。结果应用自制的能经阴道接触胎儿头皮的脑电电极设计合理,通过其记录的脑电图符合新生儿脑电图特点;正常组、胎儿缺氧组产时脑电图波形异常率有显著性差异;胎儿酸中毒组和正常组产时脑电图有显著性差异;产时胎儿脑电图异常与新生儿预后相关。结论1、自制的经阴道胎儿脑电电极可准确地采集胎儿脑电信号,且不损伤胎儿头皮,是安全、可行的;2、胎儿脑电图可用于产时胎儿状况的监护,胎儿脑电波异常可反映胎儿官内缺氧的状况,可作为产时胎儿监测的直观指标;3、产时胎儿脑电图可用于分娩时监测,可反映胎儿酸中毒的状况,4、产时胎...     

The immunological generation of a platelet-activating factor and a platet-lytic factor in the rat.

Antigen challenge of the rat peritoneal cavity which had been prepared with IgGa-rich antiserum generated activities which released [14C]-serotonin from pre-labelled human platelets. After adsorption of these activities onto Amberlite XAD-8 and elution in 80% ethanol, two factors of differing polarity were resolved by chromatography on diethylaminoethyl cellulose in organic solvents. The activity eluting in the 7:1 chloroform:methanol solvent contained a platelet-lytic factor (PLF) assessed by the parallel release of lactic acid dehydrogenase and [14C]-serotonin; the cytotoxicity of this fraction was confirmed by phase-contrast microscopy examination which demonstrated fragmentation of the exposed platelets. The activity eluting in the 1:1 methanol: aqueous 1.0 M ammonium carbonate solvent was a platelet-activating factor (PAF) as defined by release of [14C]-serotonin without lactic acid dehydrogenase. Both the lytic and the activating principles were separable from slow reacting substance of anaphylaxis and polymorphonuclear leucocyte chemotactic activity, and each presented a single activity peak of differing mobility when chromatographed on silica gel H plates. Human eosinophil phospholipase D inactivated the lytic factor by more than 85% in 2 h at 37 degrees without affecting the activity of the activating factor. The release of [14C]-serotonin induced by the PAF was not affected by the absence of calcium from the medium or by elevations in the platelet concentrations of cyclic AMP or cyclic GMP that resulted from pre-incubation of platelets with prostaglandin D2 or sodium ascorbate, respectively.     

Acquisition of Klebsiella aerogenes by neonates.

The acquisition of Klebsiella aerogenes by neonates born in hospital has been studied by sero- and klebecin typing. This organism was more commonly carried in the bowel of breast-fed babies than of bottle-fed babies. Only very rarely did babies acquire strains of K. aerogenes from their mothers. K. aerogenes was widely distributed in the ward environment and on the hands of nurses and mothers. Some strains were able to spread on the ward. These results are relevant to the control of K. aerogenes infection in maternity units.