Efficacy of ciprofloxacin in stationary-phase bacteria in vivo.

The granuloma pouch model in mice infected with Escherichia coli or Pseudomonas aeruginosa was used to investigate the bactericidal effect of ciprofloxacin in vivo on bacteria in the stationary growth phase. Ciprofloxacin caused a rapid decline in the number of colony-forming units (cfu) of E. coli shortly after initiation of therapy (40 mg/kg, intraperitoneally). Ciprofloxacin was more effective than norfloxacin or pefloxacin and comparable in efficacy to ofloxacin. The drugs penetrated well into the pouch exudate, exceeding the minimal inhibitory concentrations (MICs) of the infecting organisms. The concentrations of pefloxacin or ofloxacin were higher than those of norfloxacin or ciprofloxacin. Ciprofloxacin also showed good killing effects in pouches infected with one strain of P. aeruginosa (ICB 7453, MIC of 0.06 micrograms/ml). However, with another P. aeruginosa strain (ICB 7933), which has a MIC of 0.5 micrograms/ml, killing of stationary cells in vivo was not very pronounced. Electron microscopic evaluation of the pouch exudate revealed that phagocytosed and non-phagocytosed E. coli cells were severely damaged in comparison with untreated control cells. The earliest ultrastructural changes could be observed 15 minutes after initiation of therapy. The results demonstrate that ciprofloxacin is effective in mice for the treatment of a local inflammatory abscess harboring a stationary population of E. coli or P. aeruginosa. This specific kind of killing occurs in vivo when drug concentrations are at least eight to 10 times higher than the MIC.

Ultrastructural observations of an electron dense amorphous layer on selectively damaged endothelial cells, a possible trigger of thrombogenesis in vivo, and its inhibition by nafazatrom.

Platelet aggregation can be induced by intravascular excitation of fluoresceinisothiocyanate-dextran. Cheek pouches of untreated hamsters and of nafazatrom recipients were excised before and after exposure to the exciting light for 1, 4, 6, 10 and 25 minutes and examined electron microscopically. The first observable ultrastructural change was a swelling of endothelial cells. Polymorphonuclear leukocytes began to transmigrate into the interstitium. After 4 minutes, amorphous, optically dense substances appeared on endothelial surfaces. Only on these which platelets later became adherent. Thrombi grew until vessels were occluded. The thrombi were formed by the amorphous structure, degranulated platelets and unaffected red blood cells and leukocytes. No fibrin strands were observed. Pretreatment with 100 micrograms/kg b.w. nafazatrom, a potent antithrombotic agent which stimulates endothelial release of prostacyclin, totally abolished the occurrence of the observed amorphous epiendothelial structure as well as the occurrence of platelet thrombi.

Are yeasts in vaginal smears intracellular or extracellular?

Using light microscopic cytodiagnosis the only thing that can be said about the position of the yeast cells in relation to the epithelial cells is that the epithelial cells and the parasite are very close to one another. It is not possible to say whether both elements are only temporarily projected above one another or in direct contact. This question can only be answered unequivocally with the aid of the electron microscope. It is possible to verify that the yeast cells, despite their size, are able to invade intact epithelial cells and are capable of reproduction within these same cells.

Influence of ciprofloxacin on the ultrastructure of gram-negative and gram-positive bacteria.

Killing curves were performed in pooled active human serum using serum resistant Escherichia coli C14, Pseudomonas aeruginosa 220 and Staphylococcus aureus 25151 as test organisms. Ciprofloxacin (a quinoline-carboxylic acid derivative, Bay o 9867) was added in concentrations of 0.025-4.0 micrograms/ml. Samples were taken at different times to determine the number of viable bacteria and to prepare specimens for transmission electron microscopy. The following drug concentration-dependent results were obtained: In the early phase of the bactericidal process in Escherichia coli C14 distinct loosenings of the peripheral cytoplasm occurred. With Pseudomonas aeruginosa 220 osmiophilic condensations were detected in the less dense areas where the bacterial DNA may be located. At low concentrations, elongation of gram-negative bacterial cells was observed, whereas at higher concentrations rupture and lysis occurred. In staphylococci, severe disturbances of septum formation and surface deformations could be found; in the final stage proteolysis and empty envelopes were observed.

[Ultrastructural and microbiological studies on the activity of azlocillin against Pseudomonas aeruginosa (author's transl)]. / Ultrastrukturelle und mikrobiologische Untersuchungen zur Wirkung von Azlocillin auf Pseudomonas aeruginosa.

