Cellular retention, cytotoxicity and morphological transformation by vanadium(IV) and vanadium(V) in BALB/3T3 cell lines

Cytotoxicity, morphological transformation and cellular retention have been studied in BALB/3T3 Cl A 31-1-1 cells for ammonium or sodium vanadate [vanadium(V)] and for vanadyl sulphate [vanadium(IV)]. A morphological transformation focus assay showed transforming activity for vanadium(V) (P less than 0.005 at concentrations of 3 x 10(-6) or higher) while vanadium(IV) was not transforming in the cells. Cytotoxicity was higher for vanadium(V) than for vanadium(IV); this was particularly clear at doses from 5 x 10(-6) to 5 x 10(-5) M. The cellular retention of both vanadate and vanadyl compounds at 24, 48 and 72 h incubation was similar. At concentrations lower than 10(-6) M vanadate, the retention was linear with the dose, while at higher exposures the vanadium taken up by the cells levelled off or slightly decreased. Exposure to 10(-6) M and 10(-5) M vanadium(V) for 3 and 24 h as well as to 10(-6) M for 48 and 72 h yielded greater than 94% vanadium in the cytosol, but exposure to a toxic dose (10(-5) M) for 48 and 72 h yielded 20% vanadium associated with cellular organelles, which suggests that some sites in the cytosol become saturated with vanadium. The corresponding gel-filtration experiments indicate that a redistribution of the element among the cytosol components occurs with time.     

Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311.

C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy.     

Biochemical and cellular determinants of bleomycin cytotoxicity

Bleomycin is a mixture of cytotoxic glycopeptides which function as mininucleases, binding to DNA and producing single and double strand breaks by the formation of an activated oxygen complex. Bleomycin is an effective agent against a few human cancers, notably lymphomas, testicular and ovarian germ cell cancers and certain squamous carcinomas. Most human cancers are resistant to bleomycin a priori, however, and those which are initially sensitive frequently develop resistance to the drug during therapy. Several potential modes of resistance to bleomycin have been identified in cell culture and animal tumour models, although their relative importance in determining the responsiveness of human cancers to the drug is not well understood. Bleomycin is selectively toxic to cells in the M and G2 phases of the cell cycle, and generally more effective against actively dividing rather than resting cells. Thus, the cytokinetic state of the tumour cell population is an important determinant of drug activity. Oxygen is an essential substrate for bleomycin's action, with the degree of cytotoxicity directly related to ambient oxygen. Both acutely and chronically hypoxic cells form a substantial fraction of the cell population of many tumours, and may serve as a reservoir of cells resistant to bleomycin on this epigenetic basis. Metabolic inactivation of bleomycin is a mechanism of resistance to the drug in some cells and may influence toxicity in normal tissues. Bleomycin hydrolase activity is low in lungs and skin, the two major sites of normal tissue toxicities, and levels of this enzyme have been elevated in some but not all tumour cell lines selected for resistance to bleomycin. The capacity to repair or withstand single and double strand DNA breaks may also be an important determinant of resistance to the drug. Most yeast and mammalian cell mutants, which are hypersensitive to ionizing radiation because of defects in DNA repair, are also more sensitive to bleomycin than wild-type cells. A number of agents which interact with membranes or inhibit DNA repair, such as ethanol, lidocaine, verapamil and caffeine, have been reported to sensitize cells to bleomycin in vitro.     

Cellular F-actin levels as a marker for cellular transformation: relationship to cell division and differentiation

