Oto Zak

The role of complement in inflammation during experimental pneumococcal meningitis

The mechanism whereby an effective bactericidal inflammatory reaction develops in the subarachnoid space is not clearly defined. While normal cerebrospinal fluid is deficient in complement, immunoglobulin and leukocytes, these serum components appear in cerebrospinal fluid (CSF) during the course of bacterial meningitis. Using a rabbit model of pneumococcal meningitis we examined the role of the alternate complement pathway in three early events important to the defense of the subarachnoid space: leukocyte chemotaxis, phagocyte mediated bacterial killing, and clearance of bacterial components from the cerebrospinal fluid space. Rabbits treated with cobra venom factor to deplete complement were inoculated intracisternally with encapsulated (type II or XIX) pneumococci. Following complement depletion, there was a dramatic (at least 100-fold) decrease in the LD50 for these strains. Nevertheless, complement depletion did not affect the magnitude of CSF leucocytosis or the rate of clearance of bacterial particles from CSF. A short delay in the appearance of leukocytes in CSF was found in the absence of complement. The major effect of complement depletion, however, was to diminish the efficiency of leukocyte mediated killing of encapsulated bacteria in the CSF. Although the short delay in the onset of leukocytosis in the complement depleted animals is consistent with a chemotactic role of complement in the normal animal, the quantitatively normal leukocytosis in the complement depleted rabbits clearly indicates that important chemotaxins other than complement function in CSF. Inhibition of leukocytosis by indomethacin and diclofenac suggests that metabolite(s) of the arachidonic acid pathway may perform such a chemotactic role. A major role of complement in the defense of the subarachnoid space appears to be as an opsonin needed for the effective bactericidal activity of leukocytes. It is the lack of this function that best explains the greatly decreased LD50 value of encapsulated pneumococci in the complement depleted animal.

Systemic Neutralization of Interleukin-8 Markedly Reduces Neutrophilic Pleocytosis during Experimental Lipopolysaccharide-Induced Meningitis in Rabbits

ABSTRACT Interleukin-8 (IL-8) is elevated in the cerebrospinal fluid (CSF) of patients with meningitis and is proposed to participate in subarachnoid-space pleocytosis. However, intracisternal injection of IL-8 into rabbits failed to induce indices typical of meningitis (leukocyte, tumor necrosis factor, or protein accumulation in the CSF or histopathological changes), indicating that merely increasing the CSF level of this chemokine is insufficient to induce inflammation in this anatomical site. IL-8 treatment did not affect inflammatory responses to subsequently intracisternally administered lipopolysaccharide (LPS). IL-8 was chemotactic for rabbit neutrophils in vitro, and subcutaneous injection of IL-8 (diluted in buffer or CSF) proved the in vivo activity of this peptide and suggested the absence of an IL-8 inhibitor in normal rabbit CSF. LPS-dependent pleocytosis was only slightly diminished by intracisternally administered murine anti-rabbit IL-8 monoclonal antibody (MAb) WS-4 but was dramatically reduced by intravenously administered MAb. Therefore, elevated CSF IL-8 levels may contribute to, but cannot solely account for, neutrophil influx into the subarachnoid space during meningitis. However, inhibition of IL-8 activity of the bloodstream side of the blood-brain barrier effectively reduces pleocytosis, indicating a central role of IL-8 in neutrophil influx into CSF during bacterial meningitis. Thus, inhibition of IL-8 is a possible therapeutic target for adjunct treatment of meningitis.

Induction of meningeal inflammation by diverse bacterial cell walls.

Bacterial cell walls have been shown to be potent inflammatory agents. Cell wall components from various bacterial species can i) immunomodulate the activity of macrophages, T cells and B cells (1,2), ii) trigger the alternative pathway of complement activation (3-5) , iii) interact with polymorphonuclear leukocytes (6, 7), and iv) bind with humoral immune factors such as C-reactive protein (8). Such activities presumably contribute to inflammation provoked by cell walls in models of post4nfectious and immune diseases (9, 10). Recently, however, it has become increasingly clear that host reactions to cell walls can also contribute very significantly to the course of acute bacterial infections such as bacteremia (11-13) and meningitis (14-16).

Animal models as predictors of the safety and efficacy of antibiotics

As opposed to the testing of safety, the testing of the efficacy of antibiotics in animals is not specified in any directives or guidelines and not explicitly required by regulatory authorities. There exists, however, no doubt that in the evaluation of new compounds testing of both safety and efficacy forms an essential link between in vitro tests and clinical trials. It is inconceivable that clinicians would be prepared to conduct a trial in patients without evidence of the efficacy of the antibiotic in question in an appropriate animal model of infection. Both the models for testing safety and those for testing efficacy suffer from a number of shortcomings. If investigators are aware of these deficiencies and take them into account when interpreting the results, the predictive value of the models can be significantly enhanced.