To examine the effect of 6-[(R)-2-(2-oxo-imidazolidine-1-carboxamido)-2-phenyl-acetamido]-penicillanic acid sodium salt (azlocillin, Securopen) on the ultrastructure of Pseudomonas aeruginosa investigations were carried out by electron microscopy on thin sections and on negative-stained preparations. Depending on the concentration and the contact time of azlocillin the bacteria showed distinct alterations. The bacterial cells did not build septa and therefore only grew in length up to 100-fold that of untreated controls. The bacteria diameter remained unchanged. Survival curves showed that these bacterial filaments were unable to build colonies. They were irreversibly damaged.

[Electron microscopic and microbiological studies on the activity of mezlocillin against Escherichia coli (author's transl)]. / Elektronenmikroskopische und mikrobiologische Untersuchungen zur Wirkung von Mezlocillin auf E. coli.

Investigations by electron microscopy on thin sections and negative-stained preparations were carried out to show the effect of 6-[(R)-2-[3-methylsulfonyl-2-oxo-imidazolidine-1-carboxamido]-2-phenyl-acetamido]-penicillanic acid sodium salt (mezlocillin, Baypen) on the ultrastructures of gramnegative bacteria (Escherichia coli C 165). Distinct alterations of the bacteria were to be seen depending on concentration and cultivation time. The outer wall of the bacterial cell became irregular. Locally it detached from the plasma membrane. The normal density of the cytoplasmic structures decreased quickly. Owing to their inability to form septa the bacteria grew as extended filaments. These mezlocillin-treated cells could form filaments of up to 100 times the length of normal untreated bacteria. Those bacterial filaments, however, were unable to form colonies as was shown by survival curves carried out in parallel. The filaments, therefore, were irreversibly damaged bacterial forms.

[Acute post-traumatic lung failure - morphological evaluation (author's transl)]. / Vergleichende Morphologie des posttraumatischen Lungenversagens.

We investigated morphological findings of human biopsy material taken from lung and muscle, as well as lung tissue taken from animals in hypovolatemic-traumatic shock experiments. This was an attempt to reproduce, by way of an experimental setup with animals, the morphological changes which were formely found in the biopsies. Our morphological results lead to the assumption that the posttraumatic progressive lung failure without direct lung trauma can be divided into two major phases which are of decisive importance for the clinical development. 1. Early changes: Massive leucostasis of polymorphonuclear granulocytes with partial degranulation in the lung. In additon, a swelling of the endothelium cells, fat globules and a beginning interstitial edema were observed. In none of the cases were microthrombi found. 2. The actual "shock lung or fat embolism syndrome" which can be diagnosed also clinically 24 to 72 hours after the trauma. Morphologically predominating here are the interstitial edema and the fibrosis secondary to this and which manifests itself clinically in a disturbed gas exchange. This is accompanied by serious changes in the alveolar epithelium, deposits rich in protein as well as an increased occurence of phagocytizing macrophages in the alveoli. The early morphological changes in shock lung can most likely be influenced by therapeutical measures (such as a prompt shock treatment, a balanced restoration of the loss in blood volume and an early artificial ventilation). In this way, also the feared shock lung syndrome can be avoided. In an experimental setup with animals these early changes, especially the leucostasis, can easily by reproduced. This has become possible by the experience gained from the human lung biopsies. By way of this animal experiment new methods of treatment can be tested which might lead to a clinical progress in the prophylaxis as well as in the treatment of the progressive post-traumatic lung failure.

Bacteriological and ultrastructural studies on the effect of subinhibitory beta-lactam concentrations on intraphagocytic killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes.