Transformation is associated with profound structural and quantitative changes in the cytoskeleton. Herein we report studies using F-actin, a major cytoskeletal protein, as a quantitative marker for transformation cells, focusing on separating the effects of the cell cycle, cell differentiation, and transformation. The model system for these studies consisted of three lymphoblastic cell lines, one untransformed line (RPMI) and two transformed lines, one (HL-60) of which can be induced to differentiate and the other (Daudi) which cannot. The relation of F-actin levels to cell cycle was studied by flow cytometry with the use of fluorescein-phalloidin to label F-actin and propidium iodide to label DNA. F-actin levels in transformed Daudi and HL-60 lines were only two-thirds that of the untransformed RPMI cells. Histograms of the distribution of F-actin showed that the transformed lines consisted of two cell populations, one having an F-actin content near that of untransformed cells and the other having much less. Cell cycle analysis showed that F-actin in untransformed cells increased 10-15% as cells entered the S compartment, remaining approximately constant through G2 + M phases of the cell cycle, but in transformed cells the major increase in F-actin occurred during G2 + M phase. Double-label studies with rhodamine-phalloidin for F-actin and KI-67 monoclonal antibody for dividing cells (cells at late G1, S, G2, and M) measured with quantitative fluorescence image analysis showed that the mean F-actin content of dividing cells was twice that of nondividing cells. These results suggested that most of the cell division-related F-actin increase occurred during late G1 phase in untransformed cells. Differentiation of HL-60 cells with dimethyl sulfoxide or retinoic acid normalized the F-actin content of the nondividing cell population, but dimethyl sulfoxide and retinoic acid produced no detectable change in F-actin in the undifferentiable Daudi cells. A tumor promoter (12-O-tetradecanoylphorphol-13-acetate) inhibits differentiation of hematopoietic cells, resulted in a 32% decrease in the mean F-actin content of RPMI cells due to the appearance of a new subpopulation of low F-actin content. The 12-O-tetradecanoylphorbol-13-acetate-induced changes reversed slowly after removal of 12-O-tetradecanolyphorbol-13-acetate but more rapidly in the presence of retinoic acid. These results indicate that F-actin quantification can serve as a marker for cellular transformation and provides a tool for studying the mechanisms of cellular differentiation that may lead to a better understanding of the oncogenic process.     

Temperature dependence studies of adriamycin uptake and cytotoxicity

In order to learn whether a direct relationship exists between cellular uptake and cytotoxicity of Adriamycin, we have compared the temperature dependencies of these two processes in L1210 cells. We find that the equilibrium concentration of drug taken inside the cells varies smoothly with temperature between 37 degrees C and 0 degree C. Even at 0 degree C, however, there is still measurable uptake of the drug into cells. The cytotoxicity index (cloning in soft agar), on the other hand, does not parallel the uptake temperature dependence. Cytotoxicity rapidly diminishes as the temperature of drug exposure is lowered; at all temperatures below about 20 degrees C, Adriamycin is not active. In contrast, other cytotoxic anticancer drugs like mitomycin C, bleomycin, and ARK 73-21 (a platinum analogue) retain cytotoxic potency at low temperatures. The inability of Adriamycin to kill cells at low temperature persists even at very high drug concentrations where substantial quantities of drug enter the cells. The low temperature impotence is not a result of inoperative enzymes which could metabolize Adriamycin to an alkylating species or electron donor to oxygen, since NADH and NADPH dependent reductase activities show linear Arrhenius behavior with no indication of low temperature inactivity. Using purified L1210 plasma membranes with bound Adriamycin as a fluorescence polarization probe, we find evidence of a phase change in the cell surface occurring at the same temperature as the loss of biological activity (approximately equal to 20 degrees C). We conclude that Adriamycin induced cytotoxicity is not dictated solely by uptake, in apparent contradiction with mechanisms requiring an intracellular target. Moreover, the loss of cytotoxicity below 20 degrees C appears to be linked to a structural change in the cell surface membrane, supporting a role other than transport for this membrane in transducing Adriamycin action.     

