Klaus A. Neftel

Neutropenia after penicillins: Toxic or immune-mediated?

SummaryEight patients treated with a total of 220–550 million U penicillin-G developed neutropenia. These cases have been compared with eight patients receiving a similar dose of penicillin-G with no adverse reactions and with eight untreated subjects. All penicillin-treated patients showed raised levels of antipenicillin IgG antibodies and lymphocyte culture stimulation indices. These values were highest in the neutropenia group. Both of the two tests significantly descriminated the three groups. Antineutrophil antibodies could be detected in four of seven neutropenic patients with a staphylococcal-slide-assay while indirect immunofluorescence and microcytotoxicity tests failed to reveal these antibodies. The literature dealing with neutropenias induced by penicillin-G and its congeners is reviewed. We conclude that (1) penicillin-G in doses exceeding a total of 200 million U frequently induces neutropenia, (2) an immune-mediated pathogenesis is highly probable, (3) neutropenia after penicillins is different from two hither-to accepted types of this side effect, (4) sufficiently high amounts of penicillin-G intravenously always induce sensitization against this drug.ZusammenfassungAcht Patienten entwickelten nach total 220–550 Millionen E Penicillin-G i.v. eine Neutropenie. Wir verglichen immunologische Daten dieser Fälle mit denjenigen von acht Patienten, die mit vergleichbaren Penicillin-G-Dosen ohne ersichtliche Nebenwirkungen behandelt wurden, sowie mit denjenigen von acht unbehandelten Gesunden. Alle mit Penicillin Behandelten zeigten im Vergleich zu den Unbehandelten erhöhte Spiegel von anti-Penicillin IgG und einen stark positiven Lymphozyten-Stimulationstest mit Penicillin. Beide Untersuchungen ergaben die höchsten Werte innerhalb der Neutropeniegruppe und unterschieden alle drei Gruppen signifikant voneinander. Bei vier von sieben Neutropenikern konnten mit einem Objektträgertest unter Verwendung von Protein A enthaltenden Staphylokokken, antineutrophile Antikörper nachgewiesen werden. Mit indirekter Immunfluoreszenz und Mikrozytotoxizitätstest gelang dagegen in keinem Fall ein Antikörpernachweis. Die Literatur über Neutropenie nach Penicillin und seinen Derivaten wurde durchgesehen. Schlußfolgerungen: (1) Über 200 Mio E Penicillin-G i.v. induziert häufig eine Neutropenie, (2) ein immunvermittelter Mechanismus ist sehr wahrscheinlich, (3) die Neutropenie nach Penicillinen ist nicht identisch mit einem der zwei bisher akzeptierten Typen der medikamentös induzierten Immunneutropenie (Amidopyrin-Typ resp. Procainamid-Typ), (4) genügend hohe Dosen von Penicillin-G erzeugen immer einen positiven Lymphozyten-Stimulationstest und einen Anstieg von Penicillin-spezifischen IgG.

Adverse reactions following intravenous penicillin-G relate to degradation of the drug in vitro

SummaryWe have recently shown that high-dose intravenous therapy with penicillin-G always results in both sensitised lymphocytes and rise of anti-penicilloyl IgG antibodies. If penicillin-G is strictly given as freshly prepared bolus doses this sensitisation is prevented. In 193 patients, intravenous treatment with penicillin-G without special precautions (bolus doses stored up to 36 h at 4° C or continuous infusions) led to 8.3% definite, 6.7% probable and 14.0% possible adverse reactions. In 116 patients treated exclusively with freshly dissolved doses, 0.9% definite, 1.7% probable and 4.3% possible reactions occurred. Whereas haemolytic anaemia (7) and neutropenia (12) were observed in 19 cases of the first group no such reactions were seen in the second group. Strict application of freshly prepared single doses prevents the majority of adverse reactions following highdose intravenous penicillin-G therapy. Degradation and transformation products formed in vitro are therefore the causative agents rather than the penicillin molecule itself.