The effect of azlocillin, ticarcillin and cefsulodin, respectively, on the susceptibility of Pseudomonas aeruginosa to the antimicrobial action of human polymorphonuclear leukocytes (PMN) was investigated under two different experimental conditions. Firstly, phagocytic capacity as well as bactericidal activity of PMN were assessed in a homologous system, i.e. the clinical isolate as well as the PMN and serum were obtained from the same patient. Secondly, ultrastructural studies were performed by electron microscopy. Preincubation of bacteria with subinhibitory beta-lactam concentrations augmented the phagocytic capacity as well as the antibacterial activity of PMN; azlocillin tended to be the most effective drug in this respect. The enhanced susceptibility to leukocyte killing is not due to an increased antibacterial action of the beta-lactams in the presence of PMN. These findings suggest that a non-immunological linkage between bacteria and PMN may exist which may be based on the interaction between bacterial- and eukaryotic surface structures, respectively. It may be assumed that the antipseudomonal beta-lactam antibiotics may cause changes in the surface structures of P. aeruginosa, thus rendering them more susceptible to phagocytosis. Preliminary data indicate that the lectins on the outer membrane of P. aeruginosa are not mannose sensitive. Electron microscopic studies revealed that azlocillin pretreatment of bacteria brought about a high undulation and a disruption of the outer membrane. These morphological changes may render bacteria more vulnerable to the antimicrobial action of PMN. It may be speculated that an interference with surface adhesins and induction of morphological changes may affect engulfment and intracellular killing of bacteria.

[Comparative studies on the ultrastructure of human lung and sceletal muscle in shock. II (author's transl)]. / Vergleichende Untersuchungen der Ultrastruktur von menschlicher Lunge und Skeletmuskulatur im Schock. II.

The ultrastructure of skeletal muscle biopsies was investigated and compared to lung biopsy material of the same patient in shock. We found almost complete conformity of ultrastructural changes in the micro-vascular system. Capillary endothelia of both tissue types react in a similar way with a more or less distinct swelling of endothelial cells and rarefaction of cell structures as well as constriction of the vascular lumina. Despite an intact structure of the capillary endothelia there is development of oedema in the perivascular areas with escape of plasma components, erythrocytes and granulocytes into the muscle interstitium. Finally, more distant tissue regions are becoming oedematous. While mainly lysosomal enzymes from the large number of infiltrating granulocytes, as well as fatty globules, are made responsible for the damage in the shock lung, the damage of the capillary region in the sceletal musculature is almost certainly caused by hypoxia. We consider muscle biopsy a suitable method to obtain a better knowledge of the microcirculatory situation and of the reaction of ultrastructures in shock.

Influence of heparin, aspirin, streptokinase and factor VIII (AHF) on amorphous electron-dense substance, a mediator of platelet and thrombus adhesion in vivo.

Platelet adherence and aggregation on vessel walls are the first crucial steps in thrombogenesis and atherosclerosis. Whether platelets can be activated on damaged endothelial cells or their activation is achieved only when subendothelial structures are exposed was controversially discussed in the past. Recently, an electron-microscopic study has revealed an amorphous electron-dense substance (AEDS) after endothelial cell damage and has discussed its role as a possible trigger of thrombogenesis. The aim of the present study is to elucidate the role and origin of this substance and to investigate the influence which inhibitors of platelet function (acetylsalicylic acid), coagulation (heparin), stimulation of fibrinolysis (streptokinase) and addition of factor VIII (AHF) have on AEDS.

[Ultrastructure of the human lung in shock (author's transl)]. / Die Ultrastruktur der menschlichen Lunge im Schock. I

The material investigated was obtained by lung puncture with the aid of the Hausser needle. The puncture technique as well as the preparation of the biopsy material for electronmicroscopic diagnostics are described. The most outstanding criterion in all biopsies examined is the large number of polymorphonuclear, mainly neutrophile granulocytes in the capillary and precapillary arterioles. In contrast, hardly any platelets were found in pulmonary vessels. Also, our investigation of the material revealed no intravascular fibrin deposits while vessels are partly and sometimes completely occluded by fat droplets of different size. The vascular walls are markedly swollen. Fluid escape from smaller vessels results in an edematous swelling of varying degree in the perivascular space combined with fibrin uptake and partly or totally destroyed cell structures. The type I epithelial cells of the lung tissue are swollen and show poor cellular structures. There is in increase of the type II epithelial cells in the shock lung with their lamellary corpuscles partly transferred into the alveolar lumen. The pathomechanisms leading to these changes are discussed. We would like to point out that fibrin was never found intravascularly but was always seen in regions. These findings could indicate increased fibrinolytic activity in shock. Platelet aggregations in smaller vessels are of secondary significance in the material we examined while fat globules, however, play an important part due to their large surface extension. Our electronmicroscopic investigations prove that the lung biopsy method is of great importance for further information on the pathologenesis of early damages in the shock lung not easily discovered by light microscopy.

Induction of light hydrocarbon nephropathy by p-dichlorobenzene.