Systemic inflammation alters the inflammatory response in experimental lipopolysaccharide-induced meningitis

Experiments to evaluate the effect of the level and duration of endotoxaemia on the meningeal inflammatory response were performed in order to determine if systemic inflammation alters meningitis. Rabbits received either saline or Escherichia coli O111:B4 lipopolysacharide (LPS) intravenously at various doses (1, 3 or 10 µg) and times (−8, −2 or 0 h) before an intracisternal injection of 20 ng LPS. An intracisternal LPS injection together with saline intravenously produced a peak cerebrospinal fluid (CSF) tumour necrosis factor (TNF) level (95 ± 26 ng/ml) at 2 h and peak leucocyte level (5413 ± 764 cells/µl) at 4 h post‐injection. Blood leucocytes were slightly elevated (12 000 ± 500/µl at 0 h; 16 900 ± 280/µl at 8 h) but plasma TNF was always undetectable (< 0·05 ng/ml). Conversely, intravenous injection of 3 or 10 µg LPS 2 h prior to intracisternal LPS injection impaired pleocytosis (peak < 220 cells/µl) and delayed (∼4 h) and reduced peak CSF TNF levels (3 µg LPS 5·0 ± 1·2 ng/ml; 10 µg LPS 6·9 ± 1·9; P < 0·05). Intravenous administration of 1 µg LPS was less inhibitory to CSF inflammation, but delayed onset (peak 1100 ± 60 leucocytes/µl CSF at 8 h; 6·3 ± 0·3 ng TNF/ml CSF at 4 h; both P < 0·05). Neutropenia nadirs were dependent on LPS dose (1 µg, 4500 ± 1700; 3 µg, 1900 ± 60; 10 µg, 1100 ± 100 all at 4 h post‐intravenous dose). Peak plasma TNF levels were not dose‐dependent (> 8 ng/ml), but plasma TNF was always detectable (> 0·2 ng/ml at 10 h post‐intravenous dose). Intravenous LPS administration at 0 h also blocked pleocytosis, but the inhibitory effect was lost when administration at −8 h. In conclusion, the degree and duration of endotoxaemia affect the meningeal inflammatory response to LPS in experimental meningitis.

Penems: In Vitro and In Vivo Experiments

A mong the plethora of new antibiotics discovered in the last 10 to 15 years, the penems and carbapenems, or “non-classic” beta-lactams as they have been dubbed,1 doubtless belong to the few that not only presented an extraordinary challenge to chemists but also aroused wide interest among medical microbiologists and clinicians. Although related in denomination and structure, they are intrinsically distinct inasmuch as the carbapenems are naturally occurring fermentation products, whereas the penems are fully synthetic. The first penem (Figure 1) was synthesized at the Woodward Research Institute in Basle, Switzerland, in 1975.23 It displayed a certain, albeit unsatisfactory, activity against gram-positive bacteria, but was unstable, probably because of its acyl side chain at C-6. The synthesis of this compound, however, marked the realization of Woodward’s original idea of constructing antibiotics that would combine the beta-lactam-activating properties of the five-membered ring of the penicillins with the reactivity-enhancing double bond similarly located to that in the six-membered ring of the cephalosporins (Figure 2). It was not, however, until the molecule-stabilizing hydroxyethyl substituent at C-6, already known from thienamycin, was introduced that beta-lactamase-resistant and chemically stable penems with a promising activity profile were obtained. The penems (and carbapenems) possess a number of special antibiotic properties not shared by most other beta-lactams. Some of these are indicated in Table I and are the subject of this brief review; for the most part they are exemplified by data relating to the well-known penems SCH 29482 and SCH 34343 (Schering-Plough), FCE 22101 and FCE 22891 (Farmitalia Carlo Erba) and the carbapenem imipenem (Merck, Sharp & Dohme) (Figure 3); findings made with some new penems of Ciba-Geigy are also included.

Remarks on the screening of antibiotics for antibacterial activity

The general principles of screening antibiotics for antimicrobial activity are similar to those for screening for other pharmacological effects. The system should be adapted to the specific character of the test substance and the objectives of the program. In the screening of β-lactams, standard tests, such as determination of the MICs, effects of inoculum size or activity against systemic infection in mice, should be supplemented by less conventional studies on for instance activity against dormant bacteria or, in the case of penems or carbapenems, stability in the presence of kidney and lung dehydropeptidases.

Interaction of the novel penem CGP 31 608 and its enantiomer with type Id beta-lactamase and penicillin-binding proteins.

The novel penem CGP 31 608 (5R, 6S, 8R) and its enantiomer CGP 32 879 (5S, 6R, 8S) were shown to be essentially stable against hydrolysis by type Idβ-lactamase isolated fromPseudomonas aeruginosa18S/H. CGP 31 608 was a potent progressive inhibitor of this enzyme (I50=32μM), which was only weakly inhibited by CGP 32 879 (I50=460μM). CGP 31 608 had the highest affinity for penicillin-binding protein (PBP) 4 fromEscherichia coliK-12 (I50= 1μg/ml), followed by PBPs 2 (10μg/ml) and 1A/1Bs (100μg/ml); CGP 32 879 did not inhibit binding of14C-benzylpenicillin to the PBPs. The steric configuration of theβ-lactam nucleus of penems appears to strongly influence their affinity forβ-Iactamases and target PBPs. The balanced spectrum of CGP 31 608 may be explained by itsβ-lactamase stability and affinity for several vital PBPs.