Synergy between Trovafloxacin and Ceftriaxone against Penicillin-Resistant Pneumococci in the Rabbit Meningitis Model and In Vitro

ABSTRACT The bactericidal activities of monotherapy with trovafloxacin (−0.37 ± 0.15 Δlog10 CFU/ml · h), vancomycin (−0.32 ± 0.12 Δlog10 CFU/ml · h), and ceftriaxone (−0.36 ± 0.19 Δlog10CFU/ml · h) for the treatment of experimental meningitis in rabbits due to a clinical penicillin-resistant pneumococcal strain (MIC, 4 mg/liter) were similar. The combination of ceftriaxone with trovafloxacin considerably improved the killing rates (−0.67 ± 0.16 Δlog10 CFU/ml · h) and was slightly superior to ceftriaxone with vancomycin (killing rate, −0.53 ± 0.22 Δlog10 CFU/ml · h), the regimen most commonly used in clinical practice. In vitro, synergy was demonstrated between ceftriaxone and trovafloxacin by the checkerboard method (fractional inhibitory concentration index, 0.5) and by time-killing assays over 8 h.

Penicillin‐G degradation products inhibit in vitro granulopoiesis

Summary. 38% of penicillin‐G in solution decays at 20°C within 24 h, 50% at 37°C and 66% within 3 h at 56°C. These degraded penicillin‐G solutions strongly inhibit growth and maturation of granulocytic stem cells in vitro. Inhibitory concentrations are in the range obtainable with high dose penicillin therapy in vivo. No cytotoxicity of degraded penicillin solutions on bone marrow cells was seen over 24 h. It is suggested that penicillin‐G degradation products are responsible for severe granulo‐cytopenia observed after high dose penicillin‐G therapy.

Beta-lactam antibiotics inhibit human in vitro granulopoiesis and proliferation of some other cell types.

The antibacterial mechanisms of beta-lactam antibiotics (BLAs) are, with the exception of minor obscurities, well established. BLAs interact with enzyme proteins that are located on the outer side of the bacterial plasma membrane, the so-called penicillin-binding proteins. Some of them are essential for final steps in the synthesis of the bacterial cell wall, namely peptidoglyean crosslinking. Intrabacterial penetration of BLAs need not be postulated for explaining their antibacterial effects. Conversely, eukaryotie cells do not contain any known, specific BLA target and are generally considered to be nonsusceptible in vitro to BLAs. Accordingly, BLAs are believed to be remarkably nontoxic in humans and most adverse reactions to these drugs are thought to be immune-mediated (de Week, 1983).

Elevation of soluble Fas and soluble Fas ligand in reactive macrophage activation syndromes

Derailed T‐cell activation can give rise to life‐threatening macrophage activation, the final common pathway of the different forms of reactive macrophage activation syndromes (rMAS). Besides inappropriate activation of the immune system, impaired termination of immune responses might be another mechanism leading to rMAS. The Fas (CD95)/Fas ligand (CD95 ligand) system functions in turning off immune responses by executing activation‐induced cell death (AICD). Soluble Fas (sFas) and Fas ligand (sFasL) can interfere with their corresponding membrane‐bound counterparts, qualifying them as potential parameters of impaired immune termination. Hence, sFas and sFasL were analyzed in sera of rMAS patients. We show that soluble Fas/CD95 (sFas) is elevated >2 SD over the mean of controls in all 8 rMAS episodes studied (mean 12.08 ± 6.12 ng/mL, range 3.7–20.2; controls 2.46 ± 0.49, range 1.5–2.9). sFasL was detected during five rMAS episodes (0.70 ± 0.49 ng/mL, range 0.16–1.28; controls all below the limit of detection of 0.1). In addition, both parameters decrease during convalescence, reflecting clinical evolution. In conclusion, sFas seems to be consistently elevated during acute rMAS. sFasL is detected only in a subgroup of our adult rMAS patients extending the recent finding of sFasL elevation in a majority of children with macrophage activation syndromes (Hasegawa et al. Blood 1998;91(8):2793–2799). By interfering with AICD, sFas and sFasL might contribute to the pathogenesis of at least a subset of rMAS. Am. J. Hematol. 64:116–119, 2000.