Induction of oxidative DNA damage by arsenite and its trivalent and pentavalent methylated metabolites in cultured human cells and isolated DNA

Even though a well-known human carcinogen the underlying mechanisms of arsenic carcinogenicity are still not fully understood. For arsenite, proposed mechanisms are the interference with DNA repair processes and an increase in reactive oxygen species. Even less is known about the genotoxic potentials of its methylated metabolites monomethylarsonous [MMA(III)] and dimethylarsinous [DMA(III)] acid, monomethylarsonic [MMA(V)] and dimethylarsinic [DMA(V)] acid. Within the present study we compared the induction of oxidative DNA damage by arsenite and its methylated metabolites in cultured human cells and in isolated PM2 DNA, by frequencies of DNA strand breaks and of lesions recognized by the bacterial formamidopyrimidine-DNA glycosylase (Fpg). Only DMA(III) (> or =10 micro M) generated DNA strand breaks in isolated PM2 DNA. In HeLa S3 cells, short-term incubations (0.5-3 h) with doses as low as 10 nM arsenite induced high frequencies of Fpg-sensitive sites, whereas the induction of oxidative DNA damage after 18 h incubation was rather low. With respect to the methylated metabolites, both trivalent and pentavalent metabolites showed a pronounced induction of Fpg-sensitive sites in the nanomolar or micromolar concentration range, respectively, which was present after both short-term and long-term incubations. Furthermore MMA(III) and DMA(V) generated DNA strand breaks in a concentration-dependent manner. Taken together our results show that very low physiologically relevant doses of arsenite and the methylated metabolites induce high levels of oxidative DNA damage in cultured human cells. Thus, biomethylation of inorganic arsenic may be involved in inorganic arsenic-induced genotoxicity/carcinogenicity.     

Cellular cytotoxicity mediated by isotype-switch variants of a monoclonal antibody to human neuroblastoma

The biological property of an antibody is determined by its antigen binding characteristics and its isotype-related effector functions. We have established monoclonal antibodies of different isotypes by stepwise selection and cloning of the hybridoma CE7. The original CE7 secretes an IgG1/kappa (CE7 gamma 1) antibody that recognises a 185 kD cell surface glycoprotein expressed on all human sympatho-adrenomedullary cells. Isotype-switch variants were isolated in the following sequence: from the original CE7 gamma 1, CE7 gamma 2b variants were isolated, and from a CE7 gamma 2b variant CE7 gamma 2a variants were isolated. The antibodies of three different isotype variant cell lines possess identical antigen binding characteristics, but display distinct effector functions as demonstrated by antibody dependent cell-mediated cytotoxicity (ADCC). ADCC was performed with the neuroblastoma line IMR-32 as the target cells, and different FcR gamma positive cells were either freshly isolated from human peripheral blood leukocytes or cultured for 6-10 days and tested as potential effector cells. Tumour lysis mediated by monocyte-derived macrophages depended on the presence of CE7 gamma 2a antibodies; antibodies from the CE7 hybridomas of gamma 2b and gamma 1 isotypes were virtually inactive in ADCC assay. Pre-exposure of macrophages to rIFN-gamma enhanced their ADCC activity, a result that is compatible with the notion that the high affinity Fc IgG receptor (FcR gamma I/CD64) is involved in the triggering of ADCC in macrophages. In contrast to macrophages, mononuclear cells, nonadherent cells and monocytes displayed considerable non-specific lytic activity, which was little influenced by the presence of antibody regardless of the isotype added.     

Cellular growth and metabolic adaptations to nutrient stress environments in tumor microregions

Heterogeneity of cell subpopulation growth was significantly modulated by different oxygen and glucose environments and necrosis in multicellular tumor spheroids of rodent and human origin. PO2 profiles within spheroids measured with microelectrodes showed major differences associated with different oxygen and glucose supply conditions, indicating important interactions of these two substrates affecting oxygen consumption rates and cellular viability. Cellular interactions in association with the development of growth quiescence and differentiation changed oxygen consumption rates and slopes of PO2 profiles within spheroids. Protein synthesis in monolayer cells in culture was severely inhibited when exposed to extreme hypoxia, but certain proteins were synthesized at increased rates. Many of these oxygenated regulated proteins can also be induced by glucose deprivation. The data demonstrate cellular and subcellular changes in tumor models in vitro because of variations in oxygen and glucose supply. Many of these changes would be expected to occur in tumor microregions in vivo and could have important consequences for therapeutic responsiveness.     

