Histopathological study of experimental poststreptococcal pneumonia in mice. Group A, type 50, streptococcal infection of murine nares controls with Staphylococcus aureus and E. coli.

Microscopic methods (light and electron microscopy, histochemistry, immunohistochemistry) have been used to assess previously unknown pulmonary inflammatory responses of specific pathogen-free (SPF) mice secondary to infection via the nares by group A, type 50, streptococci suspended in saline ("strep group mice"). As controls for the strep group mice, the animals were either injected with saline alone via nares (no lesions were seen), or with Staphylococcus aureus in saline ("staph group mice") or with E. coli ("E. coli group mice"). The three different bacterial species caused clearly different histological changes in the lung. In the strep group mice, the microscopic findings were consistent with the diagnosis of lymphocytic interstitial pneumonia of bronchiolovascular bundles, secondary to exaggerated pulmonary recirculation of lymphocytes, concomitant with vasoconstrictive angiopathy of encased pulmonary artery branches and nodular inflammatory cell aggregates in lung parenchyma. These aggregates either consisted predominantly of lymphocytes, or of mixed cells (neutrophils, lymphocytes, macrophages) or of activated macrophages only. In 18 of 22 inflamed lungs of strep group mice, no bacteria could be cultured from lung tissue. In staph group mice the microscopic findings are consistent with the diagnosis of lymphocytic interstitial pneumonia of bronchiolovascular bundles, secondary to exaggerated pulmonary recirculation of lymphocytes only. In 12 of 17 inflamed lungs of staph group mice, no bacteria could be cultured from lung tissue. In E. coli group mice the microscopic findings were consistent with the diagnosis of distal terminal bronchiolitis and early pleural-based pneumonitis, in which lymphocytes and neutrophils mingled with macrophages. In 10 of 11 inflamed lungs of E. coli group mice, no bacteria could be cultured from lung tissue. The morphologic approaches described here may have potential for unravelling the complex inflammatory processes underlying different forms of interstitial and parenchymal pneumonia.

Disseminated neocortical and subcortical encephalopathy (DNSE) with widespread activation of brain macrophages: a new dementia disorder? Autopsy reports of two postmenopausal women from families with mitochondrial DNA mutations.

In this study we present 2 postmenopausal women who showed clinical symptoms that resembled those of a rather well-defined group of vascular dementia disorders, termed subcortical dementia (Binswanger disease, CADASIL). Patient 1 exhibited mitochondrial DNA (mtDNA) variants in the ND5 gene at position 13,708 and the Cytb gene at position 15,257. These DNA variants have been described in a number of neurologic disorders, but their pathogenetic potential is unclear. Patient 2 showed the same DNA alterations and an additional mtDNA variant at position 15,812 in the Cytb gene. The principal neurohistologic features of the 2 atrophic brains presented here include: subtotal selective neuronal cell loss in the cortex and, to a lesser degree, in the basal ganglia (claustrum, putamen, globus pallidus), sparing palaeocortex and periarchaeocortex, and a very characteristic and diagnostic feature was detachment of astrocytic processes from capillary walls resulting in pericapillary space formation. These pericapillary spaces were partially filled with macrophages. The spaces were not associated with total breakdown of the blood vessel walls as demonstrated by the absence of erythrocytes, lymphocytes, or polymorphonuclear leukocytes outside the vascular bed of the brain; progressive subcortical encephalopathy, as it is seen in subcortical dementia (Binswanger), but lacking arterial lipohyalinosis. The cerebral grey and white matter revealed cuffing of arteries and arterioles by adventitial macrophages. The neocortical and subcortical changes were accompanied by myriads of activated macrophages filled with lipids. The pathology of our 2 cases differs from that of other neurodegenerative disorders and we suggest the term of "disseminated neocortical and subcortical encephalopathy (DNSE) with widespread activation of brain macrophages".

Association of the LHON 13,708 and 15,257 mitochondrial DNA mutations with neurodegenerative diseases distinct from LHON.

300 patients suffering from neurodegenerative diseases distinct from Leber hereditary optic neuropathy (LHON) were screened for the presence of mitochondrial DNA mutations. We report on nine patients, eight female and one male, who all harboured mutations at positions 13,708 and 15,257 of the mitochondrial DNA. Both mutations have previously been claimed to be associated with LHON. Based on our results, these mutations occur in a number of different neurodegenerative diseases and therefore cannot be regarded as "LHON" mutations.