Effects of beta-lactams on DNA replication

In earlier studies, we have obtained evidence that beta-lactam antibiotics (BLAs) inhibit proliferation of various eukaryotic cells in vitro, including human granulopoiesis (Neftel et al., 1985), lymphoid cells (Hiigin et al., 1986), the human erythroleukemic K-562 cell (Cottagnoud and Neftel, 1986) and others. These findings can explain BLAinduced neutropenia, while other clinical consequences are as yet less clear. In liquid cultures of K-562 cells that were in some instances synchronized with aphidicolin, a reversible DNA polymerase alpha-blocker (Spadari et al., 1982), flow cytometry suggested the following conclusions (Cottagnoud et al, 1986): that BLAs act on cellular DNA synthesis and that their effect is restricted at least in part to the S-phase of the cell cycle.

Beta-lactams act on DNA synthesis in K-562 cells

Recently we have shown that beta-lactam antibiotics (BLAs) inhibit human in vitro granulopoiesis (Neftel et al., 1985) and proliferation of some other eukaryotic cells in vitro (Hiigin et al., 1986; Neftel et al., 1986) in a dose-dependent manner. Although all BLAs of the large number so far tested exhibited antiproliferative activity, the absolute inhibitory potency of individual compounds varied considerably. Nevertheless, in each system studied, a similar order of efficacy emerged: cephalosporins as a group were from 3 to more than 25 times more effective than penicillins. Clavulanic acid and N-formimidoyl-thienamycin behaved like strong cephalosporins, whereas monolactams were practically ineffective. The reproducibility of such a hierarchy of toxicity, as initially found in semi-solid agar cultures of bone marrow cells, suggests a common mechanism underlying the antiproliferative effects of BLAs in different cell culture systems. In the present work, liquid cultures of the human erythroleukemic K-562 cell were chosen as a model to elucidate further the BLA-induced proliferation inhibition. The results indicate that, at least in K-562 cells, BLAs act on DNA synthesis.

Suppressive effects of B-lactam-antibiotics on in vitro generation of cytotoxic T-cells

The in vitro effect of several compounds containing the B-lactam structure (including 6-aminopenicillanic acid, piperacillin, 7-aminocephalosporanic acid, ceftazidime and clavulanic acid) on the generation of cytotoxic lymphocytes was investigated in two different in vitro systems: (1) generation of virus specific cytotoxic T-cells and (2) proliferation of lymphocytes were assessed in secondary in vitro anti-viral immune responses. Both activities were suppressed by B-lactams in a dose-dependent manner. In these experiments, as found previously with human in vitro granulopoesis and proliferation of the human erythroleukaemic cell line K-562, cephalosporins and clavulanic acid were five to twenty times more suppressive than penicillins. Since concentrations that were effective in vitro are being achieved, particularly with cephalosporins, in the serum of patients, the possible clinical consequences of our findings may have to be taken into consideration.

Radial Segmentation of Nuclei (Rieder Cells): A Morphological Marker of T-Cell Neoplasms?

Pretreatment blood smears of adult patients with acute lymphoblastic leukemia were examined for neoplastic cells showing nuclear radial segmentation (RS). RS was present in 6 of 7 patients with immunologically proven T-cell leukemia, but not in 11 patients whose leukemic cells lacked T-cell markers. Electron microscopic studies of neoplastic cells showed multiple nuclear indentations and abundant cytoplasmic microfilaments and microtubuli in connection with pericentriolar dense material. RS formation was decreased in 1 patient after chemotherapy with a vincristine-containing regimen, while the white cell count remained unchanged. Radial segmentation (RS) of leukocyte nuclei is a well-known phenomenon, which is observed ex vivo (Rieder cells), and can be induced in vitro. Convoluted or multilobulated nuclei in lymphoid neoplasms are similar to RS nuclei both with regard to their structure and their sensitivity to spindle-blocking drugs. We propose that the nuclear alterations observed in a variety of different T-cell neoplasms are identical with RS, and suggest that RS might be a morphological marker for subsets of lymphoproliferative disorders of T-cell origin.