Fibrinolytic activity and morphological transformation of hamster embryo cells

Excretion of plasminogen activator from colonies of Syrian hamsterembryo cells has been studied after sequential exposure of thecells to benzo[a]pyrene (3 days), and 12-O-tetradecanoyl-phorbol-13-acetate(3 days). The excretion of plasminogen activator was assayedusing the fibrin/ agarose overlay technique. The frequency ofplasminogen activator-positive colonies was about two timeshigher for morphologically transformed colonies than for colonieswith normal morphology growing on the same dish. Thus, 9% ofthe transformed colonies, compared to 4% of the colonies withnormal morphology, gave clear zones of lysis in the fibrin/agaroseoverlay after 2 h of incubation. The frequency of plasminogenactivator-positive colonies on untreated dishes was 2%. Theaddition of protease inhibitors strongly reduced the formationof clear zones of lysis, while they did not affect the frequencyof morphologically transformed colonies. The data show thatthe expression of plasminogen activator is not an obligatoryevent in the process of morphological transformation.     

Ultrastructural and metabolic determinants of resistance to azo-dye susceptibility to nitrosamine carcinogenesis of the guinea-pig.

During diethylnitrosamine (DEN) administration, a distinctive difference was observed between rats and guinea-pigs in the sequence of ultrastructural changes in the hepatic endoplasmic reticulum (ER). In DEN-induced hepatic tumour cells in the guinea-pig there was extensive proliferation of the rough ER, while the smooth ER was quite sparse; in the premalignant liver the opposite was noted. This is in contrast to the rat, in which administration of either DEN or 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) brings about, in both premalignant and malignant hepatic tissue, proliferation of the smooth ER and sparsity of the rough ER. Yet, as in the rat, the number of ribosomes on the outer surface of the guinea-pig liver rough ER is greatly reduced and this is paralleled by a 49% decrease of the RNA/protein ratio as early as 4 weeks of nitrosamine administration. The decrease of RNA/protein ratio and ultrastructurally observed loss of ribosomes from the ER, following nitrosamine administration, correlate with a decrease of photometric response of microsomal suspensions to the sulphydryl probe, p-chloromercuribenzoate. While azo-dye-reductase activity is higher in untreated rats than in untreated guinea-pigs, feeding 3′-Me-DAB for 6 weeks brings about a 76% decrease in the rat, but no significant decrease in the guinea-pig, which is refractory to azo-dye carcinogenesis. Thus, the ability of the liver to inactivate the dye is greatly decreased in the rat, but not in the guinea-pig, as administration progresses toward the threshold dose for tumorigenesis. On the other hand, constitutive levels of nitrosamine dealkylase are identical in the 2 species and remain essentially unchanged following administration of DEN for 10 weeks. Inasmuch as nitrosamine dealkylation represents activating metabolism, this provides a rationale for the comparable susceptibility of the rat and guinea-pig to DEN carcinogenesis. Of the 2 enzymes in the 2 species, it is only azo-dye reductase in the guinea-pig which appears to be unregulated by glucose repression, since starvation brings about no change in this activity. Starvation-induced increase of azo-dye reductase in the rat is not influenced by administration of 3′-Me-DAB and only slightly by DEN. The starvation-induced increase of nitrosamine dealkylation is abolished, however, in both species by administration of DEN but only slightly decreased by 3′-Me-DAB.     

Bevacizumab treatment leads to observable morphological and metabolic changes in brain radiation necrosis