Anomalous diffusion of water in biological tissues.

This article deals with the characterization of biological tissues and their pathological alterations. For this purpose, diffusion is measured by NMR in the fringe field of a large superconductor with a field gradient of 50 T/m, which is rather homogenous and stable. It is due to the unprecedented properties of the gradient that we are able not only to determine the usual diffusion coefficient, but also to observe the pronounced Non-Debye feature of the relaxation function due to cellular structure. The dynamics of the probability density follow a stretched exponential or Kohlrausch-Williams-Watts function. In the long time limit the Fourier transform of the probability density follows a long-tail Lévy function, whose asymptotic is related to the fractal dimension of the underlying cellular structure. Some of the properties of Lévy walk statistics are discussed and its potential importance in understanding certain biophysical phenomena like diffusion processes in biological tissues are pointed out. We present and discuss for the first time NMR data giving evidence for Lévy processes that capture the essential features of the observed power law (scaling) dynamics of water diffusion in fresh tissue specimens: carcinomas, fibrous mastopathies, adipose and liver tissues.

Mitochondrial complex I and III mutations and neutral-lipid storage in activated mononuclear macrophages and neutrophils: a case presenting with necrotizing myopathy, poikiloderma atrophicans vasculare, and xanthogranulomatous bursitis.

We report the case of a 57-year-old woman suffering from xanthogranulomatous bursitis, necrotizing myopathy, and poikiloderma atrophicans vasculare, which are associated with marked accumulation of neutral-lipid storage phagocytes. The observed lipid storage was restricted to activated phagocytes independent of the presence of tissue necrosis and was not seen either in circulating blood leukocytes or in muscle fibers. The patient's daughter disclosed xanthomatous inflammatory reaction with profound delay of wound healing secondary to pelviscopy. Examination of the mitochondrial DNAs of the patient, her daughter, and her two grandchildren revealed two homoplasmic mutations at positions 13708 and 15257 of the mitochondrial genome. We discuss the involvement of these mutations in the pathogenesis of xanthomatous and xanthogranulomatous inflammation. Further investigations are required to test whether impairment of aerobic energy production independent from mitochondrial DNA mutations (eg, by hypoxia or microbial toxins) similarly can cause the accumulation of lipid-laden macrophages and explain the persistency of xanthogranulomatous inflammation.

Neutrophilic migration through capillarylike micropores: influence of pulmonary passage.

We report the effect of pulmonary passage on random migration and chemokinesis of neutrophils through capillarylike pores under the in vitro condition of Boyden's test. Neutrophils were isolated either from the left ventricle or from the pulmonary artery of patients who underwent coronary angiography due to suspected angina pectoris or valvular heart disease. In all 14 cases left ventricle neutrophils showed significantly enhanced chemotactic-activated migration compared with pulmonary artery neutrophils. Pulmonary passage also influenced the random migration of neutrophils, except for those derived from five patients suffering from pulmonary hypertension. Our findings might indicate that accumulation of neutrophils in the capillaries of the normal lung is counteracted by a change in neutrophilic migration behavior during pulmonary passage, thus avoiding increased neutrophilic sequestration in pulmonary microcirculation as observed in adult respiratory distress syndrome.

Identification of Legionella pneumophila in various specimens by the polymerase chain reaction.

Amplification of the mip sequence with polymerase chain reaction (PCR) proved to be specific for Legionella pneumophila. With nested PCR, the sensitivity of the test was markedly increased. The lower limit of detection for nested PCR in the aqueous medium for live and heat-inactivated dead L. pneumophilia was approximately 1-10 bacteria/ml. The sensitivity of the method, however, was reduced by a factor of 10 to 100 when the bacteria were added to homogenised pulmonary tissue. Fixing the bacteria in aqueous suspension or in tissue homogenate with buffered 4% formalin (pH 7.3) reduced the sensitivity of the PCR by a factor of about 100. After intravenous injection of heat-inactivated bacteria in mice L. pneumophila was detected in deep-frozen samples of plasma and various tissues. The molecular biological technique of nested PCR is proposed as an additional method for the diagnosis of legionella.

In situ autolysis of mouse brain: ultrastructure of mitochondria and the function of oxidative phosphorylation and mitochondrial DNA.