In order to clarify the etiology of a dose-related increase in the incidence of tubular cell adenocarcinomas of the kidney in male rats, the nephrotoxicity of p-dichlorobenzene (p-DCB) was investigated in a subchronic study. Groups of ten male and ten female Fischer 344 rats were dosed by gavage with 0 (controls), 75, 150, 300 or 600 mg p-DCB/kg/day in corn oil. Half of the animals were sacrificed after 4 weeks and the remainder after 13 weeks. Increased urinary LDH and epithelial cell excretion and exacerbation of hyaline droplet accumulation in the cytoplasm of renal cortical cells were observed in male rats over the entire dose range investigated. Tubular single cell necrosis, dilated tubules with granular cast formation in the outer zone of the medulla, were evident in male rats after 4 and 13 weeks of treatment with doses of 150-600 mg/kg/day. In female rats there was no indication of a nephrotoxic action of p-DCB. The effects on the kidney, both in their morphological characteristics and the fact that they occur exclusively in male animals, correspond to the light hydrocarbon nephropathy observed as a result of short-term treatment with a number of aliphatic and cyclic hydrocarbons. The development of cortical renal tumors seems to be associated with this kind of kidney damage which is unique to male rats. The literature on this subject generally regards these renal effects as not predictive for man.

Stable insertion of C5b-9 complement complexes into the outer membrane of serum treated, susceptible Escherichia coli cells as a prerequisite for killing.

Escherichia coli 17, a K12 derivative, was rapidly killed by human serum following a short lag period of 10 min. Stable binding of terminal C5b-9 complement complexes was investigated in time course experiments. Serum treated E. coli cells were lysed osmotically and the resulting outer and cytoplasmic membrane vesicles separated by sucrose gradient centrifugation. Exposure of E. coli 17 to serum rapidly reduced the degree of recoverability of cytoplasmic membrane vesicles. Electron microscopy revealed no interaction of C5b-9 complexes with CM vesicles. In contrast there was a clear time-dependent deposition of terminal complement complexes onto OM-vesicles. Very few complexes were detected during the prekilling phase of the reaction; initiation of the active killing phase was accompanied by a large increase in complement lesions. In contrast, no C5b-9 complexes could be visualised on outer or cytoplasmic membrane vesicles of a smooth, serum-resistant E. coli strain. We conclude that complement-mediated killing is a consequence of stable binding of C5b-9 complexes to the outer membrane of susceptible strains.

[Liver abscess in chronic granulomatosis of childhood (author's transl)]. / Leberabszess bei chronischer Granulomatose des Kindesalters.

A thirteen-year-old boy with an abscess of the liver and recurrent infections since infancy is described. Aspergillus fumigatus could be isolated from this surgical treated abscess. Polymorphonuclear leukocytes (PMN) and monocytes from the patient show a normal phagocytosis, whereas the oxygen metabolism and the intracellular killing of kastalase-positive microorganismus (Staphylococcus aureus, Candida albicans) are strikingly decreased. In contrast, the catalase-negative Pneumococcus pneumoniae is rapidly killed. The iodination (131 J) of PMN is despite phagocytosis also markedly reduced. Myeloperoxidase could be detected in the phagocytes histochemically. No morphologic abnormalities of PMN have been found by electron microscopy. A defect of the humoral of cellular immunity has been excluded. The characterized dysfunction of PMN and monocytes as well as the clinical features of the patient are consistent with a diagnosis of chronic granulomatous disease of childhood.

Electron-microscopic investigation of lung biopsies in patients with post-traumatic respiratory insufficiency.

Lung biopsies were taken from patients during the course of post-traumatic respiratory insufficiency, from 3 hours to 19 days after the onset of shock. Electron-microscopic investigations revealed that the initial phases of cellular damaged were followed by both endothelial and epithelial changes, including atelectasis, lymphatic dilation, and interstitial as well as interalveolar fibrin extravasation. Interstitial edema and extravascular migration of granulocytes, lymphocytes and mast cells are followed by fibroblastic proliferation and, as an end result, fibrosis of the alveolar septa. As a result, fibroblasts replace capillaries at the surface, where gas exchange takes place. The capillaries are forced into the depths of the interstitial tissues. At the same time, the alveolar surface becomes covered with thick-walled proliferating epithelial cell layers, which also renders gas exchange increasingly difficult. Lung function is thereby reduced.