We investigated morphological and metabolic changes of radiation necrosis (RN) of the brain following bevacizumab (BEV) treatment by using neuroimaging. Nine patients with symptomatic RN, who had already been treated with radiation therapy for malignant brain tumors (6 glioblastomas, 1 anaplastic oligodendroglioma, and 2 metastatic brain tumors), were enrolled in this prospective clinical study. RN diagnosis was neuroradiologically determined with Gd-enhanced MRI and 11C-methionine positron emission tomography (MET-PET). RN clinical and radiological changes in MRI, magnetic resonance spectroscopy (MRS) and PET were assessed following BEV therapy. Karnofsky performance status scores improved in seven patients (77.8 %). Both volumes of the Gd-enhanced area and FLAIR-high area from MRI decreased in all patients after BEV therapy and the mean size reduction rates of the lesions were 80.0 and 65.0 %, respectively. MRS, which was performed in three patients, showed a significant reduction in Cho/Cr ratio after BEV therapy. Lesion/normal tissue (L/N) ratios in MET- and 11C-choline positron emission tomography (CHO-PET) decreased in 8 (89 %) and 9 patients (100 %), respectively, and the mean L/N ratio reduction rates were 24.4 and 60.7 %, respectively. BEV-related adverse effects of grade 1 or 2 (anemia, neutropenia and lymphocytopenia) occurred in three patients. These results demonstrated that BEV therapy improved RN both clinically and radiologically. BEV therapeutic mechanisms on RN have been suggested to be related not only to the effect on vascular permeability reduction by repairing the blood–brain barrier, but also to the effect on suppression of tissue biological activity, such as immunoreactions and inflammation.     

Contribution of hydrazines-derived alkyl radicals to cytotoxicity and transformation induced in normal c-myc-overexpressing mouse fibroblasts

Several hydrazine derivatives (HD) tested so far have pharmacological activities, but many also have toxic side effects, including carcinogenesis. Their toxicity has been ascribed to carbocations (via formation of azoxy intermediates), alkyl radicals or reactive oxygen species. Cytotoxicity and transformation by carbocations is widely accepted, but the role of alkyl radicals is still questioned. We have investigated the cytotoxicity of HD to mouse fibroblasts in three activation systems in which enhanced alkyl radical formation is demonstrated by electron spin resonance/spin-trapping. Cytotoxicity was assayed by inhibition of [3H-methyl]thymidine uptake into DNA of Balb/c 3T3 and/or Myc 9E fibroblasts (normal Balb/c 3T3 cells over-expressing the c-myc proto-oncogene). Based on the results obtained in the cytotoxicity assays we also investigated the transforming potential of procarbazine (PCZ) and methylhydrazine (MeH) activated by horseradish peroxidase (HRP) using the Myc 9E cell line, which aims at the activation of a second cooperating oncogene. Our results show that: (i) cytotoxicity of HD to mouse fibroblasts is increased by HRP activation of MeH, phenelzine and PCZ, which displayed enhanced alkyl radical formation, but not of 1,2-dimethylhydrazine (DMH), which did not produce increased alkyl radical formation under these conditions; (ii) cytotoxicity of neutrophil-activated MeH (producing a 10-fold higher concentration of methyl radicals), is more pronounced than DMH; (iii) MeH and DMH activated by prolonged auto-oxidation in 24-h incubations have comparable cytotoxicity and alkyl radical formation; and (iv) PCZ and MeH activation by HRP to alkyl radicals increased the transformation induced in Myc 9E cells. Taken together, our results strongly support a role for hydrazine-derived alkyl radicals in HD- induced cytotoxicity and cell transformation.      

The intensity of vanadium(V)-induced cytotoxicity and morphological transformation in BALB/3T3 cells is dependent on glutathione-mediated bioreduction to vanadium(IV)