The effect of in situ autolysis on cerebral mitochondrial structure and function has been investigated. Mice (n = 9) were sacrificed and stored for up to 24 h under unfavorable post-mortem conditions at 25 degrees C. At different time intervals groups of three animals were submitted to post-mortem dissection and tissue from different regions of the brain was used for the preparation of "free" and synaptosomal mitochondria. On electron microscopic examination, the post-mortem period had no significant influence on mitochondrial morphology and enzymatic activities of complexes I-V of the mitochondrial oxidative phosphorylation system were still present in all the mitochondrial preparations from different regions of the brain, albeit at a reduced levels. Degradation of mitochondrial DNA was virtually absent from mitochondrial preparations during the 24-h period of autolysis, as shown by the presence of intact DNA by Southern blot and PCR analysis. Based on these results, alterations in mitochondrial DNA and deficiencies of mitochondrial respiratory complexes I-V can be recognized in cerebral tissue even after 24 h of unfavorable post-mortem storage conditions.

A fluorescence-microscopic and flow-cytometric study of HeLa cells with an experimentally induced respiratory deficiency.

HeLa cells, cultured over a long period in a medium containing low doses of ethidiumbromide, were used as a model system for flow-cytometric detection of human cells with impaired mitochondrial respiratory function. Based on laserscan and flowcytometric analysis after rhodamine 123 staining, the mitochondrial membrane potential of respiratory deficient cells seems unchanged as compared to control cells. Maintenance of this membrane potential in respiration-impaired cells requires glycolytic ATP generation, as transient inhibition of glycolysis by sodium fluoride affects rhodamine 123 accumulation in ethidiumbromide-treated cells, but not in control cells. We present a protocol which allows the detection and separation of respiratory deficient cells by flow cytometry.

Functional and molecular analysis of mitochondria in thyroid oncocytoma.

We report a functional and molecular analysis of nine oncocytic tumors of the human thyroid. In all the abundance of mitochondria observed ultrastructurally was accompanied by an increase in enzymatic activities of respiratory complexes 1 (NADH dehydrogenase), 11 (succinate dehydrogenase) IV (cytochrome c oxidase), and V (ATPase). Western blot analysis failed to detect uncoupling protein in the tumors. The elevated respiratory enzyme activities were paralleled by an increase in the mitochondrial DNA content. Restriction analysis of mitochondrial DNA gave no indication of heteroplasmy or other gross alterations. We conclude that the mitochondrial proliferation in oncocytic tumors is probably not the result of a compensatory mechanism for the deficiency in enzyme complexes of the mitochondrial respiratory chain.

[Interactions of fluconazole and phagocytic cells]. / Wechselwirkungen von Fluconazol und phagozytierenden Zellen.

Fluconazole is a triazole compound with a coefficient of distribution P at pH 7.4 of 1.6 (log P = 0.2), and has thus both hydrophilic and lipophilic properties. The physicochemical and pharmacokinetic profiles of fluconazole are clearly different from those of other azole antimycotics. 3H-labelled fluconazole penetrates very rapidly into granulocytes and monocytes (macrophages) isolated from volunteers. The concentrations of the triazole in the granulocytes are about 28% and in the macrophages about 63% higher than in the extracellular milieu. At the concentrations examined (5, 10 and 20 micrograms ml-1) fluconazole damages cells of Candida albicans which have been phagocytized by granulocytes or by macrophages. A clear synergy between fluconazole and the phagocytes can be demonstrated.

Flow cytometry as a tool to discriminate respiratory-competent and respiratory-deficient yeast cells.

The cationic lipophilic dye Rhodamine 123 (Rh123) is selectively enriched in mitochondria in a membrane potential-dependent manner. Application of drugs which interfere with the electron flow of the respiratory chain lead to a severe reduction of mitochondrial dye uptake. In this communication we show that the same effect is observed after Rh123-staining of respiratory-deficient yeast mutants. Based on this observation we used flow cytometry to discriminate respiratory-competent and respiratory-deficient yeast cells. Combined with a cell sorter we were able to selectively enrich respiring and non-respiring yeast cells, respectively, from a mixture of cells.

Interaction of fluconazole and human phagocytic cells. Uptake of the antifungal agent and its effects on the survival of ingested fungi in phagocytes.