Cytotoxicity and morphological transformation has been studiedin BALB/3T3 CI A31–1–1 mouse embryo cells for ammoniumvanadate [vanadium(V)] and vanadyl sulphate[vanadium(IV)] aloneor in combination with diethylmaleate(DEM), a cellular glutathione(GSH)-depleting agent.Cells exposed for 24 h to 10^–5 Mvanadium(V) alone or in combination with 3x10^–6M DEM showedthe characteristic hyperfine EPR signal of vanadium(IV), whichwas more obvious in the case of exposure to vanadium(V) alone.Thissuggests that the amount of vanadium(V) reduced to vanadium(IV)decreased in GSH-depleted cells. While vanadium(IV) at concentrationsof 3x10^–6M and 10^–5 M was not transforming in thecells, vanadium(V) showed neoplastic transforming activity (P<0.025 and P< 0.001 for the two doses, respectively) in comparisonto controls(vanadium unexposed cells). Cytotoxicity and morphologicaltransformation in cells exposed to vanadium(V) in combinationwith 3x^–6M DEM were significantly more intensive (P <0.005 and P < 0.01 for the two doses of vanadate tested)compared to the corresponding values observed in cells exposedto vanadium(V) alone.This suggests that the final transformingactivity response is dependent on the intracellular GSH-mediatedmechanism of reduction of vanadium(V) to vanadium (IV): (i)the extent to which vanadium(V) should be bioreduced to lesstoxic vanadium(IV) via intracellular GSH is a key point in determiningthe intensity of the observed neoplastic action; (ii) the carcinogenicpotential of vanadium(V) should be strictly dependent on itsintracellular persistence which could lead to changes in normalmetabolic patterns of vanadium(V) in the oxidized form due tolack of GSH-mediated reduction.     

The cytotoxicity of arsenic trioxide to normal hematopoietic progenitors and leukemic cells is dependent on their cell-cycle status

Arsenic trioxide (ATO) is a novel agent to treat acute promyelocytic leukemia (APL). ATO can degrade chimeric PML-RAR proteins and induce apoptosis in various cancer cells. However, its effects on primary hematopoietic CD34+ have not been examined. In this study, we compared the effects of ATO on HL60 leukemic cells and primary umbilical cord blood (UCB) CD34+ cells. HL60 cells and UCB CD34+ cells were cultured with different concentrations of ATO for up to three weeks and examined for changes of cell cycle. We found that ATO (< or = 5 microM) caused prolongation of G1/S and G2/M phase in a dose-dependent manner. The percentage of cells in G2/M increased significantly (from 8.6 to 53.8%). High-dose ATO (> or = 25 microM) caused non-specific cell death in HL60 cells without any changes in cell cycle. In contrast to HL60 cells, UCB CD34+ cells were more resistant to high-dose ATO and most ATO-resistant CD34+ cells remained in G0/G1 phase. Primary cells that were resistant to ATO were rich in CD34+ cells. We further show that the ATO resistance was not related to the expression of P-glycoprotein (MDR-1). Our results suggest that the resistance to ATO in primitive UCB CD34+ cells is most likely related to its cell-cycle status. These results could be useful to design treatments for non-APL malignancies and to enrich hematopoietic stem cells in clinically applicable settings.     

Cellular uptake and photosensitizing properties of hematoporphyrin di-ethers with similar chromatographic properties as the tumorlocalizing fraction of hematoporphyrin derivative

The di-methyl-, di-ethyl-, di-propyl-, di-normal butyl-, and di-iso-butyl-ethers of hematoporphyrin were synthesized and shown to possess chromatographic properties similar to those of the tumorlocalizing components of hematoporphyrin derivative (Hpd). The cellular uptake of these ethers, as well as their retention in cells during incubation with porphyrin free medium, increased with decreasing polarity and so did their efficiency in sensitizing cultured cells to photoinactivation. The least polar of the porphyrin ethers tested showed up to a 10-fold stronger efficiency in sensitizing cultured cells to photoinactivation than Hpd and Photofrin II (P II).     

Cellular and molecular mechanisms of metastasis as applied to carcinomatous meningitis

Cancer cells as well as bacteria metastasize to the subarachnoidal space (SAS) causing meningitis. Primary brain tumors, although not forming distant metastases, disseminate via the cerebrospinal fluid and occupy the meninges. The multistep process of cancer or bacterial dissemination is regulated through molecular crosstalk between invaders and host cells. Such crosstalks establish invasion-promoter and invasion-suppressor complexes. In carcinomatous and bacterial meningitis, the participation of host cells is prominent since leukocytes and inflammatory cytokines are the major determinants of malignancy. We propose a model in which bacterial breakdown products activate endothelial cells, a process leading to leukocyte extravasation. This initiates a cascade of inflammatory processes opening up the blood cerebrospinal fluid barrier and producing access for new invaders.