3H-labelled fluconazole (CAS 86386-73-4) very rapidly penetrated into polymorphonuclear leucocytes (PMNLs) and macrophages (monocytes) isolated from volunteers. The concentration of the antimycotic in the PMNLs was about 28% and in the macrophages even 63% above that in the extracellular medium. At the concentrations examined (5, 10 and 20 micrograms/ml) fluconazole damaged cells of Candida albicans which had been phagocytized by PMNLs or macrophages. There is an evident synergism between therapeutically attainable concentrations of fluconazole and phagocytic cells and this militates against the intracellular survival of the fungus.

Non-random spatial distribution of intermembraneous particles in red blood cell membrane.

A combined analysis using digital image processing and quantitative evaluation of computed radial distribution of freeze fracture P face particles irradiated and nonirradiated has been carried out. Important quantitative features of the arrangement of particles can be described by computing a statistical measure, the radial distribution function g(r), commonly used to study the structure of liquids. The coordinates for calculating g(r) for each sample were measured automatically by a digital image processing system. The results for the radial distribution function were found to closely resemble g(r) for fluids, indicating the presence of shortrange order. This measure distinguishes three different patterns: i) random distribution, ii) dilute gas like and iii) a liquid like model. The mean number of first nearest neighbours surrounding each particle (coordination number) was found to be of the order of three. The RBC intramembraneous particles are heterogeneous in their behavioural pattern: two thirds random; one fifth, dilute gas; and one tenth, liquid.

Interaction of azithromycin and human phagocytic cells. Uptake of the antibiotic and the effect on the survival of ingested bacteria in phagocytes.

14C-labeled azithromycin, a new macrolide antibiotic, was accumulated by various phagocytic cells isolated from volunteers or patients. The concentration of the antibiotic in monocytes, polymorphonuclear leucocytes (PMNLs), and alveolar macrophages was greater than that in the surrounding medium by a factor of between 200 and 668. Azithromycin penetrated somewhat more rapidly into PMNLs and monocytes than into alveolar macrophages. On the other hand the final concentration in the alveolar macrophages was greater by a factor of about 3 than that in the other two phagocytic cells. Staphylococcus aureus, Legionella pneumophila and Haemophilus influenzae previously taken up by the phagocytes were rapidly inactivated by low (0.031-0.5 micrograms/ml) concentrations of the antibiotic, which in the presence of the cells were subinhibitory. There is thus a clear synergism between azithromycin and the phagocytic cells which leads to increased intracellular killing of the bacteria.

The effect of antibiotics on the intracellular survival of bacteria in human phagocytic cells.

[14C]-labeled josamycin (Wilprafen) readily enters several types of human phagocytic cells-polymorphonuclear leucocytes (PMNLs), adherent monocytes and alveolar macrophages - and is accumulated by these cells to a concentration about 20 times that in the extracellular medium. Similar studies using [14C]-benzyl penicillin revealed that the beta-lactam antibiotic penetrated these cells very poorly. Low concentrations of josamycin and the various phagocytes acted synergistically to inhibit the intracellular proliferation of L. pneumophila or H. influenzae. In contrast, penicillin G was not effective against legionellae ingested by PMNLs, monocytes or alveolar macrophages, even at high concentrations. The uptake of the antibiotics apparently correlates well with its efficacy against the intracellular survival of bacterial pathogens in human phagocytic cells.

Relationships between lymphocyte cholesterol homeostasis and LDL-cholesterol.

The homeostasis of cholesterol was studied in lymphocytes freshly isolated from the blood and cultured with or without low-density lipoprotein (LDL). The content of cholesterol decreased in the lymphocytes cultured without LDL, whereas LDL substituted for cellular cholesterol losses, in spite of almost suppressed LDL-receptor and lymphocyte cholesterol synthesis. Free cholesterol was taken up from LDL mainly via cholesterol exchange and, in contrast to esterified cellular cholesterol, rapidly excreted into the medium. In vitro stimulation of lymphocyte cholesterol synthesis was correlated to the ratio of esterified to free LDL-cholesterol in the blood from which the lymphocytes had been isolated. This result probably reflects the different rates of influx and efflux of esterified or free cholesterol between plasma lipoproteins and lymphocytes. These effects should be taken into account if LDL-cholesterol is determined in plasma for the evaluation of an individual's atherosclerotic risk.